Comparative genomics of multidrug resistance-encoding IncA/C plasmids from commensal and pathogenic Escherichia coli from multiple animal sources.

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Claudia Fernández-Alarcón

Full Text Available Incompatibility group A/C (IncA/C plasmids have received recent attention for their broad host range and ability to confer resistance to multiple antimicrobial agents. Due to the potential spread of multidrug resistance (MDR phenotypes from foodborne pathogens to human pathogens, the dissemination of these plasmids represents a public health risk. In this study, four animal-source IncA/C plasmids isolated from Escherichia coli were sequenced and analyzed, including isolates from commercial dairy cows, pigs and turkeys in the U.S. and Chile. These plasmids were initially selected because they either contained the floR and tetA genes encoding for florfenicol and tetracycline resistance, respectively, and/or the bla(CMY-2 gene encoding for extended spectrum β-lactamase resistance. Overall, sequence analysis revealed that each of the four plasmids retained a remarkably stable and conserved backbone sequence, with differences observed primarily within their accessory regions, which presumably have evolved via horizontal gene transfer events involving multiple modules. Comparison of these plasmids with other available IncA/C plasmid sequences further defined the core and accessory elements of these plasmids in E. coli and Salmonella. Our results suggest that the bla(CMY-2 plasmid lineage appears to have derived from an ancestral IncA/C plasmid type harboring floR-tetAR-strAB and Tn21-like accessory modules. Evidence is mounting that IncA/C plasmids are widespread among enteric bacteria of production animals and these emergent plasmids have flexibility in their acquisition of MDR-encoding modules, necessitating further study to understand the evolutionary mechanisms involved in their dissemination and stability in bacterial populations.

Plasmid segregation mechanisms

DEFF Research Database (Denmark)

Ebersbach, G.; Gerdes, Kenn

2005-01-01

Bacterial plasmids encode partitioning (par) loci that ensure ordered plasmid segregation prior to cell division. par loci come in two types: those that encode actin-like ATPases and those that encode deviant Walker-type ATPases. ParM, the actin-like ATPase of plasmid R1, forms dynamic filaments...... that segregate plasmids paired at mid-cell to daughter cells. Like microtubules, ParM filaments exhibit dynamic instability (i.e., catastrophic decay) whose regulation is an important component of the DNA segregation process. The Walker box ParA ATPases are related to MinD and form highly dynamic, oscillating...... filaments that are required for the subcellular movement and positioning of plasmids. The role of the observed ATPase oscillation is not yet understood. However, we propose a simple model that couples plasmid segregation to ParA oscillation. The model is consistent with the observed movement...

Plasmid-Mediated Resistance to Thrombin-Induced Platelet Microbicidal Protein in Staphylococci: Role of the qacA Locus

OpenAIRE

Kupferwasser, Leon Iri; Skurray, Ronald A.; Brown, Melissa H.; Firth, Neville; Yeaman, Michael R.; Bayer, Arnold S.

1999-01-01

Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a small, cationic peptide released from rabbit platelets following thrombin stimulation. In vitro resistance to this peptide among strains of Staphylococcus aureus correlates with the survival advantage of such strains at sites of endothelial damage in humans as well as in experimental endovascular infections. The mechanisms involved in the phenotypic resistance of S. aureus to tPMP-1 are not fully delineated. The plasmid-encoded st...

Severity of nonbullous Staphylococcus aureus impetigo in children is associated with strains harboring genetic markers for exfoliative toxin B, Panton-Valentine leukocidin, and the multidrug resistance plasmid pSK41

NARCIS (Netherlands)

S. Koning (Sander); A.F. van Belkum (Alex); S.V. Snijders (Susan); J.L. Nouwen (Jan); M. op 't Veld; J.C. van der Wouden (Hans); C.M. Verduin (Cees); H.A. Verbrugh (Henri); W.B. van Leeuwen (Willem); L.W.A. van Suijlekom-Smit (Lisette)

2003-01-01

textabstractNonbullous impetigo is a common skin infection in children and is frequently caused by Staphylococcus aureus. Staphylococcal toxins and especially exfoliative toxin A are known mediators of bullous impetigo in children. It is not known whether this is also true for

Staphylococcus sciuri bacteriophages double-convert for staphylokinase and phospholipase, mediate interspecies plasmid transduction, and package mecA gene.

Science.gov (United States)

Zeman, M; Mašlaňová, I; Indráková, A; Šiborová, M; Mikulášek, K; Bendíčková, K; Plevka, P; Vrbovská, V; Zdráhal, Z; Doškař, J; Pantůček, R

2017-04-13

Staphylococcus sciuri is a bacterial pathogen associated with infections in animals and humans, and represents a reservoir for the mecA gene encoding methicillin-resistance in staphylococci. No S. sciuri siphophages were known. Here the identification and characterization of two temperate S. sciuri phages from the Siphoviridae family designated ϕ575 and ϕ879 are presented. The phages have icosahedral heads and flexible noncontractile tails that end with a tail spike. The genomes of the phages are 42,160 and 41,448 bp long and encode 58 and 55 ORFs, respectively, arranged in functional modules. Their head-tail morphogenesis modules are similar to those of Staphylococcus aureus ϕ13-like serogroup F phages, suggesting their common evolutionary origin. The genome of phage ϕ575 harbours genes for staphylokinase and phospholipase that might enhance the virulence of the bacterial hosts. In addition both of the phages package a homologue of the mecA gene, which is a requirement for its lateral transfer. Phage ϕ879 transduces tetracycline and aminoglycoside pSTS7-like resistance plasmids from its host to other S. sciuri strains and to S. aureus. Furthermore, both of the phages efficiently adsorb to numerous staphylococcal species, indicating that they may contribute to interspecies horizontal gene transfer.

A novel pAA virulence plasmid encoding toxins and two distinct variants of the fimbriae of enteroaggregative Escherichia coli

DEFF Research Database (Denmark)

Jønsson, Rie; Struve, Carsten; Boll, Erik J.

2017-01-01

phylogenetically distinct, strains harboring the major pilin subunits from both AAF/III and AAF/V. Whole-genome and plasmid sequencing revealed that in these six strains the agg3A and agg5A genes were located on a novel pAA plasmid variant. Moreover, the plasmid also encoded several other virulence genes including...... some not previously found on pAA plasmids. Thus, this plasmid endows the host strains with a remarkably high number of EAEC associated virulence genes hereby likely promoting strain pathogenicity....

pEVL: A Linear Plasmid for Generating mRNA IVT Templates With Extended Encoded Poly(A Sequences

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Alexandra E Grier

2016-01-01

Full Text Available Increasing demand for large-scale synthesis of in vitro transcribed (IVT mRNA is being driven by the increasing use of mRNA for transient gene expression in cell engineering and therapeutic applications. An important determinant of IVT mRNA potency is the 3′ polyadenosine (poly(A tail, the length of which correlates with translational efficiency. However, present methods for generation of IVT mRNA rely on templates derived from circular plasmids or PCR products, in which homopolymeric tracts are unstable, thus limiting encoded poly(A tail lengths to ≃120 base pairs (bp. Here, we have developed a novel method for generation of extended poly(A tracts using a previously described linear plasmid system, pJazz. We find that linear plasmids can successfully propagate poly(A tracts up to ≃500 bp in length for IVT mRNA production. We then modified pJazz by removing extraneous restriction sites, adding a T7 promoter sequence upstream from an extended multiple cloning site, and adding a unique type-IIS restriction site downstream from the encoded poly(A tract to facilitate generation of IVT mRNA with precisely defined encoded poly(A tracts and 3′ termini. The resulting plasmid, designated pEVL, can be used to generate IVT mRNA with consistent defined lengths and terminal residue(s.

A Ti plasmid-encoded enzyme required for degradation of mannopine is functionally homologous to the T-region-encoded enzyme required for synthesis of this opine in crown gall tumors.

OpenAIRE

Kim, K S; Chilton, W S; Farrand, S K

1996-01-01

The mocC gene encoded by the octopine/mannityl opine-type Ti plasmid pTi15955 is related at the nucleotide sequence level to mas1' encoded by the T region of this plasmid. While Mas1 is required for the synthesis of mannopine (MOP) by crown gall tumor cells, MocC is essential for the utilization of MOP by Agrobacterium spp. A cosmid clone of pTi15955, pYDH208, encodes mocC and confers the utilization of MOP on strain NT1 and on strain UIA5, a derivative of NT1 lacking the 450-kb cryptic plasm...

The Plasmid Complement of Lactococcus lactis UC509.9 Encodes Multiple Bacteriophage Resistance Systems

Science.gov (United States)

Ainsworth, Stuart; Mahony, Jennifer

2014-01-01

Lactococcus lactis subsp. cremoris strains are used globally for the production of fermented dairy products, particularly hard cheeses. Believed to be of plant origin, L. lactis strains that are used as starter cultures have undergone extensive adaptation to the dairy environment, partially through the acquisition of extrachromosomal DNA in the form of plasmids that specify technologically important phenotypic traits. Here, we present a detailed analysis of the eight plasmids of L. lactis UC509.9, an Irish dairy starter strain. Key industrial phenotypes were mapped, and genes that are typically associated with lactococcal plasmids were identified. Four distinct, plasmid-borne bacteriophage resistance systems were identified, including two abortive infection systems, AbiB and AbiD1, thereby supporting the observed phage resistance of L. lactis UC509.9. AbiB escape mutants were generated for phage sk1, which were found to carry mutations in orf6, which encodes the major capsid protein of this phage. PMID:24814781

A Ti plasmid-encoded enzyme required for degradation of mannopine is functionally homologous to the T-region-encoded enzyme required for synthesis of this opine in crown gall tumors.

Science.gov (United States)

Kim, K S; Chilton, W S; Farrand, S K

1996-06-01

The mocC gene encoded by the octopine/mannityl opine-type Ti plasmid pTi15955 is related at the nucleotide sequence level to mas1' encoded by the T region of this plasmid. While Mas1 is required for the synthesis of mannopine (MOP) by crown gall tumor cells, MocC is essential for the utilization of MOP by Agrobacterium spp. A cosmid clone of pTi15955, pYDH208, encodes mocC and confers the utilization of MOP on strain NT1 and on strain UIA5, a derivative of NT1 lacking the 450-kb cryptic plasmid pAtC58. NT1 or UIA5 harboring pYDH208 with an insertion mutation in mocC failed to utilize MOP as the sole carbon source. Plasmid pSa-C, which encodes only mocC, complemented this mutation in both strains. This plasmid also was sufficient to confer utilization of MOP on NT1 but not on UIA5. Computer analysis showed that MocC is related at the amino acid sequence level to members of the short-chain alcohol dehydrogenase family of oxidoreductases. Lysates prepared from Escherichia coli cells expressing mocC contained an enzymatic activity that oxidizes MOP to deoxyfructosyl glutamine (santhopine [SOP]) in the presence of NAD+. The reaction catalyzed by the MOP oxidoreductase is reversible; in the presence of NADH, the enzyme reduced SOP to MOP. The apparent Km values of the enzyme for MOP and SOP were 6.3 and 1.2 mM, respectively. Among analogs of MOP tested, only N-1-(1-deoxy-D-lyxityl)-L-glutamine and N-1-(1-deoxy-D-mannityl)-L-asparagine served as substrates for MOP oxidoreductase. These results indicate that mocC encodes an oxidoreductase that, as an oxidase, is essential for the catabolism of MOP. The reductase activity of this enzyme is precisely the reaction ascribed to its T-region-encoded homolog, Mas1, which is responsible for biosynthesis of mannopine in crown gall tumors.

Translational control and differential RNA decay are key elements regulating postsegregational expression of the killer protein encoded by the parB locus of plasmid R1

DEFF Research Database (Denmark)

Gerdes, K; Helin, K; Christensen, O W

1988-01-01

The parB locus of plasmid R1, which mediates plasmid stability via postsegregational killing of plasmid-free cells, encodes two genes, hok and sok. The hok gene product is a potent cell-killing protein. The hok gene is regulated at the translational level by the sok gene-encoded repressor, a small...

Evaluation of phenotypic and genotypic methods for epidemiological typing of Staphylococcus aureus isolates from bovine mastitis in Denmark

DEFF Research Database (Denmark)

Aarestrup, Frank Møller; Wegener, H. C.; Rosdahl, V. T.

1995-01-01

The value of five different typing methods (antibiogram typing, biotyping, phage typing, plasmid profiling and restriction fragment length polymorphism of the gene encoding 16S and 23S ribosomal RNA (ribotyping)), in discriminating 105 Staphylococcus aureus strains from bovine milk samples obtained....... The combination of phage, bio- or ribotyping or all three methods in combination are considered to be an efficient combination of typing methods for epidemiological investigation of S. aureus mastitis....

The multidrug resistance IncA/C transferable plasmid encodes a novel domain-swapped dimeric protein-disulfide isomerase.

Science.gov (United States)

Premkumar, Lakshmanane; Kurth, Fabian; Neyer, Simon; Schembri, Mark A; Martin, Jennifer L

2014-01-31

The multidrug resistance-encoding IncA/C conjugative plasmids disseminate antibiotic resistance genes among clinically relevant enteric bacteria. A plasmid-encoded disulfide isomerase is associated with conjugation. Sequence analysis of several IncA/C plasmids and IncA/C-related integrative and conjugative elements (ICE) from commensal and pathogenic bacteria identified a conserved DsbC/DsbG homolog (DsbP). The crystal structure of DsbP reveals an N-terminal domain, a linker region, and a C-terminal catalytic domain. A DsbP homodimer is formed through domain swapping of two DsbP N-terminal domains. The catalytic domain incorporates a thioredoxin-fold with characteristic CXXC and cis-Pro motifs. Overall, the structure and redox properties of DsbP diverge from the Escherichia coli DsbC and DsbG disulfide isomerases. Specifically, the V-shaped dimer of DsbP is inverted compared with EcDsbC and EcDsbG. In addition, the redox potential of DsbP (-161 mV) is more reducing than EcDsbC (-130 mV) and EcDsbG (-126 mV). Other catalytic properties of DsbP more closely resemble those of EcDsbG than EcDsbC. These catalytic differences are in part a consequence of the unusual active site motif of DsbP (CAVC); substitution to the EcDsbC-like (CGYC) motif converts the catalytic properties to those of EcDsbC. Structural comparison of the 12 independent subunit structures of DsbP that we determined revealed that conformational changes in the linker region contribute to mobility of the catalytic domain, providing mechanistic insight into DsbP function. In summary, our data reveal that the conserved plasmid-encoded DsbP protein is a bona fide disulfide isomerase and suggest that a dedicated oxidative folding enzyme is important for conjugative plasmid transfer.

Chromosomal and plasmid-encoded factors of Shigella flexneri induce secretogenic activity ex vivo.

Directory of Open Access Journals (Sweden)

Christina S Faherty

Full Text Available Shigella flexneri is a Gram-negative, facultative intracellular pathogen that causes millions of cases of watery or bloody diarrhea annually, resulting in significant global mortality. Watery diarrhea is thought to arise in the jejunum, and subsequent bloody diarrhea occurs as a result of invasion of the colonic epithelium. Previous literature has demonstrated that Shigella encodes enterotoxins, both chromosomally and on the 220 kilobase virulence plasmid. The ShigellaEnterotoxins 1 and 2 (ShET1 and ShET2 have been shown to increase water accumulation in the rabbit ileal loop model. In addition, these toxins increase the short circuit current in rabbit tissue mounted in Ussing chambers, which is a model for the ion exchange that occurs during watery diarrhea. In this study, we sought to validate the use of mouse jejunum in Ussing chamber as an alternative, more versatile model to study bacterial pathogenesis. In the process, we also identified enterotoxins in addition to ShET1 and ShET2 encoded by S. flexneri. Through analysis of proteins secreted from wildtype bacteria and various deletion mutants, we have identified four factors responsible for enterotoxin activity: ShET1 and Pic, which are encoded on the chromosome; ShET2 (encoded by sen or ospD3, which requires the type-III secretion system for secretion; and SepA, an additional factor encoded on the virulence plasmid. The use of mouse jejunum serves as a reliable and reproducible model to identify the enterotoxins elaborated by enteric bacteria. Moreover, the identification of all Shigella proteins responsible for enterotoxin activity is vital to our understanding of Shigella pathogenicity and to our success in developing safe and effective vaccine candidates.

Crystallization and preliminary X-ray crystallographic studies on the parD-encoded protein Kid from Escherichia coli plasmid R1

NARCIS (Netherlands)

Hargreaves, D.; Giraldo, R.; Santos-Sierra, S.; Boelens, R.; Rice, D.W.; Díaz Orejas, R.; Rafferty, J.B.

2002-01-01

DNA replication in Escherichia coli and therefore bacterial proliferation relies upon the efficient functioning of the DnaB helicase. The toxin protein Kid from the plasmid-stability system parD encoded on plasmid R1 of E. coli is thought to target and block DnaB-dependent DNA replication. The

Antimetastatic effect of PSK, a protein-bound polysaccharide, against the B16-BL6 mouse melanoma.

Science.gov (United States)

Matsunaga, K; Ohhara, M; Oguchi, Y; Iijima, H; Kobayashi, H

1996-01-01

We examined the effect of PSK, a protein-bound polysaccharide, upon in vivo metastasis and in vitro invasion of the B16-BL6 mouse melanoma cells. (1) PSK suppressed in vivo artificial and spontaneous lung metastases of B16-BL6 in C57BL/6 mice. (2) PSK in a dose-dependent fashion suppressed in vitro invasion and chemotaxis of the tumor cells using filters coated with a reconstituted basement membrane. (3) PSK had little effect on DNA synthesis in tumor cells in vitro, but suppressed tumor cell adhesion to, degradation of, and haptotaxis to components of the basement membrane. (4) PSK suppressed the binding of tumor cells to components of the basement membrane. These findings suggest that PSK may suppress metastasis through inhibition of tumor cell invasion and that this effect is the result of interactions between PSK and components of the basement membrane.

A plasmid-encoded nicotinamidase (PncA) is essential for infectivity of Borrelia burgdorferi in a mammalian host.

Science.gov (United States)

Purser, Joye E; Lawrenz, Matthew B; Caimano, Melissa J; Howell, Jerrilyn K; Radolf, Justin D; Norris, Steven J

2003-05-01

Borrelia burgdorferi, a spirochaete that causes Lyme borreliosis, contains 21 linear and circular plasmids thought to be important for survival in mammals or ticks. Our results demonstrate that the gene BBE22 encoding a nicotinamidase is capable of replacing the requirement for the 25 kb linear plasmid lp25 during mammalian infection. Transformation of B. burgdorferi lacking lp25 with a shuttle vector containing the lp25 gene BBE22 (pBBE22) restored infectivity in mice to a level comparable to that of wild-type Borrelia. This complementation also restored the growth and host adaptation of lp25-B. burgdorferi in dialysis membrane chambers (DMCs) implanted in rats. A single Cys to Ala conversion at the putative active site of BBE22 abrogated the ability of pBBE22 to re-establish infectivity or growth in DMCs. Additional Salmonella typhimurium complementation studies and enzymatic analysis demonstrated that the BBE22 gene product has nicotinamidase activity and is most probably required for the biosynthesis of NAD. These results indicate that some plasmid-encoded products fulfil physiological functions required in the enzootic cycle of pathogenic Borrelia.

Dissemination of plasmid-encoded AmpC β-lactamases in antimicrobial resistant Salmonella serotypes originating from humans, pigs and the swine environment.

Science.gov (United States)

Keelara, Shivaramu; Thakur, Siddhartha

2014-09-17

The aim of this study was to characterize and determine the inter-serovar exchange of AmpC β-lactamase conferring plasmids isolated from humans, pigs and the swine environment. Plasmids isolated from a total of 21 antimicrobial resistant (AMR) Salmonella isolates representing human clinical cases (n=6), pigs (n=6) and the swine farm environment (n=9) were characterized by replicon typing and restriction digestion, inter-serovar transferability by conjugation, and presence of AmpC β-lactamase enzyme encoding gene blaCMY-2 by southern hybridization. Based on replicon typing, the majority (17/21, 81%) of the plasmids belonged to the I1-Iγ Inc group and were between 70 and 103kb. The potential for inter-serovar plasmid transfer was further confirmed by the PCR detection of AMR genes on the plasmids isolated from trans-conjugants. Plasmids from Salmonella serovars Anatum, Ouakam, Johannesburg and Typhimurium isolated from the same cohort of pigs and their environment and S. Heidelberg from a single human clinical isolate had identical plasmids based on digestion with multiple restriction enzymes (EcoRI, HindIII and PstI) and southern blotting. We demonstrated likely horizontal inter-serovar exchange of plasmid-encoding AmpC β-lactamases resistance among MDR Salmonella serotypes isolated from pigs, swine farm environment and clinical human cases. This study provides valuable information on the role of the swine farm environment and by extension other livestock farm environments, as a potential reservoir of resistant bacterial strains that potentially transmit resistance determinants to livestock, in this case, swine, humans and possibly other hosts by horizontal exchange of plasmids. Copyright © 2014 Elsevier B.V. All rights reserved.

Effect of PS-K on long-term survival of primary lung cancer patients treated with radiation

International Nuclear Information System (INIS)

Okazaki, Atsushi; Nakajima, Nobuaki; Hayakawa, Kazushige; Saito, Yoshihiro; Mitomo, Osamu; Niibe, Hideo

1984-01-01

The effect of PS-K on long-term survival of primary lung cancer patients irradiated over 60 Gy through 1977 to 1982 was studied. PS-K was administrated orally 3.0 g, daily or intermittently in the pattern of 2 weeks per a month on patients of positive PPD skin test. All cases irradiated over 60 Gy were 174 (Group A) containing 62 cases with PS-K (Group B) and 112 cases without PS-K (Group C). Of group B, 44 cases were administrated within a month after curative irradiation (Group B1), 7 cases were administrated on time maintaining long-term good condition after irradiation (Group B2) and 11 cases were administrated after recognition of recurrence or metastasis (Group B3). Following results were obtained. 1. Obvious prolongation of survivals was recognized in the patients with PS-K after irradiation. (1) The cumulative 5 years survival rates of Group A, B 1 and C were 11.0%, 28.8% and 4.8%, respectively. (2) The cumulative 5 years survival rates of stage I,11 were 45.7% with PS-K and 10.7% without PS-K. (3) The cumulative 5 years survival rates of 21 cases matched age, sex, stage, histological type and tumor dose with and without PS-K were 37.8% and 7.2%. (4) In Group B 2, 5 cases out of 7 cases have been alived, but in Group B 3, satisfactory long-term survivals ware not obtained. 2. The necessary conditions which obtain long-term survival with PS-K were thought to be follows. One is that the tumor is brought almost to vanish by irradiation. Another is that the condition of host is superior to that of tumor in host-tumor relationship. 3. The possibility of intermittent administration of PS-K was suggested. (author)

Regulation of the pT181 encoded tetracycline resistance gene in Straphylococcus aureus

International Nuclear Information System (INIS)

Mojumdar, M.

1986-01-01

pT181 is a naturally-occurring 4437 basepair (bp) plasmid isolated from Staphylococcus aureus which encodes inducible resistance to tetracycline (Tc). The DNA sequence data has identified three open reading frames (ORFs). The largest ORF B, has been found to be responsible for the Tc resistance phenotype of pT181. Since most Tc resistance systems appear to be regulated by an effector protein and a repressor protein, several Bal 31 deletion mutants of pT181 were constructed and analyzed in an effort to identify the elements involved in Tc resistance. Two transcomplementing groups of mutants were identified within the tet gene. The mechanism of Tc resistance was studied by assaying the accumulation of [7- 3 H] Tc by Tc sensitive cells, and uninduced and induced pT181-containing cells. A sharp decrease in accumulation of the drug after an initial increase was observed in Tc induced pT181-containing cells. In vivo labeling of Bacillus subtilis minicells containing pT181 was performed with 35 S-methionine to identify the polypeptide product of the tet gene. A Tc-inducible protein having a molecular weight of approximately 50,000 daltons was detected only in B. subtilis minicells carrying pT181. Cell fractionation studies of S. aureus cells with and without pT181 showed that an approximately 28,000 daltons Tc-inducible protein was present in membranes of pT181 containing cells. The amount of TET protein in Tc induced minicells was about fifteen-fold higher than that in uninduced minicells. RNA prepared from stationary phase cells analyzed by Northern blot hybridization showed that the steady-state level of the tet mRNA in induced pT181-containing cells was bout four-fold higher than that in uninduced pT181-containing cells. When RNA synthesis was blocked with rifampicin, tet mRAN was found to be much more stable in Tc induced cells as compared to that in uninduced cells over a 30 min period

Elucidating the crucial role of poly N-acetylglucosamine from Staphylococcus aureus in cellular adhesion and pathogenesis.

Science.gov (United States)

Lin, Mei Hui; Shu, Jwu Ching; Lin, Li Ping; Chong, Kowit Yu; Cheng, Ya Wen; Du, Jia Fu; Liu, Shih-Tung

2015-01-01

Staphylococcus aureus is an important pathogen that forms biofilms on the surfaces of medical implants. Biofilm formation by S. aureus is associated with the production of poly N-acetylglucosamine (PNAG), also referred to as polysaccharide intercellular adhesin (PIA), which mediates bacterial adhesion, leading to the accumulation of bacteria on solid surfaces. This study shows that the ability of S. aureus SA113 to adhere to nasal epithelial cells is reduced after the deletion of the ica operon, which contains genes encoding PIA/PNAG synthesis. However, this ability is restored after a plasmid carrying the entire ica operon is transformed into the mutant strain, S. aureus SA113Δica, showing that the synthesis of PIA/PNAG is important for adhesion to epithelial cells. Additionally, S. carnosus TM300, which does not produce PIA/PNAG, forms a biofilm and adheres to epithelial cells after the bacteria are transformed with a PIA/PNAG-expressing plasmid, pTXicaADBC. The adhesion of S. carnosus TM300 to epithelial cells is also demonstrated by adding purified exopolysaccharide (EPS), which contains PIA/PNAG, to the bacteria. In addition, using a mouse model, we find that the abscess lesions and bacterial burden in lung tissues is higher in mice infected with S. aureus SA113 than in those infected with the mutant strain, S. aureus SA113Δica. The results indicate that PIA/PNAG promotes the adhesion of S. aureus to human nasal epithelial cells and lung infections in a mouse model. This study elucidates a mechanism that is important to the pathogenesis of S. aureus infections.

Effect of Promoter Region Mutations and mgrA Overexpression on Transcription of norA, Which Encodes a Staphylococcus aureus Multidrug Efflux Transporter

OpenAIRE

Kaatz, Glenn W.; Thyagarajan, Rama V.; Seo, Susan M.

2005-01-01

NorA is a Staphylococcus aureus multidrug transporter that confers resistance to structurally distinct compounds. The MgrA global regulatory protein is reported to augment norA expression when mgrA is overexpressed from an undefined plasmid-based promoter. Further details about norA regulatory mechanisms are scant. A chromosomal norA::lacZ transcriptional fusion was constructed in different S. aureus strains, and allele replacement was used to define the relevance of promoter region sequences...

1H, 13C and 15N backbone resonance assignment of the arsenate reductase from Staphylococcus aureus in its reduced state

NARCIS (Netherlands)

Jacobs, D.M.; Messens, J.; Wechselberger, R.W.|info:eu-repo/dai/nl/304829005; Brosens, E.; Willem, R.; Wyns, L.; Martins, J.C.

2001-01-01

In S. aureus, resistance to the metal(III)oxyanions arsenite As(III)O− 2 and antimonite Sb(III)O− 2 is mediated by two proteins, ArsB and ArsR, encoded in the ars operon of plasmid pI258 (Silver, 1999). ArsR acts as the transcription repressor, which is de-repressed in the presence of intracellular

The MazEF Toxin-Antitoxin System Alters the β-Lactam Susceptibility of Staphylococcus aureus.

Directory of Open Access Journals (Sweden)

Christopher F Schuster

Full Text Available Toxin-antitoxin (TA systems are genetic elements of prokaryotes which encode a stable toxin and an unstable antitoxin that can counteract toxicity. TA systems residing on plasmids are often involved in episomal maintenance whereas those on chromosomes can have multiple functions. The opportunistic pathogen Staphylococcus aureus possesses at least four different families of TA systems but their physiological roles are elusive. The chromosomal mazEF system encodes the RNase toxin MazF and the antitoxin MazE. In the light of ambiguity regarding the cleavage activity, we here verify that MazF specifically targets UACAU sequences in S. aureus in vivo. In a native strain background and under non-stress conditions, cleavage was observed in the absence or presence of mazE. Transcripts of spa (staphylococcal protein A and rsbW (anti-σB factor were cut, but translational reporter fusions indicated that protein levels of the encoded products were unaffected. Despite a comparable growth rate as the wild-type, an S. aureus mazEF deletion mutant was more susceptible to β-lactam antibiotics, which suggests that further genes, putatively involved in the antibiotic stress response or cell wall synthesis or turnover, are controlled by this TA system.

Putative DNA-dependent RNA polymerase in Mitochondrial Plasmid of Paramecium caudatum Stock GT704

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Trina Ekawati Tallei

2015-10-01

Full Text Available Mitochondria of Paramecium caudatum stock GT704 has a set of four kinds of linear plasmids with sizes of 8.2, 4.1, 2.8 and 1.4 kb. The plasmids of 8.2 and 2.8 kb exist as dimers consisting of 4.1- and 1.4-kb monomers, respectively. The plasmid 2.8 kb, designated as pGT704-2.8, contains an open reading frame encodes for putative DNA-dependent RNA polymerase (RNAP. This study reveals that this RNAP belongs to superfamily of DNA/RNA polymerase and family of T7/T3 single chain RNA polymerase and those of mitochondrial plasmid of fungi belonging to Basidiomycota and Ascomycota. It is suggested that RNAP of pGT704-2.8 can perform transcription without transcription factor as promoter recognition. Given that only two motifs were found, it could not be ascertained whether this RNAP has a full function independently or integrated with mtDNA in carrying out its function.

Plasmid and chromosome partitioning: surprises from phylogeny

DEFF Research Database (Denmark)

Gerdes, Kenn; Møller-Jensen, Jakob; Bugge Jensen, Rasmus

2000-01-01

Plasmids encode partitioning genes (par) that are required for faithful plasmid segregation at cell division. Initially, par loci were identified on plasmids, but more recently they were also found on bacterial chromosomes. We present here a phylogenetic analysis of par loci from plasmids and chr...

Acetic acid increases the phage-encoded enterotoxin A expression in Staphylococcus aureus

Directory of Open Access Journals (Sweden)

da Silva Ayla

2010-05-01

Full Text Available Abstract Background The effects of acetic acid, a common food preservative, on the bacteriophage-encoded enterotoxin A (SEA expression and production in Staphylococcus aureus was investigated in pH-controlled batch cultures carried out at pH 7.0, 6.5, 6.0, 5.5, 5.0, and 4.5. Also, genomic analysis of S. aureus strains carrying sea was performed to map differences within the gene and in the temperate phage carrying sea. Results The sea expression profile was similar from pH 7.0 to 5.5, with the relative expression peaking in the transition between exponential and stationary growth phase and falling during stationary phase. The levels of sea mRNA were below the detection limit at pH 5.0 and 4.5, confirmed by very low SEA levels at these pH values. The level of relative sea expression at pH 6.0 and 5.5 were nine and four times higher, respectively, in the transitional phase than in the exponential growth phase, compared to pH 7.0 and pH 6.5, where only a slight increase in relative expression in the transitional phase was observed. Furthermore, the increase in sea expression levels at pH 6.0 and 5.5 were observed to be linked to increased intracellular sea gene copy numbers and extracellular sea-containing phage copy numbers. The extracellular SEA levels increased over time, with highest levels produced at pH 6.0 in the four growth phases investigated. Using mitomycin C, it was verified that SEA was at least partially produced as a consequence of prophage induction of the sea-phage in the three S. aureus strains tested. Finally, genetic analysis of six S. aureus strains carrying the sea gene showed specific sea phage-groups and two versions of the sea gene that may explain the different sea expression and production levels observed in this study. Conclusions Our findings suggest that the increased sea expression in S. aureus caused by acetic acid induced the sea-encoding prophage, linking SEA production to the lifecycle of the phage.

Identification, characterization and preliminary X-ray diffraction analysis of the rolling-circle replication initiator protein from plasmid pSTK1

International Nuclear Information System (INIS)

Carr, Stephen B.; Mecia, Lauren B.; Phillips, Simon E. V.; Thomas, Christopher D.

2013-01-01

A proteolytically stable fragment of a plasmid replication initiation protein from the thermophile G. stearothermophilus has been biochemically characterized, crystallized and diffraction data collected to a resolution of 2.5 Å. Antibiotic resistance in bacterial pathogens poses an ever-increasing risk to human health. In antibiotic-resistant strains of Staphylococcus aureus this resistance often resides in extra-chromosomal plasmids, such as those of the pT181 family, which replicate via a rolling-circle mechanism mediated by a plasmid-encoded replication initiation protein. Currently, there is no structural information available for the pT181-family Rep proteins. Here, the crystallization of a catalytically active fragment of a homologous replication initiation protein from the thermophile Geobacillus stearothermophilus responsible for the replication of plasmid pSTK1 is reported. Crystals of the RepSTK1 fragment diffracted to a resolution of 2.5 Å and belonged to space group P2 1 2 1 2 1

Typing of Panton-Valentine Leukocidin-encoding Phages and lukSF-PV Gene Sequence Variation in Staphylococcus aureus from China

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Huanqiang Zhao

2016-08-01

Full Text Available Panton-Valentine leucocidin (PVL, encoded by lukSF-PV genes, a bi-component and pore-forming toxin, is carried by different staphylococcal bacteriophages. The prevalence of PVL in Staphylococcus aureus (S. aureus have been reported around the globe. However, the data on PVL-encoding phage types, lukSF-PV gene variation and chromosomal phage insertion sites for PVL-positive S. aureus are limited, especially in China. In order to obtain a more complete understanding of the molecular epidemiology of PVL-positive S. aureus, an integrated and modified PCR-based scheme was applied to detect the PVL-encoding phage types. Phage insertion locus and the lukSF-PV variant were determined by PCR and sequencing. Meanwhile, the genetic background was characterized by staphylococcal cassette chromosome mec (SCCmec typing, staphylococcal protein A (spa gene polymorphisms typing, pulsed-field gel electrophoresis (PFGE typing, accessory gene regulator (agr locus typing and multilocus sequence typing (MLST. Seventy eight (78/1175, 6.6% isolates possessed the lukSF-PV genes and 59.0% (46/78 of PVL-positive strains belonged to CC59 lineage. Eight known different PVL-encoding phage types were detected, and Φ7247PVL/ΦST5967PVL (n=13 and ΦPVL (n=12 were the most prevalent among them. While 25 (25/78, 32.1% isolates, belonging to ST30 and ST59 clones, were unable to be typed by the modified PCR-based scheme. Single nucleotide polymorphisms (SNPs were identified at five locations in the lukSF-PV genes, two of which were non-synonymous. Maximum-likelihood tree analysis of attachment sites sequences detected six SNP profiles for attR and eight for attL, respectively. In conclusion, the PVL-positive S. aureus mainly harbored Φ7247PVL/ΦST5967PVL and ΦPVL in the regions studied. lukSF-PV gene sequences, PVL-encoding phages and phage insertion locus generally varied with lineages. Moreover, PVL-positive clones that have emerged worldwide likely carry distinct phages.

Antibiotic resistance plasmids of Staphylococcus aureus and their clinical importance

International Nuclear Information System (INIS)

Lacey, R.W.

1975-01-01

A variety of plasmids were isolated physically, and most antibiotic resistance is thought to be plasmid mediated. A number of characters (e.g., resistance to erythromycin or methicillin, and production of pigment) are determined by genes that do not give clear indications of either plasmid or chromosomal location. Although the formation of a particular plasmid is probably, even in bacterial terms, a very rare event, once formed such an element can spread rapidly among the bacterial population. The spectacular increase in the incidence of penicillinase-producing hospital strains in the late 1940's could have been due in part to this process. Evidence is stronger, however, for the intercell transfer of recently isolated plasmids coding for resistance to fusidic acid (and penicillinase production), or for neomycin, or for tetracycline resistance. Study of bacterial plasmids can resolve fundamental biochemical problems, and give some insight into the life of the cell at the molecular level. But the immediate application of the study of staphylococcal plasmids may be directed towards improving the effectiveness of antibiotic therapy. The most important aspect of future anti-staphylococcal chemotherapy should thus be the limitation of the use of antibiotics, particularly for application to the skin and nose. (U.S.)

Elucidating the crucial role of poly N-acetylglucosamine from Staphylococcus aureus in cellular adhesion and pathogenesis.

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Mei Hui Lin

Full Text Available Staphylococcus aureus is an important pathogen that forms biofilms on the surfaces of medical implants. Biofilm formation by S. aureus is associated with the production of poly N-acetylglucosamine (PNAG, also referred to as polysaccharide intercellular adhesin (PIA, which mediates bacterial adhesion, leading to the accumulation of bacteria on solid surfaces. This study shows that the ability of S. aureus SA113 to adhere to nasal epithelial cells is reduced after the deletion of the ica operon, which contains genes encoding PIA/PNAG synthesis. However, this ability is restored after a plasmid carrying the entire ica operon is transformed into the mutant strain, S. aureus SA113Δica, showing that the synthesis of PIA/PNAG is important for adhesion to epithelial cells. Additionally, S. carnosus TM300, which does not produce PIA/PNAG, forms a biofilm and adheres to epithelial cells after the bacteria are transformed with a PIA/PNAG-expressing plasmid, pTXicaADBC. The adhesion of S. carnosus TM300 to epithelial cells is also demonstrated by adding purified exopolysaccharide (EPS, which contains PIA/PNAG, to the bacteria. In addition, using a mouse model, we find that the abscess lesions and bacterial burden in lung tissues is higher in mice infected with S. aureus SA113 than in those infected with the mutant strain, S. aureus SA113Δica. The results indicate that PIA/PNAG promotes the adhesion of S. aureus to human nasal epithelial cells and lung infections in a mouse model. This study elucidates a mechanism that is important to the pathogenesis of S. aureus infections.

Plasmid profiles as indicators of the source of contamination of Staphylococcus aureus endemic within poultry processing plants.

Science.gov (United States)

Dodd, C E; Chaffey, B J; Waites, W M

1988-06-01

A total of 530 strains of Staphylococcus aureus were isolated from the defeathering machinery of a chicken processing plant and from neck skin samples of carcasses at different stages of processing in two visits 4 weeks apart. Eleven different plasmid profiles were detected in the isolates, eight being common to both visits. The plasmid profiles of the strains forming the majority of the population on the freshly slaughtered birds were rarely present in the strains isolated from the pluckers (except at the entry to the first plucker) and were present in only a small proportion of the strains isolated from carcasses after plucking. However, the profiles from the strains isolated from the pluckers on both visits were different from those forming the majority of the population on the incoming birds but formed the major part of the carcass flora after plucking, suggesting that such strains were endemic. These strains were found as a small proportion of the isolates made from the incoming birds, suggesting that this was the route by which the endemic strains were introduced into the plant. Such endemic strains exhibited a clumping growth, even in liquid shake culture, which may have made it easier for them to become established on the pluckers and to resist cleaning and disinfection. This clumping phenotype was correlated with the presence of a 7.5-megadalton plasmid.

Contribution of plasmid-encoded peptidase S8 (PrtP) to adhesion and transit in the gut of Lactococcus lactis IBB477 strain.

Science.gov (United States)

Radziwill-Bienkowska, Joanna Maria; Robert, Véronique; Drabot, Karolina; Chain, Florian; Cherbuy, Claire; Langella, Philippe; Thomas, Muriel; Bardowski, Jacek Karol; Mercier-Bonin, Muriel; Kowalczyk, Magdalena

2017-07-01

The ability of Lactococcus lactis to adhere to the intestinal mucosa can potentially prolong the contact with the host, and therefore favour its persistence in the gut. In the present study, the contribution of plasmid-encoded factors to the adhesive and transit properties of the L. lactis subsp. cremoris IBB477 strain was investigated. Plasmid-cured derivatives as well as deletion mutants were obtained and analysed. Adhesion tests were performed using non-coated polystyrene plates, plates coated with mucin or fibronectin and mucus-secreting HT29-MTX intestinal epithelial cells. The results indicate that two plasmids, pIBB477a and b, are involved in adhesion of the IBB477 strain. One of the genes localised on plasmid pIBB477b (AJ89_14230), which encodes cell wall-associated peptidase S8 (PrtP), mediates adhesion of the IBB477 strain to bare, mucin- and fibronectin-coated polystyrene, as well as to HT29-MTX cells. Interactions between bacteria and mucus secreted by HT29-MTX cells were further investigated by fluorescent staining and confocal microscopy. Confocal images showed that IBB477 forms dense clusters embedded in secreted mucus. Finally, the ability of IBB477 strain and its ΔprtP deletion mutant to colonise the gastrointestinal tract of conventional C57Bl/6 mice was determined. Both strains were present in the gut for up to 72 h. In summary, adhesion and persistence of IBB477 were analysed by in vitro and in vivo approaches, respectively. Our studies revealed that plasmidic genes encoding cell surface proteins are more involved in the adhesion of IBB477 strain than in the ability to confer a selective advantage in the gut.

Limited Dissemination of Extended-Spectrum β-Lactamase- and Plasmid-Encoded AmpC-Producing Escherichia coli from Food and Farm Animals, Sweden.

Science.gov (United States)

Börjesson, Stefan; Ny, Sofia; Egervärn, Maria; Bergström, Jakob; Rosengren, Åsa; Englund, Stina; Löfmark, Sonja; Byfors, Sara

2016-04-01

Extended-spectrum β-lactamase (ESBL)- and plasmid-encoded ampC (pAmpC)-producing Enterobacteriaceae might spread from farm animals to humans through food. However, most studies have been limited in number of isolates tested and areas studied. We examined genetic relatedness of 716 isolates from 4,854 samples collected from humans, farm animals, and foods in Sweden to determine whether foods and farm animals might act as reservoirs and dissemination routes for ESBL/pAmpC-producing Escherichia coli. Results showed that clonal spread to humans appears unlikely. However, we found limited dissemination of genes encoding ESBL/pAmpC and plasmids carrying these genes from foods and farm animals to healthy humans and patients. Poultry and chicken meat might be a reservoir and dissemination route to humans. Although we found no evidence of clonal spread of ESBL/pAmpC-producing E. coli from farm animals or foods to humans, ESBL/pAmpC-producing E. coli with identical genes and plasmids were present in farm animals, foods, and humans.

Novel plasmids and resistance phenotypes in Yersinia pestis: unique plasmid inventory of strain Java 9 mediates high levels of arsenic resistance.

Science.gov (United States)

Eppinger, Mark; Radnedge, Lyndsay; Andersen, Gary; Vietri, Nicholas; Severson, Grant; Mou, Sherry; Ravel, Jacques; Worsham, Patricia L

2012-01-01

Growing evidence suggests that the plasmid repertoire of Yersinia pestis is not restricted to the three classical virulence plasmids. The Java 9 strain of Y. pestis is a biovar Orientalis isolate obtained from a rat in Indonesia. Although it lacks the Y. pestis-specific plasmid pMT, which encodes the F1 capsule, it retains virulence in mouse and non-human primate animal models. While comparing diverse Y. pestis strains using subtractive hybridization, we identified sequences in Java 9 that were homologous to a Y. enterocolitica strain carrying the transposon Tn2502, which is known to encode arsenic resistance. Here we demonstrate that Java 9 exhibits high levels of arsenic and arsenite resistance mediated by a novel promiscuous class II transposon, named Tn2503. Arsenic resistance was self-transmissible from Java 9 to other Y. pestis strains via conjugation. Genomic analysis of the atypical plasmid inventory of Java 9 identified pCD and pPCP plasmids of atypical size and two previously uncharacterized cryptic plasmids. Unlike the Tn2502-mediated arsenic resistance encoded on the Y. enterocolitica virulence plasmid; the resistance loci in Java 9 are found on all four indigenous plasmids, including the two novel cryptic plasmids. This unique mobilome introduces more than 105 genes into the species gene pool. The majority of these are encoded by the two entirely novel self-transmissible plasmids, which show partial homology and synteny to other enterics. In contrast to the reductive evolution in Y. pestis, this study underlines the major impact of a dynamic mobilome and lateral acquisition in the genome evolution of the plague bacterium.

Novel plasmids and resistance phenotypes in Yersinia pestis: unique plasmid inventory of strain Java 9 mediates high levels of arsenic resistance.

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Mark Eppinger

Full Text Available Growing evidence suggests that the plasmid repertoire of Yersinia pestis is not restricted to the three classical virulence plasmids. The Java 9 strain of Y. pestis is a biovar Orientalis isolate obtained from a rat in Indonesia. Although it lacks the Y. pestis-specific plasmid pMT, which encodes the F1 capsule, it retains virulence in mouse and non-human primate animal models. While comparing diverse Y. pestis strains using subtractive hybridization, we identified sequences in Java 9 that were homologous to a Y. enterocolitica strain carrying the transposon Tn2502, which is known to encode arsenic resistance. Here we demonstrate that Java 9 exhibits high levels of arsenic and arsenite resistance mediated by a novel promiscuous class II transposon, named Tn2503. Arsenic resistance was self-transmissible from Java 9 to other Y. pestis strains via conjugation. Genomic analysis of the atypical plasmid inventory of Java 9 identified pCD and pPCP plasmids of atypical size and two previously uncharacterized cryptic plasmids. Unlike the Tn2502-mediated arsenic resistance encoded on the Y. enterocolitica virulence plasmid; the resistance loci in Java 9 are found on all four indigenous plasmids, including the two novel cryptic plasmids. This unique mobilome introduces more than 105 genes into the species gene pool. The majority of these are encoded by the two entirely novel self-transmissible plasmids, which show partial homology and synteny to other enterics. In contrast to the reductive evolution in Y. pestis, this study underlines the major impact of a dynamic mobilome and lateral acquisition in the genome evolution of the plague bacterium.

Complete Genome Sequence of the Quality Control Strain Staphylococcus aureus subsp. aureus ATCC 25923.

Science.gov (United States)

Treangen, Todd J; Maybank, Rosslyn A; Enke, Sana; Friss, Mary Beth; Diviak, Lynn F; Karaolis, David K R; Koren, Sergey; Ondov, Brian; Phillippy, Adam M; Bergman, Nicholas H; Rosovitz, M J

2014-11-06

Staphylococcus aureus subsp. aureus ATCC 25923 is commonly used as a control strain for susceptibility testing to antibiotics and as a quality control strain for commercial products. We present the completed genome sequence for the strain, consisting of the chromosome and a 27.5-kb plasmid. Copyright © 2014 Treangen et al.

Reconstruction of mreB expression in Staphylococcus aureus via a collection of new integrative plasmids.

Science.gov (United States)

Yepes, Ana; Koch, Gudrun; Waldvogel, Andrea; Garcia-Betancur, Juan-Carlos; Lopez, Daniel

2014-07-01

Protein localization has been traditionally explored in unicellular organisms, whose ease of genetic manipulation facilitates molecular characterization. The two rod-shaped bacterial models Escherichia coli and Bacillus subtilis have been prominently used for this purpose and have displaced other bacteria whose challenges for genetic manipulation have complicated any study of cell biology. Among these bacteria is the spherical pathogenic bacterium Staphylococcus aureus. In this report, we present a new molecular toolbox that facilitates gene deletion in staphylococci in a 1-step recombination process and additional vectors that facilitate the insertion of diverse reporter fusions into newly identified neutral loci of the S. aureus chromosome. Insertion of the reporters does not add any antibiotic resistance genes to the chromosomes of the resultant strains, thereby making them amenable for further genetic manipulations. We used this toolbox to reconstitute the expression of mreB in S. aureus, a gene that encodes an actin-like cytoskeletal protein which is absent in coccal cells and is presumably lost during the course of speciation. We observed that in S. aureus, MreB is organized in discrete structures in association with the membrane, leading to an unusual redistribution of the cell wall material. The production of MreB also caused cell enlargement, but it did not revert staphylococcal shape. We present interactions of MreB with key staphylococcal cell wall-related proteins. This work facilitates the use S. aureus as a model system in exploring diverse aspects of cellular microbiology. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Type 3 fimbriae, encoded by the conjugative plasmid pOLA52, enhance biofilm formation and transfer frequencies in Enterobacteriaceae strains

DEFF Research Database (Denmark)

Burmølle, Mette; Bahl, Martin Iain; Jensen, Lars Bogø

2008-01-01

pathogenic, members of the family Enterobacteriaceae, including Klebsiella pneumoniae, Salmonella Typhimurium, Kluyvera sp. and Enterobacter aerogenes, pOLA52 facilitated increased biofilm formation. pOLA52 is believed to represent the first example of a conjugative plasmid encoding type 3 fimbriae...

Impact of co-carriage of IncA/C plasmids with additional plasmids on the transfer of antimicrobial resistance in Salmonella enterica isolates.

Science.gov (United States)

Han, Jing; Pendleton, Sean J; Deck, Joanna; Singh, Ruby; Gilbert, Jeffrey; Johnson, Timothy J; Sanad, Yasser M; Nayak, Rajesh; Foley, Steven L

2018-04-20

Antimicrobial resistance in Salmonella enterica is often plasmid encoded. A key resistance plasmid group is the incompatibility group (Inc) A/C plasmids that often carry multiple resistance determinants. Previous studies showed that IncA/C plasmids were often co-located with other plasmids. The current study was undertaken to evaluate the impact of plasmid co-carriage on antimicrobial resistance and plasmid transfer. A total of 1267 Salmonella isolates, representing multiple serotypes and sources were previously subjected to susceptibility testing and 251 isolates with resistance to at least 5 antimicrobial agents were identified for further study. Each isolate was subjected to PCR-based replicon typing, and those with IncA/C plasmids were selected for plasmid isolation, PCR-based mapping of IncA/C plasmid backbone genes, and conjugation assays to evaluate resistance plasmid transferability. Of the 87 identified IncA/C positive isolates, approximately 75% carried a plasmid with another identified replicon type, with the most common being I1 (39%), FIA, FIIA, FIB and HI2 (each 15%). PCR-based mapping indicated significant diversity in IncA/C backbone content, especially in regions encoding transfer-associated and hypothetical proteins. Conjugation experiments showed that nearly 68% of the isolates transferred resistance plasmids, with 90% containing additional identified plasmids or larger (>50 kb) non-typeable plasmids. The majority of IncA/C-positive strains were able to conjugally transfer antimicrobial resistance to the recipient, encoded by IncA/C and/or co-carried plasmids. These findings highlight the importance of co-located plasmids for resistance dissemination either by directly transferring resistance genes or by potentially providing the needed conjugation machinery for IncA/C plasmid transfer. Copyright © 2018. Published by Elsevier B.V.

Space-Time Trellis Coded 8PSK Schemes for Rapid Rayleigh Fading Channels

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Salam A. Zummo

2002-05-01

Full Text Available This paper presents the design of 8PSK space-time (ST trellis codes suitable for rapid fading channels. The proposed codes utilize the design criteria of ST codes over rapid fading channels. Two different approaches have been used. The first approach maximizes the symbol-wise Hamming distance (HD between signals leaving from or entering to the same encoder′s state. In the second approach, set partitioning based on maximizing the sum of squared Euclidean distances (SSED between the ST signals is performed; then, the branch-wise HD is maximized. The proposed codes were simulated over independent and correlated Rayleigh fading channels. Coding gains up to 4 dB have been observed over other ST trellis codes of the same complexity.

Immunization with plasmid DNA encoding the hemagglutinin and the nucleoprotein confers robust protection against a lethal canine distemper virus challenge.

Science.gov (United States)

Dahl, Lotte; Jensen, Trine Hammer; Gottschalck, Elisabeth; Karlskov-Mortensen, Peter; Jensen, Tove Dannemann; Nielsen, Line; Andersen, Mads Klindt; Buckland, Robin; Wild, T Fabian; Blixenkrone-Møller, Merete

2004-09-09

We have investigated the protective effect of immunization of a highly susceptible natural host of canine distemper virus (CDV) with DNA plasmids encoding the viral nucleoprotein (N) and hemagglutinin (H). The combined intradermal and intramuscular routes of immunization elicited high virus-neutralizing serum antibody titres in mink (Mustela vison). To mimic natural exposure, we also conducted challenge infection by horizontal transmission from infected contact animals. Other groups received a lethal challenge infection by administration to the mucosae of the respiratory tract and into the muscle. One of the mink vaccinated with N plasmid alone developed severe disease after challenge. In contrast, vaccination with the H plasmid together with the N plasmid conferred solid protection against disease and we were unable to detect CDV infection in PBMCs or in different tissues after challenge. Our findings show that DNA immunization by the combined intradermal and intramuscular routes can confer solid protective immunity against naturally transmitted morbillivirus infection and disease.

The immunomodulator PSK induces in vitro cytotoxic activity in tumour cell lines via arrest of cell cycle and induction of apoptosis

International Nuclear Information System (INIS)

Jiménez-Medina, Eva; Berruguilla, Enrique; Romero, Irene; Algarra, Ignacio; Collado, Antonia; Garrido, Federico; Garcia-Lora, Angel

2008-01-01

Protein-bound polysaccharide (PSK) is derived from the CM-101 strain of the fungus Coriolus versicolor and has shown anticancer activity in vitro and in in vivo experimental models and human cancers. Several randomized clinical trials have demonstrated that PSK has great potential in adjuvant cancer therapy, with positive results in the adjuvant treatment of gastric, esophageal, colorectal, breast and lung cancers. These studies have suggested the efficacy of PSK as an immunomodulator of biological responses. The precise molecular mechanisms responsible for its biological activity have yet to be fully elucidated. The in vitro cytotoxic anti-tumour activity of PSK has been evaluated in various tumour cell lines derived from leukaemias, melanomas, fibrosarcomas and cervix, lung, pancreas and gastric cancers. Tumour cell proliferation in vitro was measured by BrdU incorporation and viable cell count. Effect of PSK on human peripheral blood lymphocyte (PBL) proliferation in vitro was also analyzed. Studies of cell cycle and apoptosis were performed in PSK-treated cells. PSK showed in vitro inhibition of tumour cell proliferation as measured by BrdU incorporation and viable cell count. The inhibition ranged from 22 to 84%. Inhibition mechanisms were identified as cell cycle arrest, with cell accumulation in G 0 /G 1 phase and increase in apoptosis and caspase-3 expression. These results indicate that PSK has a direct cytotoxic activity in vitro, inhibiting tumour cell proliferation. In contrast, PSK shows a synergistic effect with IL-2 that increases PBL proliferation. These results indicate that PSK has cytotoxic activity in vitro on tumour cell lines. This new cytotoxic activity of PSK on tumour cells is independent of its previously described immunomodulatory activity on NK cells

Functional characterization of a cadmium resistance operon in Staphylococcus aureus ATCC12600: CadC does not function as a repressor.

Science.gov (United States)

Hoogewerf, Arlene J; Dyk, Lisa A Van; Buit, Tyler S; Roukema, David; Resseguie, Emily; Plaisier, Christina; Le, Nga; Heeringa, Lee; Griend, Douglas A Vander

2015-02-01

Sequencing of a cadmium resistance operon from a Staphylococcus aureus ATCC12600 plasmid revealed that it is identical to a cadCA operon found in MRSA strains. Compared to plasmid-cured and cadC-mutant strains, cadC-positive ATCC12600 cells had increased resistance to cadmium (1 mg ml(-1) cadmium sulfate) and zinc (4 mg ml(-1) zinc sulfate), but not to other metal ions. After growth in media containing 20 µg ml(-1) cadmium sulfate, cadC-mutant cells contained more intracellular cadmium than cadC-positive ATCC12600 cells, suggesting that cadC absence results in impaired cadmium efflux. Electrophoretic mobility shift assays were performed with CadC proteins encoded by the S. aureus ATCC12600 plasmid and by the cadC gene of pI258, which is known to act as a transcriptional repressor and shares only 47% protein sequence identity with ATCC12600 CadC. Mobility shifts occurred when pI258 CadC protein was incubated with the promoter DNA-regions from the pI258 and S. aureus ATCC12600 cadCA operons, but did not occur with S. aureus ATCC12600 CadC protein, indicating that the ATCC12600 CadC protein does not interact with promoter region DNA. This cadCA operon, found in MRSA strains and previously functionally uncharacterized, increases resistance to cadmium and zinc by an efflux mechanism, and CadC does not function as a transcriptional repressor. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The immunomodulator PSK induces in vitro cytotoxic activity in tumour cell lines via arrest of cell cycle and induction of apoptosis

Directory of Open Access Journals (Sweden)

Garrido Federico

2008-03-01

Full Text Available Abstract Background Protein-bound polysaccharide (PSK is derived from the CM-101 strain of the fungus Coriolus versicolor and has shown anticancer activity in vitro and in in vivo experimental models and human cancers. Several randomized clinical trials have demonstrated that PSK has great potential in adjuvant cancer therapy, with positive results in the adjuvant treatment of gastric, esophageal, colorectal, breast and lung cancers. These studies have suggested the efficacy of PSK as an immunomodulator of biological responses. The precise molecular mechanisms responsible for its biological activity have yet to be fully elucidated. Methods The in vitro cytotoxic anti-tumour activity of PSK has been evaluated in various tumour cell lines derived from leukaemias, melanomas, fibrosarcomas and cervix, lung, pancreas and gastric cancers. Tumour cell proliferation in vitro was measured by BrdU incorporation and viable cell count. Effect of PSK on human peripheral blood lymphocyte (PBL proliferation in vitro was also analyzed. Studies of cell cycle and apoptosis were performed in PSK-treated cells. Results PSK showed in vitro inhibition of tumour cell proliferation as measured by BrdU incorporation and viable cell count. The inhibition ranged from 22 to 84%. Inhibition mechanisms were identified as cell cycle arrest, with cell accumulation in G0/G1 phase and increase in apoptosis and caspase-3 expression. These results indicate that PSK has a direct cytotoxic activity in vitro, inhibiting tumour cell proliferation. In contrast, PSK shows a synergistic effect with IL-2 that increases PBL proliferation. Conclusion These results indicate that PSK has cytotoxic activity in vitro on tumour cell lines. This new cytotoxic activity of PSK on tumour cells is independent of its previously described immunomodulatory activity on NK cells.

Typing of Panton-Valentine Leukocidin-Encoding Phages and lukSF-PV Gene Sequence Variation in Staphylococcus aureus from China.

Science.gov (United States)

Zhao, Huanqiang; Hu, Fupin; Jin, Shu; Xu, Xiaogang; Zou, Yuhan; Ding, Baixing; He, Chunyan; Gong, Fang; Liu, Qingzhong

2016-01-01

Panton-Valentine leukocidin (PVL, encoded by lukSF-PV genes), a bi-component and pore-forming toxin, is carried by different staphylococcal bacteriophages. The prevalence of PVL in Staphylococcus aureus has been reported around the globe. However, the data on PVL-encoding phage types, lukSF-PV gene variation and chromosomal phage insertion sites for PVL-positive S. aureus are limited, especially in China. In order to obtain a more complete understanding of the molecular epidemiology of PVL-positive S. aureus, an integrated and modified PCR-based scheme was applied to detect the PVL-encoding phage types. Phage insertion locus and the lukSF-PV variant were determined by PCR and sequencing. Meanwhile, the genetic background was characterized by staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) gene polymorphisms typing, pulsed-field gel electrophoresis (PFGE) typing, accessory gene regulator (agr) locus typing and multilocus sequence typing (MLST). Seventy eight (78/1175, 6.6%) isolates possessed the lukSF-PV genes and 59.0% (46/78) of PVL-positive strains belonged to CC59 lineage. Eight known different PVL-encoding phage types were detected, and Φ7247PVL/ΦST5967PVL (n = 13) and ΦPVL (n = 12) were the most prevalent among them. While 25 (25/78, 32.1%) isolates, belonging to ST30, and ST59 clones, were unable to be typed by the modified PCR-based scheme. Single nucleotide polymorphisms (SNPs) were identified at five locations in the lukSF-PV genes, two of which were non-synonymous. Maximum-likelihood tree analysis of attachment sites sequences detected six SNP profiles for attR and eight for attL, respectively. In conclusion, the PVL-positive S. aureus mainly harbored Φ7247PVL/ΦST5967PVL and ΦPVL in the regions studied. lukSF-PV gene sequences, PVL-encoding phages, and phage insertion locus generally varied with lineages. Moreover, PVL-positive clones that have emerged worldwide likely carry distinct phages.

[Replication of Streptomyces plasmids: the DNA nucleotide sequence of plasmid pSB 24.2].

Science.gov (United States)

Bolotin, A P; Sorokin, A V; Aleksandrov, N N; Danilenko, V N; Kozlov, Iu I

1985-11-01

The nucleotide sequence of DNA in plasmid pSB 24.2, a natural deletion derivative of plasmid pSB 24.1 isolated from S. cyanogenus was studied. The plasmid amounted by its size to 3706 nucleotide pairs. The G-C composition was equal to 73 per cent. The analysis of the DNA structure in plasmid pSB 24.2 revealed the protein-encoding sequence of DNA, the continuity of which was significant for replication of the plasmid containing more than 1300 nucleotide pairs. The analysis also revealed two A-T-rich areas of DNA, the G-C composition of which was less than 55 per cent and a DNA area with a branched pin structure. The results may be of value in investigation of plasmid replication in actinomycetes and experimental cloning of DNA with this plasmid as a vector.

Nopaline-type Ti plasmid of Agrobacterium encodes a VirF-like functional F-box protein.

Science.gov (United States)

Lacroix, Benoît; Citovsky, Vitaly

2015-11-20

During Agrobacterium-mediated genetic transformation of plants, several bacterial virulence (Vir) proteins are translocated into the host cell to facilitate infection. One of the most important of such translocated factors is VirF, an F-box protein produced by octopine strains of Agrobacterium, which presumably facilitates proteasomal uncoating of the invading T-DNA from its associated proteins. The presence of VirF also is thought to be involved in differences in host specificity between octopine and nopaline strains of Agrobacterium, with the current dogma being that no functional VirF is encoded by nopaline strains. Here, we show that a protein with homology to octopine VirF is encoded by the Ti plasmid of the nopaline C58 strain of Agrobacterium. This protein, C58VirF, possesses the hallmarks of functional F-box proteins: it contains an active F-box domain and specifically interacts, via its F-box domain, with SKP1-like (ASK) protein components of the plant ubiquitin/proteasome system. Thus, our data suggest that nopaline strains of Agrobacterium have evolved to encode a functional F-box protein VirF.

Construction of adiponectin-encoding plasmid DNA and gene therapy of non-obese type 2 diabetes mellitus.

Science.gov (United States)

Nan, Mei Hua; Park, Jeong-Sook; Myung, Chang-Seon

2010-01-01

Adiponectin (ADN), an insulin-sensitizing adipokine, stimulates glucose uptake, inhibits gluconeogenesis, and plays an important role in improving insulin sensitivity. Since blood levels of ADN are low in type 2 diabetes mellitus (DM), this study was designed to investigate the therapeutic effectiveness of increasing the ADN level through injection of plasmid DNA encoding ADN in type 2 DM. A non-obese type 2 DM mouse model was established via combined administration of streptozotocin with nicotinamide and exhibited significantly higher plasma glucose concentration and insulin resistance compared with normal controls according to oral glucose tolerance and insulin challenge tests. Plasmid DNA encoding mouse ADN from differentiated NIH3T3 adipocytes was constructed in pVAX1 (pVAX/ADN). Transfection of pVAX/ADN into various cell lines including HeLa, HT22, HEK293, HepG2, and SK-Hep1 cells, increased ADN mRNA expression levels in a dose-dependent manner. The administration of pVAX/ADN into non-obese type 2 DM mice via tail vein significantly increased the blood level of ADN and decreased the plasma glucose concentration. Moreover, the parameters related to insulin resistance (HOMA-IR) and insulin sensitivity (QUICKI) were significantly improved. These results suggest that ADN gene therapy could be a clinically effective tool for the treatment of type 2 DM.

Effect of cytokine-encoding plasmid delivery on immune response to Japanese encephalitis virus DNA vaccine in mice.

Science.gov (United States)

Bharati, Kaushik; Appaiahgari, Mohan Babu; Vrati, Sudhanshu

2005-01-01

We have previously shown that immunization of mice with plasmid pMEa synthesizing Japanese encephalitis virus (JEV) envelope protein induced anti-JEV humoral and cellular immune responses. We now show that intra-muscular co-administration of mice with pMEa and pGM-CSF, encoding murine granulocyte-macrophage colony-stimulating factor or pIL-2, encoding murine interleukin-2 given 4 days after pMEa, augmented anti-JEV antibody titers. This did not enhance the level of protection in immunized mice against JEV. However, intra-dermal co-administration of pMEa and pGM-CSF in mice using the gene gun, enhanced anti-JEV antibody titers resulting in an increased level of protection in mice against lethal JEV challenge.

Characterization of a Staphylococcal Plasmid Related to pUB110 and Carrying Two Novel Genes, vatC and vgbB, Encoding Resistance to Streptogramins A and B and Similar Antibiotics

Science.gov (United States)

Allignet, Jeanine; Liassine, Nadia; El Solh, Névine

1998-01-01

We isolated and sequenced a plasmid, named pIP1714 (4,978 bp), which specifies resistance to streptogramins A and B and the mixture of these compounds. pIP1714 was isolated from a Staphylococcus cohnii subsp. cohnii strain found in the environment of a hospital where pristinamycin was extensively used. Resistance to both compounds and related antibiotics is encoded by two novel, probably cotranscribed genes, (i) vatC, encoding a 212-amino-acid (aa) acetyltransferase that inactivates streptogramin A and that exhibits 58.2 to 69.8% aa identity with the Vat, VatB, and SatA proteins, and (ii) vgbB, encoding a 295-aa lactonase that inactivates streptogramin B and that shows 67% aa identity with the Vgb lactonase. pIP1714 includes a 2,985-bp fragment also found in two rolling-circle replication and mobilizable plasmids, pUB110 and pBC16, from gram-positive bacteria. In all three plasmids, the common fragment was delimited by two direct repeats of four nucleotides (GGGC) and included (i) putative genes closely related to repB, which encodes a replication protein, and to pre(mob), which encodes a protein required for conjugative mobilization and site-specific recombination, and (ii) sequences very similar to the double- and single-strand origins (dso, ssoU) and the recombination site, RSA. The antibiotic resistance genes repB and pre(mob) carried by each of these plasmids were found in the same transcriptional orientation. PMID:9661023

Characterization of a staphylococcal plasmid related to pUB110 and carrying two novel genes, vatC and vgbB, encoding resistance to streptogramins A and B and similar antibiotics.

Science.gov (United States)

Allignet, J; Liassine, N; el Solh, N

1998-07-01

We isolated and sequenced a plasmid, named pIP1714 (4,978 bp), which specifies resistance to streptogramins A and B and the mixture of these compounds. pIP1714 was isolated from a Staphylococcus cohnii subsp. cohnii strain found in the environment of a hospital where pristinamycin was extensively used. Resistance to both compounds and related antibiotics is encoded by two novel, probably cotranscribed genes, (i) vatC, encoding a 212-amino-acid (aa) acetyltransferase that inactivates streptogramin A and that exhibits 58.2 to 69.8% aa identity with the Vat, VatB, and SatA proteins, and (ii) vgbB, encoding a 295-aa lactonase that inactivates streptogramin B and that shows 67% aa identity with the Vgb lactonase. pIP1714 includes a 2,985-bp fragment also found in two rolling-circle replication and mobilizable plasmids, pUB110 and pBC16, from gram-positive bacteria. In all three plasmids, the common fragment was delimited by two direct repeats of four nucleotides (GGGC) and included (i) putative genes closely related to repB, which encodes a replication protein, and to pre(mob), which encodes a protein required for conjugative mobilization and site-specific recombination, and (ii) sequences very similar to the double- and single-strand origins (dso, ssoU) and the recombination site, RSA. The antibiotic resistance genes repB and pre(mob) carried by each of these plasmids were found in the same transcriptional orientation.

Simulation Research of PSK Blindness Remodulation%相移键控信号盲解调的仿真分析

Institute of Scientific and Technical Information of China (English)

王启峰; 刘涛; 饶德虎; 李仙茂

2011-01-01

对相移键控信号调制解调原理进行了分析,并对未知信号的载波频率和码速率估计方法进行了分析与比较,推导出了相移键控和振幅相移联合键控信号盲解调的公式,并结合4PSK,8PSK,16PSK和16QAM信号进行盲解调仿真,在不同信噪比下对不同类型的相移键控信号盲解调正确解调波特率的影响进行了分析,并给出了分析结果,给通信对抗侦察提供了理论指导.%The analysis is fulfilled about principle of PSK signal. And comparison of carrier wave frequency and code velocity estimation method is jmpleted. Formula of PSK remodulation is deduced. The simulation and analysis of4PSK, 8PSK, 16PSK and 16QAM blindness remodulation are fin hed. The result of right remodulation probability at the relevant signal noise rate is educed by computer simulation. A direction reconnaissance of com unication electronic warfare is given.

Molecular characterization of a new efficiently transducing bacteriophage identified in meticillin-resistant Staphylococcus aureus.

Science.gov (United States)

Varga, Marian; Pantůček, Roman; Růžičková, Vladislava; Doškař, Jirˇí

2016-01-01

In Staphylococcus aureus, generalized transduction mediated by temperate bacteriophages represents a highly efficient way of transferring antibiotic resistance genes between strains. In the present study, we identified and characterized in detail a new efficiently transducing bacteriophage of the family Siphoviridae, designated ϕJB, which resides as a prophage in the meticillin-resistant S. aureus (MRSA) strain Jevons B. Whole-genome sequencing followed by detailed in silico analysis uncovered a linear dsDNA genome consisting of 43 ,12 bp and comprising 70 ORFs, of which ∼40 encoded proteins with unknown function. A global genome alignment of ϕJB and other efficiently transducing phages ϕ11, ϕ53, ϕ80, ϕ80α and ϕNM4 showed a high degree of homology with ϕNM4 and substantial differences with regard to other phages. Using a model transduction system with a well-defined donor and recipient, ϕJB transferred the tetracycline resistance plasmid pT181 and a penicillinase plasmid with outstanding frequencies, beating most of the above-mentioned phages by an order of magnitude. Moreover, ϕJB demonstrated high frequencies of transferring antibiotic resistance plasmids even upon induction from a lysogenic donor strain. Considering such transducing potential, ϕJB and related bacteriophages may serve as a suitable tool for elucidating the nature of transduction and its contribution to the spread of antibiotic resistance genes in naturally occurring MRSA populations.

STUDI KUALITATIF MENGENAI PERSEPSI DAN PERILAKU SEKSUAL WANITA PEKERJA SEKS KOMERSIAL (PSK DALAM UPAYA PENCEGAHAN IMS DI KOTA SEMARANG TAHUN 2012

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Ratu Matahari

2015-04-01

Full Text Available Pendahuluan: Peningkatan jumlah kasus IMS di Kota Semarangdengan jumlah kasus IMS pada tahun 2009 tercatat mencapai 2.471 kasusdan jumlah kasus IMS pada tahun 2011 adalah 2473 kasus. Tujuan: Mendeskripsikan persepsi dan perilaku seksual wanita Pekerja Seks Komersial (PSK di Lokalisasi Sunan Kuning terhadap upaya pencegahan Infeksi Menular Seksual (IMS di Kota Semarang. Metode: Penelitian ini dilakukan dengan menggunakan teknik wawancara mendalam (indepth interview pada enam PSK yang mengalami IMS dan mewawancarai dua kelompok diskusi (FGD, seorang mucikari, dan seorang petugas lapangan (PL sebagai triangulasi. Analisis data menggunakan analisis isi (content analysis. Hasil: Pola Pengetahuan PSK dan persepsi PSK terhadap IMS juga sudah baik, tetapi perilaku PSK dalam upaya mencegah penularan IMS masih belum bisa dikatakan baik karena penggunaan kondom diantara pekerja seks komersial pada saat melakukan hubungan seksual dengan pelanggannya masih rendah. Tidak adanya dukungan dari mucikari dalam meningkatkan perilaku pencegahan IMS. Hal ini menunjukkan bahwa kepedulian terhadap kesehatan diri sendiri masih rendah. Kesimpulan: Perilaku pencegahan PSK terhadap penularan IMS belum baik. Perlu diadakan pelatihan dengan metode role playing kepada para PSK dan mucikari untuk meningkatkan kepedulian mereka terhadap kesehatan sehingga diharapkan akan terjadi perubahan perilaku baru dalam upaya pencegahan penularan IMS terhadap diri mereka sendiri atau pelanggan.  Kata kunci: Persepsi, pekerja seks komersial, IMS, Kota Semarang

High diversity of genes and plasmids encoding resistance to third-generation cephalosporins and quinolones in clinical Escherichia coli from commercial poultry flocks in Italy

DEFF Research Database (Denmark)

Niero, Giulia; Bortolaia, Valeria; Vanni, Michele

2018-01-01

= 98) and layers (n = 22) between 2008 and 2012. 3GC-resistant isolates were screened for extended-spectrum and AmpC β-lactamase (ESBL/AmpC), while all isolates were tested for plasmid-mediated quinolone resistance (PMQR) genes. ESBL/AmpC- and PMQR-positive isolates were typed by pulsed-field gel......% of isolates from turkeys, broilers and layers, respectively. We identified seven ESBL/AmpC-encoding plasmid types, usually conjugative (78%), with a marked prevalence of IncI1/pST3 plasmids carrying blaCTX-M-1. PMQR occurred less frequently among isolates from turkeys (0.9%) compared to those from broilers (5......%) and layers (4%). The PMQR genes qnrS, qnrB19 and oqxA/B were located on three plasmid types and two non-typeable plasmids, mostly (85%) conjugative. ESBL/AmpC- and PMQR-positive isolates were genetically unrelated and 64% of them were additionally resistant to aminoglycosides, sulfonamides and tetracyclines...

X-ray crystal structure of the passenger domain of plasmid encoded toxin(Pet), an autotransporter enterotoxin from enteroaggregative Escherichia coli (EAEC)

Energy Technology Data Exchange (ETDEWEB)

Domingo Meza-Aguilar, J. [Departamento de Salud Pública Facultad de Medicina UNAM, Ciudad Universitaria Coyoacán 04510, D.F. (Mexico); Laboratorio de Patogenicidad Bacteriana, Unidad de Hemato Oncología e Investigación, Hospital Infantil de México Federico Gómez 06720, D.F. (Mexico); Fromme, Petra [Department of Chemistry and Biochemistry, Arizona State University, Physical Sciences BLDG D-102, Tempe, AZ 85287 (United States); Torres-Larios, Alfredo [Instituto de Fisiología Celular UNAM, Ciudad Universitaria Coyoacán 04510, D.F. (Mexico); Mendoza-Hernández, Guillermo [Instituto de Química UNAM, Ciudad Universitaria Coyoacán 04510, D.F (Mexico); Hernandez-Chiñas, Ulises [Departamento de Salud Pública Facultad de Medicina UNAM, Ciudad Universitaria Coyoacán 04510, D.F. (Mexico); Laboratorio de Patogenicidad Bacteriana, Unidad de Hemato Oncología e Investigación, Hospital Infantil de México Federico Gómez 06720, D.F. (Mexico); Arreguin-Espinosa de los Monteros, Roberto A. [Instituto de Química UNAM, Ciudad Universitaria Coyoacán 04510, D.F (Mexico); and others

2014-03-07

Highlights: • X-ray crystal structure of the passenger domain of Plasmid encoded toxin at 2.3 Å. • Structural differences between Pet passenger domain and EspP protein are described. • High flexibility of the C-terminal beta helix is structurally assigned. - Abstract: Autotransporters (ATs) represent a superfamily of proteins produced by a variety of pathogenic bacteria, which include the pathogenic groups of Escherichia coli (E. coli) associated with gastrointestinal and urinary tract infections. We present the first X-ray structure of the passenger domain from the Plasmid-encoded toxin (Pet) a 100 kDa protein at 2.3 Å resolution which is a cause of acute diarrhea in both developing and industrialized countries. Pet is a cytoskeleton-altering toxin that induces loss of actin stress fibers. While Pet (pdb code: 4OM9) shows only a sequence identity of 50% compared to the closest related protein sequence, extracellular serine protease plasmid (EspP) the structural features of both proteins are conserved. A closer structural look reveals that Pet contains a β-pleaded sheet at the sequence region of residues 181–190, the corresponding structural domain in EspP consists of a coiled loop. Secondary, the Pet passenger domain features a more pronounced beta sheet between residues 135 and 143 compared to the structure of EspP.

Simple method for identification of plasmid-coded proteins

International Nuclear Information System (INIS)

Sancar, A.; Hack, A.M.; Rupp, W.D.

1979-01-01

Proteins encoded by plasmid DNA are specifically labeled in uv-irradiated cells of Escherichia coli carrying recA and uvrA mutations because extensive degradation of the chromosome DNA occurs concurrently with amplification of plasmid DNA

Divergently overlapping cis-encoded antisense RNA regulating toxin-antitoxin systems from E. coli: hok/sok, ldr/rdl, symE/symR.

Science.gov (United States)

Kawano, Mitsuoki

2012-12-01

Toxin-antitoxin (TA) systems are categorized into three classes based on the type of antitoxin. In type I TA systems, the antitoxin is a small antisense RNA that inhibits translation of small toxic proteins by binding to the corresponding mRNAs. Those type I TA systems were originally identified as plasmid stabilization modules rendering a post-segregational killing (PSK) effect on the host cells. The type I TA loci also exist on the Escherichia coli chromosome but their biological functions are less clear. Genetic organization and regulatory elements of hok/sok and ldr/rdl families are very similar and the toxins are predicted to contain a transmembrane domain, but otherwise share no detectable sequence similarity. This review will give an overview of the type I TA modules of E. coli K-12, especially hok/sok, ldr/rdl and SOS-inducible symE/symR systems, which are regulated by divergently overlapping cis-encoded antisense RNAs.

Antimicrobial resistance patterns and plasmid profiles of ...

African Journals Online (AJOL)

Objectives: To determine the frequency of resistance of Staphylococcus aureus to various antimicrobial agents, and the relationship between antimicrobial resistance of the isolates and carriage of plasmids. Design: A random sampling of milk and meat samples was carried out. Setting: Milk was collected from various dairy ...

PSK, a biological response modifier, modifies p53 expression, mitosis and apoptosis in X-ray irradiated mouse embryos. Possible cellular mechanism of the anti-teratogenic effect

International Nuclear Information System (INIS)

Kagohashi, Yukiko; Naora, Hiroyuki; Otani, Hiroki

2002-01-01

We previously showed that PSK, a biological response modifier, suppressed X-ray irradiation induced ocular anomalies in mouse embryos. In the present study, in mouse embryos irradiated at E7.5, PSK, when administered immediately after irradiation, suppressed mitosis and increased apoptosis as compared with embryos not treated with PSK at 12 hrs after irradiation. In the irradiated embryos, p53, which is normally expressed at a high level in early embryos, increased at 6 hrs and decreased at 12 hrs after irradiation. In the irradiated and PSK-treated embryos, the p53 level did not change at 6 hrs, increased at 12 hrs and decreased at 24 hrs after irradiation. This timing of PSK-induced delayed increase of p53 coincided with that of the PSK-induced decrease in mitosis and increase in apoptosis. These results suggested that PSK modified the p53 level and affected cell proliferation and apoptosis, which might contribute to the suppression of teratogenesis. (author)

A plasmid toolkit for cloning chimeric cDNAs encoding customized fusion proteins into any Gateway destination expression vector

Science.gov (United States)

2013-01-01

Background Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. Results We present a versatile cloning toolkit for constructing fully-customizable three-part fusion proteins based on the MultiSite Gateway cloning system. The fusion protein components are encoded in the three plasmids integral to the kit. These can recombine with any purposely-engineered destination vector that uses a heterologous promoter external to the Gateway cassette, leading to the in-frame cloning of an ORF of interest flanked by two functional modules. In contrast to previous systems, a third part becomes available for peptide-encoding as it no longer needs to contain a promoter, resulting in an increased number of possible fusion combinations. We have constructed the kit’s component plasmids and demonstrate its functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently-transfected cells. Conclusions We have developed a toolkit for creating fusion proteins with customized N- and C-term modules from Gateway entry clones encoding ORFs of interest. Importantly, our method allows entry clones obtained from ORFeome

Geographical variation in the presence of genes encoding superantigenic exotoxins and beta-hemolysin among Staphylococcus aureus isolated from bovine mastitis in Europe and USA

DEFF Research Database (Denmark)

Larsen, H. D.; Aarestrup, Frank Møller; Jensen, N. E.

2002-01-01

The object was to examine the geographical variation in the presence of superantigenic exotoxins and beta-hemolysin among epidemiologically independent Staphyirrcoccus aureus isolates from bovine mastitis. A total of 462 S. aureus isolates from nine European countries and USA were examined...... for the individual exotoxins. The genes encoding enterotoxin C, TSST-1, and enterotoxin D were the most common superantigens. The present and earlier studies demonstrate that the superantigenic exotoxins that were investigated in this study, do not play a role in the pathogenesis of bovine S. aureus mastitis...... regions in the beta-hemolysin encoding gene of the Norwegian isolates is suggested, and should be investigated further. The consistent presence of beta-hemolysin suggests that this factor, or a co-existing gene correlated to beta-hemolysin, may be an active virulence factor in the pathogenesis of bovine S...

Characterization of plasmids in a human clinical strain of Lactococcus garvieae.

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Mónica Aguado-Urda

Full Text Available The present work describes the molecular characterization of five circular plasmids found in the human clinical strain Lactococcus garvieae 21881. The plasmids were designated pGL1-pGL5, with molecular sizes of 4,536 bp, 4,572 bp, 12,948 bp, 14,006 bp and 68,798 bp, respectively. Based on detailed sequence analysis, some of these plasmids appear to be mosaics composed of DNA obtained by modular exchange between different species of lactic acid bacteria. Based on sequence data and the derived presence of certain genes and proteins, the plasmid pGL2 appears to replicate via a rolling-circle mechanism, while the other four plasmids appear to belong to the group of lactococcal theta-type replicons. The plasmids pGL1, pGL2 and pGL5 encode putative proteins related with bacteriocin synthesis and bacteriocin secretion and immunity. The plasmid pGL5 harbors genes (txn, orf5 and orf25 encoding proteins that could be considered putative virulence factors. The gene txn encodes a protein with an enzymatic domain corresponding to the family actin-ADP-ribosyltransferases toxins, which are known to play a key role in pathogenesis of a variety of bacterial pathogens. The genes orf5 and orf25 encode two putative surface proteins containing the cell wall-sorting motif LPXTG, with mucin-binding and collagen-binding protein domains, respectively. These proteins could be involved in the adherence of L. garvieae to mucus from the intestine, facilitating further interaction with intestinal epithelial cells and to collagenous tissues such as the collagen-rich heart valves. To our knowledge, this is the first report on the characterization of plasmids in a human clinical strain of this pathogen.

A combined approach of hollow microneedles and nanocarriers for skin immunization with plasmid DNA encoding ovalbumin

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Pamornpathomkul B

2017-01-01

Full Text Available Boonnada Pamornpathomkul,1 Adisak Wongkajornsilp,2 Wanida Laiwattanapaisal,3 Theerasak Rojanarata,1 Praneet Opanasopit,1 Tanasait Ngawhirunpat1 1Department of Pharmaceutical Technology, Faculty of Pharmacy, Pharmaceutical Development of Green Innovations Group, Silpakorn University, Nakhon Pathom, 2Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 3Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand Abstract: The aim of this study was to investigate the use of different types of microneedles (MNs and nanocarriers for in vitro skin permeation and in vivo immunization of plasmid DNA encoding ovalbumin (pOVA. In vitro skin permeation studies indicated that hollow MNs had a superior enhancing effect on skin permeation compared with solid MN patches, electroporation (EP patches, the combination of MN and EP patches, and untreated skin. Upon using hollow MNs combined with nanocarriers for pOVA delivery, the skin permeation was higher than for the delivery of naked pOVA, as evidenced by the increased amount of pOVA in Franz diffusion cells and immunoglobulin G (IgG antibody responses. When the hollow MNs were used for the delivery of nanocarrier:pOVA complexes into the skin of mice, they induced a stronger IgG immune response than conventional subcutaneous (SC injections. In addition, immunization of mice with the hollow MNs did not induce signs of skin infection or pinpoint bleeding. Accordingly, the hollow MNs combined with a nanocarrier delivery system is a promising approach for delivering pOVA complexes to the skin for promoting successful immunization. Keywords: hollow microneedle, solid microneedle, electroporation, plasmid DNA encoding ovalbumin, skin immunization, nanocarrier

A conjugative 38 kB plasmid is present in multiple subspecies of Xylella fastidiosa.

Science.gov (United States)

Rogers, Elizabeth E; Stenger, Drake C

2012-01-01

A ≈ 38kB plasmid (pXF-RIV5) was present in the Riv5 strain of Xylella fastidiosa subsp. multiplex isolated from ornamental plum in southern California. The complete nucleotide sequence of pXF-RIV5 is almost identical to that of pXFAS01 from X. fastidiosa subsp. fastidiosa strain M23; the two plasmids vary at only 6 nucleotide positions. BLAST searches and phylogenetic analyses indicate pXF-RIV5 and pXFAS01 share some similarity to chromosomal and plasmid (pXF51) sequences of X. fastidiosa subsp. pauca strain 9a5c and more distant similarity to plasmids from a wide variety of bacteria. Both pXF-RIV5 and pXFAS01 encode homologues of a complete Type IV secretion system involved in conjugation and DNA transfer among bacteria. Mating pair formation proteins (Trb) from Yersinia pseudotuberculosis IP31758 are the mostly closely related non-X. fastidiosa proteins to most of the Trb proteins encoded by pXF-RIV5 and pXFAS01. Unlike many bacterial conjugative plasmids, pXF-RIV5 and pXFAS01 do not carry homologues of known accessory modules that confer selective advantage on host bacteria. However, both plasmids encode seven hypothetical proteins of unknown function and possess a small transposon-associated region encoding a putative transposase and associated factor. Vegetative replication of pXF-RIV5 and pXFAS01 appears to be under control of RepA protein and both plasmids have an origin of DNA replication (oriV) similar to that of pRP4 and pR751 from Escherichia coli. In contrast, conjugative plasmids commonly encode TrfA and have an oriV similar to those found in IncP-1 incompatibility group plasmids. The presence of nearly identical plasmids in single strains from two distinct subspecies of X. fastidiosa is indicative of recent horizontal transfer, probably subsequent to the introduction of subspecies fastidiosa to the United States in the late 19(th) century.

A conjugative 38 kB plasmid is present in multiple subspecies of Xylella fastidiosa.

Directory of Open Access Journals (Sweden)

Elizabeth E Rogers

Full Text Available A ≈ 38kB plasmid (pXF-RIV5 was present in the Riv5 strain of Xylella fastidiosa subsp. multiplex isolated from ornamental plum in southern California. The complete nucleotide sequence of pXF-RIV5 is almost identical to that of pXFAS01 from X. fastidiosa subsp. fastidiosa strain M23; the two plasmids vary at only 6 nucleotide positions. BLAST searches and phylogenetic analyses indicate pXF-RIV5 and pXFAS01 share some similarity to chromosomal and plasmid (pXF51 sequences of X. fastidiosa subsp. pauca strain 9a5c and more distant similarity to plasmids from a wide variety of bacteria. Both pXF-RIV5 and pXFAS01 encode homologues of a complete Type IV secretion system involved in conjugation and DNA transfer among bacteria. Mating pair formation proteins (Trb from Yersinia pseudotuberculosis IP31758 are the mostly closely related non-X. fastidiosa proteins to most of the Trb proteins encoded by pXF-RIV5 and pXFAS01. Unlike many bacterial conjugative plasmids, pXF-RIV5 and pXFAS01 do not carry homologues of known accessory modules that confer selective advantage on host bacteria. However, both plasmids encode seven hypothetical proteins of unknown function and possess a small transposon-associated region encoding a putative transposase and associated factor. Vegetative replication of pXF-RIV5 and pXFAS01 appears to be under control of RepA protein and both plasmids have an origin of DNA replication (oriV similar to that of pRP4 and pR751 from Escherichia coli. In contrast, conjugative plasmids commonly encode TrfA and have an oriV similar to those found in IncP-1 incompatibility group plasmids. The presence of nearly identical plasmids in single strains from two distinct subspecies of X. fastidiosa is indicative of recent horizontal transfer, probably subsequent to the introduction of subspecies fastidiosa to the United States in the late 19(th century.

Plasmid Conjugation in E. coli and Drug Resistance | Igwe ...

African Journals Online (AJOL)

This study aimed at determining the antibiotics susceptibility pattern of E. coli isolates claimed to be multidrug resistance using disc diffusion method. It also determined the presence of transferable resistance plasmids through conjugation and evaluated the medical significance of plasmid encoding E. coli and drug ...

Transfer of Antibiotic Resistance in Staphylococcus aureus

DEFF Research Database (Denmark)

Haaber, Jakob; Penadés, José R; Ingmer, Hanne

2017-01-01

Staphylococcus aureus is a serious human pathogen with remarkable adaptive powers. Antibiotic-resistant clones rapidly emerge mainly by acquisition of antibiotic-resistance genes from other S. aureus strains or even from other genera. Transfer is mediated by a diverse complement of mobile genetic...... of plasmids that can be transferred by conjugation and the efficiency with which transduction occurs. Here, we review the main routes of antibiotic resistance gene transfer in S. aureus in the context of its biology as a human commensal and a life-threatening pathogen. Staphylococcus aureus cells...... are effective in exchanging mobile genetic elements, including antibiotic-resistance genes.During colonization or infection of host organisms, the exchange appears to be particularly effective.Bacteriophage-mediated transfer involves both transduction and autotransduction, which may enable lysogenic S. aureus...

Characterization of Plasmid pPO1 from the Hyperacidophile Picrophilus oshimae

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Angel Angelov

2011-01-01

Full Text Available Picrophilus oshimae and Picrophilus torridus are free-living, moderately thermophilic and acidophilic organisms from the lineage of Euryarchaeota. With a pH optimum of growth at pH 0.7 and the ability to even withstand molar concentrations of sulphuric acid, these organisms represent the most extreme acidophiles known. So far, nothing is known about plasmid biology in these hyperacidophiles. Also, there are no genetic tools available for this genus. We have mobilized the 7.6 Kbp plasmid from P. oshimae in E. coli by introducing origin-containing transposons and described the plasmid in terms of its nucleotide sequence, copy number in the native host, mode of replication, and transcriptional start sites of the encoded ORFs. Plasmid pPO1 may encode a restriction/modification system in addition to its replication functions. The information gained from the pPO1 plasmid may prove useful in developing a cloning system for this group of extreme acidophiles.

Presence of Glycopeptide-Encoding Plasmids in Enterococcal Isolates from Food and Humans in Denmark

DEFF Research Database (Denmark)

Migura, Lourdes Garcia; Valenzuela, Antonio Jesus Sanchez; Jensen, Lars Bogø

2011-01-01

developed techniques for classification of plasmids. Replicons associated with sex pheromone-inducible plasmids were detected in all GR E. faecalis, whereas GR Enterococcus faecium contained plasmids known to be widely distributed among enterococci. vanA resistance is common in E. faecium isolates from meat...... and animals in Europe and is rarely found in E. faecalis. This article describes the first characterization of MGE from vanA mediated E. faecalis, thus linking this resistance genotype to pheromone responding plasmids....

Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:H- Isolates from Czech Patients with Novel Plasmid Composition Not Previously Seen in German Isolates.

Science.gov (United States)

Bauwens, Andreas; Marejková, Monika; Middendorf-Bauchart, Barbara; Prager, Rita; Kossow, Annelene; Zhang, Wenlan; Karch, Helge; Mellmann, Alexander; Bielaszewska, Martina

2017-12-01

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H - strains, first identified in Germany, have emerged as important pathogens throughout Europe. Besides chromosomally encoded Shiga toxin 2a (the major virulence factor), several putative virulence loci, including the hly , etp , and sfp operons, encoding EHEC hemolysin, type II secretion system proteins, and Sfp fimbriae, respectively, are located on the 121-kb plasmid pSFO157 in German strains. Here we report novel SF EHEC O157:H - strains isolated from patients in the Czech Republic. These strains share the core genomes and chromosomal virulence loci encoding toxins ( stx 2a and the cdtV -ABC operon) and adhesins ( eae -γ, efa1 , lpfA O157OI-141 , and lpfA O157OI-154 ) with German strains but differ essentially in their plasmids. In contrast to all previously detected SF EHEC O157:H - strains, the Czech strains carry two plasmids, of 79 kb and 86 kb. The 79-kb plasmid harbors the sfp operon, but neither of the plasmids contains the hly and etp operons. Sequence analyses demonstrated that the 79-kb plasmid (pSFO157 258/98-1) evolved from pSFO157 of German strains by deletion of a 41,534-bp region via homologous recombination, resulting in loss of the hly and etp operons. The 86-kb plasmid (pSFO157 258/98-2) displays 98% sequence similarity to a 92.7-kb plasmid of an extraintestinal pathogenic E. coli bloodstream isolate. Our finding of this novel plasmid composition in SF EHEC O157:H - strains extends the evolutionary history of EHEC O157 plasmids. Moreover, the unique molecular plasmid characteristics permit the identification of such strains, thereby facilitating further investigations of their geographic distribution, clinical significance, and epidemiology. IMPORTANCE Since their first identification in Germany in 1989, sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H - (nonmotile) strains have emerged as important causes of the life-threatening disease hemolytic

Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:H− Isolates from Czech Patients with Novel Plasmid Composition Not Previously Seen in German Isolates

Science.gov (United States)

Bauwens, Andreas; Marejková, Monika; Middendorf-Bauchart, Barbara; Prager, Rita; Kossow, Annelene; Zhang, Wenlan; Karch, Helge

2017-01-01

ABSTRACT Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H− strains, first identified in Germany, have emerged as important pathogens throughout Europe. Besides chromosomally encoded Shiga toxin 2a (the major virulence factor), several putative virulence loci, including the hly, etp, and sfp operons, encoding EHEC hemolysin, type II secretion system proteins, and Sfp fimbriae, respectively, are located on the 121-kb plasmid pSFO157 in German strains. Here we report novel SF EHEC O157:H− strains isolated from patients in the Czech Republic. These strains share the core genomes and chromosomal virulence loci encoding toxins (stx2a and the cdtV-ABC operon) and adhesins (eae-γ, efa1, lpfAO157OI-141, and lpfAO157OI-154) with German strains but differ essentially in their plasmids. In contrast to all previously detected SF EHEC O157:H− strains, the Czech strains carry two plasmids, of 79 kb and 86 kb. The 79-kb plasmid harbors the sfp operon, but neither of the plasmids contains the hly and etp operons. Sequence analyses demonstrated that the 79-kb plasmid (pSFO157 258/98-1) evolved from pSFO157 of German strains by deletion of a 41,534-bp region via homologous recombination, resulting in loss of the hly and etp operons. The 86-kb plasmid (pSFO157 258/98-2) displays 98% sequence similarity to a 92.7-kb plasmid of an extraintestinal pathogenic E. coli bloodstream isolate. Our finding of this novel plasmid composition in SF EHEC O157:H− strains extends the evolutionary history of EHEC O157 plasmids. Moreover, the unique molecular plasmid characteristics permit the identification of such strains, thereby facilitating further investigations of their geographic distribution, clinical significance, and epidemiology. IMPORTANCE Since their first identification in Germany in 1989, sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H− (nonmotile) strains have emerged as important causes of the life-threatening disease hemolytic

Investigation of diversity of plasmids carrying the blaTEM-52 gene

DEFF Research Database (Denmark)

Bielak, Eliza Maria; Bergenholtz, Rikke D.; Jørgensen, Mikael Skaanning

2011-01-01

OBJECTIVES: To investigate the diversity of plasmids that carry blaTEM-52 genes among Escherichia coli and Salmonella enterica originating from animals, meat products and humans. METHODS: A collection of 22 blaTEM-52-encoding plasmids was characterized by restriction fragment length polymorphism...... of self-transfer to a plasmid-free E. coli recipient. CONCLUSIONS: The blaTEM-52 gene found in humans could have been transmitted on transferable plasmids originating from animal sources. Some of the blaTEM-52 plasmids carry replicons that differ from the classical ones. Two novel replicons were detected...

Chlamydial plasmids and bacteriophages.

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Pawlikowska-Warych, Małgorzata; Śliwa-Dominiak, Joanna; Deptuła, Wiesław

2015-01-01

Chlamydia are absolute pathogens of humans and animals; despite being rather well recognised, they are still open for discovery. One such discovery is the occurrence of extrachromosomal carriers of genetic information. In prokaryotes, such carriers include plasmids and bacteriophages, which are present only among some Chlamydia species. Plasmids were found exclusively in Chlamydia (C.) trachomatis, C. psittaci, C. pneumoniae, C. suis, C. felis, C. muridarum and C. caviae. In prokaryotic organisms, plasmids usually code for genes that facilitate survival of the bacteria in the environment (although they are not essential). In chlamydia, their role has not been definitely recognised, apart from the fact that they participate in the synthesis of glycogen and encode proteins responsible for their virulence. Furthermore, in C. suis it was evidenced that the plasmid is integrated in a genomic island and contains the tetracycline-resistance gene. Bacteriophages specific for chlamydia (chlamydiaphages) were detected only in six species: C. psittaci, C. abortus, C. felis, C. caviae C. pecorum and C. pneumoniae. These chlamydiaphages cause inhibition of the developmental cycle, and delay transformation of reticulate bodies (RBs) into elementary bodies (EBs), thus reducing the possibility of infecting other cells in time. Plasmids and bacteriophages can be used in the diagnostics of chlamydioses; although especially in the case of plasmids, they are already used for detection of chlamydial infections. In addition, bacteriophages could be used as therapeutic agents to replace antibiotics, potentially addressing the problem of increasing antibiotic-resistance among chlamydia.

A plasmid-encoded UmuD homologue regulates expression of Pseudomonas aeruginosa SOS genes.

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Díaz-Magaña, Amada; Alva-Murillo, Nayeli; Chávez-Moctezuma, Martha P; López-Meza, Joel E; Ramírez-Díaz, Martha I; Cervantes, Carlos

2015-07-01

The Pseudomonas aeruginosa plasmid pUM505 contains the umuDC operon that encodes proteins similar to error-prone repair DNA polymerase V. The umuC gene appears to be truncated and its product is probably not functional. The umuD gene, renamed umuDpR, possesses an SOS box overlapped with a Sigma factor 70 type promoter; accordingly, transcriptional fusions revealed that the umuDpR gene promoter is activated by mitomycin C. The predicted sequence of the UmuDpR protein displays 23 % identity with the Ps. aeruginosa SOS-response LexA repressor. The umuDpR gene caused increased MMC sensitivity when transferred to the Ps. aeruginosa PAO1 strain. As expected, PAO1-derived knockout lexA-  mutant PW6037 showed resistance to MMC; however, when the umuDpR gene was transferred to PW6037, MMC resistance level was reduced. These data suggested that UmuDpR represses the expression of SOS genes, as LexA does. To test whether UmuDpR exerts regulatory functions, expression of PAO1 SOS genes was evaluated by reverse transcription quantitative PCR assays in the lexA-  mutant with or without the pUC_umuD recombinant plasmid. Expression of lexA, imuA and recA genes increased 3.4-5.3 times in the lexA-  mutant, relative to transcription of the corresponding genes in the lexA+ strain, but decreased significantly in the lexA- /umuDpR transformant. These results confirmed that the UmuDpR protein is a repressor of Ps. aeruginosa SOS genes controlled by LexA. Electrophoretic mobility shift assays, however, did not show binding of UmuDpR to 5' regions of SOS genes, suggesting an indirect mechanism of regulation.

Plasmid Complement of Lactococcus lactis NCDO712 Reveals a Novel Pilus Gene Cluster.

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Tarazanova, Mariya; Beerthuyzen, Marke; Siezen, Roland; Fernandez-Gutierrez, Marcela M; de Jong, Anne; van der Meulen, Sjoerd; Kok, Jan; Bachmann, Herwig

2016-01-01

Lactococcus lactis MG1363 is an important gram-positive model organism. It is a plasmid-free and phage-cured derivative of strain NCDO712. Plasmid-cured strains facilitate studies on molecular biological aspects, but many properties which make L. lactis an important organism in the dairy industry are plasmid encoded. We sequenced the total DNA of strain NCDO712 and, contrary to earlier reports, revealed that the strain carries 6 rather than 5 plasmids. A new 50-kb plasmid, designated pNZ712, encodes functional nisin immunity (nisCIP) and copper resistance (lcoRSABC). The copper resistance could be used as a marker for the conjugation of pNZ712 to L. lactis MG1614. A genome comparison with the plasmid cured daughter strain MG1363 showed that the number of single nucleotide polymorphisms that accumulated in the laboratory since the strains diverted more than 30 years ago is limited to 11 of which only 5 lead to amino acid changes. The 16-kb plasmid pSH74 was found to contain a novel 8-kb pilus gene cluster spaCB-spaA-srtC1-srtC2, which is predicted to encode a pilin tip protein SpaC, a pilus basal subunit SpaB, and a pilus backbone protein SpaA. The sortases SrtC1/SrtC2 are most likely involved in pilus polymerization while the chromosomally encoded SrtA could act to anchor the pilus to peptidoglycan in the cell wall. Overexpression of the pilus gene cluster from a multi-copy plasmid in L. lactis MG1363 resulted in cell chaining, aggregation, rapid sedimentation and increased conjugation efficiency of the cells. Electron microscopy showed that the over-expression of the pilus gene cluster leads to appendices on the cell surfaces. A deletion of the gene encoding the putative basal protein spaB, by truncating spaCB, led to more pilus-like structures on the cell surface, but cell aggregation and cell chaining were no longer observed. This is consistent with the prediction that spaB is involved in the anchoring of the pili to the cell.

The role of the Staphylococcal VraTSR regulatory system on vancomycin resistance and vanA operon expression in vancomycin-resistant Staphylococcus aureus.

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Qureshi, Nadia K; Yin, Shaohui; Boyle-Vavra, Susan

2014-01-01

Vancomycin is often the preferred treatment for invasive methicillin-resistant Staphylococcus aureus (MRSA) infection. With the increase in incidence of MRSA infections, the use of vancomycin has increased and, as feared, isolates of vancomycin-resistant Staphylococcus aureus (VRSA) have emerged. VRSA isolates have acquired the entercoccal vanA operon contained on transposon (Tn) 1546 residing on a conjugal plasmid. VraTSR is a vancomycin and β-lactam-inducible three-component regulatory system encoded on the S. aureus chromosome that modulates the cell-wall stress response to cell-wall acting antibiotics. Mutation in vraTSR has shown to increase susceptibility to β-lactams and vancomycin in clinical VISA strains and in recombinant strain COLVA-200 which expresses a plasmid borne vanA operon. To date, the role of VraTSR in vanA operon expression in VRSA has not been demonstrated. In this study, the vraTSR operon was deleted from the first clinical VRSA strain (VRS1) by transduction with phage harvested from a USA300 vraTSR operon deletion strain. The absence of the vraTSR operon and presence of the vanA operon were confirmed in the transductant (VRS1Δvra) by PCR. Broth MIC determinations, demonstrated that the vancomycin MIC of VRS1Δvra (64 µg/ml) decreased by 16-fold compared with VRS1 (1024 µg/ml). The effect of the vraTSR operon deletion on expression of the van gene cluster (vanA, vanX and vanR) was examined by quantitative RT-PCR using relative quantification. A 2-5-fold decreased expression of the vanA operon genes occured in strain VRS1Δvra at stationary growth phase compared with the parent strain, VRS1. Both vancomycin resistance and vancomycin-induced expression of vanA and vanR were restored by complementation with a plasmid harboring the vraTSR operon. These findings demonstrate that expression in S. aureus of the horizontally acquired enterococcal vanA gene cluster is enhanced by the staphylococcal three-component cell wall stress regulatory

Spreading of genes encoding enterotoxins, haemolysins, adhesin and biofilm among methicillin resistant Staphylococcus aureus strains with staphylococcal cassette chromosome mec type IIIA isolated from burn patients.

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Motallebi, Mitra; Jabalameli, Fereshteh; Asadollahi, Kheirollah; Taherikalani, Morovat; Emaneini, Mohammad

2016-08-01

The emergence of antibiotic-resistant Staphylococcus aureus in particular methicillin-resistant S. aureus (MRSA) is an important concern in burn medical centers either in Iran or worldwide. A total of 128 S. aureus isolates were collected from wound infection of burn patients during June 2013 to June 2014. Multiplex-polymerase chain reaction (MPCR) assay was performed for the characterization of the staphylococcal cassette chromosome mec (SCCmec). Genes encoding virulence factors and biofilm were targeted by PCR. Of 128 S. aureus isolates, 77 (60.1%) isolates were MRSA. Fifty four (70.1%) isolates were identified as SCCmec type IIIA. The most frequently detected toxin genes among MRSA isolates with SCCmec type IIIA were sea (64.1%) and hla (51.8%). The rate of coexistence of sea with hla and sea with hla and hlb was 37% and12.9%, respectively. The sec, eta, tst, pvl, hla and hlb genes were not detected in any of the MRSA isolates. The most prevalent genes encoding biofilm was eno, found in 61.1% of isolates, followed by fib and icaA found in 48.1% and 38.8% of the isolates, respectively. The rate of coexistence of fib + eno + icaA + icaD and fib + eno was 20.3% and 9.2%, respectively. The ebps gene was not detected in any of the isolates. In conclusion, our study indicated that the sea, hla, fib and icaA were most frequent genes encoding virulence factors among MRSA with SCCmec type IIIA isolated from burn wound infection. Moreover, the results of this study shows that the rate of coexistence of genes encoding different virulence factor were high. Copyright © 2016 Elsevier Ltd. All rights reserved.

Effect of concomitant use of immunomodulator (OK-432 and/or PSK) on advanced lung cancer (squamous cell carcinoma and adenocarcinoma) treated with radiation with combined chemotherapy

International Nuclear Information System (INIS)

Ogawa, Yasuhiro; Kimura, Shuji; Imajo, Yoshinari

1982-01-01

Between 1975 and 1979, 209 cases of primary lung cancer admitted to the department of radiology were treated with radiation with combined chemotherapy. OK-432 and/or PSK as an immunomodulator was administered to 130 of these cases, and survival curves were evaluated between the patients with OK-432 and/or PSK and those without immunomodulator. In 61 cases (squamous cell carcinoma and adenocarcinoma) in stage III (UICC, 1978), fifty percent survival period was found to be 12.5 months for 16 cases with OK-432, 13.5 months for 9 cases with OK-432 and PSK, 9.0 months for 18 cases with PSK, and 8.0 months for 18 cases without immunotherapy, respectively. (author)

KPC-mediated resistance in Klebsiella pneumoniae in two hospitals in Padua, Italy, June 2009-December 2011: massive spreading of a KPC-3-encoding plasmid and involvement of non-intensive care units

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Richter Sara N

2012-07-01

Full Text Available Abstract Background Klebsiella pneumoniae carbapenemases (KPCs producing bacteria have emerged as a cause of multidrug-resistant nosocomial infections worldwide. KPCs are plasmid-encoded enzymes capable of hydrolysing a broad spectrum of beta-lactams, including carbapenems and monobactams, therefore worryingly limiting antimicrobial treatment options. Analysis of circulating bacterial strains and KPC alleles may help understanding the route of KPC dissemination and therefore help containing the infection. Methods KPC-producing Klebsiella pneumoniae dissemination in two 1580- and 300- bed hospitals in Padua, Italy, from initial outbreak in 2009 to late 2011 was analysed. Molecular and clinical epidemiology, including bacterial strains, KPC-encoding plasmid sequences and associated resistance genes, involved hospital wards and relocation of patients were described. Routine antimicrobial susceptibility testing and MIC of carbapenems on clinical isolates were performed. Detection of resistance genes was obtained by PCR and sequencing. MLST, PFGE and ERIC were used for molecular genotyping. Plasmid analysis was obtained by digestion with restriction enzymes and deep sequencing. Results KPC-positive clinical samples were isolated from nearly 200 patients. In the initial outbreak intensive care units were almost exclusively involved, while medical, surgical and long-term wards were successively massively concerned. Analysis of KPC alleles, plasmids and bacterial sequence types (STs indicated that during the initial outbreak KPC-3 in ST258 and KPC-2 in ST147 were each confined in one of the two surveilled hospitals. While KPC-2 dissemination was effectively contained, KPC-3 in ST258 cross-spreading was observed. The simultaneous presence of two carbapenemases, VIM-1 and KPC-2, in the same isolate was also observed in three patients. Total sequencing of plasmid content of two KPC-3 strains showed novel association of resistance plasmids. Conclusions The

Multiple drug resistant carbapenemases producing Acinetobacter baumannii isolates harbours multiple R-plasmids

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Rajagopalan Saranathan

2014-01-01

Full Text Available Background & objectives: The nosocomial human pathogen Acinetobacter baumannii has high propensity to develop resistance to antimicrobials and to become multidrug resistant (MDR, consequently complicating the treatment. This study was carried out to investigate the presence of resistant plasmids (R-plasmids among the clinical isolates of A. baumannii. In addition, the study was performed to check the presence of common β-lactamases encoding genes on these plasmids. Methods: A total of 55 clinical isolates of A. baumannii were included in the study and all were subjected to plasmid DNA isolation, followed by PCR to check the presence of resistance gene determinants such as blaOXA-23 , blaOXA-51, blaOXA-58 and blaIMP-1 on these plasmids that encode for oxacillinase (OXA and metallo-β-lactamase (MBL type of carbapenemases. Plasmid curing experiments were carried out on selected isolates using ethidium bromide and acridine orange as curing agents and the antibiotic resistance profiles were evaluated before and after curing. Results: All the isolates were identified as A. baumannii by 16SrDNA amplification and sequencing. Plasmid DNA isolated from these isolates showed the occurrence of multiple plasmids with size ranging from 500bp to ≥ 25 kb. The percentage of blaOXA-51 and blaOXA-23 on plasmids were found to be 78 and 42 per cent, respectively and 20 isolates (36% carried blaIMP-1 gene on plasmids. Significant difference was observed in the antibiograms of plasmid cured isolates when compared to their parental ones. The clinical isolates became susceptible to more than two antibiotic classes after curing of plasmids indicating plasmid borne resistance. Interpretation & conclusions: Our study determined the plasmid mediated resistance mechanisms and occurrence of different resistance genes on various plasmids isolated from MDR A. baumannii. The present findings showed the evidence for antibiotic resistance mediated through multiple plasmids in

Effect of concomitant use of immunomodulator (OK-432 and/or PSK) on primary lung cancer (stages III, IV) treated with radiation combined with chemotherapy

International Nuclear Information System (INIS)

Kimura, S.; Ogawa, Y.; Imajo, Y.

1982-01-01

OK-432 (a lyophilized preparation of a low virulent strain, Su, of Streptococcus hemolytics (Group A) treated with penicillin G) and/or PSK (a protein-bound polysaccharide prepared from Coriolus versicolor) as an immunomodulator was administered to 209 patients of advanced lung cancer treated with radiation with combined chemotherapy. Their effects on immunological parameters and patients' survival periods were examined. Suppression of both PHA (phytohemagglutinin) skin test activities and lymphocyte blastoid transformation with PHA were reduced in the immunomodulator administered group compared with the non-administered group. Survival curves were evaluated between the patients with OK-432 and/or PSK and those without immunomodulator. It was suggested that OK-432 and/or PSK combined with radiation with combined chemotherapy was effective for the elongation of the survival period. These results indicated that the long-term administration of OK-432 and/or PSK was recommended for patients with advanced lung cancer. (author)

Plasmid-Mediated Quinolone Resistance in Shigella flexneri Isolated From Macaques

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Anthony J. Mannion

2018-03-01

Full Text Available Non-human primates (NHPs for biomedical research are commonly infected with Shigella spp. that can cause acute dysentery or chronic episodic diarrhea. These animals are often prophylactically and clinically treated with quinolone antibiotics to eradicate these possible infections. However, chromosomally- and plasmid-mediated antibiotic resistance has become an emerging concern for species in the family Enterobacteriaceae. In this study, five individual isolates of multi-drug resistant Shigella flexneri were isolated from the feces of three macaques. Antibiotic susceptibility testing confirmed resistance or decreased susceptibility to ampicillin, amoxicillin-clavulanic acid, cephalosporins, gentamicin, tetracycline, ciprofloxacin, enrofloxacin, levofloxacin, and nalidixic acid. S. flexneri isolates were susceptible to trimethoprim-sulfamethoxazole, and this drug was used to eradicate infection in two of the macaques. Plasmid DNA from all isolates was positive for the plasmid-encoded quinolone resistance gene qnrS, but not qnrA and qnrB. Conjugation and transformation of plasmid DNA from several S. flexneri isolates into antibiotic-susceptible Escherichia coli strains conferred the recipients with resistance or decreased susceptibility to quinolones and beta-lactams. Genome sequencing of two representative S. flexneri isolates identified the qnrS gene on a plasmid-like contig. These contigs showed >99% homology to plasmid sequences previously characterized from quinolone-resistant Shigella flexneri 2a and Salmonella enterica strains. Other antibiotic resistance genes and virulence factor genes were also identified in chromosome and plasmid sequences in these genomes. The findings from this study indicate macaques harbor pathogenic S. flexneri strains with chromosomally- and plasmid-encoded antibiotic resistance genes. To our knowledge, this is the first report of plasmid-mediated quinolone resistance in S. flexneri isolated from NHPs and warrants

Large IncHI2-plasmids encode extended-spectrum β-lactamases (ESBLs) in Enterobacter spp. bloodstream isolates, and support ESBL-transfer to Escherichia coli.

Science.gov (United States)

Nilsen, E; Haldorsen, B C; Sundsfjord, A; Simonsen, G S; Ingebretsen, A; Naseer, U; Samuelsen, O

2013-11-01

We investigated the prevalence of extended-spectrum β-lactamases (ESBLs) in Enterobacter spp. bloodstream isolates from 19 hospital laboratories in Norway during 2011. A total of 62/230 (27%) isolates were resistant to third-generation cephalosporins and four (1.7%) were ESBL-positive; blaCTX -M-15 (n = 3) and blaSHV -12 (n = 1). This is comparable to the prevalence of ESBLs in clinical isolates of Escherichia coli and Klebsiella pneumoniae in Norway during the same period. All ESBL-positive isolates were multidrug resistant (MDR) and harboured plasmid-mediated quinolone resistance. Three isolates supported transfer of large IncHI2-plasmids harbouring ESBL- and MDR-encoding genes to E. coli recipients by in vitro conjugation. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.

THE ENDOGENOUS BACILLUS-SUBTILIS (NATTO) PLASMIDS PTA1015 AND PTA1040 CONTAIN SIGNAL PEPTIDASE-ENCODING GENES - IDENTIFICATION OF A NEW STRUCTURAL MODULE ON CRYPTIC PLASMIDS

NARCIS (Netherlands)

MEIJER, WJJ; DEJONG, A; BEA, G; WISMAN, A; TJALSMA, H; VENEMA, G; BRON, S; MAARTEN, J; VANDIJL, JM

Various strains of Bacillus subtilis (natto) contain small cryptic plasmids that replicate via the rolling-circle mechanism. Like plasmids from other Gram-positive bacteria, these plasmids are composed of several distinct structural modules. A new structural module was identified on the B. subtilis

SVD-BASED TRANSMIT BEAMFORMING FOR VARIOUS MODULATIONS WITH CONVOLUTION ENCODING

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M. Raja

2011-09-01

Full Text Available This paper present a new beamforming technique using singular value decomposition (SVD for closed loop Multiple-input, multiple-output (MIMO wireless systems with various modulation techniques such as BPSK, 16-QAM, 16-PSK, DPSK and PAM along with convolution encoder. The channel matrix is decomposed into a number of independent orthogonal modes of excitation, which refer to as eigenmodes of the channel. Transmit precoding is performed by multiplying the input symbols with unitary matrix to produce the transmit beamforming, and the precoded symbols are transmitted over Rayleigh fading channel. At the receiver, combining process is performed by using maximum ratio combiner (MRC, and the receiver shaping is performed to retrieve the original input symbols by multiplying the received signal with conjugate transpose of the unitary matrix. Furthermore, the expressions for average bit error rate (BER for M-PSK and average BER for M-QAM are derived. The superiority of the proposed work is proved by simulation results and the proposed work is compared to the other beamforming methods.

trans-Acting Virulence Functions of the Octopine Ti Plasmid from Agrobacterium tumefaciens

NARCIS (Netherlands)

Hille, Jacques; Kan, Jan van; Schilperoort, Rob

1984-01-01

All Ti plasmid-encoded virulence functions that were studied act in trans. An octopine Ti plasmid-specific vir operon, called vir-O, located on an EcoRI restriction fragment has been characterized. Sequences with promoter activity in Escherichia coli were identified for a second vir operon, called

Optical phase-modulated radio-over-fiber links with k-means algorithm for digital demodulation of 8PSK subcarrier multiplexed signals

DEFF Research Database (Denmark)

Guerrero Gonzalez, Neil; Zibar, Darko; Yu, Xianbin

2010-01-01

A k-means algorithm for phase recovery of three, 50 Mbaud, 8PSK subcarrier multiplexed signals at 5 GHz for optical phase-modulated radio-over-fiber is proposed and experimentally demonstrated after 40 km of single mode fiber transmission......A k-means algorithm for phase recovery of three, 50 Mbaud, 8PSK subcarrier multiplexed signals at 5 GHz for optical phase-modulated radio-over-fiber is proposed and experimentally demonstrated after 40 km of single mode fiber transmission...

Characterization of a Staphylococcal Plasmid Related to pUB110 and Carrying Two Novel Genes, vatC and vgbB, Encoding Resistance to Streptogramins A and B and Similar Antibiotics

OpenAIRE

Allignet, Jeanine; Liassine, Nadia; El Solh, Névine

1998-01-01

We isolated and sequenced a plasmid, named pIP1714 (4,978 bp), which specifies resistance to streptogramins A and B and the mixture of these compounds. pIP1714 was isolated from a Staphylococcus cohnii subsp. cohnii strain found in the environment of a hospital where pristinamycin was extensively used. Resistance to both compounds and related antibiotics is encoded by two novel, probably cotranscribed genes, (i) vatC, encoding a 212-amino-acid (aa) acetyltransferase that inactivates streptogr...

The Collagen-Binding Adhesin Is a Virulence Factor in Staphylococcus aureus Keratitis

Science.gov (United States)

Rhem, Marcus N.; Lech, Elizabeth M.; Patti, Joseph M.; McDevitt, Damien; Höök, Magnus; Jones, Dan B.; Wilhelmus, Kirk R.

2000-01-01

A collagen-binding strain of Staphylococcus aureus produced suppurative inflammation in a rabbit model of soft contact lens-associated bacterial keratitis more often than its collagen-binding-negative isogenic mutant. Reintroduction of the cna gene on a multicopy plasmid into the mutant helped it regain its corneal adherence and infectivity. The topical application of a collagen-binding peptide before bacterial challenge decreased S. aureus adherence to deepithelialized corneas. These data suggest that the collagen-binding adhesin is involved in the pathogenesis of S. aureus infection of the cornea. PMID:10816547

PCR detection of staphylococcal enterotoxin genes in Staphylococcus aureus strains isolated from raw and pasteurized milk.

Science.gov (United States)

Rall, V L M; Vieira, F P; Rall, R; Vieitis, R L; Fernandes, A; Candeias, J M G; Cardoso, K F G; Araújo, J P

2008-12-10

Milk is considered a nutritious food because it contains several important nutrients including proteins and vitamins. Conversely, it can be a vehicle for several pathogenic bacteria such as Staphylococcus aureus. This study aimed to analyze the frequency of genes encoding the staphylococcal enterotoxins (SEs) SEA, SEB, SEC, SED, SEE, SEG, SEH, SEI and SEJ in S. aureus strains isolated from raw or pasteurized bovine milk. S. aureus was found in 38 (70.4%) out of 54 raw milk samples at concentrations of up to 8.9 x 10(5) CFU/ml. This microorganism was present in eight samples of pasteurized milk before the expiration date and in 11 samples analyzed on the expiration date. Of the 57 strains studied, 68.4% were positive for one or more genes encoding the enterotoxins, and 12 different genotypes were identified. The gene coding for enterotoxin A, sea, was the most frequent (16 strains, 41%), followed by sec (8 strains, 20.5%), sed (5 strains, 12.8%), seb (3 strains, 7.7%) and see (2 strains, 5.1%). Among the genes encoding the other enterotoxins, seg was the most frequently observed (11 strains, 28.2%), followed by sei (10 strains) and seh and sej (3 strains each). With the recent identification of new SEs, the perceived frequency of enterotoxigenic strains has increased, suggesting that the pathogenic potential of staphylococci may be higher than previously thought; however, further studies are required to assess the expression of these new SEs by S. aureus, and their impact in foodborne disease. The quality of Brazilian milk is still low, and efforts from the government and the entire productive chain are required to attain consumer safety.

Plasmids foster diversification and adaptation of bacterial populations in soil.

Science.gov (United States)

Heuer, Holger; Smalla, Kornelia

2012-11-01

It is increasingly being recognized that the transfer of conjugative plasmids across species boundaries plays a vital role in the adaptability of bacterial populations in soil. There are specific driving forces and constraints of plasmid transfer within bacterial communities in soils. Plasmid-mediated genetic variation allows bacteria to respond rapidly with adaptive responses to challenges such as irregular antibiotic or metal concentrations, or opportunities such as the utilization of xenobiotic compounds. Cultivation-independent detection and capture of plasmids from soil bacteria, and complete sequencing have provided new insights into the role and ecology of plasmids. Broad host range plasmids such as those belonging to IncP-1 transfer a wealth of accessory functions which are carried by similar plasmid backbones. Plasmids with a narrower host range can be more specifically adapted to particular species and often transfer genes which complement chromosomally encoded functions. Plasmids seem to be an ancient and successful strategy to ensure survival of a soil population in spatial and temporal heterogeneous conditions with various environmental stresses or opportunities that occur irregularly or as a novel challenge in soil. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Design and evaluation of protein expression in a recombinant plasmid encoding epitope gp 350/220 of the Epstein-Barr virus (EBV)

Science.gov (United States)

Himmah, Karimatul; Dluha, Nurul; Anyndita, Nadya V. M.; Rifa'i, Muhaimin; Widodo

2017-05-01

The Epstein - Barr virus (EBV) causes severe infections that may lead to cancers such as nasopharyngeal carcinoma. Development of effective EBV vaccines is necessary to prevent the virus spreading throughout the community. TheEBV has a surface protein gp 350/220, which serves as an antigen to help interact with host cells. Epitopes of the protein can potentially serve as bases for a vaccine. In a previous study, we have found a conserved epitope of gp 350/220 from all strains EBV through an in silico approach. The aim of this study is to design and overproduce a recombinant peptide of epitope gp 350/220 in E. coli. DNA encoding the conserved epitope was synthesized and cloned into plasmid pET-22b(+); the recombinant plasmid was transformed into E. coli strains DH5α and BL21. The transformed plasmid DNA was isolated and confirmed by restriction using XbaI and PstI enzymes followed by DNA sequencing. Protein expression was induced by isopropyl-D-thiogalactopyranoside (IPTG) with final concentrations of 0.1, 0.2, 1, and 2 mM in consecutive times. An osmotic shock method was used to isolate protein from periplasmic fraction of E. coli DH5α and BL21. The SDS-PAGE analysis was carried out to detect peptide target (3.4 kDa). Based on this result, the induction process did not work properly, and thus needs further investigation.

Plasmid-associated sensitivity of Bacillus thuringiensis to UV light

International Nuclear Information System (INIS)

Benoit, T.G.; Wilson, G.R.; Bull, D.L.; Aronson, A.I.

1990-01-01

Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores

Multidrug Efflux Pumps in Staphylococcus aureus: an Update

Science.gov (United States)

Costa, Sofia Santos; Viveiros, Miguel; Amaral, Leonard; Couto, Isabel

2013-01-01

The emergence of infections caused by multi- or pan-resistant bacteria in the hospital or in the community settings is an increasing health concern. Albeit there is no single resistance mechanism behind multiresistance, multidrug efflux pumps, proteins that cells use to detoxify from noxious compounds, seem to play a key role in the emergence of these multidrug resistant (MDR) bacteria. During the last decades, experimental data has established their contribution to low level resistance to antimicrobials in bacteria and their potential role in the appearance of MDR phenotypes, by the extrusion of multiple, unrelated compounds. Recent studies suggest that efflux pumps may be used by the cell as a first-line defense mechanism, avoiding the drug to reach lethal concentrations, until a stable, more efficient alteration occurs, that allows survival in the presence of that agent. In this paper we review the current knowledge on MDR efflux pumps and their intricate regulatory network in Staphylococcus aureus, a major pathogen, responsible from mild to life-threatening infections. Particular emphasis will be given to the potential role that S. aureus MDR efflux pumps, either chromosomal or plasmid-encoded, have on resistance towards different antimicrobial agents and on the selection of drug - resistant strains. We will also discuss the many questions that still remain on the role of each specific efflux pump and the need to establish appropriate methodological approaches to address all these questions. PMID:23569469

Multidrug Efflux Pumps in Staphylococcus aureus: an Update.

Science.gov (United States)

Costa, Sofia Santos; Viveiros, Miguel; Amaral, Leonard; Couto, Isabel

2013-01-01

The emergence of infections caused by multi- or pan-resistant bacteria in the hospital or in the community settings is an increasing health concern. Albeit there is no single resistance mechanism behind multiresistance, multidrug efflux pumps, proteins that cells use to detoxify from noxious compounds, seem to play a key role in the emergence of these multidrug resistant (MDR) bacteria. During the last decades, experimental data has established their contribution to low level resistance to antimicrobials in bacteria and their potential role in the appearance of MDR phenotypes, by the extrusion of multiple, unrelated compounds. Recent studies suggest that efflux pumps may be used by the cell as a first-line defense mechanism, avoiding the drug to reach lethal concentrations, until a stable, more efficient alteration occurs, that allows survival in the presence of that agent. In this paper we review the current knowledge on MDR efflux pumps and their intricate regulatory network in Staphylococcus aureus, a major pathogen, responsible from mild to life-threatening infections. Particular emphasis will be given to the potential role that S. aureus MDR efflux pumps, either chromosomal or plasmid-encoded, have on resistance towards different antimicrobial agents and on the selection of drug - resistant strains. We will also discuss the many questions that still remain on the role of each specific efflux pump and the need to establish appropriate methodological approaches to address all these questions.

Comparative Sequence Analysis of Multidrug-Resistant IncA/C Plasmids from Salmonella enterica.

Science.gov (United States)

Hoffmann, Maria; Pettengill, James B; Gonzalez-Escalona, Narjol; Miller, John; Ayers, Sherry L; Zhao, Shaohua; Allard, Marc W; McDermott, Patrick F; Brown, Eric W; Monday, Steven R

2017-01-01

Determinants of multidrug resistance (MDR) are often encoded on mobile elements, such as plasmids, transposons, and integrons, which have the potential to transfer among foodborne pathogens, as well as to other virulent pathogens, increasing the threats these traits pose to human and veterinary health. Our understanding of MDR among Salmonella has been limited by the lack of closed plasmid genomes for comparisons across resistance phenotypes, due to difficulties in effectively separating the DNA of these high-molecular weight, low-copy-number plasmids from chromosomal DNA. To resolve this problem, we demonstrate an efficient protocol for isolating, sequencing and closing IncA/C plasmids from Salmonella sp. using single molecule real-time sequencing on a Pacific Biosciences (Pacbio) RS II Sequencer. We obtained six Salmonella enterica isolates from poultry, representing six different serovars, each exhibiting the MDR-Ampc resistance profile. Salmonella plasmids were obtained using a modified mini preparation and transformed with Escherichia coli DH10Br. A Qiagen Large-Construct kit™ was used to recover highly concentrated and purified plasmid DNA that was sequenced using PacBio technology. These six closed IncA/C plasmids ranged in size from 104 to 191 kb and shared a stable, conserved backbone containing 98 core genes, with only six differences among those core genes. The plasmids encoded a number of antimicrobial resistance genes, including those for quaternary ammonium compounds and mercury. We then compared our six IncA/C plasmid sequences: first with 14 IncA/C plasmids derived from S. enterica available at the National Center for Biotechnology Information (NCBI), and then with an additional 38 IncA/C plasmids derived from different taxa. These comparisons allowed us to build an evolutionary picture of how antimicrobial resistance may be mediated by this common plasmid backbone. Our project provides detailed genetic information about resistance genes in

Comparative Sequence Analysis of Multidrug-Resistant IncA/C Plasmids from Salmonella enterica

Directory of Open Access Journals (Sweden)

Maria Hoffmann

2017-08-01

Full Text Available Determinants of multidrug resistance (MDR are often encoded on mobile elements, such as plasmids, transposons, and integrons, which have the potential to transfer among foodborne pathogens, as well as to other virulent pathogens, increasing the threats these traits pose to human and veterinary health. Our understanding of MDR among Salmonella has been limited by the lack of closed plasmid genomes for comparisons across resistance phenotypes, due to difficulties in effectively separating the DNA of these high-molecular weight, low-copy-number plasmids from chromosomal DNA. To resolve this problem, we demonstrate an efficient protocol for isolating, sequencing and closing IncA/C plasmids from Salmonella sp. using single molecule real-time sequencing on a Pacific Biosciences (Pacbio RS II Sequencer. We obtained six Salmonella enterica isolates from poultry, representing six different serovars, each exhibiting the MDR-Ampc resistance profile. Salmonella plasmids were obtained using a modified mini preparation and transformed with Escherichia coli DH10Br. A Qiagen Large-Construct kit™ was used to recover highly concentrated and purified plasmid DNA that was sequenced using PacBio technology. These six closed IncA/C plasmids ranged in size from 104 to 191 kb and shared a stable, conserved backbone containing 98 core genes, with only six differences among those core genes. The plasmids encoded a number of antimicrobial resistance genes, including those for quaternary ammonium compounds and mercury. We then compared our six IncA/C plasmid sequences: first with 14 IncA/C plasmids derived from S. enterica available at the National Center for Biotechnology Information (NCBI, and then with an additional 38 IncA/C plasmids derived from different taxa. These comparisons allowed us to build an evolutionary picture of how antimicrobial resistance may be mediated by this common plasmid backbone. Our project provides detailed genetic information about

Virulence Factor Genes in Staphylococcus aureus Isolated From Diabetic Foot Soft Tissue and Bone Infections.

Science.gov (United States)

Víquez-Molina, Gerardo; Aragón-Sánchez, Javier; Pérez-Corrales, Cristian; Murillo-Vargas, Christian; López-Valverde, María Eugenia; Lipsky, Benjamin A

2018-03-01

The aim of this study is to describe the presence of genes encoding for 4 virulence factors (pvl, eta, etb, and tsst), as well as the mecA gene conferring resistance to beta-lactam antibiotics, in patients with diabetes and a staphylococcal foot infection. We have also analyzed whether isolates of Staphylococcus aureus from bone infections have a different profile for these genes compared with those from exclusively soft tissue infections. In this cross-sectional study of a prospectively recruited series of patients admitted to the Diabetic Foot Unit, San Juan de Dios Hospital, San José, Costa Rica with a moderate or severe diabetic foot infection (DFI), we collected samples from infected soft tissue and from bone during debridement. During the study period (June 1, 2014 to May 31, 2016), we treated 379 patients for a DFI. S aureus was isolated from 101 wound samples, of which 43 were polymicrobial infections; we only included the 58 infections that were monomicrobial S aureus for this study. Infections were exclusively soft tissue in 17 patients (29.3%) while 41 (70.7%) had bone involvement (osteomyelitis). The mecA gene was detected in 35 cases (60.3%), pvl gene in 4 cases (6.9%), and tsst gene in 3 (5.2%). We did not detect etA and etB in any of the cases. There were no differences in the profile of S aureus genes encoding for virulence factors (pvl, etA, etB, and tsst) recovered from DFIs between those with just soft tissue compared to those with osteomyelitis. However, we found a significantly higher prevalence of pvl+ strains of S aureus associated with soft tissue compared with bone infections. Furthermore, we observed a significantly longer time to healing among patients infected with mecA+ (methicillin-resistant) S aureus (MRSA).

Characterization of colonizing Staphylococcus aureus isolated from surgical wards' patients in a Nigerian university hospital.

Directory of Open Access Journals (Sweden)

Deboye O Kolawole

Full Text Available In contrast to developed countries, only limited data on the prevalence, resistance and clonal structure of Staphylococcus aureus are available for African countries. Since S. aureus carriage is a risk factor for postoperative wound infection, patients who had been hospitalized in surgical wards in a Nigerian University Teaching Hospital were screened for S. aureus carriage. All S. aureus isolates were genotyped (spa, agr and assigned to multilocus sequence types (MLST. Species affiliation, methicillin-resistance, and the possession of pyrogenic toxin superantigens (PTSAg, exfoliative toxins (ETs and Panton-Valentine Leukocidin (PVL were analyzed. Of 192 patients screened, the S. aureus carrier rate was 31.8 % (n = 61. Of these isolates, 7 (11.5% were methicillin-resistant (MRSA. The isolates comprised 24 spa types. The most frequent spa types were t064, t084, t311, and t1931, while the most prevalent MLST clonal complexes were CC5 and CC15. The most frequent PTSAg genes detected were seg/sei (41.0% followed by seb (29.5%, sea (19.7%, seh (14.7% and sec (11.5. The difference between the possession of classical and newly described PTSAg genes was not significant (63.9% versus 59.0% respectively; P = 0.602. PVL encoding genes were found in 39.3% isolates. All MRSA isolates were PVL negative, SCCmec types I and VI in MLST CC 5 and CC 30, respectively. Typing of the accessory gene regulator (agr showed the following distribution: agr group 1 (n = 20, group II (n = 17, group III (n = 14 and group IV (n = 10. Compared to European data, enterotoxin gene seb and PVL-encoding genes were more prevalent in Nigerian methicillin-susceptible S. aureus isolates, which may therefore act as potential reservoir for PVL and PTSAg genes.

Characterization of Endogenous Plasmids from Lactobacillus salivarius UCC118▿ †

OpenAIRE

Fang, Fang; Flynn, Sarah; Li, Yin; Claesson, Marcus J.; van Pijkeren, Jan-Peter; Collins, J. Kevin; van Sinderen, Douwe; O'Toole, Paul W.

2008-01-01

The genome of Lactobacillus salivarius UCC118 comprises a 1.83-Mb chromosome, a 242-kb megaplasmid (pMP118), and two smaller plasmids of 20 kb (pSF118-20) and 44 kb (pSF118-44). Annotation and bioinformatic analyses suggest that both of the smaller plasmids replicate by a theta replication mechanism. Furthermore, it appears that they are transmissible, although neither possesses a complete set of conjugation genes. Plasmid pSF118-20 encodes a toxin-antitoxin system composed of pemI and pemK h...

Identification and Characterization of Linezolid-Resistant cfr-Positive Staphylococcus aureus USA300 Isolates from a New York City Medical Center

Science.gov (United States)

Zuill, Douglas E.; Scharn, Caitlyn R.; Deane, Jennifer; Sahm, Daniel F.; Goering, Richard V.; Jenkins, Stephen G.; Shaw, Karen J.

2014-01-01

The cfr gene was identified in three linezolid-resistant USA300 methicillin-resistant Staphylococcus aureus (MRSA) isolates collected over a 3-day period at a New York City medical center in 2011 as part of a routine surveillance program. Each isolate possessed a plasmid containing a pSCFS3-like cfr gene environment. Transformation of the cfr-bearing plasmids into the S. aureus ATCC 29213 background recapitulated the expected Cfr antibiogram, including resistance to linezolid, tiamulin, clindamycin, and florfenicol and susceptibility to tedizolid. PMID:25136008

Broad-Host-Range IncP-1 plasmids and their resistance potential

Directory of Open Access Journals (Sweden)

Magdalena ePopowska

2013-03-01

Full Text Available The plasmids of the incompatibility group IncP-1, also called IncP, as extrachromosomal genetic elements can transfer and replicate virtually in all Gram-negative bacteria. They are composed of backbone genes that encode a variety of essential functions and accessory genes that have implications for human health and environmental bioremediation. Broad-host-range IncP plasmids are known to spread genes between distinct phylogenetic groups of bacteria. These genes often code for resistances to a broad spectrum of antibiotics, heavy metals and quaternary ammonium compounds used as disinfectants. The backbone of these plasmids carries modules that enable them to effectively replicate, move to a new host via conjugative transfer and to be stably maintained in bacterial cells. The adaptive, resistance and virulence genes are mainly located on mobile genetic elements integrated between the functional plasmid backbone modules. Environmental studies have demonstrated the wide distribution of IncP-like replicons in manure, soils and wastewater treatment plants. They also are present in strains of pathogenic or opportunistic bacteria, which can be a cause for concern, because they may encode multiresistance. Their broad distribution suggests that IncP plasmids play a crucial role in bacterial adaptation by utilizing horizontal gene transfer. This review summarizes the variety of genetic information and physiological functions carried by IncP plasmids, which can contribute to the spread of antibiotic and heavy metal resistance while also mediating the process of bioremediation of pollutants. Due to the location of the resistance genes on plasmids with a broad host range and the presence of transposons carrying these genes it seems that the spread of these genes would be possible and quite hazardous in infection control. Future studies are required to determine the level of risk of the spread of resistance genes located on these plasmids.

Effect on Antibody and T-Cell Responses of Mixing Five GMP-Produced DNA Plasmids and Administration With Plasmid Expressing GM-CSF

National Research Council Canada - National Science Library

Sedegah, M; Charoenvit, Y; Aguiar, J; Sacci, J; Hedstrom, R; Kumar, S; Belmonte, A; Lanar, DE; Jones, TR; Abot, E

2004-01-01

.... In preparation for a clinical trial, we assessed the immunogenicity of GMP-produced plasmids encoding five Plasmodium falciparum proteins, PfCSP, PfSSP2, PfEXP1, PfLSA1, and PfLSA3, given as a mixture, or alone...

16-level differential phase shift keying (D16PSK) in direct detection optical communication systems

DEFF Research Database (Denmark)

Sambaraju, R.; Tokle, Torger; Jensen, J.B.

2006-01-01

Optical 16-level differential phase shift keying (D16PSK) carrying four bits for every symbol is proposed for direct detection optical communication systems. Transmitter and receiver schematics are presented, and the receiver sensitivity is discussed. We numerically investigate the impact...

The response of a linear monostable system and its application in parameters estimation for PSK signals

International Nuclear Information System (INIS)

Duan, Chaowei; Zhan, Yafeng

2016-01-01

The output characteristics of a linear monostable system driven with a periodic signal and an additive white Gaussian noise are studied in this paper. Theoretical analysis shows that the output signal-to-noise ratio (SNR) decreases monotonously with the increasing noise intensity but the output SNR-gain is stable. Inspired by this high SNR-gain phenomenon, this paper applies the linear monostable system in the parameters estimation algorithm for phase shift keying (PSK) signals and improves the estimation performance. - Highlights: • The response of a linear monostable system driven with a periodic signal and an additive white Gaussian noise is analyzed. • The optimal parameter of this linear monostable system to maximum the output SNR-gain is obtained. • Application of this linear monostable system in parameters estimation algorithm for PSK signals obtains performance improvement.

Prevalence of genes encoding extracellular virulence factors among meticillin-resistant Staphylococcus aureus isolates from the University Hospital, Olomouc, Czech Republic.

Science.gov (United States)

Sauer, P; Síla, J; Stosová, T; Vecerová, R; Hejnar, P; Vágnerová, I; Kolár, M; Raclavsky, V; Petrzelová, J; Lovecková, Y; Koukalová, D

2008-04-01

A rather fast and complicated progression of an infection caused by some strains of Staphylococcus aureus could be associated with the expression and co-action of virulence factor complexes in these strains. This study screened the antibiotic susceptibility and prevalence of virulence markers in isolates of meticillin-resistant S. aureus (MRSA) obtained from patients hospitalized at the University Hospital in Olomouc, Czech Republic. A total of 100 isolates was screened for 13 genes encoding extracellular virulence determinants (tst, pvl, eta, etb, sea, seb, sec, sed, see, seg, seh, sei and sej) and for their distribution in sample types. Eighty-nine isolates were positive for at least one of the genes. Genes for etb, pvl, see and seh were not detected in any of the MRSA isolates. No statistically significant differences in the occurrence of the determinants studied among sample types were found.

Characterization of Endogenous Plasmids from Lactobacillus salivarius UCC118▿ †

Science.gov (United States)

Fang, Fang; Flynn, Sarah; Li, Yin; Claesson, Marcus J.; van Pijkeren, Jan-Peter; Collins, J. Kevin; van Sinderen, Douwe; O'Toole, Paul W.

2008-01-01

The genome of Lactobacillus salivarius UCC118 comprises a 1.83-Mb chromosome, a 242-kb megaplasmid (pMP118), and two smaller plasmids of 20 kb (pSF118-20) and 44 kb (pSF118-44). Annotation and bioinformatic analyses suggest that both of the smaller plasmids replicate by a theta replication mechanism. Furthermore, it appears that they are transmissible, although neither possesses a complete set of conjugation genes. Plasmid pSF118-20 encodes a toxin-antitoxin system composed of pemI and pemK homologs, and this plasmid could be cured when PemI was produced in trans. The minimal replicon of pSF118-20 was determined by deletion analysis. Shuttle vector derivatives of pSF118-20 were generated that included the replication region (pLS203) and the replication region plus mobilization genes (pLS208). The plasmid pLS203 was stably maintained without selection in Lactobacillus plantarum, Lactobacillus fermentum, and the pSF118-20-cured derivative strain of L. salivarius UCC118 (strain LS201). Cloning in pLS203 of genes encoding luciferase and green fluorescent protein, and expression from a constitutive L. salivarius promoter, demonstrated the utility of this vector for the expression of heterologous genes in Lactobacillus. This study thus expands the knowledge base and vector repertoire of probiotic lactobacilli. PMID:18390685

Staphylococcus aureus in Some Brazilian Dairy Industries: Changes of Contamination and Diversity

DEFF Research Database (Denmark)

Dittmann, Karen Kiesbye; Chaul, Luiza T.; Lee, Sarah H. I.

2017-01-01

Staphylococcus aureus, a major food-poisoning pathogen, is a common contaminant in dairy industries worldwide, including in Brazil. We determined the occurrence of S. aureus in five dairies in Brazil over 8 months. Of 421 samples, 31 (7.4%) were positive for S. aureus and prevalence varied from 0.......8% of strains being sensitive to all antibiotic classes and no Methicillin-resistant S. aureus (MRSA) strains were found. The enterotoxin-encoding genes involved in food-poisoning, e.g., sea, sed, see, and seg were targeted by PCR. The two toxin-encoding genes, sed and see, were not detected. Only three strains...... contamination....

Development of electrochemical reporter assay using HeLa cells transfected with vector plasmids encoding various responsive elements

Energy Technology Data Exchange (ETDEWEB)

Shiku, Hitoshi, E-mail: shiku@bioinfo.che.tohoku.ac.jp [Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-Aoba, Sendai 980-8579 (Japan); Takeda, Michiaki; Murata, Tatsuya [Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-Aoba, Sendai 980-8579 (Japan); Akiba, Uichi; Hamada, Fumio [Graduate School of Engineering and Resource Science, Akita University, 1-1 Tegata gakuen-machi, Akita 010-8502 (Japan); Matsue, Tomokazu, E-mail: matsue@bioinfo.che.tohoku.ac.jp [Graduate School of Environmental Studies, Tohoku University, 6-6-11-604 Aramaki-Aoba, Sendai 980-8579 (Japan)

2009-04-27

Electrochemical assay using HeLa cell lines transfected with various plasmid vectors encoding SEAP (secreted alkaline phosphatase) as the reporter has been performed by using SECM (scanning electrochemical microscopy). The plasmid vector contains different responsive elements that include GRE (glucocorticoid response elements), CRE (cAMP responsive elements), or {kappa}B (binding site for NF{kappa}B (nuclear factor kappa B)) upstream of the SEAP sequence. The transfected HeLa cells were patterned on a culture dish in a 4 x 4 array of circles of diameter 300 {mu}m by using the PDMS (poly(dimethylsiloxane)) stencil technique. The cellular array was first exposed to 100 ng mL{sup -1} dexamethasone, 10 ng mL{sup -1} forskolin, or 100 ng mL{sup -1} TNF-{alpha} (tumor necrosis factor {alpha}) after which it was further cultured in an RPMI culture medium for 6 h. After incubation, the cellular array was soaked in a measuring solution containing 4.7 mM PAPP (p-aminophenylphosphate) at pH 9.5, following which electrochemical measurements were performed immediately within 40 min. The SECM method allows parallel evaluation of different cell lines transfected with pGRE-SEAP, pCRE-SEAP, and pNF{kappa}B-SEAP patterned on the same solid support for detection of the oxidation current of PAP (p-aminophenol) flux produced from only 300 HeLa cells in each stencil pattern. The results of the SECM method were highly sensitive as compared to those obtained from the conventional CL (chemiluminescence) protocol with at least 5 x 10{sup 4} cells per well.

Ti plasmid-encoded genes responsible for catabolism of the crown gall opine mannopine by Agrobacterium tumefaciens are homologs of the T-region genes responsible for synthesis of this opine by the plant tumor.

Science.gov (United States)

Kim, K S; Farrand, S K

1996-06-01

Agrobacterium tumefaciens NT1 harboring pSaB4, which contains the 14-kb BamHI fragment 4 from the octopine/mannityl opine-type Ti plasmid pTi15955, grew well with agropine (AGR) but slowly with mannopine (MOP) as the sole carbon source. When a second plasmid encoding a dedicated transport system for MOP was introduced, these cells grew well with both AGR and MOP. Transposon insertion mutagenesis and subcloning identified a 5.7-kb region of BamHI fragment 4 that encodes functions required for the degradation of MOP. DNA sequence analysis revealed seven putative genes in this region: mocD (moc for mannityl opine catabolism) and mocE, oriented from right to left, and mocRCBAS, oriented from left to right. Significant identities exist at the nucleotide and derived amino acid sequence levels between these moc genes and the mas genes that are responsible for opine biosynthesis in crown gall tumors. MocD is a homolog of Mas2, the anabolic conjugase encoded by mas2'. MocE and MocC are related to the amino half and the carboxyl half, respectively, of Mas1 (MOP reductase), the second enzyme for MOP biosynthesis. These results indicate that the moc and mas genes evolved from a common origin. MocR and MocS are related to each other and to a putative repressor for the AGR degradation system encoded by the rhizogenic plasmid pRiA4. MocB and MocA are homologs of 6-phosphogluconate dehydratase and glucose-6-phosphate dehydrogenase, respectively. Mutations in mocD and mocE, but not mocC, are suppressed by functions encoded by the chromosome or the 450-kb megaplasmid present in many Agrobacterium isolates. We propose that moc genes derived from genes located elsewhere in the bacterial genome and that the tumor-expressed mas genes evolved from the bacterial moc genes.

The 380 kb pCMU01 plasmid encodes chloromethane utilization genes and redundant genes for vitamin B12- and tetrahydrofolate-dependent chloromethane metabolism in Methylobacterium extorquens CM4: a proteomic and bioinformatics study.

Directory of Open Access Journals (Sweden)

Sandro Roselli

Full Text Available Chloromethane (CH3Cl is the most abundant volatile halocarbon in the atmosphere and contributes to the destruction of stratospheric ozone. The only known pathway for bacterial chloromethane utilization (cmu was characterized in Methylobacterium extorquens CM4, a methylotrophic bacterium able to utilize compounds without carbon-carbon bonds such as methanol and chloromethane as the sole carbon source for growth. Previous work demonstrated that tetrahydrofolate and vitamin B12 are essential cofactors of cmuA- and cmuB-encoded methyltransferases of chloromethane dehalogenase, and that the pathway for chloromethane utilization is distinct from that for methanol. This work reports genomic and proteomic data demonstrating that cognate cmu genes are located on the 380 kb pCMU01 plasmid, which drives the previously defined pathway for tetrahydrofolate-mediated chloromethane dehalogenation. Comparison of complete genome sequences of strain CM4 and that of four other M. extorquens strains unable to grow with chloromethane showed that plasmid pCMU01 harbors unique genes without homologs in the compared genomes (bluB2, btuB, cobA, cbiD, as well as 13 duplicated genes with homologs of chromosome-borne genes involved in vitamin B12-associated biosynthesis and transport, or in tetrahydrofolate-dependent metabolism (folC2. In addition, the presence of both chromosomal and plasmid-borne genes for corrinoid salvaging pathways may ensure corrinoid coenzyme supply in challenging environments. Proteomes of M. extorquens CM4 grown with one-carbon substrates chloromethane and methanol were compared. Of the 49 proteins with differential abundance identified, only five (CmuA, CmuB, PurU, CobH2 and a PaaE-like uncharacterized putative oxidoreductase are encoded by the pCMU01 plasmid. The mainly chromosome-encoded response to chloromethane involves gene clusters associated with oxidative stress, production of reducing equivalents (PntAA, Nuo complex, conversion of

Proteome data from a host-pathogen interaction study with Staphylococcus aureus and human lung epithelial cells

Directory of Open Access Journals (Sweden)

Kristin Surmann

2016-06-01

Full Text Available To simultaneously obtain proteome data of host and pathogen from an internalization experiment, human alveolar epithelial A549 cells were infected with Staphylococcus aureus HG001 which carried a plasmid (pMV158GFP encoding a continuously expressed green fluorescent protein (GFP. Samples were taken hourly between 1.5 h and 6.5 h post infection. By fluorescence activated cell sorting GFP-expressing bacteria could be enriched from host cell debris, but also infected host cells could be separated from those which did not carry bacteria after contact (exposed. Additionally, proteome data of A549 cells which were not exposed to S. aureus but underwent the same sample processing steps are provided as a control. Time-resolved changes in bacterial protein abundance were quantified in a label-free approach. Proteome adaptations of host cells were monitored by comparative analysis to a stable isotope labeled cell culture (SILAC standard. Proteins were extracted from the cells, digested proteolytically, measured by nanoLC–MS/MS, and subsequently identified by database search and then quantified. The data presented here are related to a previously published research article describing the interplay of S. aureus HG001 and human epithelial cells (Surmann et al., 2015 [1]. They have been deposited to the ProteomeXchange platform with the identifiers PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002384 for the S. aureus HG001 proteome dataset and PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002388 for the A549 proteome dataset.

Pharmaceutical development of the plasmid DNA vaccine pDERMATT

NARCIS (Netherlands)

Quaak, S.G.L.

2009-01-01

The discovery of tumor specific antigens and self tolerance mechanisms against these antigens led to the assumption that antigens circulating at sufficient concentration levels could break this self tolerance mechanism and evoke immunological antitumor effects. pDERMATT (plasmid DNA encoding

Staphylococcus aureus innate immune evasion is lineage-specific: a bioinfomatics study.

Science.gov (United States)

McCarthy, Alex J; Lindsay, Jodi A

2013-10-01

Staphylococcus aureus is a major human pathogen, and is targeted by the host innate immune system. In response, S. aureus genomes encode dozens of secreted proteins that inhibit complement, chemotaxis and neutrophil activation resulting in successful evasion of innate immune responses. These proteins include immune evasion cluster proteins (IEC; Chp, Sak, Scn), staphylococcal superantigen-like proteins (SSLs), phenol soluble modulins (PSMs) and several leukocidins. Biochemical studies have indicated that genetic variants of these proteins can have unique functions. To ascertain the scale of genetic variation in secreted immune evasion proteins, whole genome sequences of 88 S. aureus isolates, representing 25 clonal complex (CC) lineages, in the public domain were analysed across 43 genes encoding 38 secreted innate immune evasion protein complexes. Twenty-three genes were variable, with between 2 and 15 variants, and the variants had lineage-specific distributions. They include genes encoding Eap, Ecb, Efb, Flipr/Flipr-like, Hla, Hld, Hlg, Sbi, Scin-B/C and 13 SSLs. Most of these protein complexes inhibit complement, chemotaxis and neutrophil activation suggesting that isolates from each S. aureus lineage respond to the innate immune system differently. In contrast, protein complexes that lyse neutrophils (LukSF-PVL, LukMF, LukED and PSMs) were highly conserved, but can be carried on mobile genetic elements (MGEs). MGEs also encode proteins with narrow host-specificities arguing that their acquisition has important roles in host/environmental adaptation. In conclusion, this data suggests that each lineage of S. aureus evades host immune responses differently, and that isolates can adapt to new host environments by acquiring MGEs and the immune evasion protein complexes that they encode. Cocktail therapeutics that targets multiple variant proteins may be the most appropriate strategy for controlling S. aureus infections. Copyright © 2013 Elsevier B.V. All rights

Invasive Staphylococcus aureus infection in an African adolescent ...

African Journals Online (AJOL)

Staphylococcus aureus remains an important cause of mortality, in the community and health care set-ups. S. aureus strains with genes encoding lethal toxins and culture negative sepsis augment the diagnostic challenge in resource limited settings. With a growing rate of resistance to the causative bacteria and atypical ...

Estimating the Transfer Range of Plasmids Encoding Antimicrobial Resistance in a Wastewater Treatment Plant Microbial Community

DEFF Research Database (Denmark)

Li, Liguan; Dechesne, Arnaud; He, Zhiming

2018-01-01

sludge microbial community was challenged in standardized filter matings with one of three multidrug resistance plasmids (pKJK5, pB10, and RP4) harbored by Escherichia coli or Pseudomonas putida. Different donor–plasmid combinations had distinct transfer frequencies, ranging from 3 to 50 conjugation...... events per 100000 cells of the WWTP microbial community. In addition, transfer was observed to a broad phylogenetic range of 13 bacterial phyla with several taxa containing potentially pathogenic species. Preferential transfer to taxa belonging to the predicted evolutionary host range of the plasmids...... ARG transmission. However, the contribution of microbial communities in WWTPs to ARG dissemination remains poorly understood. Here, we examined for the first time plasmid permissiveness of an activated sludge microbial community by utilizing an established fluorescent bioreporter system. The activated...

Presence and analysis of plasmids in human and animal associated arcobacter species.

Directory of Open Access Journals (Sweden)

Laid Douidah

Full Text Available In this study, we report the screening of four Arcobacter species for the presence of small and large plasmids. Plasmids were present in 9.9% of the 273 examined strains. One Arcobacter cryaerophilus and four Arcobacter butzleri plasmids were selected for further sequencing. The size of three small plasmids isolated from A. butzleri and the one from A. cryaerophilus strains ranged between 4.8 and 5.1 kb, and the size of the large plasmid, isolated from A. butzleri, was 27.4 kbp. The G+C content of all plasmids ranged between 25.4% and 26.2%. A total of 95% of the large plasmid sequence represents coding information, which contrasts to the 20 to 30% for the small plasmids. Some of the open reading frames showed a high homology to putative conserved domains found in other related organisms, such as replication, mobilization and genes involved in type IV secretion system. The large plasmid carried 35 coding sequences, including seven genes in a contiguous region of 11.6 kbp that encodes an orthologous type IV secretion system found in the Wolinella succinogenes genome, Helicobacter pylori and Campylobacter jejuni plasmids, which makes this plasmid interesting for further exploration.

Ber analysis of iterative turbo encoded miso wireless communication system under implementation of q-ostbc scheme

OpenAIRE

Kabir, M. Hasnat; Ullah, Shaikh Enayet; Zaman, Mustari; Rashed, Md. Golam

2012-01-01

In this paper, a comprehensive study has been made to evaluate the performance of a MISO wireless communication system. The 4-by-1 spatially multiplexed Turbo encoded system under investigation incorporates Quasi-orthogonal space-time block coding (Q-STBC) and ML signal detection schemes under QPSK, QAM, 16PSK and 16QAM digital modulations. The simulation results elucidate that a significant improvement of system performance is achieved in QAM modulation. The results are also indicative of no...

Dynamics in copy numbers of five plasmids of a dairy Lactococcus lactis in dairy-related conditions including near-zero growth rates.

Science.gov (United States)

van Mastrigt, Oscar; Lommers, Marcel M A N; de Vries, Yorick C; Abee, Tjakko; Smid, Eddy J

2018-03-23

Lactic acid bacteria can carry multiple plasmids affecting their performance in dairy fermentations. The expression of plasmid-encoded genes and the activity of the corresponding proteins is severely affected by changes in the number of plasmid copies. We studied the impact of growth rate on dynamics of plasmid copy numbers at high growth rates in chemostat cultures and down to near-zero growth rates in retentostat cultures. Five plasmids of the dairy strain Lactococcus lactis FM03-V1 were selected which varied in size (3 to 39 kb), in replication mechanism (theta or rolling-circle) and in putative (dairy-associated) functions. Copy numbers ranged from 1.5 to 40.5 and the copy number of theta-type replicating plasmids were negatively correlated to the plasmid size. Despite the extremely wide range of growth rates (0.0003 h -1 to 0.6 h -1 ), copy numbers of the five plasmids were stable and only slightly increased at near-zero growth rates showing that the plasmid replication rate was strictly controlled. One low-copy number plasmid, carrying a large exopolysaccharide gene cluster, was segregationally unstable during retentostat cultivations reflected in complete loss of the plasmid in one of the retentostat cultures. The copy number of the five plasmids was also hardly affected by varying the pH value, nutrient limitation or presence of citrate (maximum 2.2-fold) signifying the stability in copy number of the plasmids. Importance Lactococcus lactis is extensively used in starter cultures for dairy fermentations. Important traits for growth and survival of L. lactis in dairy fermentations are encoded by genes located on plasmids, such as genes involved in lactose and citrate metabolism, protein degradation and oligopeptide uptake and bacteriophage resistance. Because the number of plasmid copies could affect the expression of plasmid-encoded genes, it is important to know the factors that influence the plasmid copy numbers. We monitored plasmid copy numbers of L

Gram-scale production of plasmid pUDK-HGF with current good manufacturing practices for gene therapy of critical limb ischemia.

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Hu, ChunSheng; Cheng, XiaoChen; Lu, YuXin; Wu, ZuZe; Zhang, QingLin

2016-11-16

The demand of a plasmid encoding human hepatocyte growth factor gene (pUDK-HGF) in large quantities at high purity and concentration has increased for gene therapy of critical limb ischemia (CLI) in clinical trials. In this article, we produced pUDK-HGF in compliance with current good manufacturing practices at gram scale. The process included a 50-L batch fermentation, continuous alkaline lysis, and integrated three-step chromatography on Sepharose 6 Fast Flow, PlasmidSelect Xtra, and Source 15Q. The production process has been scaled up to yield 4.24 ± 0.41 g of pharmaceutical pUDK-HGF from 1.0 kg bacterial cell paste and the overall yield reached range from 58.37 to 66.70%. The final pUDK-HGF product exhibited high purity with supercoiled percentage of > 95.8% and undetectable residual RNA, contaminated protein, and bacterial endotoxin. The phase I clinical study indicates that intramuscular injection of pUDK-HGF is safe, well tolerated, and may provide symptomatic relief to CLI patients. These results show that our manufacturing process of pUDK-HGF is efficient in producing pharmaceutical-grade plasmid DNA and is safe for clinical applications.

Two-Stage n-PSK Partitioning Carrier Phase Recovery Scheme for Circular mQAM Coherent Optical Systems

DEFF Research Database (Denmark)

Rodrigo Navarro, Jaime; Kakkar, Aditya; Pang, Xiaodan

2016-01-01

(ΔνTs) tolerance is achieved compared to the single-stage n-PSK partitioning scheme. Superior performance in the ΔνTs tolerance compared to the blind phase search algorithm is also reported. The relative improvements with respect to other CPR schemes are also validated experimentally for a 28-Gbaud C......A novel two-stage n-PSK partitioning carrier phase recovery (CPR) scheme for circular multilevel quadrature amplitude modulation (C-mQAM) constellations is presented. The first stage of the algorithm provides an initial rough estimation of the received constellation, which is utilized in the second...... stage for CPR. The performance of the proposed algorithm is studied through extensive simulations at the forward error correction bit error rate targets of 3.8 × 10−3 and 1 × 10−2 and is compared with different CPR algorithms. A significant improvement in the combined linewidth symbol duration product...

Action of cholecalciferol and alpha-tocopherol on Staphylococcus aureus efflux pumps.

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Tintino, Saulo R; Morais-Tintino, Cícera D; Campina, Fábia F; Pereira, Raimundo L; Costa, Maria do S; Braga, Maria Flaviana B M; Limaverde, Paulo W; Andrade, Jacqueline C; Siqueira-Junior, José P; Coutinho, Henrique Douglas Melo; Balbino, Valdir Q; Leal-Balbino, Tereza C; Ribeiro-Filho, Jaime; Quintans-Júnior, Lucindo J

2016-01-01

Alpha-tocopherol is one the most abundant and biologically active isoforms of vitamin E. This compound is a potent antioxidant and one of most studied isoforms of vitamin E. Vitamin D3 (cholecalciferol) is an important nutrient for calcium homeostasis and bone health, that has also been recognized as a potent modulator of the immune response. Methicillin-resistant Staphylococcus aureus (MRSA) is the most important causative agent of both nosocomial and community-acquired infections. The aim of this study was to evaluate the inhibitory effect of alpha-tocopherol and cholecalciferol on both S. aureus and multidrug resistant S. aureus efflux pumps. The RN4220 strain has the plasmid pUL5054 that is the carrier of gene that encodes the macrolide resistance protein (an efflux pump) MsrA; the IS-58 strain possesses the TetK tetracycline efflux protein in its genome and the 1199B strain resists to hydrophilic fluoroquinolones via a NorA-mediated mechanism. The antibacterial activity was evaluated by determining the Minimal Inhibitory Concentration (MIC) and a possible inhibition of efflux pumps was associated to a reduction of the MIC. In this work we observed that in the presence of the treatments there was a decrease in the MIC for the RN4220 and IS-58 strains, suggesting that the substances presented an inhibitory effect on the efflux pumps of these strains. Significant efforts have been done to identify efflux pump inhibitors (EPIs) from natural sources and, therefore, the antibacterial properties of cholecalciferol and alpha-tocopherol might be attributed to a direct effect on the bacterial cell depending on their amphipathic structure.

Characterization of pLAC1, a cryptic plasmid isolated from Lactobacillus acidipiscis and comparative analysis with its related plasmids.

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Asteri, Ioanna-Areti; Papadimitriou, Konstantinos; Boutou, Effrossyni; Anastasiou, Rania; Pot, Bruno; Vorgias, Constantinos E; Tsakalidou, Effie

2010-07-15

The pLAC1 plasmid of Lactobacillus acidipiscis ACA-DC 1533, a strain isolated from traditional Kopanisti cheese, was characterised. Nucleotide sequence analysis revealed a circular molecule of 3478bp with a G+C content of 37.2%. Ab initio annotation indicated four putative open reading frames (orfs). orf1 and orf4 were found to encode a replication initiation protein (Rep) and a mobilization protein (Mob), respectively. The deduced products of orf2 and orf3 revealed no significant homology to other known proteins. However, in silico examination of the plasmid sequence supported the existence of a novel operon that includes rep, orf2 and orf3 in pLAC1 and that this operon is highly conserved also in plasmids pLB925A02, pSMA23, pLC88 and pC7. RT-PCR experiments allowed us to verify that these three genes are co-transcribed as a single polycistronic mRNA species. Furthermore, phylogenetic analysis of pLAC1 Rep and Mob proteins demonstrated that they may have derived from different plasmid origins, suggesting that pLAC1 is a product of a modular evolution process. Comparative analysis of full length nucleotide sequences of pLAC1 and related Lactobacillus plasmids showed that pLAC1 shares a very similar replication backbone with pLB925A02, pSMA23 and pLC88. In contrast, mob of pLAC1 was almost identical with the respective gene of plasmids pLAB1000, pLB4 and pPB1. These findings lead to the conclusion that pLAC1 acquired mob probably via an ancestral recombination event. Our overall work highlights the importance of characterizing plasmids deriving from non-starter 'wild' isolates in order to better appreciate plasmid divergence and evolution of lactic acid bacteria. 2010 Elsevier B.V. All rights reserved.

Spread of Staphylococcus aureus resistant to penicillin and tetracycline within and between dairy herds

DEFF Research Database (Denmark)

Waage, S.; Bjorland, J.; Caugant, D. A.

2002-01-01

One hundred and seven bovine isolates of penicillin and tetracycline resistant Staphylococcus aureus, recovered from 25 different dairy herds in various parts of Norway, were characterized using antimicrobial susceptibility testing, multilocus enzyme electrophoresis, ribotyping, plasmid analysis ...

The phytosulfokine (PSK) receptor is capable of guanylate cyclase activity and enabling cyclic GMP-dependent signaling in plants

KAUST Repository

Kwezi, Lusisizwe; Ruzvidzo, Oziniel; Wheeler, Janet I.; Govender, Kershini; Iacuone, Sylvana; Thompson, Philip E.; Gehring, Christoph A; Irving, Helen R.

2011-01-01

Phytosulfokines (PSKs) are sulfated pentapeptides that stimulate plant growth and differentiation mediated by the PSK receptor (PSKR1), which is a leucine-rich repeat receptor-like kinase. We identified a putative guanylate cyclase (GC) catalytic center in PSKR1 that is embedded within the kinase domain and hypothesized that the GC works in conjunction with the kinase in downstream PSK signaling. We expressed the recombinant complete kinase (cytoplasmic) domain of AtPSKR1 and show that it has serine/threonine kinase activity using the Ser/Thr peptide 1 as a substrate with an approximate Km of 7.5 μM and Vmax of 1800 nmol min-1 mg-1 of protein. This same recombinant protein also has GC activity in vitro that is dependent on the presence of either Mg2+ or Mn2+. Overexpression of the full-length AtPSKR1 receptor in Arabidopsis leaf protoplasts raised the endogenous basal cGMP levels over 20-fold, indicating that the receptor has GC activity in vivo. In addition, PSK-α itself, but not the non-sulfated backbone, induces rapid increases in cGMP levels in protoplasts. Together these results indicate that the PSKR1 contains dual GC and kinase catalytic activities that operate in vivo and that this receptor constitutes a novel class of enzymes with overlapping catalytic domains. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

The phytosulfokine (PSK) receptor is capable of guanylate cyclase activity and enabling cyclic GMP-dependent signaling in plants

KAUST Repository

Kwezi, Lusisizwe

2011-04-19

Phytosulfokines (PSKs) are sulfated pentapeptides that stimulate plant growth and differentiation mediated by the PSK receptor (PSKR1), which is a leucine-rich repeat receptor-like kinase. We identified a putative guanylate cyclase (GC) catalytic center in PSKR1 that is embedded within the kinase domain and hypothesized that the GC works in conjunction with the kinase in downstream PSK signaling. We expressed the recombinant complete kinase (cytoplasmic) domain of AtPSKR1 and show that it has serine/threonine kinase activity using the Ser/Thr peptide 1 as a substrate with an approximate Km of 7.5 μM and Vmax of 1800 nmol min-1 mg-1 of protein. This same recombinant protein also has GC activity in vitro that is dependent on the presence of either Mg2+ or Mn2+. Overexpression of the full-length AtPSKR1 receptor in Arabidopsis leaf protoplasts raised the endogenous basal cGMP levels over 20-fold, indicating that the receptor has GC activity in vivo. In addition, PSK-α itself, but not the non-sulfated backbone, induces rapid increases in cGMP levels in protoplasts. Together these results indicate that the PSKR1 contains dual GC and kinase catalytic activities that operate in vivo and that this receptor constitutes a novel class of enzymes with overlapping catalytic domains. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

Plasmid-Mediated Resistance in Enterobacteriaceae Changing Landscape and Implications for Therapy

NARCIS (Netherlands)

Schultsz, Constance; Geerlings, Suzanne

2012-01-01

Antimicrobial resistance is increasing worldwide, and pathogenic microorganism's that are resistant to all available antimicrobial agents are increasingly reported. Emerging plasmid-encoded extended-spectrum beta-lactamases (ESBLs) and carbapenemases are increasingly reported worldwide.

Acquisition of Carbapenem Resistance by Plasmid-Encoded-AmpC-Expressing Escherichia coli.

Science.gov (United States)

van Boxtel, Ria; Wattel, Agnes A; Arenas, Jesús; Goessens, Wil H F; Tommassen, Jan

2017-01-01

Although AmpC β-lactamases can barely degrade carbapenems, if at all, they can sequester them and prevent them from reaching their targets. Thus, carbapenem resistance in Escherichia coli and other Enterobacteriaceae can result from AmpC production and simultaneous reduction of antibiotic influx into the periplasm by mutations in the porin genes. Here we investigated the route and genetic mechanisms of acquisition of carbapenem resistance in a clinical E. coli isolate carrying bla CMY-2 on a plasmid by selecting for mutants that are resistant to increasing concentrations of meropenem. In the first step, the expression of OmpC, the only porin produced in the strain under laboratory conditions, was lost, leading to reduced susceptibility to meropenem. In the second step, the expression of the CMY-2 β-lactamase was upregulated, leading to resistance to meropenem. The loss of OmpC was due to the insertion of an IS1 element into the ompC gene or to frameshift mutations and premature stop codons in this gene. The bla CMY-2 gene was found to be located on an IncIγ plasmid, and overproduction of the CMY-2 enzyme resulted from an increased plasmid copy number due to a nucleotide substitution in the inc gene. The clinical relevance of these genetic mechanisms became evident from the analysis of previously isolated carbapenem-resistant clinical isolates, which appeared to carry similar mutations. Copyright © 2016 American Society for Microbiology.

Bacterial mitosis: Partitioning protein ParA oscillates in spiral-shaped structures and positions plasmids at mid-cell

DEFF Research Database (Denmark)

Ebersbach, G.; Gerdes, Kenn

2004-01-01

The par2 locus of Escherichia coli plasmid pB171 encodes oscillating ATPase ParA, DNA binding protein ParB and two cis-acting DNA regions to which ParB binds (parC1 and parC2). Three independent techniques were used to investigate the subcellular localization of plasmids carrying par2. In cells......A-GFP oscillated in spiral-shaped structures. Amino acid substitutions in ParA simultaneously abolished ParA spiral formation, oscillation and either plasmid localization or plasmid separation at mid-cell. Therefore, our results suggest that ParA spirals position plasmids at the middle of the bacterial nucleoid...

Protocol for Evaluating the Permissiveness of Bacterial Communities Toward Conjugal Plasmids by Quantification and Isolation of Transconjugants

DEFF Research Database (Denmark)

Klümper, Uli; Dechesne, Arnaud; Smets, Barth F.

2014-01-01

may encode catabolic pathways, virulence factors, and antibiotic or metal resistances, it is of environmental, evolutionary, and medical relevance to track and monitor the fate of plasmids in mixed microbial community. When assessing the short-term and long-term implications of conjugal plasmid...... a gfp-tagged plasmid in a mCherry red fluorescently tagged donor strain repressing gfp expression. We take advantage of fluorescent marker genes to microscopically detect plasmid transfer events and use subsequent high-throughput fluorescence-activated cell sorting (FACS) to isolate...

The complete sequence and comparative analysis of a multidrug- resistance and virulence multireplicon IncFII plasmid pEC302/04 from an extraintestinal pathogenic Escherichia coli EC302/04 indicate extensive diversity of IncFII plasmids

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Wing Sze eHo

2016-01-01

Full Text Available Extraintestinal pathogenic Escherichia coli (ExPEC that causes extraintestinal infections often harbor plasmids encoding fitness traits such as resistance and virulence determinants that are of clinical importance. We determined the complete nucleotide sequence of plasmid pEC302/04 from a multidrug-resistant E. coli EC302/04 which was isolated from the tracheal aspirate of a patient in Malaysia. In addition, we also performed comparative sequence analyses of 18 related IncFIIA plasmids to determine the phylogenetic relationship and diversity of these plasmids. The 140,232 bp pEC302/04 is a multireplicon plasmid that bears three replication systems (FII, FIA and FIB with subtype of F2:A1:B1. The plasmid is self-transmissible with a complete transfer region. pEC302/04 also carries antibiotic resistance genes such as blaTEM-1 and a class I integron containing sul1, cml and aadA resistance genes, conferring multidrug resistance (MDR to its host, E. coli EC302/04. Besides, two iron acquisition systems (SitABCD and IutA-IucABCD which are the conserved virulence determinants of ExPEC-colicin V or B and M (ColV/ColBM-producing plasmids were identified in pEC302/04. Multiple toxin-antitoxin (TA-based addiction systems (i.e., PemI/PemK, VagC/VagD, CcdA/CcdB, and Hok/Sok and a plasmid partitioning system, ParAB and PsiAB, which are important for plasmid maintenance were also found.Comparative plasmid analysis revealed only one conserved gene, the repA1 as the core genome, showing that there is an extensive diversity among the IncFIIA plasmids. The phylogenetic relationship of 18 IncF plasmids based on the core regions revealed that ColV/ColBM-plasmids and non-ColV/ColBM plasmids were separated into two distinct groups. These plasmids, which carry highly diverse genetic contents, are also mosaic in nature. The atypical combination of genetic materials, i.e., the MDR- and ColV/ColBM-plasmid-virulence encoding regions in a single ExPEC plasmid is rare but of

The Complete Sequence and Comparative Analysis of a Multidrug-Resistance and Virulence Multireplicon IncFII Plasmid pEC302/04 from an Extraintestinal Pathogenic Escherichia coli EC302/04 Indicate Extensive Diversity of IncFII Plasmids.

Science.gov (United States)

Ho, Wing Sze; Yap, Kien-Pong; Yeo, Chew Chieng; Rajasekaram, Ganeswrie; Thong, Kwai Lin

2015-01-01

Extraintestinal pathogenic Escherichia coli (ExPEC) that causes extraintestinal infections often harbor plasmids encoding fitness traits such as resistance and virulence determinants that are of clinical importance. We determined the complete nucleotide sequence of plasmid pEC302/04 from a multidrug-resistant E. coli EC302/04 which was isolated from the tracheal aspirate of a patient in Malaysia. In addition, we also performed comparative sequence analyses of 18 related IncFIIA plasmids to determine the phylogenetic relationship and diversity of these plasmids. The 140,232 bp pEC302/04 is a multireplicon plasmid that bears three replication systems (FII, FIA, and FIB) with subtype of F2:A1:B1. The plasmid is self-transmissible with a complete transfer region. pEC302/04 also carries antibiotic resistance genes such as bla TEM-1 and a class I integron containing sul1, cml and aadA resistance genes, conferring multidrug resistance (MDR) to its host, E. coli EC302/04. Besides, two iron acquisition systems (SitABCD and IutA-IucABCD) which are the conserved virulence determinants of ExPEC-colicin V or B and M (ColV/ColBM)-producing plasmids were identified in pEC302/04. Multiple toxin-antitoxin (TA)-based addiction systems (i.e., PemI/PemK, VagC/VagD, CcdA/CcdB, and Hok/Sok) and a plasmid partitioning system, ParAB, and PsiAB, which are important for plasmid maintenance were also found. Comparative plasmid analysis revealed only one conserved gene, the repA1 as the core genome, showing that there is an extensive diversity among the IncFIIA plasmids. The phylogenetic relationship of 18 IncF plasmids based on the core regions revealed that ColV/ColBM-plasmids and non-ColV/ColBM plasmids were separated into two distinct groups. These plasmids, which carry highly diverse genetic contents, are also mosaic in nature. The atypical combination of genetic materials, i.e., the MDR- and ColV/ColBM-plasmid-virulence encoding regions in a single ExPEC plasmid is rare but of clinical

Possibilities for Advanced Encoding Techniques at Signal Transmission in the Optical Transmission Medium

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Filip Čertík

2016-01-01

Full Text Available This paper presents a possible simulation of negative effects in the optical transmission medium and an analysis for the utilization of different signal processing techniques at the optical signal transmission. An attention is focused on the high data rate signal transmission in the optical fiber influenced by linear and nonlinear environmental effects presented by the prepared simulation model. The analysis includes possible utilization of OOK, BPSK, DBPSK, BFSK, QPSK, DQPSK, 8PSK, and 16QAM modulation techniques together with RS, BCH, and LDPC encoding techniques for the signal transmission in the optical fiber. Moreover, the prepared simulation model is compared with real optical transmission systems. In the final part, a comparison of the selected modulation techniques with different encoding techniques and their implementation in real transmission systems is shown.

Dendritic cell mediated delivery of plasmid DNA encoding LAMP/HIV-1 Gag fusion immunogen enhances T cell epitope responses in HLA DR4 transgenic mice.

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Gregory G Simon

2010-01-01

Full Text Available This report describes the identification and bioinformatics analysis of HLA-DR4-restricted HIV-1 Gag epitope peptides, and the application of dendritic cell mediated immunization of DNA plasmid constructs. BALB/c (H-2d and HLA-DR4 (DRA1*0101, DRB1*0401 transgenic mice were immunized with immature dendritic cells transfected by a recombinant DNA plasmid encoding the lysosome-associated membrane protein-1/HIV-1 Gag (pLAMP/gag chimera antigen. Three immunization protocols were compared: 1 primary subcutaneous immunization with 1x10(5 immature dendritic cells transfected by electroporation with the pLAMP/gag DNA plasmid, and a second subcutaneous immunization with the naked pLAMP/gag DNA plasmid; 2 primary immunization as above, and a second subcutaneous immunization with a pool of overlapping peptides spanning the HIV-1 Gag sequence; and 3 immunization twice by subcutaneous injection of the pLAMP/gag DNA plasmid. Primary immunization with pLAMP/gag-transfected dendritic cells elicited the greatest number of peptide specific T-cell responses, as measured by ex vivo IFN-gamma ELISpot assay, both in BALB/c and HLA-DR4 transgenic mice. The pLAMP/gag-transfected dendritic cells prime and naked DNA boost immunization protocol also resulted in an increased apparent avidity of peptide in the ELISpot assay. Strikingly, 20 of 25 peptide-specific T-cell responses in the HLA-DR4 transgenic mice contained sequences that corresponded, entirely or partially to 18 of the 19 human HLA-DR4 epitopes listed in the HIV molecular immunology database. Selection of the most conserved epitope peptides as vaccine targets was facilitated by analysis of their representation and variability in all reported sequences. These data provide a model system that demonstrates a the superiority of immunization with dendritic cells transfected with LAMP/gag plasmid DNA, as compared to naked DNA, b the value of HLA transgenic mice as a model system for the identification and evaluation

Comparative genomics of an IncA/C multidrug resistance plasmid from Escherichia coli and Klebsiella isolates from intensive care unit patients and the utility of whole-genome sequencing in health care settings.

Science.gov (United States)

Hazen, Tracy H; Zhao, LiCheng; Boutin, Mallory A; Stancil, Angela; Robinson, Gwen; Harris, Anthony D; Rasko, David A; Johnson, J Kristie

2014-08-01

The IncA/C plasmids have been implicated for their role in the dissemination of β-lactamases, including gene variants that confer resistance to expanded-spectrum cephalosporins, which are often the treatment of last resort against multidrug-resistant, hospital-associated pathogens. A bla(FOX-5) gene was detected in 14 Escherichia coli and 16 Klebsiella isolates that were cultured from perianal swabs of patients admitted to an intensive care unit (ICU) of the University of Maryland Medical Center (UMMC) in Baltimore, MD, over a span of 3 years. Four of the FOX-encoding isolates were obtained from subsequent samples of patients that were initially negative for an AmpC β-lactamase upon admission to the ICU, suggesting that the AmpC β-lactamase-encoding plasmid was acquired while the patient was in the ICU. The genomes of five E. coli isolates and six Klebsiella isolates containing bla(FOX-5) were selected for sequencing based on their plasmid profiles. An ∼ 167-kb IncA/C plasmid encoding the FOX-5 β-lactamase, a CARB-2 β-lactamase, additional antimicrobial resistance genes, and heavy metal resistance genes was identified. Another FOX-5-encoding IncA/C plasmid that was nearly identical except for a variable region associated with the resistance genes was also identified. To our knowledge, these plasmids represent the first FOX-5-encoding plasmids sequenced. We used comparative genomics to describe the genetic diversity of a plasmid encoding a FOX-5 β-lactamase relative to the whole-genome diversity of 11 E. coli and Klebsiella isolates that carry this plasmid. Our findings demonstrate the utility of whole-genome sequencing for tracking of plasmid and antibiotic resistance gene distribution in health care settings. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Genomic analyses of the Chlamydia trachomatis core genome show an association between chromosomal genome, plasmid type and disease

NARCIS (Netherlands)

Versteeg, Bart; Bruisten, Sylvia M.; Pannekoek, Yvonne; Jolley, Keith A.; Maiden, Martin C. J.; van der Ende, Arie; Harrison, Odile B.

2018-01-01

Background: Chlamydia trachomatis (Ct) plasmid has been shown to encode genes essential for infection. We evaluated the population structure of Ct using whole-genome sequence data (WGS). In particular, the relationship between the Ct genome, plasmid and disease was investigated. Results: WGS data

Identification of novel substrates of Shigella T3SA through analysis of its virulence plasmid-encoded secretome

Science.gov (United States)

Pinaud, Laurie; Ferrari, Mariana L.; Friedman, Robin; Jehmlich, Nico; von Bergen, Martin; Phalipon, Armelle; Sansonetti, Philippe J.

2017-01-01

Many human Gram-negative bacterial pathogens express a Type Three Secretion Apparatus (T3SA), including among the most notorious Shigella spp., Salmonella enterica, Yersinia enterocolitica and enteropathogenic Escherichia coli (EPEC). These bacteria express on their surface multiple copies of the T3SA that mediate the delivery into host cells of specific protein substrates critical to pathogenesis. Shigella spp. are Gram-negative bacterial pathogens responsible for human bacillary dysentery. The effector function of several Shigella T3SA substrates has largely been studied but their potential cellular targets are far from having been comprehensively delineated. In addition, it is likely that some T3SA substrates have escaped scrutiny as yet. Indeed, sequencing of the virulence plasmid of Shigella flexneri has revealed numerous open reading frames with unknown functions that could encode additional T3SA substrates. Taking advantage of label-free mass spectrometry detection of proteins secreted by a constitutively secreting strain of S. flexneri, we identified five novel substrates of the T3SA. We further confirmed their secretion through the T3SA and translocation into host cells using β-lactamase assays. The coding sequences of two of these novel T3SA substrates (Orf13 and Orf131a) have a guanine-cytosine content comparable to those of T3SA components and effectors. The three other T3SA substrates identified (Orf48, Orf86 and Orf176) have significant homology with antitoxin moieties of type II Toxin-Antitoxin systems usually implicated in the maintenance of low copy plasmids. While Orf13 and Orf131a might constitute new virulence effectors contributing to S. flexneri pathogenicity, potential roles for the translocation into host cells of antitoxins or antitoxin-like proteins during Shigella infection are discussed. PMID:29073283

Identification of novel substrates of Shigella T3SA through analysis of its virulence plasmid-encoded secretome.

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Laurie Pinaud

Full Text Available Many human Gram-negative bacterial pathogens express a Type Three Secretion Apparatus (T3SA, including among the most notorious Shigella spp., Salmonella enterica, Yersinia enterocolitica and enteropathogenic Escherichia coli (EPEC. These bacteria express on their surface multiple copies of the T3SA that mediate the delivery into host cells of specific protein substrates critical to pathogenesis. Shigella spp. are Gram-negative bacterial pathogens responsible for human bacillary dysentery. The effector function of several Shigella T3SA substrates has largely been studied but their potential cellular targets are far from having been comprehensively delineated. In addition, it is likely that some T3SA substrates have escaped scrutiny as yet. Indeed, sequencing of the virulence plasmid of Shigella flexneri has revealed numerous open reading frames with unknown functions that could encode additional T3SA substrates. Taking advantage of label-free mass spectrometry detection of proteins secreted by a constitutively secreting strain of S. flexneri, we identified five novel substrates of the T3SA. We further confirmed their secretion through the T3SA and translocation into host cells using β-lactamase assays. The coding sequences of two of these novel T3SA substrates (Orf13 and Orf131a have a guanine-cytosine content comparable to those of T3SA components and effectors. The three other T3SA substrates identified (Orf48, Orf86 and Orf176 have significant homology with antitoxin moieties of type II Toxin-Antitoxin systems usually implicated in the maintenance of low copy plasmids. While Orf13 and Orf131a might constitute new virulence effectors contributing to S. flexneri pathogenicity, potential roles for the translocation into host cells of antitoxins or antitoxin-like proteins during Shigella infection are discussed.

Analysis of mutational resistance to trimethoprim in Staphylococcus aureus by genetic and structural modelling techniques.

Science.gov (United States)

Vickers, Anna A; Potter, Nicola J; Fishwick, Colin W G; Chopra, Ian; O'Neill, Alex J

2009-06-01

This study sought to expand knowledge on the molecular mechanisms of mutational resistance to trimethoprim in Staphylococcus aureus, and the fitness costs associated with resistance. Spontaneous trimethoprim-resistant mutants of S. aureus SH1000 were recovered in vitro, resistance genotypes characterized by DNA sequencing of the gene encoding the drug target (dfrA) and the fitness of mutants determined by pair-wise growth competition assays with SH1000. Novel resistance genotypes were confirmed by ectopic expression of dfrA alleles in a trimethoprim-sensitive S. aureus strain. Molecular models of S. aureus dihydrofolate reductase (DHFR) were constructed to explore the structural basis of trimethoprim resistance, and to rationalize the observed in vitro fitness of trimethoprim-resistant mutants. In addition to known amino acid substitutions in DHFR mediating trimethoprim resistance (F(99)Y and H(150)R), two novel resistance polymorphisms (L(41)F and F(99)S) were identified among the trimethoprim-resistant mutants selected in vitro. Molecular modelling of mutated DHFR enzymes provided insight into the structural basis of trimethoprim resistance. Calculated binding energies of the substrate (dihydrofolate) for the mutant and wild-type enzymes were similar, consistent with apparent lack of fitness costs for the resistance mutations in vitro. Reduced susceptibility to trimethoprim of DHFR enzymes carrying substitutions L(41)F, F(99)S, F(99)Y and H(150)R appears to result from structural changes that reduce trimethoprim binding to the enzyme. However, the mutations conferring trimethoprim resistance are not associated with fitness costs in vitro, suggesting that the survival of trimethoprim-resistant strains emerging in the clinic may not be subject to a fitness disadvantage.

Identification of dfrA14 in two distinct plasmids conferring trimethoprim resistance in Actinobacillus pleuropneumoniae.

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Bossé, Janine T; Li, Yanwen; Walker, Stephanie; Atherton, Tom; Fernandez Crespo, Roberto; Williamson, Susanna M; Rogers, Jon; Chaudhuri, Roy R; Weinert, Lucy A; Oshota, Olusegun; Holden, Matt T G; Maskell, Duncan J; Tucker, Alexander W; Wren, Brendan W; Rycroft, Andrew N; Langford, Paul R

2015-08-01

The objective of this study was to determine the distribution and genetic basis of trimethoprim resistance in Actinobacillus pleuropneumoniae isolates from pigs in England. Clinical isolates collected between 1998 and 2011 were tested for resistance to trimethoprim and sulphonamide. The genetic basis of trimethoprim resistance was determined by shotgun WGS analysis and the subsequent isolation and sequencing of plasmids. A total of 16 (out of 106) A. pleuropneumoniae isolates were resistant to both trimethoprim (MIC >32 mg/L) and sulfisoxazole (MIC ≥256 mg/L), and a further 32 were resistant only to sulfisoxazole (MIC ≥256 mg/L). Genome sequence data for the trimethoprim-resistant isolates revealed the presence of the dfrA14 dihydrofolate reductase gene. The distribution of plasmid sequences in multiple contigs suggested the presence of two distinct dfrA14-containing plasmids in different isolates, which was confirmed by plasmid isolation and sequencing. Both plasmids encoded mobilization genes, the sulphonamide resistance gene sul2, as well as dfrA14 inserted into strA, a streptomycin-resistance-associated gene, although the gene order differed between the two plasmids. One of the plasmids further encoded the strB streptomycin-resistance-associated gene. This is the first description of mobilizable plasmids conferring trimethoprim resistance in A. pleuropneumoniae and, to our knowledge, the first report of dfrA14 in any member of the Pasteurellaceae. The identification of dfrA14 conferring trimethoprim resistance in A. pleuropneumoniae isolates will facilitate PCR screens for resistance to this important antimicrobial. © The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

The large universal Pantoea plasmid LPP-1 plays a major role in biological and ecological diversification

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De Maayer Pieter

2012-11-01

Full Text Available Abstract Background Pantoea spp. are frequently isolated from a wide range of ecological niches and have various biological roles, as plant epi- or endophytes, biocontrol agents, plant-growth promoters or as pathogens of both plant and animal hosts. This suggests that members of this genus have undergone extensive genotypic diversification. One means by which this occurs among bacteria is through the acquisition and maintenance of plasmids. Here, we have analyzed and compared the sequences of a large plasmid common to all sequenced Pantoea spp. Results and discussion The Large PantoeaPlasmids (LPP-1 of twenty strains encompassing seven different Pantoea species, including pathogens and endo-/epiphytes of a wide range of plant hosts as well as insect-associated strains, were compared. The LPP-1 plasmid sequences range in size from ~281 to 794 kb and carry between 238 and 750 protein coding sequences (CDS. A core set of 46 proteins, encompassing 2.2% of the total pan-plasmid (2,095 CDS, conserved among all LPP-1 plasmid sequences, includes those required for thiamine and pigment biosynthesis. Phylogenetic analysis reveals that these plasmids have arisen from an ancestral plasmid, which has undergone extensive diversification. Analysis of the proteins encoded on LPP-1 also showed that these plasmids contribute to a wide range of Pantoea phenotypes, including the transport and catabolism of various substrates, inorganic ion assimilation, resistance to antibiotics and heavy metals, colonization and persistence in the host and environment, pathogenesis and antibiosis. Conclusions LPP-1 is universal to all Pantoea spp. whose genomes have been sequenced to date and is derived from an ancestral plasmid. LPP-1 encodes a large array of proteins that have played a major role in the adaptation of the different Pantoea spp. to their various ecological niches and their specialization as pathogens, biocontrol agents or benign saprophytes found in many diverse

Plasmids encoding PKI(1-31), a specific inhibitor of cAMP-stimulated gene expression, inhibit the basal transcriptional activity of some but not all cAMP-regulated DNA response elements in JEG-3 cells.

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Grove, J R; Deutsch, P J; Price, D J; Habener, J F; Avruch, J

1989-11-25

Plasmids that encode a bioactive amino-terminal fragment of the heat-stable inhibitor of the cAMP-dependent protein kinase, PKI(1-31), were employed to characterize the role of this protein kinase in the control of transcriptional activity mediated by three DNA regulatory elements in the JEG-3 human placental cell line. The 5'-flanking sequence of the human collagenase gene contains the heptameric sequence, 5'-TGAGTCA-3', previously identified as a "phorbol ester" response element. Reporter genes containing either the intact 1.2-kilobase 5'-flanking sequence from the human collagenase gene or just the 7-base pair (bp) response element, when coupled to an enhancerless promoter, each exhibit both cAMP and phorbol ester-stimulated expression in JEG-3 cells. Cotransfection of either construct with plasmids encoding PKI(1-31) inhibits cAMP-stimulated but not basal- or phorbol ester-stimulated expression. Pretreatment of cells with phorbol ester for 1 or 2 days abrogates completely the response to rechallenge with phorbol ester but does not alter the basal expression of either construct; cAMP-stimulated expression, while modestly inhibited, remains vigorous. The 5'-flanking sequence of the human chorionic gonadotropin-alpha subunit (HCG alpha) gene has two copies of the sequence, 5'-TGACGTCA-3', contained in directly adjacent identical 18-bp segments, previously identified as a cAMP-response element. Reporter genes containing either the intact 1.5 kilobase of 5'-flanking sequence from the HCG alpha gene, or just the 36-bp tandem repeat cAMP response element, when coupled to an enhancerless promoter, both exhibit a vigorous cAMP stimulation of expression but no response to phorbol ester in JEG-3 cells. Cotransfection with plasmids encoding PKI(1-31) inhibits both basal and cAMP-stimulated expression in a parallel fashion. The 5'-flanking sequence of the human enkephalin gene mediates cAMP-stimulated expression of reporter genes in both JEG-3 and CV-1 cells. Plasmids

Comparative genomics of the IncA/C multidrug resistance plasmid family.

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Fricke, W Florian; Welch, Timothy J; McDermott, Patrick F; Mammel, Mark K; LeClerc, J Eugene; White, David G; Cebula, Thomas A; Ravel, Jacques

2009-08-01

Multidrug resistance (MDR) plasmids belonging to the IncA/C plasmid family are widely distributed among Salmonella and other enterobacterial isolates from agricultural sources and have, at least once, also been identified in a drug-resistant Yersinia pestis isolate (IP275) from Madagascar. Here, we present the complete plasmid sequences of the IncA/C reference plasmid pRA1 (143,963 bp), isolated in 1971 from the fish pathogen Aeromonas hydrophila, and of the cryptic IncA/C plasmid pRAx (49,763 bp), isolated from Escherichia coli transconjugant D7-3, which was obtained through pRA1 transfer in 1980. Using comparative sequence analysis of pRA1 and pRAx with recent members of the IncA/C plasmid family, we show that both plasmids provide novel insights into the evolution of the IncA/C MDR plasmid family and the minimal machinery necessary for stable IncA/C plasmid maintenance. Our results indicate that recent members of the IncA/C plasmid family evolved from a common ancestor, similar in composition to pRA1, through stepwise integration of horizontally acquired resistance gene arrays into a conserved plasmid backbone. Phylogenetic comparisons predict type IV secretion-like conjugative transfer operons encoded on the shared plasmid backbones to be closely related to a group of integrating conjugative elements, which use conjugative transfer for horizontal propagation but stably integrate into the host chromosome during vegetative growth. A hipAB toxin-antitoxin gene cluster found on pRA1, which in Escherichia coli is involved in the formation of persister cell subpopulations, suggests persistence as an early broad-spectrum antimicrobial resistance mechanism in the evolution of IncA/C resistance plasmids.

Identification of oriT and a recombination hot spot in the IncA/C plasmid backbone.

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Hegyi, Anna; Szabó, Mónika; Olasz, Ferenc; Kiss, János

2017-09-06

Dissemination of multiresistance has been accelerating among pathogenic bacteria in recent decades. The broad host-range conjugative plasmids of the IncA/C family are effective vehicles of resistance determinants in Gram-negative bacteria. Although more than 150 family members have been sequenced to date, their conjugation system and other functions encoded by the conserved plasmid backbone have been poorly characterized. The key cis-acting locus, the origin of transfer (oriT), has not yet been unambiguously identified. We present evidence that IncA/C plasmids have a single oriT locus immediately upstream of the mobI gene encoding an indispensable transfer factor. The fully active oriT spans ca. 150-bp AT-rich region overlapping the promoters of mobI and contains multiple inverted and direct repeats. Within this region, the core domain of oriT with reduced but detectable transfer activity was confined to a 70-bp segment containing two inverted repeats and one copy of a 14-bp direct repeat. In addition to oriT, a second locus consisting of a 14-bp imperfect inverted repeat was also identified, which mimicked the function of oriT but which was found to be a recombination site. Recombination between two identical copies of these sites is RecA-independent, requires a plasmid-encoded recombinase and resembles the functioning of dimer-resolution systems.

DNA sequence analysis of plasmids from multidrug resistant Salmonella enterica serotype Heidelberg isolates.

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Jing Han

Full Text Available Salmonella enterica serovar Heidelberg is among the most detected serovars in swine and poultry, ranks among the top five serotypes associated with human salmonellosis and is disproportionately associated with invasive infections and mortality in humans. Salmonella are known to carry plasmids associated with antimicrobial resistance and virulence. To identify plasmid-associated genes in multidrug resistant S. enterica serovar Heidelberg, antimicrobial resistance plasmids from five isolates were sequenced using the 454 LifeSciences pyrosequencing technology. Four of the isolates contained incompatibility group (Inc A/C multidrug resistance plasmids harboring at least eight antimicrobial resistance genes. Each of these strains also carried a second resistance plasmid including two IncFIB, an IncHI2 and a plasmid lacking an identified Inc group. The fifth isolate contained an IncI1 plasmid, encoding resistance to gentamicin, streptomycin and sulfonamides. Some of the IncA/C plasmids lacked the full concert of transfer genes and yet were able to be conjugally transferred, likely due to the transfer genes carried on the companion plasmids in the strains. Several non-IncA/C resistance plasmids also carried putative virulence genes. When the sequences were compared to previously sequenced plasmids, it was found that while all plasmids demonstrated some similarity to other plasmids, they were unique, often due to differences in mobile genetic elements in the plasmids. Our study suggests that Salmonella Heidelberg isolates harbor plasmids that co-select for antimicrobial resistance and virulence, along with genes that can mediate the transfer of plasmids within and among other bacterial isolates. Prevalence of such plasmids can complicate efforts to control the spread of S. enterica serovar Heidelberg in food animal and human populations.

Molecular Cloning, Expression and Characterization of Plasmid Encoding Rhomboid 4 (ROM4 of Tachyzoite of Toxoplasma gondii RH Strain

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Mohammad Taghi RAHIMI

2017-12-01

Full Text Available AbstractBackground: The objective of this study was to clone, express and characterize the gene encoding rhomboid 4 (ROM4 proteins, a vital gene in surface adhesion and host cell invasion process of tachyzoite of T. gondii in an appropriate expression vector and eukaryotic cell for production of recombinant protein.Methods: Toxoplasma RNA was isolated from tachyzoites (RH strain and complementary DNA was synthesized. Oligonucleotide primer pair was designed based on Toxoplasma ROM4 gene sequence with XhoI and EcoRI restriction sites at 5´ end of forward and reverse primers, respectively. ROM4 gene was amplified by PCR, cloned into pTG19-T vector and the recombinant plasmid was sequenced. The gene was subcloned into pcDNA3 plasmid and expressed in CHO cells as eukaryotic cell. SDS-PAGE and western blotting were performed for protein determination and verification.Results: Cloning of ROM4 gene in pTG19-T vector was confirmed by colony-PCR and enzymatic digestion. The results of enzymatic digestion and gene sequencing confirmed successful cloning and subcloning procedures. The nucleotide sequence of the cloned ROM4 gene showed 99% homology compared to the corresponding sequences of original gene. SDS-PAGE and western blotting analyses of the purified protein revealed a single band having expected size of 65 kDa.Conclusion: This eukaryotic expression system is an appropriate system for high-level recombinant protein production of ROM4 gene from T. gondii tachyzoites used as antigenic component for serological assay and vaccine development.

Expression, purification, and activity assay of peptide deformylase from Escherichia coli and Staphylococcus aureus.

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Che, Xuchun; Hu, Jinwei; Wang, Lijuan; Zhu, Zhifeng; Xu, Qiong; Lv, Junqiang; Fu, Zheng; Sun, Yajun; Sun, Jia; Lin, Gang; Lu, Rong; Yao, Zhi

2011-11-01

Peptide deformylase (PDF) is considered an attractive target for screening novel antibiotics. The PDF from Escherichia coli and Staphylococcus aureus are representative of the gram-negative species type of PDF (type I PDF) and the gram-positive species type of PDF (type II PDF), respectively. They could be used for screening broad-spectrum antibiotics. Herein, we cloned the def gene by PCR, inserted it into plasmid pET-22b-def, and transformed the plasmid into E. coli BL21 (DE3) cells, then the cells were induced by IPTG to express PDF. E. coli Ni(2+)-PDF was extracted and purified by ion-exchange chromatography and gel filtration chromatography. S. aureus PDFs were extracted and purified using the MagExtractor kit. The nickel form of S. aureus PDF was obtained by adding NiCl(2) to all reagents used for purification. Iron-enriched S. aureus PDF was obtained by adding FeCl(3) to the growth medium for E. coli BL21 (DE3) cells and adding FeCl(3) and catalase to all reagents used for purification. The activities of PDFs were analyzed, compared, and grouped according to the experimental conditions that produced optimal activity, and we used actinonin as an inhibitor of PDF and calculated the IC(50) value. We obtained high expression of E. coli and S. aureus PDF with high activity and stability. The function of PDFs was inhibited by actinonin in a dose-dependent manner. Results may be helpful for future mechanistic investigations of PDF as well as high-throughput screening for other PDF inhibitors.

Characterization and plasmid elimination of NDM-1-producing Acinetobacter calcoaceticus from China.

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Yang Sun

Full Text Available The presence of multidrug-resistant bacterial pathogens in the environment poses a serious threat to public health. The opportunistic Acinetobacter spp. are among the most prevalent causes of nosocomial infections. Here, we performed complete genome sequencing of the Acinetobacter calcoaceticus strain XM1570, which was originally cultivated from the sputum of a patient diagnosed with pneumonia in Xiamen in 2010. We identified carbapenem resistance associated gene bla(NDM-1 located on a 47.3-kb plasmid. Three methods--natural reproduction, sodium dodecyl sulfate treatment and nalidixic acid treatment--were used to eliminate the bla(NDM-1-encoding plasmid, which achieved elimination rates of 3.32% (10/301, 83.78% (278/332, and 84.17% (298/354, respectively. Plasmid elimination dramatically increased antibiotic sensitivity, reducing the minimum bacteriostatic concentration of meropenem from 256 µg/ml in the clinical strain to 0.125 µg/ml in the plasmid-eliminated strain. Conjugation transfer assays showed that the bla(NDM-1-containing plasmid could be transferred into Escherichia coli DH5α:pBR322 in vitro as well as in vivo in mice. The bla(NDM-1 genetic environment was in accordance with that of other bla(NDM-1 genes identified from India, Japan, and Hong-Kong. The multilocus sequence type of the isolate was identified as ST-70. Two novel genes encoding intrinsic OXA and ADC were identified and named as OXA-417 and ADC-72. The finding of bla(NDM-1 in species like A. calcoaceticus demonstrates the wide spread of this gene in gram-negative bacteria which is possible by conjugative plasmid transfer. The results of this study may help in the development of a treatment strategy for controlling NDM-1 bacterial infection and transmission.

The broad-host-range plasmid pSFA231 isolated from petroleum-contaminated sediment represents a new member of the PromA plasmid family.

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Li, Xiaobin; Top, Eva M; Wang, Yafei; Brown, Celeste J; Yao, Fei; Yang, Shan; Jiang, Yong; Li, Hui

2014-01-01

A self-transmissible broad-host-range (BHR) plasmid pSFA231 was isolated from petroleum-contaminated sediment in Shen-fu wastewater irrigation zone, China, using the triparental mating exogenous plasmid capture method. Based on its complete sequence the plasmid has a size of 41.5 kb and codes for 50 putative open reading frames (orfs), 29 of which represent genes involved in replication, partitioning and transfer functions of the plasmid. Phylogenetic analysis grouped pSFA231 into the newly defined PromA plasmid family, which currently includes five members. Further comparative genomic analysis shows that pSFA231 shares the common backbone regions with the other PromA plasmids, i.e., genes involved in replication, maintenance and control, and conjugative transfer. Nevertheless, phylogenetic divergence was found in specific gene products. We propose to divide the PromA group into two subgroups, PromA-α (pMRAD02, pSB102) and PromA-β (pMOL98, pIPO2T, pSFA231, pTer331), based on the splits network analysis of the RepA protein. Interestingly, a cluster of hypothetical orfs located between parA and traA of pSFA231 shows high similarity with the corresponding regions on pMOL98, pIPO2T, and pTer331, suggesting these hypothetical orfs may represent "essential" plasmid backbone genes for the PromA-β subgroup. Alternatively, they may also be accessory genes that were first acquired and then stayed as the plasmid diverged. Our study increases the available collection of complete genome sequences of BHR plasmids, and since pSFA231 is the only characterized PromA plasmid from China, our findings also enhance our understanding of the genetic diversity of this plasmid group in different parts of the world.

The broad-host-range plasmid pSFA231 isolated from petroleum-contaminated sediment represents a new member of the PromA plasmid family

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Xiaobin eLi

2015-01-01

Full Text Available A self-transmissible broad-host-range (BHR plasmid pSFA231 was isolated from petroleum-contaminated sediment in Shen-fu wastewater irrigation zone, China, using the triparental mating exogenous plasmid capture method. Based on its complete sequence the plasmid has a size of 41.5 kb and codes for 50 putative open reading frames (orfs, 28 of which represent genes involved in replication, partitioning and transfer functions of the plasmid. Phylogenetic analysis grouped pSFA231 into the newly defined PromA plasmid family, which currently includes five members. Further comparative genomic analysis shows that pSFA231 shares the common backbone regions with the other PromA plasmids, i.e., genes involved in replication, maintenance and control, and conjugative transfer. Nevertheless, phylogenetic divergence was found in specific gene products. We propose to divide the PromA group into two subgroups, PromA-α (pMRAD02, pSB102 and PromA-β (pMOL98, pIPO2T, pSFA231, pTer331, based on the splits network analysis of the RepA protein. Interestingly, a cluster of hypothetical orfs located between parA and traA of pSFA231 shows high similarity with the corresponding regions on pMOL98, pIPO2T and pTer331, suggesting these hypothetical orfs may represent ‘‘essential’’ plasmid backbone genes for the PromA-β subgroup. Alternatively, they may also be accessory genes that were first acquired and then stayed as the plasmid diverged. Our study increases the available collection of complete genome sequences of BHR plasmids, and since pSFA231 is the only characterized PromA plasmid from China, our findings also enhance our understanding of the genetic diversity of this plasmid group in different parts of the world.

Microarray analysis of toxicogenomic effects of Ortho-phenylphenol in Staphylococcus aureus

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Toghrol Freshteh

2008-09-01

Full Text Available Abstract Background Staphylococcus aureus (S. aureus, is responsible for many infectious diseases, ranging from benign skin infections to life-threatening endocarditis and toxic shock syndrome. Ortho-phenylphenol (OPP is an antimicrobial agent and an active ingredient of EPA-registered disinfectants with wide human exposure in various agricultural, hospital and veterinary disinfectant products. Despite many uses, an understanding of a cellular response to OPP and it's mechanism of action, targeted genes, and the connectivity between targeted genes and the rest of cell metabolism remains obscure. Results Herein, we performed a genome-wide transcriptome analysis of the cellular responses of S. aureus when exposed to 0.82 mM of OPP for 20 and 60 min. Our data indicated that OPP downregulated the biosynthesis of many amino acids, which are required for protein synthesis. In particular, the genes encoding the enzymes of the diaminopimelate (DAP pathway which results in lysine biosynthesis were significantly downregualted. Intriguingly, we revealed that the transcription of genes encoding ribosomal proteins was upregulated by OPP and at the same time, the genes encoding iron acquisition and transport were downregulated. The genes encoding virulence factors were upregulated and genes encoding phospholipids were downregulated upon 20 min exposure to OPP. Conclusion By using microarray analysis that enables us to simultaneously and globally examine the complete transcriptome during cellular responses, we have revealed novel information regarding the mode of action of OPP on Staphylococcus: OPP inhibits anabolism of many amino acids and highly downregulates the genes that encode the enzymes involved in the DAP pathway. Lysine and DAP are essential for building up the peptidoglycan cell wall. It was concluded that the mode of action of OPP is similar to the mechanism of action of some antibiotics. The discovery of this phenomenon provides useful

Restriction Fragment Length Polymorphisms of Virulence Plasmids in Rhodococcus equi

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Takai, Shinji; Shoda, Masato; Sasaki, Yukako; Tsubaki, Shiro; Fortier, Guillaume; Pronost, Stephane; Rahal, Karim; Becu, Teotimo; Begg, Angela; Browning, Glenn; Nicholson, Vivian M.; Prescott, John F.

1999-01-01

Virulent Rhodococcus equi, which is a well-known cause of pyogranulomatous pneumonia in foals, possesses a large plasmid encoding virulence-associated 15- to 17-kDa antigens. Foal and soil isolates from five countries—Argentina, Australia, Canada, France, and Japan—were investigated for the presence of 15- to 17-kDa antigens by colony blotting, using the monoclonal antibody 10G5, and the gene coding for 15- to 17-kDa antigens by PCR. Plasmid DNAs extracted from positive isolates were digested with restriction endonucleases BamHI, EcoRI, EcoT22I, and HindIII, and the digestion patterns that resulted divided the plasmids of virulent isolates into five closely related types. Three of the five types had already been reported in Canadian and Japanese isolates, and the two new types had been found in French and Japanese isolates. Therefore, we tentatively designated these five types 85-kb type I (pREAT701), 85-kb type II (a new type), 87-kb type I (EcoRI and BamHI type 2 [V. M. Nicholson and J. F. Prescott, J. Clin. Microbiol. 35:738–740, 1997]), 87-kb type II (a new type), and 90-kb (pREL1) plasmids. The 85-kb type I plasmid was found in isolates from Argentina, Australia, Canada, and France. Plasmid 87-kb type I was isolated in specimens from Argentina, Canada, and France. The 85-kb type II plasmid appeared in isolates from France. On the other hand, plasmids 87-kb type II and 90-kb were found only in isolates from Japan. These results revealed geographic differences in the distribution of the virulence plasmids found in the five countries and suggested that the restriction fragment length polymorphism of virulence plasmids might be useful to elucidate the molecular epidemiology of virulent R. equi in the world. PMID:10488224

Molecular and phenotypic characteristics of methicillin-resistant Staphylococcus aureus isolated from hospitalized patients.

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de Oliveira, Caio Ferreira; Morey, Alexandre Tadachi; Santos, Jussevania Pereira; Gomes, Ludmila Vilela Pereira; Cardoso, Juscélio Donizete; Pinge-Filho, Phileno; Perugini, Márcia Regina Eches; Yamauchi, Lucy Megumi; Yamada-Ogatta, Sueli Fumie

2015-07-30

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading causes of infections acquired in both community and hospital settings. In this study, MRSA isolated from different sources of hospitalized patients was characterized by molecular and phenotypic methods. A total of 123 S. aureus isolates were characterized according to their genetic relatedness by repetitive element sequence based-PCR (REP-PCR), in vitro antimicrobial susceptibility profile, SCCmec typing and presence of seven virulence factor-encoding genes. REP-PCR fingerprinting showed low relatedness between the isolates, and the predominance of one specific lineage or clonal group was not observed. All isolates were susceptible to teicoplanin and linezolide. All isolates were resistant to cefoxitin and penicillin, and the majority were also resistant to one or more other antimicrobials. Fifty isolates (41.7%) were intermediately resistant to vancomycin. Most isolates harbored SCCmec type II (53.7%), followed by type I (22.8%), type IV (8.1%) and type III (1.6%). All isolates harbored at least two virulence factor-encoding genes, and the prevalence was as follows: coa, 100%; icaA, 100%; hla, 13.0%; hlb, 91.1%, hld, 91.1%; lukS-PV and lukF-PV, 2.4%; and tst, 34.1%. A positive association with the presence of hla and SCCmec type II, and tst and SCCmec type I was observed. This study showed the high virulence potential of multidrug-resistant MRSA circulating in a teaching hospital. A high prevalence of MRSA showing intermediate vancomycin resistance was also observed, indicating the urgent need to improve strategies for controlling the use of antimicrobials for appropriate management of S. aureus infections.

The effect of structural design parameters on FPGA-based feed-forward space-time trellis coding-orthogonal frequency division multiplexing channel encoders

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Passas, Georgios; Freear, Steven; Fawcett, Darren

2010-08-01

Orthogonal frequency division multiplexing (OFDM)-based feed-forward space-time trellis code (FFSTTC) encoders can be synthesised as very high speed integrated circuit hardware description language (VHDL) designs. Evaluation of their FPGA implementation can lead to conclusions that help a designer to decide the optimum implementation, given the encoder structural parameters. VLSI architectures based on 1-bit multipliers and look-up tables (LUTs) are compared in terms of FPGA slices and block RAMs (area), as well as in terms of minimum clock period (speed). Area and speed graphs versus encoder memory order are provided for quadrature phase shift keying (QPSK) and 8 phase shift keying (8-PSK) modulation and two transmit antennas, revealing best implementation under these conditions. The effect of number of modulation bits and transmit antennas on the encoder implementation complexity is also investigated.

Analysis of plasmid profiling as a method for rapid differentiation of food-associated Clostridium perfringens strains.

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Jones, M K; Iwanejko, L A; Longden, M S

1989-09-01

Plasmid analysis of over 120 strains of Clostridium perfringens, isolated during food-poisoning incidents and from animal carcasses and food constituents with no association with food poisoning, showed the potential of plasmid profiling as a means of differentiating epidemiologically related strains. On average 65% of freshly isolated strains contained one or more plasmids which could be used in the analysis. Comparison of profiles of strains from unrelated sources or unrelated strains from the same source showed a particularly wide variety of plasmid profiles. Thus the possibility that epidemiologically-unrelated strains might possess similar profiles appears to be very low in this organism. Analysis of serologically-related strains from the same source revealed similar plasmid profiles in all the plasmid-bearing strains examined. A high proportion (71%) of fresh and well-characterized food-poisoning strains possessed plasmids of 6.2 kb in size (compared with 19% of non-food-poisoning strains). The possible role of these plasmids is discussed, since the structural gene encoding the enterotoxin type A was not present on any of the plasmids in the food-poisoning strains tested.

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks.

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Chen, Yuan-Jyue; Rao, Sundipta D; Seelig, Georg

2015-11-25

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

Identification of novel Clostridium perfringens type E strains that carry an iota toxin plasmid with a functional enterotoxin gene.

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Kazuaki Miyamoto

Full Text Available Clostridium perfringens enterotoxin (CPE is a major virulence factor for human gastrointestinal diseases, such as food poisoning and antibiotic associated diarrhea. The CPE-encoding gene (cpe can be chromosomal or plasmid-borne. Recent development of conventional PCR cpe-genotyping assays makes it possible to identify cpe location (chromosomal or plasmid in type A isolates. Initial studies for developing cpe genotyping assays indicated that all cpe-positive strains isolated from sickened patients were typable by cpe-genotypes, but surveys of C. perfringens environmental strains or strains from feces of healthy people suggested that this assay might not be useful for some cpe-carrying type A isolates. In the current study, a pulsed-field gel electrophoresis Southern blot assay showed that four cpe-genotype untypable isolates carried their cpe gene on a plasmid of ∼65 kb. Complete sequence analysis of the ∼65 kb variant cpe-carrying plasmid revealed no intact IS elements and a disrupted cytosine methyltransferase (dcm gene. More importantly, this plasmid contains a conjugative transfer region, a variant cpe gene and variant iota toxin genes. The toxin genes encoded by this plasmid are expressed based upon the results of RT-PCR assays. The ∼65 kb plasmid is closely related to the pCPF4969 cpe plasmid of type A isolates. MLST analyses indicated these isolates belong to a unique cluster of C. perfringens. Overall, these isolates carrying a variant functional cpe gene and iota toxin genes represent unique type E strains.

Differences in the stability of the plasmids of Yersinia pestis cultures in vitro: impact on virulence

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TC Leal-Balbino

2004-11-01

Full Text Available Plasmid and chromosomal genes encode determinants of virulence for Yersinia pestis, the causative agent of plague. However, in vitro, Y. pestis genome is very plastic and several changes have been described. To evaluate the alterations in the plasmid content of the cultures in vitro and the impact of the alterations to their pathogenicity, three Y. pestis isolates were submitted to serial subculture, analysis of the plasmid content, and testing for the presence of characteristic genes in each plasmid of colonies selected after subculture. Different results were obtained with each strain. The plasmid content of one of them was shown to be stable; no apparent alteration was produced through 32 subcultures. In the other two strains, several alterations were observed. LD50 in mice of the parental strains and the derived cultures with different plasmid content were compared. No changes in the virulence plasmid content could be specifically correlated with changes in the LD50.

Staphylococcus aureus Enterotoxin A Gene Isolated From Raw Red Meat and Poultry in Tehran, Iran

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Mohammad Hossein Sarrafzadeh Zargar

2014-07-01

Full Text Available Background: Staphylococcus aureus is the most prevalent infectious agent of food materials. Enterotoxin producing types of S. aureus cause well-known food-borne disease. Staphylococcal Enterotoxin A (SEA is the most important agent of gastroenteritis. Objectives: The present study aimed to screen the raw meat samples collected from different regions of Tehran for S. aureus infection and type of encoding enterotoxin. Materials and Methods: Hundred and eighty six meat samples were collected randomly from city dealers and transferred to laboratory within screw cap containers. The samples were first cultured according to the standard bacteriological methods and then S. aureus isolates were identified using standard bacteriological tests. The isolates were subjected to Polymerase Chain Reaction (PCR to detect gene encoding SEA. Results: Staphylococcus aureus isolated from 29 (15.6% meat samples including beef 14.8%, raw lamb 15%, raw chicken 15.7% and raw turkey 16.6%. Using special primer sets proved that the species isolated from five samples (two raw chicken, two raw beef and one raw turkey encoded enterotoxin A. Conclusions: Although staphylococcal contamination within food material is more or less a routine, but detection of enterotoxin encoding species from raw meat samples is alarming for health authorities. These data highlight the importance of periodic surveillance of raw meat distributed among ordinary consumers.

Characterization of a Large Antibiotic Resistance Plasmid Found in Enteropathogenic Escherichia coli Strain B171 and Its Relatedness to Plasmids of Diverse E. coli and Shigella Strains.

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Hazen, Tracy H; Michalski, Jane; Nagaraj, Sushma; Okeke, Iruka N; Rasko, David A

2017-09-01

Enteropathogenic Escherichia coli (EPEC) is a leading cause of severe infantile diarrhea in developing countries. Previous research has focused on the diversity of the EPEC virulence plasmid, whereas less is known regarding the genetic content and distribution of antibiotic resistance plasmids carried by EPEC. A previous study demonstrated that in addition to the virulence plasmid, reference EPEC strain B171 harbors a second, larger plasmid that confers antibiotic resistance. To further understand the genetic diversity and dissemination of antibiotic resistance plasmids among EPEC strains, we describe the complete sequence of an antibiotic resistance plasmid from EPEC strain B171. The resistance plasmid, pB171_90, has a completed sequence length of 90,229 bp, a GC content of 54.55%, and carries protein-encoding genes involved in conjugative transfer, resistance to tetracycline ( tetA ), sulfonamides ( sulI ), and mercury, as well as several virulence-associated genes, including the transcriptional regulator hha and the putative calcium sequestration inhibitor ( csi ). In silico detection of the pB171_90 genes among 4,798 publicly available E. coli genome assemblies indicates that the unique genes of pB171_90 ( csi and traI ) are primarily restricted to genomes identified as EPEC or enterotoxigenic E. coli However, conserved regions of the pB171_90 plasmid containing genes involved in replication, stability, and antibiotic resistance were identified among diverse E. coli pathotypes. Interestingly, pB171_90 also exhibited significant similarity with a sequenced plasmid from Shigella dysenteriae type I. Our findings demonstrate the mosaic nature of EPEC antibiotic resistance plasmids and highlight the need for additional sequence-based characterization of antibiotic resistance plasmids harbored by pathogenic E. coli . Copyright © 2017 American Society for Microbiology.

Typing of Panton-Valentine leukocidin-encoding phages carried by methicillin-susceptible and methicillin-resistant Staphylococcus aureus from Italy.

Science.gov (United States)

Sanchini, A; Del Grosso, M; Villa, L; Ammendolia, M G; Superti, F; Monaco, M; Pantosti, A

2014-11-01

Panton-Valentine leukocidin (PVL) is the hallmark of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) but can also be found in methicillin-susceptible S. aureus (MSSA) sharing pathogenic and epidemiological characteristics of CA-MRSA. PVL is encoded by two co-transcribed genes that are carried by different staphylococcal bacteriophages. We applied an extended PCR-based typing scheme for the identification of two morphological groups (elongated-head group and icosahedral-head group I phages) and specific PVL phage types in S. aureus isolates recovered in Italy. We examined 48 PVL-positive isolates (25 MSSA and 23 MRSA) collected from different hospital laboratories from April 2005 to May 2011. spa typing, multilocus sequence typing and staphylococcal cassette chromosome mec typing were applied to categorize the isolates. Phage typeability was 48.0% in MSSA and 91.3% in MRSA, highlighting the limitation of the PCR typing scheme when applied to PVL-positive MSSA. Five different PVL phages and two variants of a known phage were detected, the most prevalent being ΦSa2usa, recovered in 15 out of 48 (31.2%) isolates, and carried by both MSSA and MRSA belonging to CC8 and CC5. The recently described ΦTCH60 was recovered in four isolates. A PVL phage (ΦSa119) from an ST772 MRSA, that was not detected using the previous typing scheme, was sequenced, and new primers were designed for the identification of the icosahedral-head group II PVL phages present in ST772 and ST59 MRSA. A comprehensive PVL-phage typing can contribute to the understanding of the epidemiology and evolution of PVL-positive MSSA and MRSA. © 2014 The Authors Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.

Cloning and DNA sequence of the mercuric- and organomercurial-resistance determinants of plasmid pDU1358

International Nuclear Information System (INIS)

Griffin, H.G.; Foster, T.J.; Silver, S.; Misra, T.K.

1987-01-01

The broad-spectrum mercurial-resistance plasmid pDU1358 was analyzed by cloning the resistance determinants and preparing a physical and genetic map of a 45-kilobase (kb) region of the plasmid that contains two separate mercurial-resistance operons that mapped about 20 kb apart. One encoded narrow-spectrum mercurial resistance to Hg 2+ and a few organomercurials; the other specified broad-spectrum resistance to phenylmercury and additional organomercurials. Each determinant governed mercurial transport functions. Southern DNA x DNA hybridization experiments using gene-specific probes from the plasmid R100 mer operon indicated close homology with the R100 deteminant. The 2153 base pairs of the promoter-distal part of the broad-spectrum Hg 2+ -resistance operon of pDU1358 were sequenced. This region included the 3'-terminal part of the merA gene, merD, unidentified reading frame URF1, and a part of URF2 homologous to previously sequenced determinants of plasmid R100. Between the merA and merD genes, an open reading frame encoding a 212 amino acid polypeptide was identified as the merB gene that determines the enzyme organomercurial lyase that cleaves the C-Hg bond of phenylmercury

NDM-1 encoded by a pNDM-HN380-like plasmid pNDM-BJ03 in clinical Enterobacter cloacae.

Science.gov (United States)

Lü, Yang; Liu, Wei; Liang, Hui; Zhao, Shulong; Zhang, Wei; Liu, Jia; Jin, Cheng; Hu, Hongyan

2018-02-01

A carbapenemase-producing Enterobacter cloacae hhy03 with a bla NDM-1 and bla SHV-12 -coharboring plasmid was isolated from a sputum specimen of a patient. This is the third nucleotide sequence report of bla NDM-1 -harboring plasmid from Enterobacter cloacae that have caused lethal infections in China, indicating the spread of NDM-1 by IncX3 plasmid between Enterobacteriaceae. Copyright © 2017. Published by Elsevier Inc.

Characterization of an IncA/C Multidrug Resistance Plasmid in Vibrio alginolyticus.

Science.gov (United States)

Ye, Lianwei; Li, Ruichao; Lin, Dachuan; Zhou, Yuanjie; Fu, Aisi; Ding, Qiong; Chan, Edward Wai Chi; Yao, Wen; Chen, Sheng

2016-05-01

Cephalosporin-resistant Vibrio alginolyticus was first isolated from food products, with β-lactamases encoded by blaPER-1, blaVEB-1, and blaCMY-2 being the major mechanisms mediating their cephalosporin resistance. The complete sequence of a multidrug resistance plasmid, pVAS3-1, harboring the blaCMY-2 and qnrVC4 genes was decoded in this study. Its backbone exhibited genetic homology to known IncA/C plasmids recoverable from members of the family Enterobacteriaceae, suggesting its possible origin in Enterobacteriaceae. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Conjugative plasmid pAW63 brings new insights into the genesis of the Bacillus anthracis virulence plasmid pXO2 and of the Bacillus thuringiensis plasmid pBT9727

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Mahillon Jacques

2005-07-01

Full Text Available Abstract Background Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis belong to the genetically close-knit Bacillus cereus sensu lato group, a family of rod-shaped Gram-positive bacteria. pAW63 is the first conjugative plasmid from the B. cereus group to be completely sequenced. Results The 71,777 bp nucleotide sequence of pAW63 reveals a modular structure, including a 42 kb tra region encoding homologs of the Type IV secretion systems components VirB11, VirB4 and VirD4, as well as homologs of Gram-positive conjugation genes from Enterococcus, Lactococcus, Listeria, Streptococcus and Staphylococcus species. It also firmly establishes the existence of a common backbone between pAW63, pXO2 from Bacillus anthracis and pBT9727 from the pathogenic Bacillus thuringiensis serovar konkukian strain 97-27. The alignment of these three plasmids highlights the presence of well conserved segments, in contrast to distinct regions of high sequence plasticity. The study of their specific differences has provided a three-point reference framework that can be exploited to formulate solid hypotheses concerning the functionalities and the molecular evolution of these three closely related plasmids. This has provided insight into the chronology of their divergence, and led to the discovery of two Type II introns on pAW63, matching copies of the mobile element IS231L in different loci of pXO2 and pBT9727, and the identification on pXO2 of a 37 kb pathogenicity island (PAI containing the anthrax capsule genes. Conclusion The complete sequence determination of pAW63 has led to a functional map of the plasmid yielding insights into its conjugative apparatus, which includes T4SS-like components, as well as its resemblance to other large plasmids of Gram-positive bacteria. Of particular interest is the extensive homology shared between pAW63 and pXO2, the second virulence plasmid of B. anthracis, as well as pBT9727 from the pathogenic strain B. thuringiensis

Comparative genomic and plasmid analysis of beer-spoiling and non-beer-spoiling Lactobacillus brevis isolates.

Science.gov (United States)

Bergsveinson, Jordyn; Ziola, Barry

2017-12-01

Beer-spoilage-related lactic acid bacteria (BSR LAB) belong to multiple genera and species; however, beer-spoilage capacity is isolate-specific and partially acquired via horizontal gene transfer within the brewing environment. Thus, the extent to which genus-, species-, or environment- (i.e., brewery-) level genetic variability influences beer-spoilage phenotype is unknown. Publicly available Lactobacillus brevis genomes were analyzed via BlAst Diagnostic Gene findEr (BADGE) for BSR genes and assessed for pangenomic relationships. Also analyzed were functional coding capacities of plasmids of LAB inhabiting extreme niche environments. Considerable genetic variation was observed in L. brevis isolated from clinical samples, whereas 16 candidate genes distinguish BSR and non-BSR L. brevis genomes. These genes are related to nutrient scavenging of gluconate or pentoses, mannose, and metabolism of pectin. BSR L. brevis isolates also have higher average nucleotide identity and stronger pangenome association with one another, though isolation source (i.e., specific brewery) also appears to influence the plasmid coding capacity of BSR LAB. Finally, it is shown that niche-specific adaptation and phenotype are plasmid-encoded for both BSR and non-BSR LAB. The ultimate combination of plasmid-encoded genes dictates the ability of L. brevis to survive in the most extreme beer environment, namely, gassed (i.e., pressurized) beer.

Plasmids in Gram negatives: molecular typing of resistance plasmids.

Science.gov (United States)

Carattoli, Alessandra

2011-12-01

A plasmid is defined as a double stranded, circular DNA molecule capable of autonomous replication. By definition, plasmids do not carry genes essential for the growth of host cells under non-stressed conditions but they have systems which guarantee their autonomous replication also controlling the copy number and ensuring stable inheritance during cell division. Most of the plasmids confer positively selectable phenotypes by the presence of antimicrobial resistance genes. Plasmids evolve as an integral part of the bacterial genome, providing resistance genes that can be easily exchanged among bacteria of different origin and source by conjugation. A multidisciplinary approach is currently applied to study the acquisition and spread of antimicrobial resistance in clinically relevant bacterial pathogens and the established surveillance can be implemented by replicon typing of plasmids. Particular plasmid families are more frequently detected among Enterobacteriaceae and play a major role in the diffusion of specific resistance genes. For instance, IncFII, IncA/C, IncL/M, IncN and IncI1 plasmids carrying extended-spectrum beta-lactamase genes and acquired AmpC genes are currently considered to be "epidemic resistance plasmids", being worldwide detected in Enterobacteriaceae of different origin and sources. The recognition of successful plasmids is an essential first step to design intervention strategies preventing their spread. Copyright © 2011 Elsevier GmbH. All rights reserved.

Analytical Investigations on Carrier Phase Recovery in Dispersion-Unmanaged n-PSK Coherent Optical Communication Systems

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Tianhua Xu

2016-09-01

Full Text Available Using coherent optical detection and digital signal processing, laser phase noise and equalization enhanced phase noise can be effectively mitigated using the feed-forward and feed-back carrier phase recovery approaches. In this paper, theoretical analyses of feed-back and feed-forward carrier phase recovery methods have been carried out in the long-haul high-speed n-level phase shift keying (n-PSK optical fiber communication systems, involving a one-tap normalized least-mean-square (LMS algorithm, a block-wise average algorithm, and a Viterbi-Viterbi algorithm. The analytical expressions for evaluating the estimated carrier phase and for predicting the bit-error-rate (BER performance (such as the BER floors have been presented and discussed in the n-PSK coherent optical transmission systems by considering both the laser phase noise and the equalization enhanced phase noise. The results indicate that the Viterbi-Viterbi carrier phase recovery algorithm outperforms the one-tap normalized LMS and the block-wise average algorithms for small phase noise variance (or effective phase noise variance, while the one-tap normalized LMS algorithm shows a better performance than the other two algorithms for large phase noise variance (or effective phase noise variance. In addition, the one-tap normalized LMS algorithm is more sensitive to the level of modulation formats.

Characterization of epidemic IncI1-Iγ plasmids harboring ambler class A and C genes in Escherichia coli and Salmonella enterica from animals and humans.

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Smith, Hilde; Bossers, Alex; Harders, Frank; Wu, Guanghui; Woodford, Neil; Schwarz, Stefan; Guerra, Beatriz; Rodríguez, Irene; van Essen-Zandbergen, Alieda; Brouwer, Michael; Mevius, Dik

2015-09-01

The aim of the study was to identify the plasmid-encoded factors contributing to the emergence and spread of epidemic IncI1-Iγ plasmids obtained from Escherichia coli and Salmonella enterica isolates from animal and human reservoirs. For this, 251 IncI1-Iγ plasmids carrying various extended-spectrum β-lactamase (ESBL) or AmpC β-lactamase genes were compared using plasmid multilocus sequence typing (pMLST). Thirty-two of these plasmids belonging to different pMLST types were sequenced using Roche 454 and Illumina platforms. Epidemic IncI1-Iγ plasmids could be assigned to various dominant clades, whereas rarely detected plasmids clustered together as a distinct clade. Similar phylogenetic trees were obtained using only the plasmid backbone sequences, showing that the differences observed between the plasmids belonging to distinct clades resulted mainly from differences between their backbone sequences. Plasmids belonging to the various clades differed particularly in the presence/absence of genes encoding partitioning and addiction systems, which contribute to stable inheritance during cell division and plasmid maintenance. Despite this, plasmids belonging to the various phylogenetic clades also showed marked resistance gene associations, indicating the circulation of successful plasmid-gene combinations. The variation in traY and excA genes found in IncI1-Iγ plasmids is conserved within pMLST sequence types and plays a role in incompatibility, although functional study is needed to elucidate the role of these genes in plasmid epidemiology. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Effects of deoxycycline induced lentivirus encoding FasL gene on ...

African Journals Online (AJOL)

Abstract. Fas/Fas ligand (FasL)-mediated apoptosis plays a critical role in deletion of activated T cells. This study aimed to construct the lentivirus encoding FasL gene induced by deoxycycline and evaluate its effects on apoptosis of Th1 cells. A plasmid expression system encoding FasL was constructed through utilizing the ...

Characterization of epidemic IncI1-Iγ plasmids harboring ambler class A and C genes in Escherichia coli and Salmonella enterica from animals and humans

NARCIS (Netherlands)

Smith, Hilde; Bossers, Alex; Harders, Frank; Wu, Guanghui; Woodford, Neil; Schwarz, Stefan; Guerra, Beatriz; Rodríguez, Irene; van Essen-Zandbergen, Alieda; Brouwer, Michael; Mevius, Dik

The aim of the study was to identify the plasmid-encoded factors contributing to the emergence and spread of epidemic IncI1-Iγ plasmids obtained from Escherichia coli and Salmonella enterica isolates from animal and human reservoirs. For this, 251 IncI1-Iγ plasmids carrying various extended-spectrum

Response of Methicillin-Resistant Staphylococcus aureus to Amicoumacin A

OpenAIRE

Lama, Amrita; Pané-Farré, Jan; Chon, Tai; Wiersma, Anna M.; Sit, Clarissa S.; Vederas, John C.; Hecker, Michael; Nakano, Michiko M.

2012-01-01

Amicoumacin A exhibits strong antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA), hence we sought to uncover its mechanism of action. Genome-wide transcriptome analysis of S. aureus COL in response to amicoumacin A showed alteration in transcription of genes specifying several cellular processes including cell envelope turnover, cross-membrane transport, virulence, metabolism, and general stress response. The most highly induced gene was lrgA, encoding an antiho...

Insights into dynamics of mobile genetic elements in hyperthermophilic environments from five new Thermococcus plasmids.

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Mart Krupovic

Full Text Available Mobilome of hyperthermophilic archaea dwelling in deep-sea hydrothermal vents is poorly characterized. To gain insight into genetic diversity and dynamics of mobile genetic elements in these environments we have sequenced five new plasmids from different Thermococcus strains that have been isolated from geographically remote hydrothermal vents. The plasmids were ascribed to two subfamilies, pTN2-like and pEXT9a-like. Gene content and phylogenetic analyses illuminated a robust connection between pTN2-like plasmids and Pyrococcus abyssi virus 1 (PAV1, with roughly half of the viral genome being composed of genes that have homologues in plasmids. Unexpectedly, pEXT9a-like plasmids were found to be closely related to the previously sequenced plasmid pMETVU01 from Methanocaldococcus vulcanius M7. Our data suggests that the latter observation is most compatible with an unprecedented horizontal transfer of a pEXT9a-like plasmid from Thermococcales to Methanococcales. Gene content analysis revealed that thermococcal plasmids encode Hfq-like proteins and toxin-antitoxin (TA systems of two different families, VapBC and RelBE. Notably, although abundant in archaeal genomes, to our knowledge, TA and hfq-like genes have not been previously found in archaeal plasmids or viruses. Finally, the plasmids described here might prove to be useful in developing new genetic tools for hyperthermophiles.

Plasmids and rickettsial evolution: insight from Rickettsia felis.

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Joseph J Gillespie

2007-03-01

Full Text Available The genome sequence of Rickettsia felis revealed a number of rickettsial genetic anomalies that likely contribute not only to a large genome size relative to other rickettsiae, but also to phenotypic oddities that have confounded the categorization of R. felis as either typhus group (TG or spotted fever group (SFG rickettsiae. Most intriguing was the first report from rickettsiae of a conjugative plasmid (pRF that contains 68 putative open reading frames, several of which are predicted to encode proteins with high similarity to conjugative machinery in other plasmid-containing bacteria.Using phylogeny estimation, we determined the mode of inheritance of pRF genes relative to conserved rickettsial chromosomal genes. Phylogenies of chromosomal genes were in agreement with other published rickettsial trees. However, phylogenies including pRF genes yielded different topologies and suggest a close relationship between pRF and ancestral group (AG rickettsiae, including the recently completed genome of R. bellii str. RML369-C. This relatedness is further supported by the distribution of pRF genes across other rickettsiae, as 10 pRF genes (or inactive derivatives also occur in AG (but not SFG rickettsiae, with five of these genes characteristic of typical plasmids. Detailed characterization of pRF genes resulted in two novel findings: the identification of oriV and replication termination regions, and the likelihood that a second proposed plasmid, pRFdelta, is an artifact of the original genome assembly.Altogether, we propose a new rickettsial classification scheme with the addition of a fourth lineage, transitional group (TRG rickettsiae, that is unique from TG and SFG rickettsiae and harbors genes from possible exchanges with AG rickettsiae via conjugation. We offer insight into the evolution of a plastic plasmid system in rickettsiae, including the role plasmids may have played in the acquirement of virulence traits in pathogenic strains, and the

Subtle genetic changes enhance virulence of methicillin resistant and sensitive Staphylococcus aureus

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Hawes Alicia C

2007-11-01

Full Text Available Abstract Background Community acquired (CA methicillin-resistant Staphylococcus aureus (MRSA increasingly causes disease worldwide. USA300 has emerged as the predominant clone causing superficial and invasive infections in children and adults in the USA. Epidemiological studies suggest that USA300 is more virulent than other CA-MRSA. The genetic determinants that render virulence and dominance to USA300 remain unclear. Results We sequenced the genomes of two pediatric USA300 isolates: one CA-MRSA and one CA-methicillin susceptible (MSSA, isolated at Texas Children's Hospital in Houston. DNA sequencing was performed by Sanger dideoxy whole genome shotgun (WGS and 454 Life Sciences pyrosequencing strategies. The sequence of the USA300 MRSA strain was rigorously annotated. In USA300-MRSA 2658 chromosomal open reading frames were predicted and 3.1 and 27 kilobase (kb plasmids were identified. USA300-MSSA contained a 20 kb plasmid with some homology to the 27 kb plasmid found in USA300-MRSA. Two regions found in US300-MRSA were absent in USA300-MSSA. One of these carried the arginine deiminase operon that appears to have been acquired from S. epidermidis. The USA300 sequence was aligned with other sequenced S. aureus genomes and regions unique to USA300 MRSA were identified. Conclusion USA300-MRSA is highly similar to other MRSA strains based on whole genome alignments and gene content, indicating that the differences in pathogenesis are due to subtle changes rather than to large-scale acquisition of virulence factor genes. The USA300 Houston isolate differs from another sequenced USA300 strain isolate, derived from a patient in San Francisco, in plasmid content and a number of sequence polymorphisms. Such differences will provide new insights into the evolution of pathogens.

Carriage of extended-spectrum beta-lactamase-plasmids does not reduce fitness but enhances virulence in some strains of pandemic E. coli lineages

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Katharina eSchaufler

2016-03-01

Full Text Available Pathogenic ESBL-producing E. coli lineages occur frequently worldwide, not only in a human health context but in animals and the environment, also in settings with low antimicrobial pressures. This study investigated the fitness costs of ESBL-plasmids and their influence on chromosomally encoded features associated with virulence, such as those involved in the planktonic and sessile behaviors of ST131 and ST648 E. coli. ESBL-plasmid-carrying wild-type E. coli strains, their corresponding ESBL-plasmid-cured variants (PCV, and complementary ESBL-carrying transformants were comparatively analyzed using growth curves, Omnilog® phenotype microarray (PM assays, macrocolony and biofilm formation, swimming motility, and RNA sequence analysis. Growth curves and PM results pointed towards similar growth and metabolic behaviors among the strains. Phenotypic differences in some strains were detected, including enhanced curli fimbriae and/or cellulose production as well as a reduced swimming capacity of some ESBL-carrying strains, as compared to their respective PCVs. RNA sequencing mostly confirmed the phenotypic results, suggesting that the chromosomally encoded csgD pathway is a key factor involved. These results contradict the hypothesis that ESBL-plasmid-carriage leads to a fitness loss in ESBL-carrying strains. Instead, the results indicate an influence of some ESBL-plasmids on chromosomally encoded features associated with virulence in some E. coli strains. In conclusion, apart from antibiotic resistance selective advantages, ESBL-plasmid-carriage may also lead to enhanced virulence or adaption to specific habitats in some strains of pandemic ESBL-producing E. coli lineages.

Novel RepA-MCM proteins encoded in plasmids pTAU4, pORA1 and pTIK4 from Sulfolobus neozealandicus

DEFF Research Database (Denmark)

Greve, B.; Jensen, S.; Phan, H.

2005-01-01

Three plasmids isolated from the crenarchaeal thermoacidophile Sulfolobus neozealandicus were characterized. Plasmids pTAU4 (7,192 bp), pORA1 (9,689 bp) and pTIK4 (13,638 bp) show unusual properties that distinguish them from previously characterized cryptic plasmids of the genus Sulfolobus. Plas...

Comparative proteomics of Staphylococcus aureus and the response of methicillin-resistant and methicillin-sensitive strains to Triton X-100

DEFF Research Database (Denmark)

Cordwell, Stuart J; Larsen, Martin Røssel; Cole, Rebecca T

2002-01-01

profiles of S. aureus strains COL (methicillin-resistant) and 8325 (methicillin-sensitive). Reference mapping via this approach identified 377 proteins that corresponded to 266 distinct ORFs. Amongst these identified proteins were 14 potential virulence factors. The production of 41 'hypothetical' proteins....... Comparative maps were used to characterize the S. aureus response to treatment with Triton X-100 (TX-100), a detergent that has been shown to reduce methicillin resistance independently of an interaction with the mecA-encoded penicillin-binding protein 2a. In response to growth of the bacteria in the presence...... of TX-100, 44 protein spots showed altered levels of abundance, and 11 of these spots were found only in COL. The products of genes regulated by sigma(B) (the alternative sigma factor), including Asp23 and three proteins of unknown function, and SarA (a regulator of virulence genes) were shown...

In Silico Detection and Typing of Plasmids using PlasmidFinder and Plasmid Multilocus Sequence Typing

DEFF Research Database (Denmark)

Carattoli, Alessandra; Zankari, Ea; García-Fernández, Aurora

2014-01-01

In the work presented here, we designed and developed two easy-to-use Web tools for in silico detection and characterization of whole-genome sequence (WGS) and whole-plasmid sequence data from members of the family Enterobacteriaceae. These tools will facilitate bacterial typing based on draft...... genomes of multidrug-resistant Enterobacteriaceae species by the rapid detection of known plasmid types. Replicon sequences from 559 fully sequenced plasmids associated with the family Enterobacteriaceae in the NCBI nucleotide database were collected to build a consensus database for integration...... sequences identified in the 559 fully sequenced plasmids. For plasmid multilocus sequence typing (pMLST) analysis, a database that is updated weekly was generated from www.pubmlst.org and integrated into a Web tool called pMLST. Both databases were evaluated using draft genomes from a collection...

Treatment with Cefotaxime Affects Expression of Conjugation Associated Proteins and Conjugation Transfer Frequency of an IncI1 Plasmid in Escherichia coli

DEFF Research Database (Denmark)

Møller, Thea S B; Liu, Gang; Boysen, Anders

2017-01-01

research suggests that the effect of antibiotic treatment on plasmid conjugation frequencies, and hence the spread of resistance plasmids, may have been overestimated. We addressed the question by quantifying transfer proteins and conjugation frequencies of a blaCTX-M-1 encoding IncI1 resistance plasmid....... The frequency of plasmid conjugation, measured in an antibiotic free environment, increased significantly when the donor was pre-grown in broth containing CTX compared to growth without this drug, regardless of whether blaCTX-M-1 was located on the plasmid or in trans on the chromosome. The results shows...

AAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines.

Science.gov (United States)

Luo, Yuxia; Frederick, Amy; Martin, John M; Scaria, Abraham; Cheng, Seng H; Armentano, Donna; Wadsworth, Samuel C; Vincent, Karen A

2017-06-01

Adeno-associated virus (AAV) producer cell lines are created via transfection of HeLaS3 cells with a single plasmid containing three components (the vector sequence, the AAV rep and cap genes, and a selectable marker gene). As this plasmid contains both the cis (Rep binding sites) and trans (Rep protein encoded by the rep gene) elements required for site-specific integration, it was predicted that plasmid integration might occur within the AAVS1 locus on human chromosome 19 (chr19). The objective of this study was to investigate whether integration in AAVS1 might be correlated with vector yield. Plasmid integration sites within several independent cell lines were assessed via Southern, fluorescence in situ hybridization (FISH) and PCR analyses. In the Southern analyses, the presence of fragments detected by both rep- and AAVS1-specific probes suggested that for several mid- and high-producing lines, plasmid DNA had integrated into the AAVS1 locus. Analysis with puroR and AAVS1-specific probes suggested that integration in AAVS1 was a more widespread phenomenon. High-producing AAV2-secreted alkaline phosphatase (SEAP) lines (masterwell 82 [MW82] and MW278) were evaluated via FISH using probes specific for the plasmid, AAVS1, and a chr19 marker. FISH analysis detected two plasmid integration sites in MW278 (neither in AAVS1), while a total of three sites were identified in MW82 (two in AAVS1). An inverse PCR assay confirmed integration within AAVS1 for several mid- and high-producing lines. In summary, the FISH, Southern, and PCR data provide evidence of site-specific integration of the plasmid within AAVS1 in several AAV producer cell lines. The data also suggest that integration in AAVS1 is a general phenomenon that is not necessarily restricted to high producers. The results also suggest that plasmid integration within the AAVS1 locus is not an absolute requirement for a high vector yield.

Protein-Nanocrystal Conjugates Support a Single Filament Polymerization Model in R1 Plasmid Segregation

Energy Technology Data Exchange (ETDEWEB)

Choi, Charina L.; Claridge, Shelley A.; Garner, Ethan C.; Alivisatos, A. Paul; Mullins, R. Dyche

2008-07-15

To ensure inheritance by daughter cells, many low-copy number bacterial plasmids, including the R1 drug-resistance plasmid, encode their own DNA segregation systems. The par operon of plasmid R1 directs construction of a simple spindle structure that converts free energy of polymerization of an actin-like protein, ParM, into work required to move sister plasmids to opposite poles of rod-shaped cells. The structures of individual components have been solved, but little is known about the ultrastructure of the R1 spindle. To determine the number of ParM filaments in a minimal R1 spindle, we used DNA-gold nanocrystal conjugates as mimics of the R1 plasmid. Wefound that each end of a single polar ParM filament binds to a single ParR/parC-gold complex, consistent with the idea that ParM filaments bind in the hollow core of the ParR/parC ring complex. Our results further suggest that multifilament spindles observed in vivo are associated with clusters of plasmidssegregating as a unit.

Two-Stage n-PSK Partitioning Carrier Phase Recovery Scheme for Circular mQAM Coherent Optical Systems

Directory of Open Access Journals (Sweden)

Jaime Rodrigo Navarro

2016-06-01

Full Text Available A novel two-stage n-PSK partitioning carrier phase recovery (CPR scheme for circular multilevel quadrature amplitude modulation (C-mQAM constellations is presented. The first stage of the algorithm provides an initial rough estimation of the received constellation, which is utilized in the second stage for CPR. The performance of the proposed algorithm is studied through extensive simulations at the forward error correction bit error rate targets of 3.8 × 10−3 and 1 × 10−2 and is compared with different CPR algorithms. A significant improvement in the combined linewidth symbol duration product (ΔνTs tolerance is achieved compared to the single-stage n-PSK partitioning scheme. Superior performance in the ΔνTs tolerance compared to the blind phase search algorithm is also reported. The relative improvements with respect to other CPR schemes are also validated experimentally for a 28-Gbaud C-16QAM back-to-back transmission system. The computational complexity of the proposed CPR scheme is studied, and reduction factors of 24.5 | 30.1 and 59.1 | 63.3 are achieved for C-16QAM and C-64QAM, respectively, compared to single-stage BPS in the form of multipliers | adders.

PCR-Based Analysis of ColE1 Plasmids in Clinical Isolates and Metagenomic Samples Reveals Their Importance as Gene Capture Platforms

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Manuel Ares-Arroyo

2018-03-01

Full Text Available ColE1 plasmids are important vehicles for the spread of antibiotic resistance in the Enterobacteriaceae and Pasteurellaceae families of bacteria. Their monitoring is essential, as they harbor important resistant determinants in humans, animals and the environment. In this work, we have analyzed ColE1 replicons using bioinformatic and experimental approaches. First, we carried out a computational study examining the structure of different ColE1 plasmids deposited in databases. Bioinformatic analysis of these ColE1 replicons revealed a mosaic genetic structure consisting of a host-adapted conserved region responsible for the housekeeping functions of the plasmid, and a variable region encoding a wide variety of genes, including multiple antibiotic resistance determinants. From this exhaustive computational analysis we developed a new PCR-based technique, targeting a specific sequence in the conserved region, for the screening, capture and sequencing of these small plasmids, either specific for Enterobacteriaceae or specific for Pasteurellaceae. To validate this PCR-based system, we tested various collections of isolates from both bacterial families, finding that ColE1 replicons were not only highly prevalent in antibiotic-resistant isolates, but also present in susceptible bacteria. In Pasteurellaceae, ColE1 plasmids carried almost exclusively antibiotic resistance genes. In Enterobacteriaceae, these plasmids encoded a large range of traits, including not only antibiotic resistance determinants, but also a wide variety of genes, showing the huge genetic plasticity of these small replicons. Finally, we also used a metagenomic approach in order to validate this technique, performing this PCR system using total DNA extractions from fecal samples from poultry, turkeys, pigs and humans. Using Illumina sequencing of the PCR products we identified a great diversity of genes encoded by ColE1 replicons, including different antibiotic resistance

A universal encoding scheme for MIMO transmission using a single active element for PSK modulation schemes

DEFF Research Database (Denmark)

Alrabadi, Osama; Papadias, C.B.; Kalis, A.

2009-01-01

A universal scheme for encoding multiple symbol streams using a single driven element (and consequently a single radio frequency (RF) frontend) surrounded by parasitic elements (PE) loaded with variable reactive loads, is proposed in this paper. The proposed scheme is based on creating a MIMO sys...

eap Gene as novel target for specific identification of Staphylococcus aureus.

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Hussain, Muzaffar; von Eiff, Christof; Sinha, Bhanu; Joost, Insa; Herrmann, Mathias; Peters, Georg; Becker, Karsten

2008-02-01

The cell surface-associated extracellular adherence protein (Eap) mediates adherence of Staphylococcus aureus to host extracellular matrix components and inhibits inflammation, wound healing, and angiogenesis. A well-characterized collection of S. aureus and non-S. aureus staphylococcal isolates (n = 813) was tested for the presence of the Eap-encoding gene (eap) by PCR to investigate the use of the eap gene as a specific diagnostic tool for identification of S. aureus. Whereas all 597 S. aureus isolates were eap positive, this gene was not detectable in 216 non-S. aureus staphylococcal isolates comprising 47 different species and subspecies of coagulase-negative staphylococci and non-S. aureus coagulase-positive or coagulase-variable staphylococci. Furthermore, non-S. aureus isolates did not express Eap homologs, as verified on the transcriptional and protein levels. Based on these data, the sensitivity and specificity of the newly developed PCR targeting the eap gene were both 100%. Thus, the unique occurrence of Eap in S. aureus offers a promising tool particularly suitable for molecular diagnostics of this pathogen.

Lysionotin attenuates Staphylococcus aureus pathogenicity by inhibiting α-toxin expression.

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Teng, Zihao; Shi, Dongxue; Liu, Huanyu; Shen, Ziying; Zha, Yonghong; Li, Wenhua; Deng, Xuming; Wang, Jianfeng

2017-09-01

α-Toxin, one of the best known pore-forming proteins produced by Staphylococcus aureus (S. aureus), is a critical virulence factor in multiple infections. The necessity of α-toxin for S. aureus pathogenicity suggests that this toxin is an important target for the development of a potential treatment strategy. In this study, we showed that lysionotin, a natural compound, can inhibit the hemolytic activity of culture supernatants by S. aureus by reducing α-toxin expression. Using real-time PCR analysis, we showed that transcription of hla (the gene encoding α-toxin) and agr (the locus regulating hla) was significantly inhibited by lysionotin. Lactate dehydrogenase and live/dead assays indicated that lysionotin effectively protected human alveolar epithelial cells against S. aureus, and in vivo studies also demonstrated that lysionotin can protect mice from pneumonia caused by S. aureus. These findings suggest that lysionotin is an efficient inhibitor of α-toxin expression and shows significant protection against S. aureus in vitro and in vivo. This study supports a potential strategy for the treatment of S. aureus infection by inhibiting the expression of virulence factors and indicates that lysionotin may be a potential treatment for S. aureus pneumonia.

Decreased uptake of cadmium by a resistant strain of Staphylococcus aureus

Energy Technology Data Exchange (ETDEWEB)

Chopra, I

1971-01-01

Penicillinase plasmids, and some related extrachromosomal elements in Staphylococcus aureus, can specify resistance to inorganic ions, including Hg/sup 2 +/ and Cd/sup 2 +/. There is some tentative evidence that resistance to Hg/sup 2 +/ ions is due to the impermeability of the cells to the ions and not to a higher concentration of free -SH groups in resistance to cadmium ions is unknown. Experiments are described which show that there is a markedly decreased rate of uptake of Cd/sup 2 +/ ions by resistant cells when compared with strains that lack the cad-r gene. The uptake of Cd/sup 2 +/ ions has been studied in the cadmium resistant Staphylococcus aureus strain 8325 (..cap alpha...i/sup +/p/sup +/.cad-r.mer-r) and in its cadmium-sensitive derivative, strain 8324(N). Strain 8325(N) was obtained from 8325 (..cap alpha...i/sup +/.cad-r.mer-r) by isolating a variant which had spontaneously lost the ..cap alpha.. penicillinase plasmid specifying resistance to Cd/sup 2 +/ ions. Cadmium uptake was estimated by adding /sup 115m/CdCl/sub 2/ (final concentration: 10/sup -4/M) to exponentially growing cultures of the sensitive and resistant strains and following the uptake of radioactivity.

Colonization of nursing professionals by Staphylococcus aureus La colonización de los profesionales de enfermería por Staphylococcus aureus A colonização dos profissionais de enfermagem por Staphylococcus aureus

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Josely Pinto de Moura

2011-04-01

Full Text Available This cross-sectional study aimed to investigate the presence of Staphylococcus aureus in the saliva of the nursing team of a teaching hospital in the interior of São Paulo State. Three saliva samples were collected from 351 individuals with an interval of two months between each collection. All ethical aspects were considered. In 867 (82.3% cultures there was no identification of Staphylococcus aureus in the saliva, in 88 (17.7% cultures Staphylococcus aureus was isolated, 26 (2.5% of which were resistant to methicillin. The prevalence of professionals colonized by Staphylococcus aureus was 41.0% (144/351, of which 7.1% (25/351 were characterized as methicillin-resistant Staphylococcus aureus. Transient carriers represented 81.2% and persistent carriers 18.8%. Resistance to mupirocin was 73.1% of MRSA and 9.3% of MSSA. The results demonstrate that it is the nurse and nursing technician that are the professional categories most susceptible to MRSA. Broader discussion on the thematic and interventions are needed.Se trata de un estudio transversal que tuvo como objetivo investigar la presencia de Staphylococcus aureus en la saliva del equipo de enfermería de un hospital escuela del interior del estado de Sao Paulo. Fueron recolectadas tres muestras de saliva de 351 individuos con intervalo de dos meses. Todos los aspectos éticos fueron contemplados. En 867 (82,3% culturas no hubo identificación de Staphylococcus aureus en la saliva, en 88 (17,7% culturas fue aislado Staphylococcus aureus, siendo 26 (2,5% resistentes a la meticilina. La prevalencia de profesionales colonizados por Staphylococcus aureus fue de 41,0% (144/351, de los cuales 7,1% (25/351 fueron caracterizados como Staphylococcus aureus resistentes a la meticilina. Los portadores transitorios representaron 81,2% y los persistentes 18,8%. La resistencia a la mupirocina fue de 73,1% entre los resistentes a la meticilina y 9,3% en los sensibles a la meticilina. Los resultados

Mechanism of antagonistic effects of Andrographis paniculata methanolic extract against Staphylococcus aureus.

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Hussain, Roslinah Mohamad; Razak, Zayan Nabilah Rasyidah Abd; Saad, Wan Mazlina Md; Mustakim, Maimunah

2017-07-01

To investigate the effects of Andrographis paniculata (Burm.f.) Wall. Ex Nees (A. paniculata) on expressions and activities of catalase, superoxide dismutase and alkylhydroperoxide reductase C in Staphylococcus aureus (S. aureus) with respect to its survival in vitro. Antioxidative property of methanolic leaves extract of A. paniculata (0.06 mg/mL). Minimum inhibitory concentration (MIC) was determined by its ability to reduce hydrogen peroxide (H 2 O 2 ) toxicity against S. aureus ATCC 25923 [(3.8 × 10 8 ) cfu/mL]. Effects of the extract on expressions of katA (encoding catalase), sodA and sodM [encoding superoxide dismutases (SODs)], and ahpC [encoding alkylhydroperoxide reductase C (AhpC)] in S. aureus were determined by RT-qPCR and corresponding enzyme activity assays were performed. Nitroblue tetrazolium reduction (NBT) assay was performed to determine effects of the extract on intracellular and extracellular levels of O 2- in S. aureus. Cells challenged with 7.5 mmol/L H 2 O 2 showed 0% survival in 30 min whereas 25% survived after treatment with the extract and H 2 O 2 . Cells that were treated with the extract alone had 43% survival in the same exposure period. Expressions of sodA and sodM genes in extract-treated cells were lowered 0.8-fold and 0.7-fold, respectively with decrease in total SOD activity of 26.8 U compared to untreated cells, 32.4 U (P paniculata methanolic leaves extract (0.06 mg/mL) reduce H 2 O 2 toxicity and more importantly, was in itself effectively inhibitory against S. aureus. Further, our observations suggest that a probable mode of its inhibitory mechanism against S. aureus is by reducing total SOD activity through downregulation of sodA and sodM expressions. Copyright © 2017 Hainan Medical University. Production and hosting by Elsevier B.V. All rights reserved.

Cloning and Sequencing of Gene Encoding Outer Membrane Lipoprotein LipL41 of Leptospira Interrogans Serovar Grippotyphosa

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M.S. Soltani

2014-12-01

Full Text Available Background: Leptospirosis is an infectious bacterial disease caused by pathogenic serovars of Leptospira. Development of reliable and applicable diagnostic test and also recombinant vaccine for this disease require specific antigens that are highly conserved among diverse pathogenic leptospiral serovars. Outer membrane proteins(OMPs of leptospira are effective antigens which can stimulate remarkable immune responses during infection, among them LipL41 is an immunogenic lipoprotein which is present only in pathogenic serovars so it could be regarded as a good candidate for vaccine development and diagnostic method. In order to identify genetic conservation of the lipL41 gene, we cloned and sequenced this gen from Leptospira interrogans serovar vaccinal and field of Grippotyphosa. Materials and Methods: Leptospira interrogans serovar vaccinal Grippotyphosa (RTCC2808 and serovar field Grippotyphosa (RTCC2825were used to inoculate into the selective culture medium(EMJH. The genomic DNA was extracted by standard phenol-chloroform method. The lipL41 gene were amplified by specific primers and cloned into pTZ57R/T vector and transformed into the competent E. coli (Top10 cells. the extracted recombinant plasmid were sequenced. And the related sequences were subjected to homology analysis by comparing them to sequences in the Genbank database. Results: PCR amplification of the lipL41 gene resulted in the 1065 bp PCR product. DNA sequence analysis revealed that lipL41 gene between serovar vaccinal Grippotyphosa (RTCC2808and serovar field Grippotyphosa (RTCC2825 in Iran was 100%. It was also showed that the lipL41 gene had high identity (96%-100% with other pathogenic serovars submitted in Genbank database. Conclusion: The results of this study showed that the lipL41 gene was highly conserved among various pathogenic Leptospira serovars( >95.9 % identity. Hence the cloned gene could be further used for expression of recombinant protein for serodiagnosis

Communications of Staphylococcus aureus and non-aureus Staphylococcus species from bovine intramammary infections and teat apex colonization.

Science.gov (United States)

Mahmmod, Yasser S; Klaas, Ilka Christine; Svennesen, Line; Pedersen, Karl; Ingmer, Hanne

2018-05-16

The role of non-aureus staphylococci (NAS) in the risk of acquisition of intramammary infections with Staphylococcus aureus is vague and still under debate. The objectives of this study were to (1) investigate the distribution patterns of NAS species from milk and teat skin in dairy herds with automatic milking systems, and (2) examine if the isolated NAS influences the expression of S. aureus virulence factors controlled by the accessory gene regulator (agr) quorum sensing system. In 8 herds, 14 to 20 cows with elevated somatic cell count were randomly selected for teat skin swabbing and aseptic quarter foremilk samples from right hind and left front quarters. Teat skin swabs were collected using the modified wet-dry method and milk samples were taken aseptically for bacterial culture. Colonies from quarters with suspicion of having NAS in milk or teat skin samples (or both) were subjected to MALDI-TOF assay for species identification. To investigate the interaction between S. aureus and NAS, 81 isolates NAS were subjected to a qualitative β-galactosidase reporter plate assay. In total, 373 NAS isolates were identified representing 105 from milk and 268 from teat skin of 284 quarters (= 142 cows). Sixteen different NAS species were identified, 15 species from teat skin and 10 species from milk. The most prevalent NAS species identified from milk were Staphylococcus epidermidis (50%), Staphylococcus haemolyticus (15%), and Staphylococcus chromogenes (11%), accounting for 76%. Meanwhile, the most prevalent NAS species from teat skin were Staphylococcus equorum (43%), S. haemolyticus (16%), and Staphylococcus cohnii (14%), accounting for 73%. Using reporter gene fusions monitoring transcriptional activity of key virulence factors and regulators, we found that out of 81 supernatants of NAS isolates, 77% reduced expression of hla, encoding a-hemolysin, 70% reduced expression of RNAIII, the key effector molecule of agr, and 61% reduced expression of spa encoding

Role of enzymes of homologous recombination in illegitimate plasmid recombination in Bacillus subtilis

NARCIS (Netherlands)

Meima, R; Haijema, BJ; Haan, GJ; Venema, G; Bron, S

The structural stability of plasmid pGP1, which encodes a fusion between the penicillinase gene (penP) of Bacillus licheniformis and the Escherichia coli lacZ gene, was investigated in Bacillus subtilis strains expressing mutated subunits of the ATP-dependent nuclease, AddAB, and strains lacking the

Evaluation of approaches to monitor Staphylococcus aureus virulence factor expression during human disease.

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Wouter Rozemeijer

Full Text Available Staphylococcus aureus is a versatile pathogen of medical significance, using multiple virulence factors to cause disease. A prophylactic S. aureus 4-antigen (SA4Ag vaccine comprising capsular polysaccharide (types 5 and 8 conjugates, clumping factor A (ClfA and manganese transporter C (MntC is under development. This study was designed to characterize S. aureus isolates recovered from infected patients and also to investigate approaches for examining expression of S. aureus vaccine candidates and the host response during human infection. Confirmation of antigen expression in different disease states is important to support the inclusion of these antigens in a prophylactic vaccine. Hospitalized patients with diagnosed S. aureus wound (27 or bloodstream (24 infections were enrolled. Invasive and nasal carriage S. aureus isolates were recovered and characterized for genotypic diversity. S. aureus antigen expression was evaluated directly by real-time, quantitative, reverse-transcriptase PCR (qRT-PCR analysis and indirectly by serology using a competitive Luminex immunoassay. Study isolates were genotypically diverse and all had the genes encoding the antigens present in the SA4Ag vaccine. S. aureus nasal carriage was detected in 55% of patients, and in those subjects 64% of the carriage isolates matched the invasive strain. In swab samples with detectable S. aureus triosephosphate isomerase housekeeping gene expression, RNA transcripts encoding the S. aureus virulence factors ClfA, MntC, and capsule polysaccharide were detected by qRT-PCR. Antigen expression was indirectly confirmed by increases in antibody titer during the course of infection from acute to convalescent phase. Demonstration of bacterial transcript expression together with immunological response to the SA4Ag antigens in a clinically relevant patient population provides support for inclusion of these antigens in a prophylactic vaccine.

Evaluation of approaches to monitor Staphylococcus aureus virulence factor expression during human disease.

Science.gov (United States)

Rozemeijer, Wouter; Fink, Pamela; Rojas, Eduardo; Jones, C Hal; Pavliakova, Danka; Giardina, Peter; Murphy, Ellen; Liberator, Paul; Jiang, Qin; Girgenti, Douglas; Peters, Remco P H; Savelkoul, Paul H M; Jansen, Kathrin U; Anderson, Annaliesa S; Kluytmans, Jan

2015-01-01

Staphylococcus aureus is a versatile pathogen of medical significance, using multiple virulence factors to cause disease. A prophylactic S. aureus 4-antigen (SA4Ag) vaccine comprising capsular polysaccharide (types 5 and 8) conjugates, clumping factor A (ClfA) and manganese transporter C (MntC) is under development. This study was designed to characterize S. aureus isolates recovered from infected patients and also to investigate approaches for examining expression of S. aureus vaccine candidates and the host response during human infection. Confirmation of antigen expression in different disease states is important to support the inclusion of these antigens in a prophylactic vaccine. Hospitalized patients with diagnosed S. aureus wound (27) or bloodstream (24) infections were enrolled. Invasive and nasal carriage S. aureus isolates were recovered and characterized for genotypic diversity. S. aureus antigen expression was evaluated directly by real-time, quantitative, reverse-transcriptase PCR (qRT-PCR) analysis and indirectly by serology using a competitive Luminex immunoassay. Study isolates were genotypically diverse and all had the genes encoding the antigens present in the SA4Ag vaccine. S. aureus nasal carriage was detected in 55% of patients, and in those subjects 64% of the carriage isolates matched the invasive strain. In swab samples with detectable S. aureus triosephosphate isomerase housekeeping gene expression, RNA transcripts encoding the S. aureus virulence factors ClfA, MntC, and capsule polysaccharide were detected by qRT-PCR. Antigen expression was indirectly confirmed by increases in antibody titer during the course of infection from acute to convalescent phase. Demonstration of bacterial transcript expression together with immunological response to the SA4Ag antigens in a clinically relevant patient population provides support for inclusion of these antigens in a prophylactic vaccine.

The response of a linear monostable system and its application in parameters estimation for PSK signals

Science.gov (United States)

Duan, Chaowei; Zhan, Yafeng

2016-03-01

The output characteristics of a linear monostable system driven with a periodic signal and an additive white Gaussian noise are studied in this paper. Theoretical analysis shows that the output signal-to-noise ratio (SNR) decreases monotonously with the increasing noise intensity but the output SNR-gain is stable. Inspired by this high SNR-gain phenomenon, this paper applies the linear monostable system in the parameters estimation algorithm for phase shift keying (PSK) signals and improves the estimation performance.

Eliminação de resistência a drogas por fluorquinolonas em Staphylococcus aureus de origem bovina Elimination of resistance to drugs by fluoroquinolones in bovine strains of Staphylococcus aureus

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Maria S.V. Pereira

2004-03-01

Full Text Available Cepas de Staphylococcus aureus de origem bovina foram submetidas ao tratamento com quatro fluoquinolonas na concentração subinibitória (1/2 x CMI, para avaliar a influência desses agentes sobre plasmídios. A ciprofloxacina mostrou ser a fluorquinolona mais eficiente, eliminando marcas de resistência para estreptomicina, tetraciclina, penicilina e cádmio. A norfloxacina e a pefloxacina eliminaram resistência para penicilina e tetraciclina, respectivamente; no entanto, não foi evidenciada a eliminação de plasmídio com ofloxacina. Os resultados confirmam a eficácia das fluor-quinolonas em eliminar plasmídios de resistência mostrando a importância desses estudos como contribuição para o entendimento da prevenção de linhagens multiresistentes, uma vez que as quinolonas em concentrações subinibitórias podem aumentar a sensibilidade das linhagens a outros agentes antimicrobianos.Bovine strains of Staphylococcus aureus were submitted to treatment with four fluoro-quinolones in subinhibitory concentrations (1/2 x MICs to evaluate their influence on the curing of plasmids. Ciprofloxacin showed to be the most efficient by eliminating resistance to streptomycin, tetracyclin, penicillin, and cadmium nitrate. Norfloxacin and pefloxacin eliminated penicillin- and tetracyclin-resistance respectively. Otherwise, plasmids elimination by ofloxacin was not evidenced. The results obtained in this study confirm the potential of fluoroquinolones to eliminate antibiotic-resistant plasmids, and showed to be a valuable contribution for the prevention of multi-resistant strains, and may even enhance their sensitivity to other chemotherapeutic agents.

High Prevalence of Plasmid-Mediated Quinolone Resistance and IncQ Plasmids Carrying qnrS2 Gene in Bacteria from Rivers near Hospitals and Aquaculture in China.

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Yanping Wen

Full Text Available Effluents from hospital and aquaculture are considered important sources of quinolone resistance. However, little information is available on the impact of this effluent on nearby rivers. In this study, 188 ciprofloxacin-resistant bacterial isolates obtained from rivers near hospitals and aquaculture were screened for plasmid-mediated quinolone resistance (PMQR genes. Species identification, antibiotic susceptibility testing, and PMQR gene transferability assessment were conducted for PMQR-positive bacteria. Representative qnrS2-encoding plasmids were subsequently sequenced using a primer-walking approach. In total, 44 isolates (23.4% were positive for qnr genes (16 qnrB2, 3 qnrS1, and 25 qnrS2 and 32 isolates (17.0% were positive for aac(6'-Ib-cr. Other PMQR genes were not detected. The qnrB2 and aac(6'-Ib-cr genes had a higher prevalence in aquaculture samples than in hospital samples, and were significantly associated with Enterobacteriaceae (p < 0.05. In contrast, the prevalence of qnrS2 was not site-related, but was significantly associated with Aeromonas spp. (p < 0.05. All PMQR isolates were resistant to three or more classes of antibiotics. Eleven qnrS2-harboring plasmids from Aeromonas spp., including a novel conjugative plasmid pHP18, were selected for sequencing. These plasmids were small in size (6,388-16,197 bp and belonged to the IncQ or IncU plasmid family, with qnrS2 being part of a mobile insertion cassette. Taken together, our findings suggest that aquaculture is a possible source for aac(6'-Ib-cr and qnrB2 dissemination, and demonstrate the ubiquity of qnrS2 in aquatic environments. Finally, Aeromonas spp. served as vectors for qnrS2 with the help of IncQ-type plasmids.

Cross-Talk between Staphylococcus aureus and Other Staphylococcal Species via the agr Quorum Sensing System

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Jaime Canovas

2016-11-01

Full Text Available Staphylococci are associated with both humans and animals. While most are non-pathogenic colonizers, Staphylococcus aureus is an opportunistic pathogen capable of causing severe infections. S. aureus virulence is controlled by the agr quorum sensing system responding to secreted auto-inducing peptides (AIPs sensed by AgrC, a two component histidine kinase. agr loci are found also in other staphylococcal species and for Staphylococcus epidermidis, the encoded AIP represses expression of agr regulated virulence genes in S. aureus. In this study we aimed to better understand the interaction between staphylococci and S. aureus, and show that this interaction may eventually lead to the identification of new anti-virulence candidates to target S. aureus infections. Here we show that culture supernatants of 37 out of 52 staphylococcal isolates representing 17 different species inhibit S. aureus agr. The dog pathogen, Staphylococcus schleiferi, expressed the most potent inhibitory activity and was active against all four agr classes found in S. aureus. By employing a S. aureus strain encoding a constitutively active AIP receptor we show that the activity is mediated via agr. Subsequent cloning and heterologous expression of the S. schleiferi AIP in S. aureus demonstrated that this molecule was likely responsible for the inhibitory activity, and further proof was provided when pure synthetic S. schleiferi AIP was able to completely abolish agr induction of an S. aureus reporter strain. To assess impact on S. aureus virulence, we co-inoculated S. aureus and S. schleiferi in vivo in the Galleria mellonella wax moth larva, and found that expression of key S. aureus virulence factors was abrogated. Our data show that the S. aureus agr locus is highly responsive to other staphylococcal species suggesting that agr is an inter-species communication system. Based on these results we speculate that interactions between S. aureus and other colonizing staphylococci

The SXT conjugative element and linear prophage N15 encode toxin-antitoxin-stabilizing systems homologous to the tad-ata module of the Paracoccus aminophilus plasmid pAMI2.

Science.gov (United States)

Dziewit, Lukasz; Jazurek, Magdalena; Drewniak, Lukasz; Baj, Jadwiga; Bartosik, Dariusz

2007-03-01

A group of proteic toxin-antitoxin (TA) cassettes whose representatives are widely distributed among bacterial genomes has been identified. These cassettes occur in chromosomes, plasmids, bacteriophages, and noncomposite transposons, as well as in the SXT conjugative element of Vibrio cholerae. The following four homologous loci were subjected to detailed comparative studies: (i) tad-ata from plasmid pAMI2 of Paracoccus aminophilus (the prototype of this group), (ii) gp49-gp48 from the linear bacteriophage N15 of Escherichia coli, (iii) s045-s044 from SXT, and (iv) Z3230-Z3231 from the genomic island of enterohemorrhagic Escherichia coli O157:H7 strain EDL933. Functional analysis revealed that all but one of these loci (Z3230-Z3231) are able to stabilize heterologous replicons, although the host ranges varied. The TA cassettes analyzed have the following common features: (i) the toxins are encoded by the first gene of each operon; (ii) the antitoxins contain a predicted helix-turn-helix motif of the XRE family; and (iii) the cassettes have two promoters that are different strengths, one which is located upstream of the toxin gene and one which is located upstream of the antitoxin gene. All four toxins tested are functional in E. coli; overexpression of the toxins (in the absence of antitoxin) results in a bacteriostatic effect manifested by elongation of bacterial cells and growth arrest. The toxins have various effects on cell viability, which suggests that they may recognize different intracellular targets. Preliminary data suggest that different cellular proteases are involved in degradation of antitoxins encoded by the loci analyzed.

A study of Staphylococcus aureusnasal carriage, antibacterial resistance and virulence factor encoding genes in a tertiary care hospital, Kayseri, Turkey.

Science.gov (United States)

Oguzkaya-Artan, M; Artan, C; Baykan, Z; Sakalar, C; Turan, A; Aksu, H

2015-01-01

This study was to determine the virulence encoding genes, and the antibiotic resistance patterns of the Staphylococcus aureus isolates, which were isolated from the nasal samples of chest clinic patients. The nasal samples of the in-patients (431) and out-patients (1857) in Kayseri Training and Research Hospital's Chest Clinic, Kayseri, Turkey, were cultured on CHROMagar (Biolife, Italiana) S. aureus, and subcultured on sheep blood agar for the isolation of S. aureus. Disc diffusion method was used for antimicrobial susceptibility testing. The occurrence of the staphylococcal virulence encoding genes (enterotoksins [sea, seb, sec, see, seg, seh, sei, sej], fibronectin-binding proteins A, B [fnbA, fnbB], toxic shock syndrome toxin-1 [tst]) were detected by polymerase chain reaction. Forty-five of the 55 (81.8%) S. aureus isolates from inpatients, and 319 (90.6%) isolates from tested 352 out-patient's isolates were suspected to all the antibiotics tested. methicillin-resistant S. aureus (MRSA) was detected in 1.2% of S. aureus isolates. Rifampin, trimethoprim-sulfamethoxazole, clindamycin, erythromycin, gentamicin resistance rates were 1.2%, 1.7%, 2.0%, 8.8%, and 1.2%, respectively. The isolates were susceptible to teicoplanin and vancomycin. The genes most frequently found were tst (92.7%), seg (85.8%), sea (83.6%), fnbA (70.9%). There was no statistical significance detected between MRSA and mecA-negative S. aureus isolates in encoding genes distribution (P > 0.05). Our results show that virulence factor encoding genes were prevalent in patients with S. aureus carriage, whereas antibiotic resistance was low. These virulence determinants may increase the risk for subsequent invasive infections in carriers.

Characterization of class 1 integrons associated with R-plasmids in clinical Aeromonas salmonicida isolates from various geographical areas

DEFF Research Database (Denmark)

Schmidt, A.S.; Bruun, Morten Sichlau; Larsen, J.L.

2001-01-01

Class 1 integrons were found in 26 of 40 antibiotic-resistant isolates of the fish pathogen Aeromonas salmonicida from Northern Europe and North America. Three different dhfr genes, conferring trimethoprim resistance, and one ant(3 " )1a aminoglycoside resistance gene were identified as gene...... inserts. The gene cassettes tended to be conserved among isolates from a particular geographical area. Nineteen isolates transferred R- plasmids carrying different tet determinants to Escherichia coli in filter mating assays, and in 15 cases, the class 1 integrons were co-transferred. Transferable...... sulphadiazine, trimethoprim and streptomycin resistances were invariably encoded by integrons. It thus appears that integron-encoded antibiotic resistance genes contribute substantially to the horizontal spread of antimicrobial resistance within this species, being associated with conjugative plasmids....

Prevalence and characterization of methicillin-resistant Staphylococcus aureus carrying mecA or mecC and methicillin-susceptible Staphylococcus aureus in dairy sheep farms in central Italy.

Science.gov (United States)

Giacinti, G; Carfora, V; Caprioli, A; Sagrafoli, D; Marri, N; Giangolini, G; Amoruso, R; Iurescia, M; Stravino, F; Dottarelli, S; Feltrin, F; Franco, A; Amatiste, S; Battisti, A

2017-10-01

Between January and May 2012, a total of 286 bulk tank milk samples from dairy sheep farms located in central Italy were tested for the presence of Staphylococcus aureus. One hundred fifty-three samples were positive for S. aureus (53.5%), with an average count of 2.53 log cfu/mL. A total of 679 S. aureus colonies were screened for methicillin resistance by the cefoxitin disk diffusion test, and 104 selected cefoxitin-susceptible isolates were also tested for their susceptibility to other antimicrobials representative of the most relevant classes active against Staphylococcus spp. by using the Kirby-Bauer disk diffusion method. Two methicillin-resistant Staphylococcus aureus (MRSA) isolates, carrying respectively the mecA and the mecC genes, were detected in 2 samples from 2 different farms (prevalence 0.7%). The mecA-positive MRSA isolate was blaZ positive, belonged to spa type t127, sequence type (ST)1, clonal complex (CC)1, carried a staphylococcal cassette chromosome mec (SCCmec) type IVa, and was phenotypically resistant to all the β-lactams tested and to erythromycin, streptomycin, kanamycin, and tetracycline. The mecC-positive MRSA isolate was negative for the chromosomally or plasmid-associated blaZ gene but positive for the blaZ allotype associated with SCCmec XI (blaZ-SCCmecXI), belonged to spa type 843, ST(CC)130, carried a SCCmec type XI, and was resistant only to β-lactams. Both MRSA were negative for the presence of specific immune-evasion and virulence genes such as those coding for the Panton-Valentine leucocidin, the toxic shock syndrome toxin 1, and the immune evasion cluster genes. Regarding the presence of the major S. aureus enterotoxin genes, the mecC-positive MRSA tested negative, whereas the ST (CC)1 mecA-positive MRSA harbored the seh gene. Among the 104 methicillin-susceptible S. aureus isolates examined for antimicrobial susceptibility, 63 (60.58%) were susceptible to all the antimicrobials tested, and 41 (39.42%) were resistant to at

Unraveling the regulatory network of IncA/C plasmid mobilization: When genomic islands hijack conjugative elements.

Science.gov (United States)

Carraro, Nicolas; Matteau, Dominick; Burrus, Vincent; Rodrigue, Sébastien

2015-01-01

Conjugative plasmids of the A/C incompatibility group (IncA/C) have become substantial players in the dissemination of multidrug resistance. These large conjugative plasmids are characterized by their broad host-range, extended spectrum of antimicrobials resistance, and prevalence in enteric bacteria recovered from both environmental and clinical settings. Until recently, relatively little was known about the basic biology of IncA/C plasmids, mostly because of the hindrance of multidrug resistance for molecular biology experiments. To circumvent this issue, we previously developed pVCR94ΔX, a convenient prototype that codes for a reduced set of antibiotic resistances. Using pVCR94ΔX, we then characterized the regulatory pathway governing IncA/C plasmid dissemination. We found that the expression of roughly 2 thirds of the genes encoded by this plasmid, including large operons involved in the conjugation process, depends on an FlhCD-like master activator called AcaCD. Beyond the mobility of IncA/C plasmids, AcaCD was also shown to play a key role in the mobilization of different classes of genomic islands (GIs) identified in various pathogenic bacteria. By doing so, IncA/C plasmids can have a considerable impact on bacterial genomes plasticity and evolution.

Genomic organization of a vancomycin-resistant staphylococcus aureus

International Nuclear Information System (INIS)

Mirani, A.Z.; Jamil, N.

2013-01-01

Objective: To study the genomic organization of vancomycin resistance in a local isolate of vancomycin resistant Staphylococcus aureus (VRSA). Study Design: Experimental study. Place and Duration of Study: Department of Microbiology, University of Karachi, January 2008 through December 2010. Methodology: A vancomycin-resistant Staphylococcus aureus (VRSA-CP2) isolate (MIC 16 mu g/ml) was isolated from a local hospital of Karachi. Species identification was confirmed by Gram staining, standard biochemical tests and PCR amplification of the nuc gene. The vancomycin MIC was re-confirmed by E-test. For the genetic determination of vancomycin resistance, in-vitro amplification of vanA cassette was performed by using plasmid DNA of CP2, CP2's transformant as template on MWG Thermo-Cycler. Amplified products of vanR, vanS, vanH, vanA, vanY, orf2, orf1D, orf2E, orf-Rev and IS element genes were subjected to Sanger's electrophoresis based sequence determination using specific primers. The Basic Local Alignment Search Tool (BLAST) algorithm was used to identify sequences in GenBank with similarities to the vanA cassette genes. Results: The vancomycin-resistant isolate CP2 was found to be resistant to oxacillin, chloramphenicol, erythromycin, rifampicin, gentamicin, tetracycline and ciprofloxacin, as well. The isolate CP2 revealed four bands: one of large molecular size approx 56.4 kb and three of small size approx 6.5 kb, approx 6.1 kb and approx 1.5 kb by agarose gel electrophoresis indicating the presence of 3 plasmids. The plasmid DNA of isolate CP2 was analyzed by PCR for the presence of the van cassettes with each of the vanA , vanB and vanC specific primers. It carried vanA cassette, which comprises of vanR, vanS, vanH, vanA, vanY, and orf2. The vanA cassette of isolate CP2 also carried an insertion element (IS). However, it did not show the PCR product for orf1. Vancomycin resistance was successfully transferred from the donor CP2 to a vancomycin-sensitive recipient S

Isolation and characterization of the pesticide-degrading plasmid pJP1 from Alcaligenes paradoxus

International Nuclear Information System (INIS)

Fisher, P.R.; Appleton, J.; Pemberton, J.M.

1978-01-01

A strain of Alcaligenes paradoxus, unable to degrade phenoxyacetic acid, was shown to degrade two synthetic derivatives of this molecule, the herbicides 2,4-dichlorophenoxyacetic acid and 2-methyl-4-chlorophenoxyacetic acid. The ability to degrade these pesticides is encoded by a 58-megadalton conjugal plasmid, pJP1

Complete nucleotide sequence of pGA45, a 140,698-bp incFIIY plasmid encoding blaIMI-3-mediated carbapenem resistance, from river sediment

Directory of Open Access Journals (Sweden)

Bingjun eDang

2016-02-01

Full Text Available Plasmid pGA45 was isolated from the sediment of Haihe River using E. coli CV601 (gfp-tagged as recipients and indigenous bacteria from sediment as donors. This plasmid confers reduced susceptibility to imipenem which belongs to carbapenem group. Plasmid pGA45 was fully sequenced on an Illumina HiSeq 2000 sequencing system. The complete sequence of plasmid pGA45 was 140,698 bp in length with an average G+C content of 52.03%. Sequence analysis shows that pGA45 belongs to incFIIY group and harbors a backbone region shares high homology and gene synteny to several other incF plasmids including pNDM1_EC14653, pYDC644, pNDM-Ec1GN574, pRJF866, pKOX_NDM1 and pP10164-NDM. In addition to the backbone region, plasmid pGA45 harbors two notable features including one blaIMI-3-containing region and one type VI secretion system region. The blaIMI-3-containing region is responsible for bacteria carbapenem resistance and the type VI secretion system region is probably involved in bacteria virulence, respectively. Plasmid pGA45 represents the first complete nucleotide sequence of the blaIMI-harboring plasmid from environment sample and the sequencing of this plasmid provided insight into the architecture used for the dissemination of blaIMI carbapenemase genes.

Isolation of temperature-sensitive mutations in murC of Staphylococcus aureus.

Science.gov (United States)

Ishibashi, Mihoko; Kurokawa, Kenji; Nishida, Satoshi; Ueno, Kohji; Matsuo, Miki; Sekimizu, Kazuhisa

2007-09-01

Enzymes in the bacterial peptidoglycan biosynthesis pathway are important targets for novel antibiotics. Of 750 temperature-sensitive (TS) mutants of Gram-positive Staphylococcus aureus, six were complemented by the murC gene, which encodes the UDP-N-acetylmuramic acid:l-alanine ligase. Each mutation resulted in a single amino acid substitution and, in all cases, the TS phenotype was suppressed by high osmotic stress. In mutant strains with the G222E substitution, a decrease in the viable cell number immediately after shift to the restrictive temperature was observed. These results suggest that S. aureus MurC protein is essential for cell growth. The MurC H343Y mutation is located in the putative alanine recognition pocket. Consistent with this, allele-specific suppression was observed of the H343Y mutation by multiple copies of the aapA gene, which encodes an alanine transporter. The results suggest an in vivo role for the H343 residue of S. aureus MurC protein in high-affinity binding to L-alanine.

Molecular Characterization of Endocarditis-Associated Staphylococcus aureus

Science.gov (United States)

Nethercott, Cara; Mabbett, Amanda N.; Totsika, Makrina; Peters, Paul; Ortiz, Juan C.; Nimmo, Graeme R.; Coombs, Geoffrey W.

2013-01-01

Infective endocarditis (IE) is a life-threatening infection of the heart endothelium and valves. Staphylococcus aureus is a predominant cause of severe IE and is frequently associated with infections in health care settings and device-related infections. Multilocus sequence typing (MLST), spa typing, and virulence gene microarrays are frequently used to classify S. aureus clinical isolates. This study examined the utility of these typing tools to investigate S. aureus epidemiology associated with IE. Ninety-seven S. aureus isolates were collected from patients diagnosed with (i) IE, (ii) bloodstream infection related to medical devices, (iii) bloodstream infection not related to medical devices, and (iv) skin or soft-tissue infections. The MLST clonal complex (CC) for each isolate was determined and compared to the CCs of members of the S. aureus population by eBURST analysis. The spa type of all isolates was also determined. A null model was used to determine correlations of IE with CC and spa type. DNA microarray analysis was performed, and a permutational analysis of multivariate variance (PERMANOVA) and principal coordinates analysis were conducted to identify genotypic differences between IE and non-IE strains. CC12, CC20, and spa type t160 were significantly associated with IE S. aureus. A subset of virulence-associated genes and alleles, including genes encoding staphylococcal superantigen-like proteins, fibrinogen-binding protein, and a leukocidin subunit, also significantly correlated with IE isolates. MLST, spa typing, and microarray analysis are promising tools for monitoring S. aureus epidemiology associated with IE. Further research to determine a role for the S. aureus IE-associated virulence genes identified in this study is warranted. PMID:23616460

Molecular characterization of endocarditis-associated Staphylococcus aureus.

Science.gov (United States)

Nethercott, Cara; Mabbett, Amanda N; Totsika, Makrina; Peters, Paul; Ortiz, Juan C; Nimmo, Graeme R; Coombs, Geoffrey W; Walker, Mark J; Schembri, Mark A

2013-07-01

Infective endocarditis (IE) is a life-threatening infection of the heart endothelium and valves. Staphylococcus aureus is a predominant cause of severe IE and is frequently associated with infections in health care settings and device-related infections. Multilocus sequence typing (MLST), spa typing, and virulence gene microarrays are frequently used to classify S. aureus clinical isolates. This study examined the utility of these typing tools to investigate S. aureus epidemiology associated with IE. Ninety-seven S. aureus isolates were collected from patients diagnosed with (i) IE, (ii) bloodstream infection related to medical devices, (iii) bloodstream infection not related to medical devices, and (iv) skin or soft-tissue infections. The MLST clonal complex (CC) for each isolate was determined and compared to the CCs of members of the S. aureus population by eBURST analysis. The spa type of all isolates was also determined. A null model was used to determine correlations of IE with CC and spa type. DNA microarray analysis was performed, and a permutational analysis of multivariate variance (PERMANOVA) and principal coordinates analysis were conducted to identify genotypic differences between IE and non-IE strains. CC12, CC20, and spa type t160 were significantly associated with IE S. aureus. A subset of virulence-associated genes and alleles, including genes encoding staphylococcal superantigen-like proteins, fibrinogen-binding protein, and a leukocidin subunit, also significantly correlated with IE isolates. MLST, spa typing, and microarray analysis are promising tools for monitoring S. aureus epidemiology associated with IE. Further research to determine a role for the S. aureus IE-associated virulence genes identified in this study is warranted.

Co-expression of an Erwinia chrysanthemi pectate lyase-encoding gene (pelE) and an E. carotovora polygalacturonase-encoding gene (peh1) in Saccharomyces cerevisiae.

Science.gov (United States)

Laing, E; Pretorius, I S

1993-05-01

A pectate lyase (PL)-encoding gene (pelE) from Erwinia chrysanthemi and a polygalacturonase (PG)-encoding gene (peh1) from E. carotovora were each inserted between a novel yeast expression-secretion cassette and a yeast gene terminator, and cloned separately into a yeast-centromeric shuttle vector (YCp50), generating recombinant plasmids pAMS12 and pAMS13. Transcription initiation signals present in the expression-secretion cassette were derived from the yeast alcohol dehydrogenase gene promoter (ADC1P), whereas the transcription termination signals were derived from the yeast tryptophan synthase gene terminator (TRP5T). Secretion of PL and PG was directed by the signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1s). A pectinase cassette comprising ADC1P-MF alpha 1s-pelE-TRP5T and ADC1P-MF alpha 1s-peh1-TRP5T was subcloned into YCp50, generating plasmid pAMS14. Subsequently, the dominant selectable Geneticin G418-resistance (GtR) marker, APH1, inserted between the yeast uridine diphosphoglucose 4-epimerase gene promoter (GAL10P) and yeast orotidine-5'-phosphate carboxylase gene terminator (URA3T), was cloned into pAMS14, resulting in plasmid pAMS15. Plasmids pAMS12, pAMS13 and pAMS14 were transformed into a laboratory strain of Saccharomyces cerevisiae, whereas pAMS15 was stably introduced into two commercial wine yeast strains. DNA-DNA and DNA-RNA hybridization analyses revealed the presence of these plasmids, and the pelE and peh1 transcripts in the yeast transformants, respectively. A polypectate agarose assay indicated the extracellular production of biologically active PL and PG by the S. cerevisiae transformants and confirmed that co-expression of the pelE and peh1 genes synergistically enhanced pectate degradation.

Nucleotide Sequence and Characterization of the Broad-Host-Range Lactococcal Plasmid pWVO1

NARCIS (Netherlands)

Leenhouts, Cornelis; Tolner, Berend; Bron, Sierd; Kok, Jan; Venema, Gerhardus; Seegers, Jozef

The nucleotide sequence of the Lactococcus lactis broad-host-range plasmid pWVO1, replicating in both gram-positive and gram-negative bacteria, was determined. This analysis revealed four open reading frames (ORFs). ORF A appeared to encode a trans-acting 26.8-kDa protein (RepA), necessary for

Metabolic activity, urease production, antibiotic resistance and virulence in dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus

Science.gov (United States)

Vandecandelaere, Ilse; Van Nieuwerburgh, Filip; Deforce, Dieter

2017-01-01

In this paper, the metabolic activity in single and dual species biofilms of Staphylococcus epidermidis and Staphylococcus aureus isolates was investigated. Our results demonstrated that there was less metabolic activity in dual species biofilms compared to S. aureus biofilms. However, this was not observed if S. aureus and S. epidermidis were obtained from the same sample. The largest effect on metabolic activity was observed in biofilms of S. aureus Mu50 and S. epidermidis ET-024. A transcriptomic analysis of these dual species biofilms showed that urease genes and genes encoding proteins involved in metabolism were downregulated in comparison to monospecies biofilms. These results were subsequently confirmed by phenotypic assays. As metabolic activity is related to acid production, the pH in dual species biofilms was slightly higher compared to S. aureus Mu50 biofilms. Our results showed that S. epidermidis ET-024 in dual species biofilms inhibits metabolic activity of S. aureus Mu50, leading to less acid production. As a consequence, less urease activity is required to compensate for low pH. Importantly, this effect was biofilm-specific. Also S. aureus Mu50 genes encoding virulence-associated proteins (Spa, SplF and Dps) were upregulated in dual species biofilms compared to monospecies biofilms and using Caenorhabditis elegans infection assays, we demonstrated that more nematodes survived when co-infected with S. epidermidis ET-024 and S. aureus mutants lacking functional spa, splF or dps genes, compared to nematodes infected with S. epidermidis ET-024 and wild- type S. aureus. Finally, S. epidermidis ET-024 genes encoding resistance to oxacillin, erythromycin and tobramycin were upregulated in dual species biofilms and increased resistance was subsequently confirmed. Our data indicate that both species in dual species biofilms of S. epidermidis and S. aureus influence each other’s behavior, but additional studies are required necessary to elucidate the exact

Adding to Yersinia enterocolitica Gene Pool Diversity: Two Cryptic Plasmids from a Biotype 1A Isolate

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Daniela Lepka

2009-01-01

Full Text Available We report the nucleotide sequence of two novel cryptic plasmids (4357 and 14 662 base pairs carried by a Yersinia enterocolitica biotype 1A strain isolated from pork. As distinguished from most biotype 1A strains, this isolate, designated 07-04449, exhibited adherence to eukaryotic cells. The smaller plasmid pYe4449-1 carries five attributable open reading frames (ORFs encoding the first CcdA/CcdB-like antitoxin/toxin system described for a Yersinia plasmid, a RepA-like replication initiation protein, and mobilizing factors MobA and MobC. The deduced amino acid sequences showed highest similarity to proteins described in Salmonella (CcdA/B, Klebsiella (RepA, and Plesiomonas (MobA/C indicating genomic fluidity among members of the Enterobacteriaceae. One additional ORF with unknown function, termed ORF5, was identified with an ancestry distinct from the rest of the plasmid. While the C+G content of ORF5 is 38.3%, the rest of pYe4449-1 shows a C+G content of 55.7%. The C+G content of the larger plasmid pYe4449-2 (54.9% was similar to that of pYe4449-1 (53.7% and differed from that of the Y. enterocolitica genome (47.3%. Of the 14 ORFs identified on pYe4449-2, only six ORFs showed significant similarity to database entries. For three of these ORFs likely functions could be ascribed: a TnpR-like resolvase and a phage replication protein, localized each on a low C+G island, and DNA primase TraC. Two ORFs of pYe4449-2, ORF3 and ORF7, seem to encode secretable proteins. Epitope-tagging of ORF3 revealed protein expression at 4°C but not at or above 27°C suggesting adaptation to a habitat outside swine. The hypothetical protein encoded by ORF7 is the member of a novel repeat protein family sharing the DxxGN(xnDxxGN motif. Our findings illustrate the exceptional gene pool diversity within the species Y. enterocolitica driven by horizontal gene transfer events.

A study of Staphylococcus aureus nasal carriage, antibacterial ...

African Journals Online (AJOL)

Aim: This study was to determine the virulence encoding genes, and the antibiotic resistance patterns of the Staphylococcus aureus isolates, which were isolated from the nasal samples of chest clinic patients. Materials and Methods: The nasal samples of the in‑patients (431) and out‑patients (1857) in Kayseri Training and ...

Two-step method for curing Escherichia coli of ColE1-derived plasmids

DEFF Research Database (Denmark)

Hove-Jensen, Bjarne

2008-01-01

To cure Escherichia coli for plasmids derived from the ColE1 replicon advantage is taken of the fact that maintenance of this replicon requires a wild-type allele of polA, encoding DNA polymerase I. Curing is achieved by cotransduction of a mutant polA allele with metE::Tn10, fadAB::Tn10 or other...

Nucleotide sequence of pOLA52: a conjugative IncX1 plasmid from Escherichia coli which enables biofilm formation and multidrug efflux

DEFF Research Database (Denmark)

Norman, Anders; Hansen, Lars H.; She, Qunxin

2008-01-01

. The plasmid was also classified as IncX1 with incompatibility testing. The conjugal transfer and plasmid maintenance regions of pOLA52 therefore seem to represent IncX1 orthologues of the well-characterized IncX2 plasmid R6K. Sequence homology searches in GenBank also suggested a considerably higher...... of type 3 fimbriae (mrkABCDF). The plasmid was found to be 51,602 bp long with 68 putative genes. About half of the plasmid constituted a conserved IncX1-type backbone with predicted regions for conjugation, replication and partitioning, as well as a toxin/antitoxin (TA) plasmid addiction system...... prevalence of IncX1 group plasmids than IncX2. The 21 kb 'genetic load' region of pOLA52 was shown to consist of a mosaic, among other things a fragmented Tn3 transposon encoding ampicillin resistance. Most notably the oqxAB and mrkABCDF cassettes were contained within two composite transposons (Tn6010...

Construction of an infectious clone of human adenovirus type 41.

Science.gov (United States)

Chen, Duo-Ling; Dong, Liu-Xin; Li, Meng; Guo, Xiao-Juan; Wang, Min; Liu, Xin-Feng; Lu, Zhuo-Zhuang; Hung, Tao

2012-07-01

Human adenovirus type 41 (HAdV-41) is well known for its fastidiousness in cell culture. To construct an infectious clone of HAdV-41, a DNA fragment containing the left and right ends of HAdV-41 as well as a kanamycin resistance gene and a pBR322 replication origin was excised from the previously constructed plasmid pAd41-GFP. Using homologous recombination, the plasmid pKAd41 was generated by co-transformation of the E. coli BJ5183 strain with this fragment and HAdV-41 genomic DNA. Virus was rescued from pKAd41-transfected 293TE7 cells, a HAdV-41 E1B55K-expressing cell line. The genomic integrity of the rescued virus was verified by restriction analysis and sequencing. Two fibers on the virion were confirmed by western blot. Immunofluorescence showed that more expression of the hexon protein could be found in 293TE7 cells than in 293 cells after HAdV-41 infection. The feature of non-lytic replication was preserved in 293TE7 cells, since very few progeny HAdV-41 viruses were released to the culture medium. These results show that pKAd41 is an effective infectious clone and suggest that the combination of pKAd41 and 293TE7 cells is an ideal system for virological study of HAdV-41.

Survey and properties of Staphylococcus aureus isolated from Japanese-style desserts.

Science.gov (United States)

Shimamura, Yuko; Kidokoro, Shiho; Murata, Masatsune

2006-07-01

We surveyed the contamination of 315 Japanese- and western-style desserts and 247 human hands by Staphylococcus aureus and other staphylococcal bacteria. The most frequently isolated staphylococcal bacterium was S. warneri, followed by S. aureus. Only 1.9% of western-style desserts were contaminated by S. aureus strains, while 19.4% and 13.0% of Japanese-style desserts and human hands respectively were contaminated. Ninety-four isolates of S. aureus were characterized as to their biological properties and enterotoxigenicity. Although staphylococcal enterotoxins (SEs) were detected by enzyme-linked immunosorbent assay in the cultured broth of all S. aureus isolates, the reversed passive latex agglutination method and the polymerase chain reaction showed only 39 (41.5%) and 40 (42.6%) samples respectively as SE-positive. The predominant type of SE was SEB (67.5%), and eight strains produced SEA. None of the S. aureus strains had penicillin-binding protein 2', showing that methicillin-resistant S. aureus was not present in the samples.

Transfection of genetically encoded photoswitchable probes for STORM imaging.

Science.gov (United States)

Bates, Mark; Jones, Sara A; Zhuang, Xiaowei

2013-06-01

Conventional fluorescence microscopy is limited by its spatial resolution, leaving many biological structures too small to be studied in detail. Stochastic optical reconstruction microscopy (STORM) is a method for superresolution fluorescence imaging based on the high accuracy localization of individual fluorophores. It uses optically switchable fluorophores: molecules that can be switched between a nonfluorescent and a fluorescent state by exposure to light. This protocol describes the transfection of genetically encoded photoswitchable probes for STORM imaging. It includes a discussion of how to choose a photoswitchable fluorescent protein; standard molecular biology techniques should be used to generate a plasmid containing the sequence of the photoswitchable protein linked to the gene of interest. Once the plasmid has been generated and has been verified, it can be introduced into cells via any standard means of gene delivery, such as lipofection or electroporation. Optimal conditions will vary considerably for different cell lines and plasmids. Here, we present an example protocol for the transfection of BS-C-1 cells with an mEos2-vimentin plasmid using the lipid-based reagent FuGENE6.

A novel plasmid-encoded serotype conversion mechanism through addition of phosphoethanolamine to the O-antigen of Shigella flexneri.

Directory of Open Access Journals (Sweden)

Qiangzheng Sun

Full Text Available Shigella flexneri is the major pathogen causing bacillary dysentery in developing countries. S. flexneri is divided into at least 16 serotypes based on the combination of antigenic determinants present in the O-antigen. All the serotypes (except for serotype 6 share a basic O-unit containing one N-acetyl-d-glucosamine and three l-rhamnose residues, whereas differences between the serotypes are conferred by phage-encoded glucosylation and/or O-acetylation. Serotype Xv is a newly emerged and the most prevalent serotype in China, which can agglutinate with both MASF IV-1 and 7,8 monoclonal antibodies. The factor responsible for the presence of MASF IV-1 (E1037 epitope has not yet been identified. In this study, we analyzed the LPS structure of serotype Xv strains and found that the MASF IV-1 positive phenotype depends on an O-antigen modification with a phosphoethanolamine (PEtN group attached at position 3 of one of the rhamnose residues. A plasmid carried gene, lpt-O (LPS phosphoethanolamine transferase for O-antigen, mediates the addition of PEtN for serotype Xv and other MASF IV-1 positive strains. These findings reveal a novel serotype conversion mechanism in S. flexneri and show the necessity of further extension of the serotype classification scheme recognizing the MASF IV-1 positive strains as distinctive subtypes.

Inhibition of Virulence Gene Expression in Staphylococcus aureus by Novel Depsipeptides from a Marine Photobacterium

Directory of Open Access Journals (Sweden)

Lone Gram

2011-12-01

Full Text Available During a global research expedition, more than five hundred marine bacterial strains capable of inhibiting the growth of pathogenic bacteria were collected. The purpose of the present study was to determine if these marine bacteria are also a source of compounds that interfere with the agr quorum sensing system that controls virulence gene expression in Staphylococcus aureus. Using a gene reporter fusion bioassay, we recorded agr interference as enhanced expression of spa, encoding Protein A, concomitantly with reduced expression of hla, encoding α-hemolysin, and rnaIII encoding RNAIII, the effector molecule of agr. A marine Photobacterium produced compounds interfering with agr in S. aureus strain 8325-4, and bioassay-guided fractionation of crude extracts led to the isolation of two novel cyclodepsipeptides, designated solonamide A and B. Northern blot analysis confirmed the agr interfering activity of pure solonamides in both S. aureus strain 8325-4 and the highly virulent, community-acquired strain USA300 (CA-MRSA. To our knowledge, this is the first report of inhibitors of the agr system by a marine bacterium.

CAPRRESI: Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion.

Science.gov (United States)

Santillán, Orlando; Ramírez-Romero, Miguel A; Dávila, Guillermo

2017-06-25

Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the original plasmid recovery method and introduces restriction enzyme digestion to ease DNA ligation reactions (required for chimera assembly). For this protocol, users clone wildtype genes into the same plasmid (pUC18 or pUC19). After the in silico selection of amino acid sequence regions where chimeras should be assembled, users obtain all the synonym DNA sequences that encode them. Ad hoc Perl scripts enable users to determine all synonym DNA sequences. After this step, another Perl script searches for restriction enzyme sites on all synonym DNA sequences. This in silico analysis is also performed using the ampicillin resistance gene (ampR) found on pUC18/19 plasmids. Users design oligonucleotides inside synonym regions to disrupt wildtype and ampR genes by PCR. After obtaining and purifying complementary DNA fragments, restriction enzyme digestion is accomplished. Chimera assembly is achieved by ligating appropriate complementary DNA fragments. pUC18/19 vectors are selected for CAPRRESI because they offer technical advantages, such as small size (2,686 base pairs), high copy number, advantageous sequencing reaction features, and commercial availability. The usage of restriction enzymes for chimera assembly eliminates the need for DNA polymerases yielding blunt-ended products. CAPRRESI is a fast and low-cost method for fusing protein-coding genes.

Cloning of a Recombinant Plasmid Encoding Thiol-Specific Antioxidant Antigen (TSA) Gene of Leishmania majorand Expression in the Chinese Hamster Ovary Cell Line.

Science.gov (United States)

Fatemeh, Ghaffarifar; Fatemeh, Tabatabaie; Zohreh, Sharifi; Abdolhosein, Dalimiasl; Mohammad Zahir, Hassan; Mehdi, Mahdavi

2012-01-01

TSA (thiol-specific antioxidant antigen) is the immune-dominant antigen of Leishmania major and is considered to be the most promising candidate molecule for a recombinant or DNA vaccine against leishmaniasis. The aim of the present work was to express a plasmid containing the TSA gene in eukaryotic cells. Genomic DNA was extracted, and the TSA gene was amplified by polymerase chain reaction (PCR). The PCR product was cloned into the pTZ57R/T vector, followed by subcloning into the eukaryotic expression vector pcDNA3 (EcoRI and HindIII sites). The recombinant plasmid was characterised by restriction digest and PCR. Eukaryotic Chinese hamster ovary cells were transfected with the plasmid containing the TSA gene. Expression of the L. major TSA gene was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting. The plasmid containing the TSA gene was successfully expressed, as demonstrated by a band of 22.1 kDa on Western blots. The plasmid containing the TSA gene can be expressed in a eukaryotic cell line. Thus, the recombinant plasmid may potentially be used as a DNA vaccine in animal models.

RepA and RepB exert plasmid incompatibility repressing the transcription of the repABC operon.

Science.gov (United States)

Pérez-Oseguera, Angeles; Cevallos, Miguel A

2013-11-01

Rhizobium etli CFN42 has a multipartite genome composed of one chromosome and six large plasmids with low copy numbers, all belonging to the repABC plasmid family. All elements essential for replication and segregation of these plasmids are encoded within the repABC operon. RepA and RepB direct plasmid segregation and are involved in the transcriptional regulation of the operon, and RepC is the initiator protein of the plasmid. Here we show that in addition to RepA (repressor) and RepB (corepressor), full transcriptional repression of the operon located in the symbiotic plasmid (pRetCFN42d) of this strain requires parS, the centromere-like sequence, and the operator sequence. However, the co-expression of RepA and RepB is sufficient to induce the displacement of the parental plasmid. RepA is a Walker-type ATPase that self associates in vivo and in vitro and binds specifically to the operator region in its RepA-ADP form. In contrast, RepA-ATP is capable of binding to non-specific DNA. RepA and RepB form high molecular weight DNA-protein complexes in the presence of ATP and ADP. RepA carrying ATP-pocket motif mutations induce full repression of the repABC operon without the participation of RepB and parS. These mutants specifically bind the operator sequence in their ATP or ADP bound forms. In addition, their expression in trans exerts plasmid incompatibility against the parental plasmid. RepA and RepB expressed in trans induce plasmid incompatibility because of their ability to repress the repABC operon and not only by their capacity to distort the plasmid segregation process. Copyright © 2013 Elsevier Inc. All rights reserved.

Comparative symbiotic plasmid analysis indicates that symbiosis gene ancestor type affects plasmid genetic evolution.

Science.gov (United States)

Wang, X; Zhao, L; Zhang, L; Wu, Y; Chou, M; Wei, G

2018-07-01

Rhizobial symbiotic plasmids play vital roles in mutualistic symbiosis with legume plants by executing the functions of nodulation and nitrogen fixation. To explore the gene composition and genetic constitution of rhizobial symbiotic plasmids, comparison analyses of 24 rhizobial symbiotic plasmids derived from four rhizobial genera was carried out. Results illustrated that rhizobial symbiotic plasmids had higher proportion of functional genes participating in amino acid transport and metabolism, replication; recombination and repair; carbohydrate transport and metabolism; energy production and conversion and transcription. Mesorhizobium amorphae CCNWGS0123 symbiotic plasmid - pM0123d had similar gene composition with pR899b and pSNGR234a. All symbiotic plasmids shared 13 orthologous genes, including five nod and eight nif/fix genes which participate in the rhizobia-legume symbiosis process. These plasmids contained nod genes from four ancestors and fix genes from six ancestors. The ancestral type of pM0123d nod genes was similar with that of Rhizobium etli plasmids, while the ancestral type of pM0123d fix genes was same as that of pM7653Rb. The phylogenetic trees constructed based on nodCIJ and fixABC displayed different topological structures mainly due to nodCIJ and fixABC ancestral type discordance. The study presents valuable insights into mosaic structures and the evolution of rhizobial symbiotic plasmids. This study compared 24 rhizobial symbiotic plasmids that included four genera and 11 species, illuminating the functional gene composition and symbiosis gene ancestor types of symbiotic plasmids from higher taxonomy. It provides valuable insights into mosaic structures and the evolution of symbiotic plasmids. © 2018 The Society for Applied Microbiology.

MvaT Family Proteins Encoded on IncP-7 Plasmid pCAR1 and the Host Chromosome Regulate the Host Transcriptome Cooperatively but Differently.

Science.gov (United States)

Yun, Choong-Soo; Takahashi, Yurika; Shintani, Masaki; Takeda, Toshiharu; Suzuki-Minakuchi, Chiho; Okada, Kazunori; Yamane, Hisakazu; Nojiri, Hideaki

2016-02-01

MvaT proteins are members of the H-NS family of proteins in pseudomonads. The IncP-7 conjugative plasmid pCAR1 carries an mvaT-homologous gene, pmr. In Pseudomonas putida KT2440 bearing pCAR1, pmr and the chromosomally carried homologous genes, turA and turB, are transcribed at high levels, and Pmr interacts with TurA and TurB in vitro. In the present study, we clarified how the three MvaT proteins regulate the transcriptome of P. putida KT2440(pCAR1). Analyses performed by a modified chromatin immunoprecipitation assay with microarray technology (ChIP-chip) suggested that the binding regions of Pmr, TurA, and TurB in the P. putida KT2440(pCAR1) genome are almost identical; nevertheless, transcriptomic analyses using mutants with deletions of the genes encoding the MvaT proteins during the log and early stationary growth phases clearly suggested that their regulons were different. Indeed, significant regulon dissimilarity was found between Pmr and the other two proteins. Transcription of a larger number of genes was affected by Pmr deletion during early stationary phase than during log phase, suggesting that Pmr ameliorates the effects of pCAR1 on host fitness more effectively during the early stationary phase. Alternatively, the similarity of the TurA and TurB regulons implied that they might play complementary roles as global transcriptional regulators in response to plasmid carriage. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET), a new method for plasmid reconstruction from whole genome sequences.

Science.gov (United States)

Lanza, Val F; de Toro, María; Garcillán-Barcia, M Pilar; Mora, Azucena; Blanco, Jorge; Coque, Teresa M; de la Cruz, Fernando

2014-12-01

Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ-proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages.

Identification of a novel vga(E) gene variant that confers resistance to pleuromutilins, lincosamides and streptogramin A antibiotics in staphylococci of porcine origin.

Science.gov (United States)

Li, Jun; Li, Beibei; Wendlandt, Sarah; Schwarz, Stefan; Wang, Yang; Wu, Congming; Ma, Zhiyong; Shen, Jianzhong

2014-04-01

To investigate the genetic basis of pleuromutilin resistance in coagulase-negative staphylococci of porcine origin that do not carry known pleuromutilin resistance genes and to determine the localization and genetic environment of the identified resistance gene. Plasmid DNA of two pleuromutilin-resistant Staphylococcus cohnii and Staphylococcus simulans isolates was transformed into Staphylococcus aureus RN4220. The identified resistance plasmids were sequenced completely. The candidate gene for pleuromutilin resistance was cloned into shuttle vector pAM401. S. aureus RN4220 transformants carrying this recombinant shuttle vector were tested for their MICs. S. cohnii isolate SA-7 and S. simulans isolate SSI1 carried the same plasmid of 5584 bp, designated pSA-7. A variant of the vga(E) gene was detected, which encodes a 524 amino acid ATP-binding cassette protein. The variant gene shared 85.7% nucleotide sequence identity and the variant protein 85.3% amino acid sequence identity with the original vga(E) gene and Vga(E) protein, respectively. The Vga(E) variant conferred cross-resistance to pleuromutilins, lincosamides and streptogramin A antibiotics. Plasmid pSA-7 showed an organization similar to that of the apmA-carrying plasmid pKKS49 from methicillin-resistant S. aureus and the dfrK-carrying plasmid pKKS966 from Staphylococcus hyicus. Sequence comparisons suggested that recombination events may have played a role in the acquisition of this vga(E) variant. A novel vga(E) gene variant was identified, which was located on a small plasmid and was not associated with the transposon Tn6133 [in contrast to the original vga(E) gene]. The plasmid location may enable its further dissemination to other staphylococci and possibly also to other bacteria.

Virulence properties of methicillin-susceptible Staphylococcus aureus food isolates encoding Panton-Valentine Leukocidin gene.

Science.gov (United States)

Sudagidan, Mert; Aydin, Ali

2010-04-15

In this study, three Panton-Valentine Leukocidin gene carrying methicillin-susceptible Staphylococcus aureus (MSSA) strains (M1-AAG42B, PY30C-b and YF1B-b) were isolated from different food samples in Kesan-Edirne, Turkey. These strains were characterized on the basis of MLST type, spa type, virulence factor gene contents, antibiotic susceptibilities against 21 antibiotics and biofilm formation. The genetic relatedness of the strains was determined by PFGE. In addition, the complete gene sequences of lukS-PV and lukF-PV were also investigated. All strains were found to be susceptible to tested antibiotics and they were mecA negative. Three strains showed the same PFGE band pattern, ST152 clonal type and t355 spa type. In the detection of virulence factor genes, sea, seb, sec, sed, see, seg, seh, sei, sej, sek, sel, sem, sen, seo, sep, seq, seu, eta, etb, set1, geh and tst genes were not detected. All strains showed the positive results for alpha- and beta-haemolysin genes (hla and hlb), protease encoding genes (sspA, sspB and aur), lukE and lukD leukocidin genes (lukED). The strains were found to be non-biofilm formers. By this study, the virulence properties of the strains were described and this is one of the first reports regarding PVL-positive MSSA strains from food. (c) 2010 Elsevier B.V. All rights reserved.

Functional properties and structural requirements of the plasmid pMV158-encoded MobM relaxase domain.

Science.gov (United States)

Fernández-López, Cris; Pluta, Radoslaw; Pérez-Luque, Rosa; Rodríguez-González, Lorena; Espinosa, Manuel; Coll, Miquel; Lorenzo-Díaz, Fabián; Boer, D Roeland

2013-07-01

A crucial element in the horizontal transfer of mobilizable and conjugative plasmids is the relaxase, a single-stranded endonuclease that nicks the origin of transfer (oriT) of the plasmid DNA. The relaxase of the pMV158 mobilizable plasmid is MobM (494 residues). In solution, MobM forms a dimer through its C-terminal domain, which is proposed to anchor the protein to the cell membrane and to participate in type 4 secretion system (T4SS) protein-protein interactions. In order to gain a deeper insight into the structural MobM requirements for efficient DNA catalysis, we studied two endonuclease domain variants that include the first 199 or 243 amino acid residues (MobMN199 and MobMN243, respectively). Our results confirmed that the two proteins behaved as monomers in solution. Interestingly, MobMN243 relaxed supercoiled DNA and cleaved single-stranded oligonucleotides harboring oriTpMV158, whereas MobMN199 was active only on supercoiled DNA. Protein stability studies using gel electrophoresis and mass spectrometry showed increased susceptibility to degradation at the domain boundary between the N- and C-terminal domains, suggesting that the domains change their relative orientation upon DNA binding. Overall, these results demonstrate that MobMN243 is capable of nicking the DNA substrate independently of its topology and that the amino acids 200 to 243 modulate substrate specificity but not the nicking activity per se. These findings suggest that these amino acids are involved in positioning the DNA for the nuclease reaction rather than in the nicking mechanism itself.

Whole-genome comparison of meticillin-resistant Staphylococcus aureus CC22 SCCmecIV from people and their in-contact pets.

Science.gov (United States)

Loeffler, Anette; McCarthy, Alex; Lloyd, David H; Musilová, Eva; Pfeiffer, Dirk U; Lindsay, Jodi A

2013-10-01

Meticillin-resistant Staphylococcus aureus (MRSA) infections remain important medical and veterinary challenges. The MRSA isolated from dogs and cats typically belong to dominant hospital-associated clones, in the UK mostly EMRSA-15 (CC22 SCCmecIV), suggesting original human-to-animal transmission. Nevertheless, little is known about host-specific genetic variation within the same S. aureus lineage. To identify host-specific variation amongst MRSA CC22 SCCmecIV by comparing isolates from pets with those from in-contact humans using whole-genome microarray. Six pairs of MRSA CC22 SCCmecIV from human carriers (owners and veterinary staff) and their respective infected in-contact pets were compared using a 62-strain whole-genome S. aureus microarray (SAM-62). The presence of putative host-specific genes was subsequently determined in a larger number of human (n = 47) and pet isolates (n = 93) by PCR screening. Variation in mobile genetic elements (MGEs) occurred frequently and appeared largely independent of host and in-contact pair. A plasmid (SAP078A) encoding heavy-metal resistance genes (arsR, arsA, cadA, cadC, mco and copB) was found in three of six human and none of six animal isolates. However, only two of four resistance genes were associated with human hosts (P = 0.015 for arsA and cadA). The variation found amongst MGEs highlights that genetic adaptation in MRSA continues. However, host-specific MGEs were not detected, which supports the hypothesis that pets may not be natural hosts of MRSA CC22 and emphasizes that rigorous hygiene measures are critical to prevent contamination and infection of dogs and cats. The host specificity of individual heavy-metal resistance genes warrants further investigation into different selection pressures in humans and animals. © 2013 ESVD and ACVD.

Plasmid Flux in Escherichia coli ST131 Sublineages, Analyzed by Plasmid Constellation Network (PLACNET), a New Method for Plasmid Reconstruction from Whole Genome Sequences

Science.gov (United States)

Garcillán-Barcia, M. Pilar; Mora, Azucena; Blanco, Jorge; Coque, Teresa M.; de la Cruz, Fernando

2014-01-01

Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ–proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages. PMID:25522143

Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET, a new method for plasmid reconstruction from whole genome sequences.

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Val F Lanza

2014-12-01

Full Text Available Bacterial whole genome sequence (WGS methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage, comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC, comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ-proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages.

Direct identification of antibiotic resistance genes on single plasmid molecules using CRISPR/Cas9 in combination with optical DNA mapping

Science.gov (United States)

Müller, Vilhelm; Rajer, Fredrika; Frykholm, Karolin; Nyberg, Lena K.; Quaderi, Saair; Fritzsche, Joachim; Kristiansson, Erik; Ambjörnsson, Tobias; Sandegren, Linus; Westerlund, Fredrik

2016-12-01

Bacterial plasmids are extensively involved in the rapid global spread of antibiotic resistance. We here present an assay, based on optical DNA mapping of single plasmids in nanofluidic channels, which provides detailed information about the plasmids present in a bacterial isolate. In a single experiment, we obtain the number of different plasmids in the sample, the size of each plasmid, an optical barcode that can be used to identify and trace the plasmid of interest and information about which plasmid that carries a specific resistance gene. Gene identification is done using CRISPR/Cas9 loaded with a guide-RNA (gRNA) complementary to the gene of interest that linearizes the circular plasmids at a specific location that is identified using the optical DNA maps. We demonstrate the principle on clinically relevant extended spectrum beta-lactamase (ESBL) producing isolates. We discuss how the gRNA sequence can be varied to obtain the desired information. The gRNA can either be very specific to identify a homogeneous group of genes or general to detect several groups of genes at the same time. Finally, we demonstrate an example where we use a combination of two gRNA sequences to identify carbapenemase-encoding genes in two previously not characterized clinical bacterial samples.

Effect of degradative plasmid CAM-OCT on responses of Pseudomonas bacteria to UV light

International Nuclear Information System (INIS)

McBeth, D.L.

1989-01-01

The effect of plasmid CAM-OCT on responses to UV irradiation was compared in Pseudomonas aeruginosa, in Pseudomonas putida, and in Pseudomonas putida mutants carrying mutations in UV response genes. CAM-OCT substantially increased both survival and mutagenesis in the two species. P. aeruginosa strains without CAM-OCT exhibited much higher UV sensitivity than did P. putida strains. UV-induced mutagenesis of plasmid-free P. putida was easily detected in three different assays (two reversion assays and one forward mutation assay), whereas UV mutagenesis of P. aeruginosa without CAM-OCT was seen only in the forward mutation assay. These results suggest major differences in DNA repair between the two species and highlight the presence of error-prone repair functions on CAM-OCT. A number of P. putida mutants carrying chromosomal mutations affecting either survival or mutagenesis after UV irradiation were isolated, and the effect of CAM-OCT on these mutants was determined. All mutations producing a UV-sensitive phenotype in P. putida were fully suppressed by the plasmid, whereas the plasmid had a more variable effect on mutagenesis mutations, suppressing some and producing no suppression of others. On the basis of the results reported here and results obtained by others with plasmids carrying UV response genes, it appears that CAM-OCT may differ either in regulation or in the number and functions of UV response genes encoded

Cross-Talk between Staphylococcus aureus and Other Staphylococcal Species via the agr Quorum Sensing System

DEFF Research Database (Denmark)

Canovas de la Nuez, Jaime; Baldry, Mara; Bojer, Martin S

2016-01-01

-inducing peptides (AIPs) sensed by AgrC, a two component histidine kinase. agr loci are found also in other staphylococcal species and for Staphylococcus epidermidis, the encoded AIP represses expression of agr regulated virulence genes in S. aureus. In this study we aimed to better understand the interaction...... between staphylococci and S. aureus, and show that this interaction may eventually lead to the identification of new anti-virulence candidates to target S. aureus infections. Here we show that culture supernatants of 37 out of 52 staphylococcal isolates representing 17 different species inhibit S. aureus...... suggesting that agr is an inter-species communication system. Based on these results we speculate that interactions between S. aureus and other colonizing staphylococci will significantly influence the ability of S. aureus to cause infection, and we propose that other staphylococci are potential sources...

Commercial biocides induce transfer of prophage Φ13 from human strains of Staphylococcus aureus to livestock CC398

DEFF Research Database (Denmark)

Tang, Yuanyue; Nielsen, Lene Nørby; Hvitved, Annemette

2017-01-01

Human strains of Staphylococcus aureus commonly carry the bacteriophage ΦSa3 that encodes immune evasion factors. Recently, this prophage has been found in livestock-associated, methicillin resistant S. aureus (MRSA) CC398 strains where it may promote human colonization. Here, we have addressed...

The complete genome sequence of Bacillus velezensis strain GH1-13 reveals agriculturally beneficial properties and a unique plasmid.

Science.gov (United States)

Kim, Sang Yoon; Song, Hajin; Sang, Mee Kyung; Weon, Hang-Yeon; Song, Jaekyeong

2017-10-10

The bacterial strain Bacillus velezensis GH1-13, isolated from rice paddy soil in Korea, has been shown to promote plant growth and have strong antagonistic activities against pathogens. Here, we report the complete genome sequence of GH1-13, revealing that it possesses a single 4,071,980-bp circular chromosome with 46.2% GC-content. The chromosome encodes 3,930 genes, and we have also identified a unique plasmid in the strain that encodes a further 104 genes (71,628bp and 31.7% GC-content). The genome was found to contain various enzyme-encoding operons, including indole-3-acetic acid (IAA) biosynthesis proteins, 2,3-butanediol dehydrogenase, various non-ribosomal peptide synthetases, and several polyketide synthases. These properties are responsible for the promotion of plant growth and the biosynthesis of secondary metabolites. They therefore have multiple beneficial effects that could be applied to agriculture. Through curing, we found that the unique plasmid of GH1-13 has important roles in the production of phytohormones, such as IAA, and in shaping phenotypic and physiological characteristics. The plasmid therefore likely influences the biological activities of GH1-13. The complete genome sequence of B. velezensis GH1-13 contributes to our understanding of this beneficial strain and will encourage research into its development for agricultural or biotechnological applications, enhancing productivity and crop quality. Copyright © 2017 Elsevier B.V. All rights reserved.

The cryptic plasmid is more important for Chlamydia muridarum to colonize the mouse gastrointestinal tract than to infect the genital tract.

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Lili Shao

Full Text Available Chlamydia has been detected in the gastrointestinal tracts of both animals and humans. However, the mechanism by which Chlamydia colonizes the gut remains unclear. Chlamydia muridarum is known to spread from the genital to the gastrointestinal tracts hematogenously. The C. muridarum plasmid is a key pathogenic determinant in the mouse upper genital tract although plasmid-deficient C. muridarum is still able to colonize the upper genital tract. We now report that plasmid-deficient C. muridarum exhibits significantly delayed/reduced spreading from the mouse genital to the gastrointestinal tracts. C. muridarum with or without plasmid maintained similar levels in the mouse circulatory system following intravenous inoculation but the hematogenous plasmid-deficient C. muridarum was significantly less efficient in colonizing the gastrointestinal tract. Consistently, plasmid-deficient C. muridarum failed to restore normal colonization in the gastrointestinal tract even after intragastric inoculation at a high dose. Thus, we have demonstrated a plasmid-dependent colonization of C. muridarum in the gastrointestinal tract, supporting the concept that C. muridarum may have acquired the plasmid for adaptation to the mouse gastrointestinal tract during oral-fecal transmission. Since the plasmid is more important for C. muridarum to colonize the gastrointestinal tract than to infect the genital tract, the current study has laid a foundation for further defining the host pathways targeted by the plasmid-encoded or -regulated chlamydial effectors.

Plasmid pORF-hTRAIL targeting to glioma using transferrin-modified polyamidoamine dendrimer

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Gao S

2015-12-01

Full Text Available Song Gao,1,* Jianfeng Li,2 Chen Jiang,2 Bo Hong,3 Bing Hao4,* 1Department of Clinical Laboratory, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, 2Key Laboratory of Smart Drug Delivery, Ministry of Education, Department of Pharmaceutics, School of Pharmacy, Fudan University, Shanghai, 3Department of Pathology, The Second Affiliated Hospital, 4Key Laboratory of Combined Multi-Organ Transplantation, Ministry of Public Health, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China *These authors contributed equally to this work Abstract: A gene drug delivery system for glioma therapy based on transferrin (Tf-modified polyamidoamine dendrimer (PAMAM was prepared. Gene drug, tumor necrosis factor-related apoptosis-inducing ligand (hTRAIL-encoding plasmid open reading frame (pORF-hTRAIL, Trail, was condensed by Tf-modified PAMAM to form nanoparticles (NPs. PAMAM-PEG-Tf/DNA NPs showed higher cellular uptake, in vitro gene expression, and cytotoxicity than PAMAM-PEG/DNA NPs in C6 cells. The in vivo targeting efficacy of NPs was visualized by ex vivo fluorescence imaging. Tf-modified NPs showed obvious glioma-targeting trend. Plasmid encoding green fluorescence protein (GFP was also condensed by modified or unmodified PAMAM to evaluate the in vivo gene expression level. The PAMAM-PEG-Tf/plasmid encoding enhanced green fluorescence protein (pEGFP NPs exhibited higher GFP expression level than PAMAM-PEG/pEGFP NPs. TUNEL assay revealed that Tf-modified NPs could induce much more tumor apoptosis. The median survival time of PAMAM-PEG-Tf/Trail-treated rats (28.5 days was longer than that of rats treated with PAMAM-PEG/Trail (25.5 days, temozolomide (24.5 days, PAMAM-PEG-Tf/pEGFP (19 days, or saline (17 days. The therapeutic effect was further confirmed by magnetic resonance imaging. This study demonstrated that targeting gene delivery system had potential application for the

Norlichexanthone Reduces Virulence Gene Expression and Biofilm Formation in Staphylococcus aureus

DEFF Research Database (Denmark)

Baldry, Mara; Nielsen, Anita; Bojer, Martin S.

2016-01-01

Staphylococcus aureus is a serious human pathogen and antibiotic resistant, community-associated strains, such as the methicillin resistant S. aureus (MRSA) strain USA300, continue to spread. To avoid resistance, anti-virulence therapy has been proposed where toxicity is targeted rather than...... viability. Previously we have shown that norlichexanthone, a small non-reduced tricyclic polyketide produced by fungi and lichens, reduces expression of hla encoding α-hemolysin as well as the regulatory RNAIII of the agr quorum sensing system in S. aureus 8325-4. The aim of the present study was to further...... SaeRS system. Our data show that norlichexanthone treatment reduces expression of key virulence factors in CA-MRSA strain USA300 via AgrA binding and represses biofilm formation....

Virulence potential of Staphylococcus aureus isolates from Buruli ulcer patients.

Science.gov (United States)

Amissah, Nana Ama; Chlebowicz, Monika A; Ablordey, Anthony; Tetteh, Caitlin S; Prah, Isaac; van der Werf, Tjip S; Friedrich, Alex W; van Dijl, Jan Maarten; Stienstra, Ymkje; Rossen, John W

2017-06-01

Buruli ulcer (BU) is a necrotizing infection of the skin and subcutaneous tissue caused by Mycobacterium ulcerans. BU wounds may also be colonized with other microorganisms including Staphylococcus aureus. This study aimed to characterize the virulence factors of S. aureus isolated from BU patients. Previously sequenced genomes of 21 S. aureus isolates from BU patients were screened for the presence of virulence genes. The results show that all S. aureus isolates harbored on their core genomes genes for known virulence factors like α-hemolysin, and the α- and β-phenol soluble modulins. Besides the core genome virulence genes, mobile genetic elements (MGEs), i.e. prophages, genomic islands, pathogenicity islands and a Staphylococcal cassette chromosome (SCC) were found to carry different combinations of virulence factors, among them genes that are known to encode factors that promote immune evasion, superantigens and Panton-Valentine Leucocidin. The present observations imply that the S. aureus isolates from BU patients harbor a diverse repertoire of virulence genes that may enhance bacterial survival and persistence in the wound environment and potentially contribute to delayed wound healing. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

The sudden dominance of blaCTX-M harbouring plasmids in Shigella spp. Circulating in Southern Vietnam.

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Nhu Thi Khanh Nguyen

2010-06-01

Full Text Available Plasmid mediated antimicrobial resistance in the Enterobacteriaceae is a global problem. The rise of CTX-M class extended spectrum beta lactamases (ESBLs has been well documented in industrialized countries. Vietnam is representative of a typical transitional middle income country where the spectrum of infectious diseases combined with the spread of drug resistance is shifting and bringing new healthcare challenges.We collected hospital admission data from the pediatric population attending the hospital for tropical diseases in Ho Chi Minh City with Shigella infections. Organisms were cultured from all enrolled patients and subjected to antimicrobial susceptibility testing. Those that were ESBL positive were subjected to further investigation. These investigations included PCR amplification for common ESBL genes, plasmid investigation, conjugation, microarray hybridization and DNA sequencing of a bla(CTX-M encoding plasmid.We show that two different bla(CTX-M genes are circulating in this bacterial population in this location. Sequence of one of the ESBL plasmids shows that rather than the gene being integrated into a preexisting MDR plasmid, the bla(CTX-M gene is located on relatively simple conjugative plasmid. The sequenced plasmid (pEG356 carried the bla(CTX-M-24 gene on an ISEcp1 element and demonstrated considerable sequence homology with other IncFI plasmids.The rapid dissemination, spread of antimicrobial resistance and changing population of Shigella spp. concurrent with economic growth are pertinent to many other countries undergoing similar development. Third generation cephalosporins are commonly used empiric antibiotics in Ho Chi Minh City. We recommend that these agents should not be considered for therapy of dysentery in this setting.

Simplified Soft-output Demapper Based on a Linear Transformation Technique for M-ary PSK

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Jianping Li

2014-10-01

Full Text Available Combining channel coding with high-order modulation schemes, namely coded modulation (CM, is an efficient digital transmission technology. CM requires the demapper to provide a soft decision bit metric as a part of the inputs to the decoder. This paper proposes an efficient soft- output demapper for M-ary PSK. This novel demodulation algorithm continues to evolve the conventional MAX-Log-MAP algorithm and summarizes the final derivation into a form of matrix multiplication. The Computational complexity for getting one bit soft value of a signal by employing the proposed algorithm remains a constant with the increase of modulation order. Meanwhile, the theoretical analysis and simulation results prove that the novel simplified soft-output demapper can obtain the same performance as MAX-Log-MAP.

Bacterial clade with the ribosomal RNA operon on a small plasmid rather than the chromosome.

Science.gov (United States)

Anda, Mizue; Ohtsubo, Yoshiyuki; Okubo, Takashi; Sugawara, Masayuki; Nagata, Yuji; Tsuda, Masataka; Minamisawa, Kiwamu; Mitsui, Hisayuki

2015-11-17

rRNA is essential for life because of its functional importance in protein synthesis. The rRNA (rrn) operon encoding 16S, 23S, and 5S rRNAs is located on the "main" chromosome in all bacteria documented to date and is frequently used as a marker of chromosomes. Here, our genome analysis of a plant-associated alphaproteobacterium, Aureimonas sp. AU20, indicates that this strain has its sole rrn operon on a small (9.4 kb), high-copy-number replicon. We designated this unusual replicon carrying the rrn operon on the background of an rrn-lacking chromosome (RLC) as the rrn-plasmid. Four of 12 strains close to AU20 also had this RLC/rrn-plasmid organization. Phylogenetic analysis showed that those strains having the RLC/rrn-plasmid organization represented one clade within the genus Aureimonas. Our finding introduces a previously unaddressed viewpoint into studies of genetics, genomics, and evolution in microbiology and biology in general.

Antimicrobial drug resistance in Staphylococcus aureus isolated from cattle in Brazil.

Science.gov (United States)

Pereira, M S; Siqueira-Júnior, J P

1995-06-01

Isolates of Staphylococcus aureus obtained from apparently healthy cattle in the State of Paraiba, Brazil were characterized in relation to resistance to 21 antimicrobial agents. Among the 46 isolates obtained, resistance to penicillin was most frequent, followed by resistance to cadmium, streptomycin, arsenate, tetracycline, mercury, erythromycin and kanamycin/neomycin. All isolates were susceptible to fusidic acid, ethidium bromide, cetrimide, chloramphenicol, benzalkonium chloride, doxycycline, gentamicin, methicillin, minocycline, novobiocin, rifamycin, tylosin and vancomycin. Only six isolates were susceptible to all the drugs tested. With respect to the antibiotics, multi-resistant isolates were uncommon. These results are probably a consequence of the peculiarities of local drug usage pressures. In relation to metal ions, resistance to mercury was rare while resistance to arsenate was relatively frequent, which contrasts with the situation for human Staph. aureus strains. After treatment with ethidium bromide, elimination of resistance to penicillin, tetracycline, streptomycin, erythromycin and cadmium was observed, which was consistent with the genetic determinants being plasmid-borne.

Sequencing of IncX-plasmids suggests ubiquity of mobile forms of a biofilm-promoting gene cassette recruited from Klebsiella pneumoniae.

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Mette Burmølle

Full Text Available Plasmids are a highly effective means with which genetic traits that influence human health, such as virulence and antibiotic resistance, are disseminated through bacterial populations. The IncX-family is a hitherto sparsely populated group of plasmids that are able to thrive within Enterobacteriaceae. In this study, a replicon-centric screening method was used to locate strains from wastewater sludge containing plasmids belonging to the IncX-family. A transposon aided plasmid capture method was then employed to transport IncX-plasmids from their original hosts (and co-hosted plasmids into a laboratory strain (Escherichia coli Genehogs® for further study. The nucleotide sequences of the three newly isolated IncX-plasmids (pLN126_33, pMO17_54, pMO440_54 and the hitherto un-sequenced type-plasmid R485 revealed a remarkable occurrence of whole or partial gene cassettes that promote biofilm-formation in Klebsiella pneumonia or E. coli, in all four instances. Two of the plasmids (R485 and pLN126_33 were shown to directly induce biofilm formation in a crystal violet retention assay in E. coli. Sequence comparison revealed that all plasmid-borne forms of the type 3 fimbriae encoding gene cassette mrkABCDF were variations of a composite transposon Tn6011 first described in the E. coli IncX plasmid pOLA52. In conclusion, IncX-plasmids isolated from Enterobacteriaceae over almost 40 years and on three different continents have all been shown to carry a type 3 fimbriae gene cassette mrkABCDF stemming from pathogenic K. pneumoniae. Apart from contributing general knowledge about IncX-plasmids, this study also suggests an apparent ubiquity of a mobile form of an important virulence factor and is an illuminating example of the recruitment, evolution and dissemination of genetic traits through plasmid-mediated horizontal gene transfer.

Sequencing of IncX-plasmids suggests ubiquity of mobile forms of a biofilm-promoting gene cassette recruited from Klebsiella pneumoniae.

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Burmølle, Mette; Norman, Anders; Sørensen, Søren J; Hansen, Lars Hestbjerg

2012-01-01

Plasmids are a highly effective means with which genetic traits that influence human health, such as virulence and antibiotic resistance, are disseminated through bacterial populations. The IncX-family is a hitherto sparsely populated group of plasmids that are able to thrive within Enterobacteriaceae. In this study, a replicon-centric screening method was used to locate strains from wastewater sludge containing plasmids belonging to the IncX-family. A transposon aided plasmid capture method was then employed to transport IncX-plasmids from their original hosts (and co-hosted plasmids) into a laboratory strain (Escherichia coli Genehogs®) for further study. The nucleotide sequences of the three newly isolated IncX-plasmids (pLN126_33, pMO17_54, pMO440_54) and the hitherto un-sequenced type-plasmid R485 revealed a remarkable occurrence of whole or partial gene cassettes that promote biofilm-formation in Klebsiella pneumonia or E. coli, in all four instances. Two of the plasmids (R485 and pLN126_33) were shown to directly induce biofilm formation in a crystal violet retention assay in E. coli. Sequence comparison revealed that all plasmid-borne forms of the type 3 fimbriae encoding gene cassette mrkABCDF were variations of a composite transposon Tn6011 first described in the E. coli IncX plasmid pOLA52. In conclusion, IncX-plasmids isolated from Enterobacteriaceae over almost 40 years and on three different continents have all been shown to carry a type 3 fimbriae gene cassette mrkABCDF stemming from pathogenic K. pneumoniae. Apart from contributing general knowledge about IncX-plasmids, this study also suggests an apparent ubiquity of a mobile form of an important virulence factor and is an illuminating example of the recruitment, evolution and dissemination of genetic traits through plasmid-mediated horizontal gene transfer.

Gene Electrotransfer of Plasmid with Tissue Specific Promoter Encoding shRNA against Endoglin Exerts Antitumor Efficacy against Murine TS/A Tumors by Vascular Targeted Effects.

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Monika Stimac

Full Text Available Vascular targeted therapies, targeting specific endothelial cell markers, are promising approaches for the treatment of cancer. One of the targets is endoglin, transforming growth factor-β (TGF-β co-receptor, which mediates proliferation, differentiation and migration of endothelial cells forming neovasculature. However, its specific, safe and long-lasting targeting remains the challenge. Therefore, in our study we evaluated the transfection efficacy, vascular targeted effects and therapeutic potential of the plasmid silencing endoglin with the tissue specific promoter, specific for endothelial cells marker endothelin-1 (ET (TS plasmid, in comparison to the plasmid with constitutive promoter (CON plasmid, in vitro and in vivo. Tissue specificity of TS plasmid was demonstrated in vitro on several cell lines, and its antiangiogenic efficacy was demonstrated by reducing tube formation of 2H11 endothelial cells. In vivo, on a murine mammary TS/A tumor model, we demonstrated good antitumor effect of gene electrotransfer (GET of either of both plasmids in treatment of smaller tumors still in avascular phase of growth, as well as on bigger tumors, already well vascularized. In support to the observations on predominantly vascular targeted effects of endoglin, histological analysis has demonstrated an increase in necrosis and a decrease in the number of blood vessels in therapeutic groups. A significant antitumor effect was observed in tumors in avascular and vascular phase of growth, possibly due to both, the antiangiogenic and the vascular disrupting effect. Furthermore, the study indicates on the potential use of TS plasmid in cancer gene therapy since the same efficacy as of CON plasmid was determined.

Gene Electrotransfer of Plasmid with Tissue Specific Promoter Encoding shRNA against Endoglin Exerts Antitumor Efficacy against Murine TS/A Tumors by Vascular Targeted Effects.

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Stimac, Monika; Dolinsek, Tanja; Lampreht, Ursa; Cemazar, Maja; Sersa, Gregor

2015-01-01

Vascular targeted therapies, targeting specific endothelial cell markers, are promising approaches for the treatment of cancer. One of the targets is endoglin, transforming growth factor-β (TGF-β) co-receptor, which mediates proliferation, differentiation and migration of endothelial cells forming neovasculature. However, its specific, safe and long-lasting targeting remains the challenge. Therefore, in our study we evaluated the transfection efficacy, vascular targeted effects and therapeutic potential of the plasmid silencing endoglin with the tissue specific promoter, specific for endothelial cells marker endothelin-1 (ET) (TS plasmid), in comparison to the plasmid with constitutive promoter (CON plasmid), in vitro and in vivo. Tissue specificity of TS plasmid was demonstrated in vitro on several cell lines, and its antiangiogenic efficacy was demonstrated by reducing tube formation of 2H11 endothelial cells. In vivo, on a murine mammary TS/A tumor model, we demonstrated good antitumor effect of gene electrotransfer (GET) of either of both plasmids in treatment of smaller tumors still in avascular phase of growth, as well as on bigger tumors, already well vascularized. In support to the observations on predominantly vascular targeted effects of endoglin, histological analysis has demonstrated an increase in necrosis and a decrease in the number of blood vessels in therapeutic groups. A significant antitumor effect was observed in tumors in avascular and vascular phase of growth, possibly due to both, the antiangiogenic and the vascular disrupting effect. Furthermore, the study indicates on the potential use of TS plasmid in cancer gene therapy since the same efficacy as of CON plasmid was determined.

A new aptamer/graphene interdigitated gold electrode piezoelectric sensor for rapid and specific detection of Staphylococcus aureus.

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Lian, Yan; He, Fengjiao; Wang, Huan; Tong, Feifei

2015-03-15

A novel aptamer/graphene interdigitated gold electrode piezoelectric sensor was developed for the rapid and specific detection of Staphylococcus aureus (S. aureus) by employing S. aureus aptamer as a biological recognition element. 4-Mercaptobenzene-diazonium tetrafluoroborate (MBDT) salt was used as a molecular cross-linking agent to chemically bind graphene to interdigital gold electrodes (IDE) that are connected to a series electrode piezoelectric quartz crystal (SPQC). S. aureus aptamers were assembly immobilized onto graphene via the π-π stacking of DNA bases. Due to the specific binding between S. aureus and aptamer, when S. aureus is present, the DNA bases interacted with the aptamer, thereby dropping the aptamer from the surface of the graphene. The electric parameters of the electrode surface was changed and resulted in the change of oscillator frequency of the SPQC. This detection was completed within 60min. The constructed sensor demonstrated a linear relationship between resonance frequency shifts with bacterial concentrations ranging from 4.1×10(1)-4.1×10(5)cfu/mL with a detection limit of 41cfu/mL. The developed strategy can detect S. aureus rapidly and specifically for clinical diagnosis and food testing. Copyright © 2014 Elsevier B.V. All rights reserved.

A prospective cohort study of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus carriage in neonates: the role of maternal carriage and phenotypic and molecular characteristics

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Lin, Jialing; Wu, Chuanan; Yan, Chunrong; Ou, Qianting; Lin, Dongxin; Zhou, Junli; Ye, Xiaohua; Yao, Zhenjiang

2018-01-01

Background Staphylococcus aureus, particularly methicillin-resistant S. aureus (MRSA), in mothers can cause serious outcomes in neonates. We aimed to elucidate the associations of S. aureus and MRSA carriage between mothers and neonates. Methods A prospective cohort study was conducted between August and November 2015 in two hospitals in Shenzhen, China. Chinese pregnant women and their neonates who met the inclusion criteria were included in this study; samples and relevant information were collected. We assessed maternal–neonatal associations by using Poisson regression models. Results Overall, 1834 mothers and their neonates were included in this study. The prevalence of isolate carriage among the mothers was as follows: S. aureus (nasal, 25.8%; vaginal, 7.3%; and nasal and vaginal, 3.3%) and MRSA (nasal, 5.7%; vaginal, 1.7%; and nasal and vaginal, 0.5%). The incidences of S. aureus and MRSA carriage among neonates were 3.3% and 0.8%, respectively. Of the 21 maternal–neonatal pairs with S. aureus carriage, 14 were concordant pairs with the same phenotypic and molecular characteristics. After adjustment, the relative risks and 95% confidence intervals (CIs) between the S. aureus carriage of neonates and nasal S. aureus carriage, vaginal S. aureus carriage, and both nasal and vaginal S. aureus carriage of mothers were 2.8 (95% CI, 1.6–4.8), 7.1 (95% CI, 4.1–12.4), and 9.6 (95% CI, 4.2–22.4), respectively. Conclusion S. aureus carriage in mothers increases the risk for neonates. PMID:29731644

Inactivation efficiency of plasmid-encoded antibiotic resistance genes during water treatment with chlorine, UV, and UV/H2O2.

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Yoon, Younggun; Chung, Hay Jung; Wen Di, Doris Yoong; Dodd, Michael C; Hur, Hor-Gil; Lee, Yunho

2017-10-15

This study assessed the inactivation efficiency of plasmid-encoded antibiotic resistance genes (ARGs) both in extracellular form (e-ARG) and present within Escherichia coli (intracellular form, i-ARG) during water treatment with chlorine, UV (254 nm), and UV/H 2 O 2 . A quantitative real-time PCR (qPCR) method was used to quantify the ARG damage to amp R (850 bp) and kan R (806 bp) amplicons, both of which are located in the pUC4K plasmid. The plate count and flow cytometry methods were also used to determine the bacterial inactivation parameters, such as culturability and membrane damage, respectively. In the first part of the study, the kinetics of E. coli inactivation and ARG damage were determined in phosphate buffered solutions. The ARG damage occurred much more slowly than E. coli inactivation in all cases. To achieve 4-log reduction of ARG concentration at pH 7, the required chlorine exposure and UV fluence were 33-72 (mg × min)/L for chlorine and 50-130 mJ/cm 2 for UV and UV/H 2 O 2 . After increasing pH from 7 to 8, the rates of ARG damage decreased for chlorine, while they did not vary for UV and UV/H 2 O 2 . The i-ARGs mostly showed lower rates of damage compared to the e-ARGs due to the protective roles of cellular components against oxidants and UV. The contribution of OH radicals to i-ARG damage was negligible in UV/H 2 O 2 due to significant OH radical scavenging by cellular components. In all cases, the ARG damage rates were similar for amp R versus kan R , except for the chlorination of e-ARGs, in which the damage to amp R occurred faster than that to kan R . Chlorine and UV dose-dependent ARG inactivation levels determined in a wastewater effluent matrix could be reasonably explained by the kinetic data obtained from the phosphate buffered solutions and the expected oxidant (chlorine and OH radicals) demands by water matrix components. These results can be useful in optimizing chlorine and UV-based disinfection systems to achieve ARG

Staphylococcus aureus Nuc2 is a functional, surface-attached extracellular nuclease.

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Megan R Kiedrowski

Full Text Available Staphylococcus aureus is a prominent bacterial pathogen that causes a diverse range of acute and chronic infections. Recently, it has been demonstrated that the secreted nuclease (Nuc enzyme is a virulence factor in multiple models of infection, and in vivo expression of nuc has facilitated the development of an infection imaging approach based on Nuc-activatable probes. Interestingly, S. aureus strains encode a second nuclease (Nuc2 that has received limited attention. With the growing interest in bacterial nucleases, we sought to characterize Nuc2 in more detail through localization, expression, and biochemical studies. Fluorescence microscopy and alkaline phosphatase localization approaches using Nuc2-GFP and Nuc2-PhoA fusions, respectively, demonstrated that Nuc2 is membrane bound with the C-terminus facing the extracellular environment, indicating it is a signal-anchored Type II membrane protein. Nuc2 enzyme activity was detectable on the S. aureus cell surface using a fluorescence resonance energy transfer (FRET assay, and in time courses, both nuc2 transcription and enzyme activity peaked in early logarithmic growth and declined in stationary phase. Using a mouse model of S. aureus pyomyositis, Nuc2 activity was detected with activatable probes in vivo in nuc mutant strains, demonstrating that Nuc2 is produced during infections. To assess Nuc2 biochemical properties, the protein was purified and found to cleave both single- and double-stranded DNA, and it exhibited thermostability and calcium dependence, paralleling the properties of Nuc. Purified Nuc2 prevented biofilm formation in vitro and modestly decreased biomass in dispersal experiments. Altogether, our findings confirm that S. aureus encodes a second, surface-attached and functional DNase that is expressed during infections and displays similar biochemical properties to the secreted Nuc enzyme.

Origin and Evolution of Rickettsial Plasmids.

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Khalid El Karkouri

Full Text Available Rickettsia species are strictly intracellular bacteria that have undergone a reductive genomic evolution. Despite their allopatric lifestyle, almost half of the 26 currently validated Rickettsia species have plasmids. In order to study the origin, evolutionary history and putative roles of rickettsial plasmids, we investigated the evolutionary processes that have shaped 20 plasmids belonging to 11 species, using comparative genomics and phylogenetic analysis between rickettsial, microbial and non-microbial genomes.Plasmids were differentially present among Rickettsia species. The 11 species had 1 to 4 plasmid (s with a size ranging from 12 kb to 83 kb. We reconstructed pRICO, the last common ancestor of the current rickettsial plasmids. pRICO was vertically inherited mainly from Rickettsia/Orientia chromosomes and diverged vertically into a single or multiple plasmid(s in each species. These plasmids also underwent a reductive evolution by progressive gene loss, similar to that observed in rickettsial chromosomes, possibly leading to cryptic plasmids or complete plasmid loss. Moreover, rickettsial plasmids exhibited ORFans, recent gene duplications and evidence of horizontal gene transfer events with rickettsial and non-rickettsial genomes mainly from the α/γ-proteobacteria lineages. Genes related to maintenance and plasticity of plasmids, and to adaptation and resistance to stress mostly evolved under vertical and/or horizontal processes. Those involved in nucleotide/carbohydrate transport and metabolism were under the influence of vertical evolution only, whereas genes involved in cell wall/membrane/envelope biogenesis, cycle control, amino acid/lipid/coenzyme and secondary metabolites biosynthesis, transport and metabolism underwent mainly horizontal transfer events.Rickettsial plasmids had a complex evolution, starting with a vertical inheritance followed by a reductive evolution associated with increased complexity via horizontal gene

Deteksi Gen Penyandi Sifat Resistensi Metisilin, Penisilin dan Tetrasiklin pada Isolat Staphylococcus aureus Asal Susu Mastitis Subklinis Sapi Perah

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Fatkhanuddin Aziz

2017-02-01

species specific gene of S. aureus and the gene encoding methicillin, penicillin and tetracycline were confirmed by PCR method. The results showed 11 isolates representing of Methicillin Susceptible Staphylococcus aureus (MSSA could be identified, wherein 5 isolates were harboring both of penicillin and tetracycline resistant genes respectively.

C68 from the Sulfolobus islandicus plasmid-virus pSSVx is a novel member of the AbrB-like transcription factor family

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Contursi, Patrizia; D'Ambrosio, Katia; Pirone, Luciano

2011-01-01

The genetic element pSSVx from Sulfolobus islandicus, strain REY15/4, is a hybrid between a plasmid and a fusellovirus. This plasmid-virus hybrid infects several species of the hyperthermophilic acidophilic crenarchaeon Sulfolobus. The open reading frame orfc68 of pSSVx encodes a 7.7 kDa protein...... factors, such as AbrB from Bacillus subtilis. Nevertheless, C68 constitutes a novel representative of this family because it shows several peculiar structural and functional features....

Human Staphylococcus aureus lineages among Zoological Park residents in Greece

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E. Drougka

2015-10-01

Full Text Available Staphylococcus aureus is a part of the microbiota flora in many animal species. The clonal spread of S. aureus among animals and personnel in a Zoological Park was investigated. Samples were collected from colonized and infected sites among 32 mammals, 11 birds and eight humans. The genes mecA, mecC, lukF/lukS-PV (encoding Panton-Valentine leukocidin, PVL and tst (toxic shock syndrome toxin-1 were investigated by PCR. Clones were defined by Multilocus Sequence Typing (MLST, spa type and Pulsed-Field Gel Electrophoresis (PFGE. Seven S. aureus isolates were recovered from four animals and one from an employee. All were mecA, mecC and tst–negative, whereas, one carried the PVL genes and was isolated from an infected Squirrel monkey. Clonal analysis revealed the occurrence of seven STs, eight PFGE and five spa types including ones of human origin. Even though a variety of genotypes were identified among S. aureus strains colonizing zoo park residents, our results indicate that colonization with human lineages has indeed occurred.

Prevalence of clonal complexes and virulence genes among commensal and invasive Staphylococcus aureus isolates in Sweden.

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Gunlög Rasmussen

Full Text Available Staphylococcus aureus encodes a remarkable number of virulence factors which may contribute to its pathogenicity and ability to cause invasive disease. The main objective of this study was to evaluate the association between S. aureus invasiveness and bacterial genotype, in terms of the presence of virulence genes and affiliation to clonal complexes. Also, the significance of different virulence genes, mainly adhesins, for the development of infective endocarditis was investigated. DNA microarray technology was used to analyze 134 S. aureus isolates, all methicillin-susceptible, derived from three groups of clinically well-characterized patients: nasal carriers (n=46, bacteremia (n=55, and bacteremia with infective endocarditis (n=33. Invasive isolates were dominant in four of the major clonal complexes: 5, 8, 15, and 25. Of the 170 virulence genes examined, those encoding accessory gene regulator group II (agr II, capsule polysaccharide serotype 5 (cap5, and adhesins such as S. aureus surface protein G (sasG and fibronectin-binding protein B (fnbB were found to be associated with invasive disease. The same was shown for the leukocidin genes lukD/lukE, as well as the genes encoding serine protease A and B (splA/splB, staphylococcal complement inhibitor (scn and the staphylococcal exotoxin-like protein (setC or selX. In addition, there was a trend of higher prevalence of certain genes or gene clusters (sasG, agr II, cap5 among isolates causing infective endocarditis compared to other invasive isolates. In most cases, the presence of virulence genes was linked to clonal complex affiliation. In conclusion, certain S. aureus clonal lineages harboring specific sets of virulence genes seem to be more successful in causing invasive disease.

Plasmid Negative Regulation of CPAF Expression Is Pgp4 Independent and Restricted to Invasive Chlamydia trachomatis Biovars

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Michael John Patton

2018-01-01

Full Text Available Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes blinding trachoma and sexually transmitted disease. C. trachomatis isolates are classified into 2 biovars—lymphogranuloma venereum (LGV and trachoma—which are distinguished biologically by their natural host cell infection tropism. LGV biovars infect macrophages and are invasive, whereas trachoma biovars infect oculo-urogenital epithelial cells and are noninvasive. The C. trachomatis plasmid is an important virulence factor in the pathogenesis of these infections. Central to its pathogenic role is the transcriptional regulatory function of the plasmid protein Pgp4, which regulates the expression of plasmid and chromosomal virulence genes. As many gene regulatory functions are post-transcriptional, we employed a comparative proteomic study of cells infected with plasmid-cured C. trachomatis serovars A and D (trachoma biovar, a L2 serovar (LGV biovar, and the L2 serovar transformed with a plasmid containing a nonsense mutation in pgp4 to more completely elucidate the effects of the plasmid on chlamydial infection biology. Our results show that the Pgp4-dependent elevations in the levels of Pgp3 and a conserved core set of chromosomally encoded proteins are remarkably similar for serovars within both C. trachomatis biovars. Conversely, we found a plasmid-dependent, Pgp4-independent, negative regulation in the expression of the chlamydial protease-like activity factor (CPAF for the L2 serovar but not the A and D serovars. The molecular mechanism of plasmid-dependent negative regulation of CPAF expression in the LGV serovar is not understood but is likely important to understanding its macrophage infection tropism and invasive infection nature.

Comparison of Biofilm Formation between Methicillin-Resistant and Methicillin-Susceptible Isolates of Staphylococcus aureus.

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Ghasemian, Abdolmajid; Najar Peerayeh, Shahin; Bakhshi, Bita; Mirzaee, Mohsen

2016-07-01

The aim of this study was to compare the biofilm formation and the prevalence of biofilm-associated genes between the isolates of methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) Staphylococcus aureus. In total, 209 S. aureus isolates were collected. The antibiotic susceptibility test was conducted using nine antibiotics according to the guidelines of Clinical and Laboratory Standards Institute. Phenotypic biofilm formation was performed with microtiter plate assay. The polymerase chain reaction was employed to detect icaA, icaD, icaB, icaC, clfA, clfB, fnbA, fnbB, fib, cna, eno, ebps, bbp, mecA, and SCCmec types as well as agr group genes with specific primers. Sixty-four (30.62%) isolates were resistant to methicillin, and 54 (83%) MRSA harbored SCCmec III. Furthermore, 122 (58.3%) isolates belonged to agr group I. Twenty-six (36.1%) MRSA and 42 (28.9%) MSSA isolates were strong biofilm producers (no significant difference). The prevalence of icaA, icaD, icaB, and icaC genes in MSSA isolates was 71, 41, 76, and 72%, respectively. The frequency of clfA, clfB, fnbA, fnbB, fib, cna, eno, ebps, and bbp in MSSA was 100, 100, 56, 46, 74, 54, 78, 11, and 1%, respectively. However, in MRSA isolates, the frequency was 97, 97, 64, 51, 76, 56, 79, and 12% with no track of bbp, respectively. Statistical difference between MSSA and MRSA regarding biofilm formation and the frequency of all biofilm-encoding genes was not significant. The majority of the S. aureus isolates harbored clfA, clfB, eno, fib, icaA, and icaD genes.

Prevalence and characterisation of Staphylococcus aureus causing community-acquired skin and soft tissue infections on Java and Bali, Indonesia.

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Santosaningsih, Dewi; Santoso, Sanarto; Setijowati, Nanik; Rasyid, Harun A; Budayanti, Nyoman S; Suata, Ketut; Widhyatmoko, Dicky B; Purwono, Priyo B; Kuntaman, Kuntaman; Damayanti, Damayanti; Prakoeswa, Cita R S; Laurens, Mitchell; van Nierop, Josephine W I; Nanninga, Geraldine L; Oudenes, Neline; de Regt, Michelle; Snijders, Susan V; Verbrugh, Henri A; Severin, Juliëtte A

2018-01-01

To define the role of Staphylococcus aureus in community settings among patients with skin and soft tissue infections (SSTI) in Indonesia. Staphylococcus aureus were cultured from anterior nares, throat and wounds of 567 ambulatory patients presenting with SSTI. The mecA gene and genes encoding Panton-Valentine leukocidin (PVL; lukF-PV and lukS-PV) and exfoliative toxin (ET; eta and etb) were determined by PCR. Clonal relatedness among methicillin-resistant S. aureus (MRSA) and PVL-positive S. aureus was analysed using multilocus variable-number tandem-repeat analysis (MLVA) typing, and multilocus sequence typing (MLST) for a subset of isolates. Staphylococcal cassette chromosome mec (SCCmec) was determined for all MRSA isolates. Moreover, determinants for S. aureus SSTI, and PVL/ET-positive vs PVL/ET-negative S. aureus were assessed. Staphylococcus aureus were isolated from SSTI wounds of 257 (45.3%) patients, eight (3.1%) of these were MRSA. Genes encoding PVL and ETs were detected in 21.8% and 17.5% of methicillin-susceptible S. aureus (MSSA), respectively. PVL-positive MRSA was not detected. Nasopharyngeal S. aureus carriage was an independent determinant for S. aureus SSTI (odds ratio [OR] 1.8). Primary skin infection (OR 5.4) and previous antibiotic therapy (OR 3.5) were associated with PVL-positive MSSA. Primary skin infection (OR 2.2) was the only factor associated with ET-positive MSSA. MLVA typing revealed two more prevalent MSSA clusters. One ST1-MRSA-SCCmec type IV isolate and a cluster of ST239-MRSA-SCCmec type III were found. Community-acquired SSTI in Indonesia was frequently caused by PVL-positive MSSA, and the hospital-associated ST239-MRSA may have spread from the hospital into the community. © 2017 John Wiley & Sons Ltd.

Yeast transformation mediated by Agrobacterium strains harboring an Ri plasmid: comparative study between GALLS of an Ri plasmid and virE of a Ti plasmid.

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Kiyokawa, Kazuya; Yamamoto, Shinji; Sato, Yukari; Momota, Naoto; Tanaka, Katsuyuki; Moriguchi, Kazuki; Suzuki, Katsunori

2012-07-01

Agrobacterium strains containing a Ti plasmid can transfer T-DNA not only to plants but also to fungi, including the yeast Saccharomyces cerevisiae. However, no Agrobacterium strain harboring an Ri plasmid has been evaluated in fungal transformation. Some Ri plasmids have GALLS , instead of virE1 and virE2. GALLS protein can functionally substitute in plant transformation for a structurally different protein VirE2. In this study, we compared the yeast transformation ability among Agrobacterium donors: a strain containing a Ti plasmid, strains harboring either an agropine-type or a mikimopine-type Ri plasmid, and a strain having a modified Ri plasmid supplemented with a Ti plasmid type virE operon. Agrobacterium strains possessing GALLS transformed yeast cells far less efficiently than the strain containing virE operon. Production of GALLS in recipient yeast cells improved the yeast transformation mediated by an Agrobacterium strain lacking neither GALLS nor virE operon. A reporter assay to detect mobilization of the proteins fused with Cre recombinase revealed that VirE2 protein is much more abundant in yeast cells than GALLS. Based on these results, we concluded that the low yeast transformability mediated by Agrobacterium strains having the Ri plasmid is because of low amount of mobilized GALLS in yeast cells. © 2012 The Authors Journal compilation © 2012 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

Staphylococcus aureus Toxins and Diabetic Foot Ulcers: Role in Pathogenesis and Interest in Diagnosis

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Dunyach-Remy, Catherine; Ngba Essebe, Christelle; Sotto, Albert; Lavigne, Jean-Philippe

2016-01-01

Infection of foot ulcers is a common, often severe and costly complication in diabetes. Diabetic foot infections (DFI) are mainly polymicrobial, and Staphylococcus aureus is the most frequent pathogen isolated. The numerous virulence factors and toxins produced by S. aureus during an infection are well characterized. However, some particular features could be observed in DFI. The aim of this review is to describe the role of S. aureus in DFI and the implication of its toxins in the establishment of the infection. Studies on this issue have helped to distinguish two S. aureus populations in DFI: toxinogenic S. aureus strains (harboring exfoliatin-, EDIN-, PVL- or TSST-encoding genes) and non-toxinogenic strains. Toxinogenic strains are often present in infections with a more severe grade and systemic impact, whereas non-toxinogenic strains seem to remain localized in deep structures and bone involving diabetic foot osteomyelitis. Testing the virulence profile of bacteria seems to be a promising way to predict the behavior of S. aureus in the chronic wounds. PMID:27399775

Reconfigurable digital receiver for 8PSK subcarrier multiplexed and 16QAM single carrier phase‐modulated radio over fiber links

DEFF Research Database (Denmark)

Guerrero Gonzalez, Neil; Zibar, Darko; Yu, Xianbin

2011-01-01

A reconfigurable digital receiver based on the k‐means algorithm is proposed for phase‐modulated subcarrier multiplexed (SCM) and quadrature amplitude‐modulated single carrier, phase‐modulated radio‐over‐fiber links. We report successful demodulation after 40 km single mode fiber transmission wit...... with three 50 Mbaud 8PSK SCM signals and a 312.5 Mbaud 16QAM single carrier. © 2011 Wiley Periodicals, Inc. Microwave Opt Technol Lett 53:1015–1018, 2011; View this article online at wileyonlinelibrary.com. DOI 10.1002/mop.25905...

Hospital clones of methicillin-resistant Staphylococcus aureus are carried by medical students even before healthcare exposure

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Ido Orlin

2017-01-01

Full Text Available Abstract Background Methicillin-resistant Staphylococcus aureus (MRSA strains are prevalent in healthcare and the community. Few studies have examined MRSA carriage among medical students. The aim of this study is to examine Staphylococcus aureus (SA carriage, and particular MRSA, over time in cohort medical students Methods Prospective collection of nasal swabs from medical students in Israel and assessment of SA carriage. Three samples were taken per student in preclinical and clinical parts of studies. Antibiotic susceptibilities were recorded and MRSA typing was performed by staphylococcal cassette chromosome mec (SCCmec types, Panton Valentine Leukocidin (PVL encoding genes, and spa types. Clonality was assessed by pulsed-field gel electrophoresis. Results Among 58 students, SA carriage rates increased from 33% to 38% to 41% at baseline (preclinical studies, 13 and 19 months (clinical studies, respectively (p = 0.07. Methicillin-susceptible SA (MSSA carriage increased in the clinical studies period (22 to 41%, p = 0.01. Overall, seven students (12% carried 13 MRSA isolates. MRSA isolates were PVL negative and were characterized as SCCmecII-t002, SCCmecIV-t032, or t12435 with untypable SCCmec. MRSA carriage during the pre-clinical studies was evident in 4/7 students. Two students carried different MRSA clones at various times and persistent MRSA carriage was noted in one student. Simultaneous carriage of MRSA and MSSA was not detected. Conclusions MSSA carriage increased during the clinical part of studies in Israeli medical students. Compared with previous reports, higher rates of MRSA carriage were evident. MRSA strains were genotypically similar to Israeli healthcare-associated clones; however, carriage occurred largely before healthcare exposure, implying community-acquisition of hospital strains.

CARTOGRAPHIE DU PLASMIDE pSU100, PLASMIDE CRYPTIQUE DE LACTOBACILLUS CASEI

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F BENSALAH

2003-06-01

Ce plasmide appelé pSU100 a été cloné dans le vecteur de transformation pUC18 au site EcoRI chez E. coli JM103. Les profils électrophorétiques de restriction obtenus par des digestions simples, doubles et triples sous l’action de 33 endonucléases, ont contribué à l’élaboration d’une carte de restriction de ce plasmide. Cinq sites uniques ont été identifiés, ainsi que d’autres sites doubles et multiples. Une étude préliminaire du rôle physiologique de ce plasmide a permis de déceler une résistance à la kanamycine.

Isolation and identification of antibiotic resistance genes in Staphylococcus aureus isolates from respiratory system infections in shahrekord, Iran

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Maryam Reisi

2014-07-01

Full Text Available   Introduction : Staphylococcus aureus is considered as one of pathogenic agents in humans, that engages different body parts including respiratory system and causes to spend lots of costs and extending patient’s treatment period. This study which is performed to separate and investigate the pattern of antibiotic resistance in Staphylococcus aureus isolates from upper respiratory system infections in Shahrekord.   Materials and methods: This study was done by sectional-descriptive method On 200 suspicious persons to the upper respiratory system infections who were referred to the Imam Ali clinic in Shahrekord in 2012. After isolation of Staphylococcus aureus from cultured nose discharges, antibiotic resistance genes were identified by polymerase chain reaction (PCR by using defined primer pairs .   Results : Among 200 investigated samples in 60 cases (30% Staphylococcus aureus infection (by culturing and PCR method was determined. Isolates showed the lowest amount of antibiotic resistance to vancomycin (0.5% and the highest amount of resistance to the penicillin G and cefotaxime (100%. mecA gene (encoding methicillin resistance with frequency of 85.18% and aacA-D gene (encoding resistance to aminoglycosides with frequency of 28.33% showed the highest and lowest frequency of antibiotic resistance genes coding in Staphylococcus aureus isolates respectively .   Discussion and conclusion : Notable prevalence of resistant Staphylococcus aureus isolates in community acquired respiratory infections, recommend continuous control necessity to impede the spreading of these bacteria and their infections.  

Control of Copper Resistance and Inorganic Sulfur Metabolism by Paralogous Regulators in Staphylococcus aureus*

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Grossoehme, Nicholas; Kehl-Fie, Thomas E.; Ma, Zhen; Adams, Keith W.; Cowart, Darin M.; Scott, Robert A.; Skaar, Eric P.; Giedroc, David P.

2011-01-01

All strains of Staphylococcus aureus encode a putative copper-sensitive operon repressor (CsoR) and one other CsoR-like protein of unknown function. We show here that NWMN_1991 encodes a bona fide Cu(I)-inducible CsoR of a genetically unlinked copA-copZ copper resistance operon in S. aureus strain Newman. In contrast, an unannotated open reading frame found between NWMN_0027 and NWMN_0026 (denoted NWMN_0026.5) encodes a CsoR-like regulator that represses expression of adjacent genes by binding specifically to a pair of canonical operator sites positioned in the NWMN_0027–0026.5 intergenic region. Inspection of these regulated genes suggests a role in assimilation of inorganic sulfur from thiosulfate and vectorial sulfur transfer, and we designate NWMN_0026.5 as CstR (CsoR-like sulfur transferase repressor). Expression analysis demonstrates that CsoR and CstR control their respective regulons in response to distinct stimuli with no overlap in vivo. Unlike CsoR, CstR does not form a stable complex with Cu(I); operator binding is instead inhibited by oxidation of the intersubunit cysteine pair to a mixture of disulfide and trisulfide linkages by a likely metabolite of thiosulfate assimilation, sulfite. CsoR is unreactive toward sulfite under the same conditions. We conclude that CsoR and CstR are paralogs in S. aureus that function in the same cytoplasm to control distinct physiological processes. PMID:21339296

Control of copper resistance and inorganic sulfur metabolism by paralogous regulators in Staphylococcus aureus.

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Grossoehme, Nicholas; Kehl-Fie, Thomas E; Ma, Zhen; Adams, Keith W; Cowart, Darin M; Scott, Robert A; Skaar, Eric P; Giedroc, David P

2011-04-15

All strains of Staphylococcus aureus encode a putative copper-sensitive operon repressor (CsoR) and one other CsoR-like protein of unknown function. We show here that NWMN_1991 encodes a bona fide Cu(I)-inducible CsoR of a genetically unlinked copA-copZ copper resistance operon in S. aureus strain Newman. In contrast, an unannotated open reading frame found between NWMN_0027 and NWMN_0026 (denoted NWMN_0026.5) encodes a CsoR-like regulator that represses expression of adjacent genes by binding specifically to a pair of canonical operator sites positioned in the NWMN_0027-0026.5 intergenic region. Inspection of these regulated genes suggests a role in assimilation of inorganic sulfur from thiosulfate and vectorial sulfur transfer, and we designate NWMN_0026.5 as CstR (CsoR-like sulfur transferase repressor). Expression analysis demonstrates that CsoR and CstR control their respective regulons in response to distinct stimuli with no overlap in vivo. Unlike CsoR, CstR does not form a stable complex with Cu(I); operator binding is instead inhibited by oxidation of the intersubunit cysteine pair to a mixture of disulfide and trisulfide linkages by a likely metabolite of thiosulfate assimilation, sulfite. CsoR is unreactive toward sulfite under the same conditions. We conclude that CsoR and CstR are paralogs in S. aureus that function in the same cytoplasm to control distinct physiological processes.

Livestock-Associated Methicillin and Multidrug Resistant Staphylococcus aureus Is Present among Industrial, Not Antibiotic-Free Livestock Operation Workers in North Carolina

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Wing, Steve; Hall, Devon; Baron, Dothula; Price, Lance B.; Larsen, Jesper; Stegger, Marc; Stewart, Jill; Heaney, Christopher D.

2013-01-01

Objectives Administration of antibiotics to food animals may select for drug-resistant pathogens of clinical significance, such as methicillin-resistant Staphylococcus aureus (MRSA). In the United States, studies have examined prevalence of MRSA carriage among individuals exposed to livestock, but prevalence of multidrug-resistant S. aureus (MDRSA) carriage and the association with livestock raised with versus without antibiotic selective pressure remains unclear. We aimed to examine prevalence, antibiotic susceptibility, and molecular characteristics of S. aureus among industrial livestock operation (ILO) and antibiotic-free livestock operation (AFLO) workers and household members in North Carolina. Methods Participants in this cross-sectional study were interviewed and provided a nasal swab for S. aureus analysis. Resulting S. aureus isolates were assessed for antibiotic susceptibility, multi-locus sequence type, and absence of the scn gene (a marker of livestock association). Results Among 99 ILO and 105 AFLO participants, S. aureus nasal carriage prevalence was 41% and 40%, respectively. Among ILO and AFLO S. aureus carriers, MRSA was detected in 7% (3/41) and 7% (3/42), respectively. Thirty seven percent of 41 ILO versus 19% of 42 AFLO S. aureus-positive participants carried MDRSA. S. aureus clonal complex (CC) 398 was observed only among workers and predominated among ILO (13/34) compared with AFLO (1/35) S. aureus-positive workers. Only ILO workers carried scn-negative MRSA CC398 (2/34) and scn-negative MDRSA CC398 (6/34), and all of these isolates were tetracycline resistant. Conclusions Despite similar S. aureus and MRSA prevalence among ILO and AFLO-exposed individuals, livestock-associated MRSA and MDRSA (tetracycline-resistant, CC398, scn-negative) were only present among ILO-exposed individuals. These findings support growing concern about antibiotics use and confinement in livestock production, raising questions about the potential for occupational

Livestock-associated methicillin and multidrug resistant Staphylococcus aureus is present among industrial, not antibiotic-free livestock operation workers in North Carolina.

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Jessica L Rinsky

Full Text Available OBJECTIVES: Administration of antibiotics to food animals may select for drug-resistant pathogens of clinical significance, such as methicillin-resistant Staphylococcus aureus (MRSA. In the United States, studies have examined prevalence of MRSA carriage among individuals exposed to livestock, but prevalence of multidrug-resistant S. aureus (MDRSA carriage and the association with livestock raised with versus without antibiotic selective pressure remains unclear. We aimed to examine prevalence, antibiotic susceptibility, and molecular characteristics of S. aureus among industrial livestock operation (ILO and antibiotic-free livestock operation (AFLO workers and household members in North Carolina. METHODS: Participants in this cross-sectional study were interviewed and provided a nasal swab for S. aureus analysis. Resulting S. aureus isolates were assessed for antibiotic susceptibility, multi-locus sequence type, and absence of the scn gene (a marker of livestock association. RESULTS: Among 99 ILO and 105 AFLO participants, S. aureus nasal carriage prevalence was 41% and 40%, respectively. Among ILO and AFLO S. aureus carriers, MRSA was detected in 7% (3/41 and 7% (3/42, respectively. Thirty seven percent of 41 ILO versus 19% of 42 AFLO S. aureus-positive participants carried MDRSA. S. aureus clonal complex (CC 398 was observed only among workers and predominated among ILO (13/34 compared with AFLO (1/35 S. aureus-positive workers. Only ILO workers carried scn-negative MRSA CC398 (2/34 and scn-negative MDRSA CC398 (6/34, and all of these isolates were tetracycline resistant. CONCLUSIONS: Despite similar S. aureus and MRSA prevalence among ILO and AFLO-exposed individuals, livestock-associated MRSA and MDRSA (tetracycline-resistant, CC398, scn-negative were only present among ILO-exposed individuals. These findings support growing concern about antibiotics use and confinement in livestock production, raising questions about the potential for

Pmr, a histone-like protein H1 (H-NS) family protein encoded by the IncP-7 plasmid pCAR1, is a key global regulator that alters host function.

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Yun, Choong-Soo; Suzuki, Chiho; Naito, Kunihiko; Takeda, Toshiharu; Takahashi, Yurika; Sai, Fumiya; Terabayashi, Tsuguno; Miyakoshi, Masatoshi; Shintani, Masaki; Nishida, Hiromi; Yamane, Hisakazu; Nojiri, Hideaki

2010-09-01

Histone-like protein H1 (H-NS) family proteins are nucleoid-associated proteins (NAPs) conserved among many bacterial species. The IncP-7 plasmid pCAR1 is transmissible among various Pseudomonas strains and carries a gene encoding the H-NS family protein, Pmr. Pseudomonas putida KT2440 is a host of pCAR1, which harbors five genes encoding the H-NS family proteins PP_1366 (TurA), PP_3765 (TurB), PP_0017 (TurC), PP_3693 (TurD), and PP_2947 (TurE). Quantitative reverse transcription-PCR (qRT-PCR) demonstrated that the presence of pCAR1 does not affect the transcription of these five genes and that only pmr, turA, and turB were primarily transcribed in KT2440(pCAR1). In vitro pull-down assays revealed that Pmr strongly interacted with itself and with TurA, TurB, and TurE. Transcriptome comparisons of the pmr disruptant, KT2440, and KT2440(pCAR1) strains indicated that pmr disruption had greater effects on the host transcriptome than did pCAR1 carriage. The transcriptional levels of some genes that increased with pCAR1 carriage, such as the mexEF-oprN efflux pump genes and parI, reverted with pmr disruption to levels in pCAR1-free KT2440. Transcriptional levels of putative horizontally acquired host genes were not altered by pCAR1 carriage but were altered by pmr disruption. Identification of genome-wide Pmr binding sites by ChAP-chip (chromatin affinity purification coupled with high-density tiling chip) analysis demonstrated that Pmr preferentially binds to horizontally acquired DNA regions. The Pmr binding sites overlapped well with the location of the genes differentially transcribed following pmr disruption on both the plasmid and the chromosome. Our findings indicate that Pmr is a key factor in optimizing gene transcription on pCAR1 and the host chromosome.

Interactions of Kid-Kis toxin-antitoxin complexes with the parD operator-promotor region of plasmid R1 are piloted by the Kis antitoxin and tuned by the stoichiometry of Kid-Kis oligomers

NARCIS (Netherlands)

Monti, M.C.; Hernandez-Arriaga, A.M.; Kamphuis, M.B.; Lopez-Villarejo, J.; Heck, A.J.R.; Boelens, R.; Diaz-Orejas, R.; van den Heuvel, R.H.H.

2007-01-01

The parD operon of Escherichia coli plasmid R1 encodes a toxin–antitoxin system, which is involved in plasmid stabilization. The toxin Kid inhibits cell growth by RNA degradation and its action is neutralized by the formation of a tight complex with the antitoxin Kis. A fascinating but poorly

Presence of antiseptic resistance genes in porcine methicillin-resistant Staphylococcus aureus.

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Wong, T Z; Zhang, M; O'Donoghue, M; Boost, M

2013-03-23

Numerous studies have documented the presence of methicillin-resistant Staphylococcus aureus (MRSA) in meat-producing animals, which has led to concern about its spread into the community. Disinfectants play an important role in reduction of contamination in both animal husbandry and food-preparation, helping control spread of organisms from foodstuffs, including raw meat. Plasmid-borne antiseptic resistance (AR) genes increasing tolerance to several disinfectants have been reported in S. aureus of human origin (qacA/B and smr) and from bovine, equine, and caprine staphylococcal isolates (qacG, qacH, and qacJ). This study investigated the presence of AR genes in porcine MRSA isolates. Plasmid DNA from 100 MRSA ST9 strains isolated from pig carcasses was amplified for the presence of AR genes. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) to benzalkonium chloride (BC) and chlorhexidine gluconate (CHX) were determined in AR gene-positive isolates. qacG was present in 45 strains, eight of which also harbored smr. No strains carried qacA/B, qacH or qacJ. Presence of smr increased MICs to both BC and CHX and MBCs of CHX, but qacG presence only resulted in elevated MBC for CHX. This is the first report of AR genes from a porcine source. AR gene positivity has previously been associated with methicillin resistance and AR gene presence in these strains may increase their ability to persist in the environment. Improved implementation of hygiene measures during transportation and pre- and post-slaughter should be considered to prevent spread in the community. Copyright © 2012 Elsevier B.V. All rights reserved.

High dose of plasmid IL-15 inhibits immune responses in an influenza non-human primates immunogenicity model

International Nuclear Information System (INIS)

Yin Jiangmei; Dai Anlan; Laddy, Dominick J.; Yan Jian; Arango, Tatiana; Khan, Amir S.; Lewis, Mark G.; Andersen, Hanne; Kutzler, Michele A.; Draghia-Akli, Ruxandra; Weiner, David B.; Boyer, Jean D.

2009-01-01

Interleukin (IL)-15, is a cytokine that is important for the maintenance of long-lasting, high-avidity T cell response to invading pathogens and has, therefore, been used in vaccine and therapeutic platforms as an adjuvant. In addition to pure protein delivery, plasmids encoding the IL-15 gene have been utilized. However, it is critical to determine the appropriate dose to maximize the adjuvanting effects. We immunized rhesus macaques with different doses of IL-15 expressing plasmid in an influenza non-human primate immunogenicity model. We found that co-immunization of rhesus macaques with a Flu DNA-based vaccine and low doses of plasmid encoding macaque IL-15 enhanced the production of IFN-γ (0.5 mg) and the proliferation of CD4 + and CD8 + T cells, as well as T CM levels in proliferating CD8 + T cells (0.25 mg). Whereas, high doses of IL-15 (4 mg) decrease the production of IFN-γ and the proliferation of CD4 + and CD8 + T cells and T CM levels in the proliferating CD4 + and CD8 + T cells. In addition, the data of hemagglutination inhibition (HI) antibody titer suggest that although not significantly different, there appears to be a slight increase in antibodies at lower doses of IL-15. Importantly, however, the higher doses of IL-15 decrease the antibody levels significantly. This study demonstrates the importance of optimizing DNA-based cytokine adjuvants.

The detection and sequencing of a broad-host-range conjugative IncP-1β plasmid in an epidemic strain of Mycobacterium abscessus subsp. bolletii.

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Sylvia Cardoso Leão

Full Text Available BACKGROUND: An extended outbreak of mycobacterial surgical infections occurred in Brazil during 2004-2008. Most infections were caused by a single strain of Mycobacterium abscessus subsp. bolletii, which was characterized by a specific rpoB sequevar and two highly similar pulsed-field gel electrophoresis (PFGE patterns differentiated by the presence of a ∼50 kb band. The nature of this band was investigated. METHODOLOGY/PRINCIPAL FINDINGS: Genomic sequencing of the prototype outbreak isolate INCQS 00594 using the SOLiD platform demonstrated the presence of a 56,267-bp [corrected] circular plasmid, designated pMAB01. Identity matrices, genetic distances and phylogeny analyses indicated that pMAB01 belongs to the broad-host-range plasmid subgroup IncP-1β and is highly related to BRA100, pJP4, pAKD33 and pB10. The presence of pMAB01-derived sequences in 41 M. abscessus subsp. bolletii isolates was evaluated using PCR, PFGE and Southern blot hybridization. Sixteen of the 41 isolates showed the presence of the plasmid. The plasmid was visualized as a ∼50-kb band using PFGE and Southern blot hybridization in 12 isolates. The remaining 25 isolates did not exhibit any evidence of this plasmid. The plasmid was successfully transferred to Escherichia coli by conjugation and transformation. Lateral transfer of pMAB01 to the high efficient plasmid transformation strain Mycobacterium smegmatis mc(2155 could not be demonstrated. CONCLUSIONS/SIGNIFICANCE: The occurrence of a broad-host-range IncP-1β plasmid in mycobacteria is reported for the first time. Thus, genetic exchange could result in the emergence of specific strains that might be better adapted to cause human disease.

Molecular and clinical characteristics of clonal complex 59 methicillin-resistant Staphylococcus aureus infections in Mainland China.

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Juan Li

Full Text Available Detailed molecular analyses of Clonal Complex 59 (CC59 methicillin-resistant Staphylococcus aureus (MRSA isolates from children in seven major cities across Mainland China were examined. A total of 110 CC59 isolates from invasive and non-invasive diseases were analyzed by multilocus sequence typing (MLST, Staphylococcus cassette chromosome mec (SCCmec typing, staphylococcal protein A (spa typing and pulsed-field gel electrophoresis (PFGE. Antibiotics susceptibilities, carriage of plasmids and 42 virulence genes and the expression of virulence factors were examined. ST59 (101/110, 91.8% was the predominant sequence type (ST, while single locus variants (SLVs belonging to ST338 (8/110, 7.3% and ST375 (1/110, 0.9% were obtained. Three SCCmec types were found, namely type III (2.7%, type IV (74.5% and type V (22.7%. Seven spa types including t437, which accounted for 87.3%, were determined. Thirteen PFGE types were obtained. PFGE types A and B were the major types totally accounting for 81.8%. The dominant clone was ST59-t437-IVa (65.5%, followed by ST59-t437-V (14.5%. The positive rate of luks-PV and lukF-PV PVL encoding (pvl gene was 55.5%. Plasmids were detected in 83.6% (92/110 of the strains. The plasmid size ranging from 23.4 kb to 50 kb was most prevalent which accounted for 83.7% (77/92. A significantly lower expression of hla was found in ST59-t437-IVa compared with ST59-t437-V. Among the 110 cases, 61.8% of the patients were less than 1 year old. A total of 90 cases (81.8% were community-associated (CA infections whereas 20 cases (18.2% were hospital-associated (HA infections. Out of the 110 patients, 36.4% (40/110 were diagnosed with invasive infectious diseases in which ST59-t437-IVa accounted for 67.5% (27/40. In brief, ST59-t437-IVa was proved as the dominant clone in CC59 MRSA strains. The carriage rate of pvl gene was high. CC59 MRSA could result in CA and HA infections. The majortiy of MRSA infection children were in young age.

Nuclear-encoded mitochondrial MTO1 and MRPL41 are regulated in an opposite epigenetic mode based on estrogen receptor status in breast cancer

International Nuclear Information System (INIS)

Kim, Tae Woo; Kim, Byungtak; Kim, Ju Hee; Kang, Seongeun; Park, Sung-Bin; Jeong, Gookjoo; Kang, Han-Sung; Kim, Sun Jung

2013-01-01

MTO1 and MRPL41 are nuclear-encoded mitochondrial genes encoding a mitochondrial tRNA-modifying enzyme and a mitochondrial ribosomal protein, respectively. Although both genes have been known to have potential roles in cancer, little is known about their molecular regulatory mechanism, particularly from an epigenetic approach. In this study, we aimed to address their epigenetic regulation through the estrogen receptor (ER) in breast cancer. Digital differential display (DDD) was conducted to identify mammary gland-specific gene candidates including MTO1 and MRPL41. Promoter CpG methylation and expression in breast cancer cell lines and tissues were examined by methylation-specific PCR and real time RT-PCR. Effect of estradiol (E2), tamoxifen, and trichostatin A (TSA) on gene expression was examined in ER + and ER- breast cancer cell lines. Chromatin immunoprecipitation and luciferase reporter assay were performed to identify binding and influencing of the ER to the promoters. Examination of both cancer tissues and cell lines revealed that the two genes showed an opposite expression pattern according to ER status; higher expression of MTO1 and MRPL41 in ER- and ER+ cancer types, respectively, and their expression levels were inversely correlated with promoter methylation. Tamoxifen, E2, and TSA upregulated MTO1 expression only in ER+ cells with no significant changes in ER- cells. However, these chemicals upregulated MRPL41 expression only in ER- cells without significant changes in ER+ cells, except for tamoxifen that induced downregulation. Chromatin immunoprecipitation and luciferase reporter assay identified binding and influencing of the ER to the promoters and the binding profiles were differentially regulated in ER+ and ER- cells. These results indicate that different epigenetic status including promoter methylation and different responses through the ER are involved in the differential expression of MTO1 and MRPL41 in breast cancer

Amphibian antimicrobial peptide fallaxin analogue FL9 affects virulence gene expression and DNA replication in Staphylococcus aureus.

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Gottschalk, Sanne; Gottlieb, Caroline T; Vestergaard, Martin; Hansen, Paul R; Gram, Lone; Ingmer, Hanne; Thomsen, Line E

2015-12-01

The rapid rise in antibiotic-resistant pathogens is causing increased health concerns, and consequently there is an urgent need for novel antimicrobial agents. Antimicrobial peptides (AMPs), which have been isolated from a wide range of organisms, represent a very promising class of novel antimicrobials. In the present study, the analogue FL9, based on the amphibian AMP fallaxin, was studied to elucidate its mode of action and antibacterial activity against the human pathogen Staphylococcus aureus. Our data showed that FL9 may have a dual mode of action against S. aureus. At concentrations around the MIC, FL9 bound DNA, inhibited DNA synthesis and induced the SOS DNA damage response, whereas at concentrations above the MIC the interaction between S. aureus and FL9 led to membrane disruption. The antibacterial activity of the peptide was maintained over a wide range of NaCl and MgCl(2) concentrations and at alkaline pH, while it was compromised by acidic pH and exposure to serum. Furthermore, at subinhibitory concentrations of FL9, S. aureus responded by increasing the expression of two major virulence factor genes, namely the regulatory rnaIII and hla, encoding α-haemolysin. In addition, the S. aureus-encoded natural tolerance mechanisms included peptide cleavage and the addition of positive charge to the cell surface, both of which minimized the antimicrobial activity of FL9. Our results add new information about FL9 and its effect on S. aureus, which may aid in the future development of analogues with improved therapeutic potential.

Prevalence and molecular characteristics of Staphylococcus aureus, including methicillin resistant strains, isolated from bulk can milk and raw milk products in pastoral communities of South-West Uganda.

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Asiimwe, Benon B; Baldan, Rossella; Trovato, Alberto; Cirillo, Daniela M

2017-06-13

Staphylococcus aureus strains are now regarded as zoonotic agents. In pastoral settings where human-animal interaction is intimate, multi-drug resistant microorganisms have become an emerging zoonotic issue of public health concern. The study of S. aureus prevalence, antimicrobial resistance and clonal lineages in humans, animals and food in African settings has great relevance, taking into consideration the high diversity of ethnicities, cultures and food habits that determine the lifestyle of the people. Little is known about milk carriage of methicillin resistant S. aureus strains (MRSA) and their virulence factors in Uganda. Here, we present the prevalence of MRSA in bulk can milk and raw milk products in pastoral communities of south-west Uganda. We also present PFGE profiles, spa-types, as well as frequency of enterotoxins genes. S. aureus was identified by the coagulase test, susceptibility testing by the Kirby-Bauer disc diffusion and E-test methods and MRSA by detection of the mecA gene and SCCmec types. The presence of Panton - Valentine Leucocidin (PVL) genes and staphylococcal enterotoxins was determined by PCR, while genotyping was by PFGE and spa typing. S. aureus were isolated from 30/148 (20.3%) milk and 11/91(12%) sour milk samples. mecA gene carriage, hence MRSA, was detected in 23/41 (56.1%) of the isolates, with 21 of the 23 (91.3%) being SCCmec type V; while up to 30/41 (73.2%) of the isolates were resistant to tetracycline. Only five isolates carried the PVL virulence gene, while PFGE typing revealed ten clusters (ranging from two seven isolates each) that comprised 83% of the sample, and only eight isolates with unique pulsotypes. The largest PFGE profile (E) consisted of seven isolates while t7753, t1398, and t2112 were the most common spa-types. Thirty seven of the 41 strains (90.2%) showed at least one of the eight enterotoxin genes tested, with sem 29 (70.7%), sei 25 (61%) and seg 21 (51.2%) being the most frequently observed genes. This

Rap phosphatase of virulence plasmid pXO1 inhibits Bacillus anthracis sporulation.

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Bongiorni, Cristina; Stoessel, Ricarda; Shoemaker, Dorinda; Perego, Marta

2006-01-01

This study shows that the Bacillus anthracis pXO1 virulence plasmid carries a Rap-Phr system, BXA0205, which regulates sporulation initiation in this organism. The BXA0205Rap protein was shown to dephosphorylate the Spo0F response regulator intermediate of the phosphorelay signal transduction system that regulates the initiation of the developmental pathway in response to environmental, metabolic, and cell cycle signals. The activity of the Rap protein was shown to be inhibited by the carboxy-terminal pentapeptide generated through an export-import processing pathway from the associated BXA0205Phr protein. Deregulation of the Rap activity by either overexpression or lack of the Phr pentapeptide resulted in severe inhibition of sporulation. Five additional Rap-Phr encoding systems were identified on the chromosome of B. anthracis, one of which, BA3790-3791, also affected sporulation initiation. The results suggest that the plasmid-borne Rap-Phr system may provide a selective advantage to the virulence of B. anthracis.

Rap Phosphatase of Virulence Plasmid pXO1 Inhibits Bacillus anthracis Sporulation†

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Bongiorni, Cristina; Stoessel, Ricarda; Shoemaker, Dorinda; Perego, Marta

2006-01-01

This study shows that the Bacillus anthracis pXO1 virulence plasmid carries a Rap-Phr system, BXA0205, which regulates sporulation initiation in this organism. The BXA0205Rap protein was shown to dephosphorylate the Spo0F response regulator intermediate of the phosphorelay signal transduction system that regulates the initiation of the developmental pathway in response to environmental, metabolic, and cell cycle signals. The activity of the Rap protein was shown to be inhibited by the carboxy-terminal pentapeptide generated through an export-import processing pathway from the associated BXA0205Phr protein. Deregulation of the Rap activity by either overexpression or lack of the Phr pentapeptide resulted in severe inhibition of sporulation. Five additional Rap-Phr encoding systems were identified on the chromosome of B. anthracis, one of which, BA3790-3791, also affected sporulation initiation. The results suggest that the plasmid-borne Rap-Phr system may provide a selective advantage to the virulence of B. anthracis. PMID:16385039

Conjugative Plasmid Transfer in Xylella fastidiosa Is Dependent on tra and trb Operon Functions

OpenAIRE

Burbank, Lindsey P.; Van Horn, Christopher R.

2017-01-01

The insect-transmitted plant pathogen Xylella fastidiosa is capable of efficient horizontal gene transfer (HGT) and recombination. Natural transformation occurs at high rates in X. fastidiosa, but there also is evidence that certain strains of X. fastidiosa carry native plasmids equipped with transfer and mobilization genes, suggesting conjugation as an additional mechanism of HGT in some instances. Two operons, tra and trb, putatively encoding a conjugative type IV secretion system, are foun...

Large-scale preparation of plasmid DNA.

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Heilig, J S; Elbing, K L; Brent, R

2001-05-01

Although the need for large quantities of plasmid DNA has diminished as techniques for manipulating small quantities of DNA have improved, occasionally large amounts of high-quality plasmid DNA are desired. This unit describes the preparation of milligram quantities of highly purified plasmid DNA. The first part of the unit describes three methods for preparing crude lysates enriched in plasmid DNA from bacterial cells grown in liquid culture: alkaline lysis, boiling, and Triton lysis. The second part describes four methods for purifying plasmid DNA in such lysates away from contaminating RNA and protein: CsCl/ethidium bromide density gradient centrifugation, polyethylene glycol (PEG) precipitation, anion-exchange chromatography, and size-exclusion chromatography.

The cost of copy number in a selfish genetic element: the 2-μm plasmid of Saccharomyces cerevisiae.

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Harrison, Ellie; Koufopanou, V; Burt, A; MacLean, R C

2012-11-01

Many autonomously replicating genetic elements exist as multiple copies within the cell. The copy number of these elements is often assumed to have important fitness consequences for both element and host, yet the forces shaping its evolution are not well understood. The 2 μm is a multicopy plasmid of Saccharomyces yeasts, encoding just four genes that are solely involved in plasmid replication. One simple model for the fitness relationship between yeasts and 2 μm is that plasmid copy number evolves as a trade-off between selection for increased vertical transmission, favouring high copy number, and selection for decreased virulence, favouring low copy number. To test this model, we experimentally manipulated the copy number of the plasmid and directly measured the fitness cost, in terms of growth rate reduction, associated with high plasmid copy number. We find that the fitness burden imposed by the 2 μm increases with plasmid copy number, such that each copy imposes a fitness burden of 0.17% (± 0.008%), greatly exceeding the cost expected for it to be stably maintained in yeast populations. Our results demonstrate the crucial importance of copy number in the evolution of yeast per 2 μm associations and pave the way for future studies examining how selection can shape the cost of multicopy elements. © 2012 The Authors. Journal of Evolutionary Biology © 2012 European Society For Evolutionary Biology.

Quantifying and resolving multiple vector transformants in S. cerevisiae plasmid libraries.

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Scanlon, Thomas C; Gray, Elizabeth C; Griswold, Karl E

2009-11-20

In addition to providing the molecular machinery for transcription and translation, recombinant microbial expression hosts maintain the critical genotype-phenotype link that is essential for high throughput screening and recovery of proteins encoded by plasmid libraries. It is known that Escherichia coli cells can be simultaneously transformed with multiple unique plasmids and thusly complicate recombinant library screening experiments. As a result of their potential to yield misleading results, bacterial multiple vector transformants have been thoroughly characterized in previous model studies. In contrast to bacterial systems, there is little quantitative information available regarding multiple vector transformants in yeast. Saccharomyces cerevisiae is the most widely used eukaryotic platform for cell surface display, combinatorial protein engineering, and other recombinant library screens. In order to characterize the extent and nature of multiple vector transformants in this important host, plasmid-born gene libraries constructed by yeast homologous recombination were analyzed by DNA sequencing. It was found that up to 90% of clones in yeast homologous recombination libraries may be multiple vector transformants, that on average these clones bear four or more unique mutant genes, and that these multiple vector cells persist as a significant proportion of library populations for greater than 24 hours during liquid outgrowth. Both vector concentration and vector to insert ratio influenced the library proportion of multiple vector transformants, but their population frequency was independent of transformation efficiency. Interestingly, the average number of plasmids born by multiple vector transformants did not vary with their library population proportion. These results highlight the potential for multiple vector transformants to dominate yeast libraries constructed by homologous recombination. The previously unrecognized prevalence and persistence of multiply

Quantifying and resolving multiple vector transformants in S. cerevisiae plasmid libraries

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Gray Elizabeth C

2009-11-01

Full Text Available Abstract Background In addition to providing the molecular machinery for transcription and translation, recombinant microbial expression hosts maintain the critical genotype-phenotype link that is essential for high throughput screening and recovery of proteins encoded by plasmid libraries. It is known that Escherichia coli cells can be simultaneously transformed with multiple unique plasmids and thusly complicate recombinant library screening experiments. As a result of their potential to yield misleading results, bacterial multiple vector transformants have been thoroughly characterized in previous model studies. In contrast to bacterial systems, there is little quantitative information available regarding multiple vector transformants in yeast. Saccharomyces cerevisiae is the most widely used eukaryotic platform for cell surface display, combinatorial protein engineering, and other recombinant library screens. In order to characterize the extent and nature of multiple vector transformants in this important host, plasmid-born gene libraries constructed by yeast homologous recombination were analyzed by DNA sequencing. Results It was found that up to 90% of clones in yeast homologous recombination libraries may be multiple vector transformants, that on average these clones bear four or more unique mutant genes, and that these multiple vector cells persist as a significant proportion of library populations for greater than 24 hours during liquid outgrowth. Both vector concentration and vector to insert ratio influenced the library proportion of multiple vector transformants, but their population frequency was independent of transformation efficiency. Interestingly, the average number of plasmids born by multiple vector transformants did not vary with their library population proportion. Conclusion These results highlight the potential for multiple vector transformants to dominate yeast libraries constructed by homologous recombination. The

Human Intestinal Cells Modulate Conjugational Transfer of Multidrug Resistance Plasmids between Clinical Escherichia coli Isolates

DEFF Research Database (Denmark)

Machado, Ana Manuel; Sommer, Morten

2014-01-01

Bacterial conjugation in the human gut microbiota is believed to play a major role in the dissemination of antibiotic resistance genes and virulence plasmids. However, the modulation of bacterial conjugation by the human host remains poorly understood and there is a need for controlled systems...... to study this process. We established an in vitro co-culture system to study the interaction between human intestinal cells and bacteria. We show that the conjugation efficiency of a plasmid encoding an extended spectrum beta-lactamase is reduced when clinical isolates of Escherichia coli are co...... of the intestinal cells exposed to bacteria leading to a two-fold reduction in conjugation efficiency. These results show that human gut epithelial cells can modulate bacterial conjugation and may have relevance to gene exchange in the gut....

Mobility of the native Bacillus subtilis conjugative plasmid pLS20 is regulated by intercellular signaling.

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Singh, Praveen K; Ramachandran, Gayetri; Ramos-Ruiz, Ricardo; Peiró-Pastor, Ramón; Abia, David; Wu, Ling J; Meijer, Wilfried J J

2013-10-01

Horizontal gene transfer mediated by plasmid conjugation plays a significant role in the evolution of bacterial species, as well as in the dissemination of antibiotic resistance and pathogenicity determinants. Characterization of their regulation is important for gaining insights into these features. Relatively little is known about how conjugation of Gram-positive plasmids is regulated. We have characterized conjugation of the native Bacillus subtilis plasmid pLS20. Contrary to the enterococcal plasmids, conjugation of pLS20 is not activated by recipient-produced pheromones but by pLS20-encoded proteins that regulate expression of the conjugation genes. We show that conjugation is kept in the default "OFF" state and identified the master repressor responsible for this. Activation of the conjugation genes requires relief of repression, which is mediated by an anti-repressor that belongs to the Rap family of proteins. Using both RNA sequencing methodology and genetic approaches, we have determined the regulatory effects of the repressor and anti-repressor on expression of the pLS20 genes. We also show that the activity of the anti-repressor is in turn regulated by an intercellular signaling peptide. Ultimately, this peptide dictates the timing of conjugation. The implications of this regulatory mechanism and comparison with other mobile systems are discussed.

Construction and Identification of a Recombinant Plasmid Encoding Echinococcus granulosus Oncosphere Antigen (EG95Abstract Background: Cystic echinococcosis (CE, as a zoonotic disease cause to health threat and economic losses. Despite implemented cont

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Nahideh MAZAHERI

2017-12-01

Full Text Available AbstractBackground: Cystic echinococcosis (CE, as a zoonotic disease cause to health threat and economic losses. Despite implemented control programs, few countries have been able to decrease or eliminate this infection. Vaccination of the intermediate host offers an additional strategy to control the parasite transmission and EG95 antigen is considered more than the others in the vaccine issue. According to the high protection induced by the EG95 recombinant vaccine, this study was designed to construct recombinant plasmid formulation of EG95 antigen.Methods: In 2015, the Echinococcus granulosus eggs were recovered from an infected dog in Parasitological laboratory of Tarbiat Modares University in Tehran, Iran. Following hatching, the oncospheres of E. granulosus were activated to increase the presence of the desired mRNA. The extracted mRNA was transcribed to the cDNA which used as template in RT-PCR. Then the EG95 gene cloned into pET28a vector and the recombinant plasmids expression was  investigated in prokaryotic and eukaryotic cells.Results:  The recombinant plasmid encoding EG95 antigen was successfully constructed and identified by PCR, restriction enzyme digestion and sequencing. In vitro expression of the EG95 antigen was confirmed in prokary­otic and eukaryotic systems by SDS-PAGE and western blotting analysis.Conclusion: Because of potential advantages of DNA vaccines, including ability to induce long-term immune responses, low production cost and stability in different temperatures, this study carried out to construct the EG95 gene into a vector. This recombinant vector can be evaluated in further studies as a DNA vaccine may provide new prospects for the development of a vaccine against cystic hydatid disease.

Molecular epidemiology of clinical and carrier strains of methicillin resistant Staphylococcus aureus (MRSA in the hospital settings of north India

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Dar Mohammad J

2006-09-01

Full Text Available Abstract Background The study was conducted between 2000 and 2003 on 750 human subjects, yielding 850 strains of staphylococci from clinical specimens (575, nasal cultures of hospitalized patients (100 and eye & nasal sources of hospital workers (50 & 125 respectively in order to determine their epidemiology, acquisition and dissemination of resistance genes. Methods Organisms from clinical samples were isolated, cultured and identified as per the standard routine procedures. Susceptibility was measured by the agar diffusion method, as recommended by the Nat ional Committee for Clinical Laboratory Standards (NCCLS. The modified method of Birnboin and Takahashi was used for isolation of plasmids from staphylococci. Pulsed-field gel electrophoresis (PFGE typing of clinical and carrier Methicillin resistant Staphylococcus aureus (MRSA strains isolated during our study was performed as described previously. Results It was shown that 35.1% of Staphylococcus aureus and 22.5% of coagulase-negative staphylococcal isolates were resistant to methicillin. Highest percentage of MRSA (35.5% was found in pus specimens (n = 151. The multiple drug resistance of all MRSA (n = 180 and Methicillin resistant Coagulase-negative Staphylococcus aureus (MRCNS (n = 76 isolates was detected. In case of both methicillin-resistant as well as methicillin-sensitive Saphylococcal isolates zero resistance was found to vancomycin where as highest resistance was found to penicillin G followed by ampicillin. It was shown that the major reservoir of methicillin resistant staphylococci in hospitals are colonized/infected inpatients and colonized hospital workers, with carriers at risk for developing endogenous infection or transmitting infection to health care workers and patients. The results were confirmed by molecular typing using PFGE by SmaI-digestion. It was shown that the resistant markers G and T got transferred from clinical S. aureus (JS-105 to carrier S. aureus (JN-49

The Frequency of Staphylococcus aureus Isolated from Endocervix of Infertile Women in Northwest Iran

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Akhi Mohammad Taghi

2017-01-01

Full Text Available Background Infertility is one of the major social issues. Due to the asymptomatic cervical infection associated with Staphylococcus aureus (S. aureus, the majority of patients remain undiagnosed. The present study intended to assess the frequency of S. aureus isolated from infertile women’s endocervix in northwest Iran. Materials and Methods In a descriptive cross sectional study, specimens were randomly collected during vagina examination using a sterile speculum and swabbing. After performance of antibiotic susceptibility testing, polymerase chain reaction (PCR was used to identify methicillin-resistance S. aureus (MRSA and toxic shock syndrome toxin-1 (TSST-1. Results About 26 (26% and 9 (9% women’s urogenital tracts were colonized by S. aureus and Candida spp., respectively, of which three (11.5% patients were infected with fungi and S. aureus, simultaneously. Antibiotic susceptibility results showed high activity of vancomycin and co-trimoxazole on isolates. Regarding PCR results, mecA sequences were detected in 7 (26.9% strains, whilst the tst gene encoding TSST-1 was not detected in any of clinical strains. Conclusion The prevalence of S. aureus was very high in infertile women. Therefore, it demands all patients undergoing infertility treatment to be investigated thoroughly for this type of infection.

Differences in virulence genes and genome patterns of mastitis-associated Staphylococcus aureus among goat, cow, and human isolates in Taiwan.

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Chu, Chishih; Wei, Yajiun; Chuang, Shih-Te; Yu, Changyou; Changchien, Chih-Hsuan; Su, Yaochi

2013-03-01

A total of 117 mastitis-associated Staphylococcus aureus isolates from cow, goat, and human patients were analyzed for differences in antibiotic susceptibility, virulence genes, and genotypes using accessory gene regulator (agr) typing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Multidrug-resistant (MDR) S. aureus were commonly found in all sources, though they were predominantly found in human and goat isolates. Almost 70% of the isolates were resistant to ampicillin and penicillin. Host-associated virulence genes were identified as follows: tst, a gene encoding toxic shock syndrome toxin, was found in goat isolates; lukED and lukM, genes encoding leukocidin, found in cow isolates; lukPV, a gene encoding leukocidin, found in human isolates; and eta, a gene encoding for exfoliative toxin, found in both human and cow isolates. All four types of hemolysin, α, β, γ, and δ, were identified in human isolates, three types (α, γ, and δ), were identified in cow isolates, and two types (α and δ) were identified in goat isolates. Agr-typing determined agr1 to be the main subtype in human and cow isolates. PFGE and MLST analysis revealed the presence of diverse genotypes among the three sources. In conclusion, mastitis-associated, genetically diverse strains of MDR S. aureus differed in virulence genes among human, cow, and goat isolates.

Conjugal properties of the Sinorhizobium meliloti plasmid mobilome.

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Pistorio, Mariano; Giusti, María A; Del Papa, María F; Draghi, Walter O; Lozano, Mauricio J; Tejerizo, Gonzalo Torres; Lagares, Antonio

2008-09-01

The biology and biochemistry of plasmid transfer in soil bacteria is currently under active investigation because of its central role in prokaryote adaptation and evolution. In this work, we examined the conjugal properties of the cryptic plasmids present in a collection of the N(2)-fixing legume-symbiont Sinorhizobium meliloti. The study was performed on 65 S. meliloti isolates recovered from 25 humic soils of Argentina, which were grouped into 22 plasmid-profile types [i.e. plasmid operational taxonomic units (OTUs)]. The cumulative Shannon index calculated for the observed plasmid profiles showed a clear saturation plateau, thus indicating an adequate representation of the S. meliloti plasmid-profile types in the isolates studied. The results show that isolates of nearly 14% of the plasmid OTUs hosted transmissible plasmids and that isolates of 29% of the plasmid OTUs were able to retransfer the previously characterized mobilizable-cryptic plasmid pSmeLPU88b to a third recipient strain. It is noteworthy that isolates belonging to 14% of the plasmid OTUs proved to be refractory to the entrance of the model plasmid pSmeLPU88b, suggesting either the presence of surface exclusion phenomena or the occurrence of restriction incompatibility with the incoming replicon. Incompatibility for replication between resident plasmids and plasmid pSmeLPU88b was observed in c. 20% of the OTUs. The results reported here reveal a widespread compatibility among the conjugal functions of the cryptic plasmids in S. meliloti, and this fact, together with the observed high proportion of existing donor genotypes, points to the extrachromosomal compartment of the species as being an extremely active plasmid mobilome.

Cloning and sequencing of Staphylococcus aureus murC, a gene essential for cell wall biosynthesis.

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Lowe, A M; Deresiewicz, R L

1999-01-01

Staphylococcus aureus is a major human pathogen that is increasingly resistant to clinically useful antimicrobial agents. While screening for S. aureus genes expressed during mammalian infection, we isolated murC. This gene encodes UDP-N-acetylmuramoyl-L-alanine synthetase, an enzyme essential for cell wall biosynthesis in a number of bacteria. S. aureus MurC has a predicted mass 49,182 Da and complements the temperature-sensitive murC mutation of E. coli ST222. Sequence data on the DNA flanking staphylococcal murC suggests that the local gene organization there parallels that found in B. subtilis, but differs from that found in gram-negative bacterial pathogens. MurC proteins represent promising targets for broad spectrum antimicrobial drug development.

pIMP-PH114 carrying bla IMP-4 in a Klebsiella pneumoniae strain is closely related to other multidrug-resistant IncA/C2 plasmids.

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Ho, Pak-Leung; Lo, Wai-U; Chan, Jane; Cheung, Yuk-Yam; Chow, Kin-Hung; Yam, Wing-Cheong; Lin, Chi-Ho; Que, Tak-Lun

2014-02-01

The IncA/C plasmids are broad host-range vehicles which have been associated with wide dissemination of CMY-2 among Enterobacteriaceae of human and animal origins. Acquired metallo-β-lactamases (MBLs) such as the IMP-type enzymes are increasingly reported in multidrug-resistant Gram-negative bacteria worldwide, particularly in Enterobacteriaceae. We described the complete sequence of the first IMP-4-encoding IncA/C2 plasmid, pIMP-PH114 (151,885 bp), from a sequence type 1 Klebsiella pneumoniae strain that was recovered from a patient who was hospitalized in the Philippines. pIMP-PH114 consists of a backbone from the IncA/C2 plasmids, with the insertion of a novel Tn21-like class 1 integron composite structure (containing the cassette array bla IMP-4-qacG-aacA4-catB3, followed by a class C β-lactamase bla DHA-1 and the mercury resistance operon, merRTPCADE) and a sul2-floR encoding region. Phylogenetic analysis of the IncA/C repA sequences showed that pIMP-PH114 formed a subgroup with other IncA/C plasmids involved in the international spread of CMY-2, TEM-24 and NDM-1. Identical bla IMP-4 arrays have been described among different Enterobacteriaceae and Acinetobacter spp. in China, Singapore and Australia but the genetic context is different. The broad host range of IncA/C plasmids may have facilitated dissemination of the bla IMP-4 arrays among different diverse groups of bacteria.

Effect of the Plasmid-DNA Vaccination on Macroscopic and Microscopic Damage Caused by the Experimental Chronic Trypanosoma cruzi Infection in the Canine Model

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Olivia Rodríguez-Morales

2013-01-01

Full Text Available The dog is considered the main domestic reservoir for Trypanosoma cruzi infection and a suitable experimental animal model to study the pathological changes during the course of Chagas disease (CD. Vaccine development is one of CD prevention methods to protect people at risk. Two plasmids containing genes encoding a trans-sialidase protein (TcSP and an amastigote-specific glycoprotein (TcSSP4 were used as DNA vaccines in a canine model. Splenomegaly was not found in either of the recombinant plasmid-immunized groups; however, cardiomegaly was absent in animals immunized only with the plasmid containing the TcSSP4 gene. The inflammation of subendocardial and myocardial tissues was prevented only with the immunization with TcSSP4 gene. In conclusion, the vaccination with these genes has a partial protective effect on the enlargement of splenic and cardiac tissues during the chronic CD and on microscopic hearth damage, since both plasmids prevented splenomegaly but only one avoided cardiomegaly, and the lesions in heart tissue of dog immunized with plasmid containing the TcSSP4 gene covered only subepicardial tissue.

Characterization of the Complete Nucleotide Sequences of IncA/C2 Plasmids Carrying In809-Like Integrons from Enterobacteriaceae Isolates of Wildlife Origin.

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Papagiannitsis, Costas C; Kutilova, Iva; Medvecky, Matej; Hrabak, Jaroslav; Dolejska, Monika

2017-09-01

A total of 18 Enterobacteriaceae (17 from gulls and 1 from a clinical sample) collected from Australia, carrying IncA/C plasmids with the IMP-encoding In809-like integrons, were studied. Seven plasmids, being representatives of different origins, plasmid sizes, replicon combinations, and resistance genes, were completely sequenced. Plasmid pEc158, identified in a clinical Escherichia coli ST752 isolate, showed extensive similarity to type 2 IncA/C 2 plasmids. pEc158 carried none of the bla CMY-2 -like region or ARI-B and ARI-A regions, while it contained a hybrid transposon structure. The six remaining plasmids, which were of wildlife origin, were highly similar to each other and probably were fusion derivatives of type 1 and type 2 A/C 2 plasmids. The latter plasmids contained an ARI-B region and hybrid transposon structures. In all plasmids, hybrid transposon structures containing In809-like integrons were inserted 3,434 bp downstream of the rhs2 start codon. In all cases, the one outermost 38-bp inverted repeat (IR) of the transposon was associated with the Tn 1696 tnp module, while the other outermost 38-bp IR of the transposon was associated with either a Tn 6317 -like module or a Tn 21 mer module. However, the internal structure of the transposon and the resistance genes were different in each plasmid. These findings indicated that, for the specific periods of time and settings, different IncA/C 2 plasmid types carrying In809-like elements circulated among isolates of wildlife and clinical origins. Additionally, they provided the basis for speculations regarding the reshuffling of IncA/C 2 plasmids with In809-like integrons and confirmed the rapid evolution of IncA/C 2 plasmid lineages. Copyright © 2017 American Society for Microbiology.

Expansion of the IncX plasmid family for improved identification and typing of novel plasmids in drug-resistant Enterobacteriaceae

DEFF Research Database (Denmark)

Johnson, Timothy J.; Bielak, Eliza Maria; Fortini, Daniela

2012-01-01

and biofilm formation. Previous plasmid-based replicon typing procedures have indicated that the prevalence of IncX plasmids is low among members of the Enterobacteriaceae. However, examination of a number of IncX-like plasmid sequences and their occurrence in various organisms suggests that IncX plasmid...

Virulence Factors of Staphylococcus aureus Isolates in an Iranian Referral Children's Hospital.

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Sabouni, Farah; Mahmoudi, Shima; Bahador, Abbas; Pourakbari, Babak; Sadeghi, Reihaneh Hosseinpour; Ashtiani, Mohammad Taghi Haghi; Nikmanesh, Bahram; Mamishi, Setareh

2014-04-01

The clinical importance of Staphylococcus aureus (S. aureus) is attributed to notable virulence factors, surface proteins, toxins, and enzymes as well as the rapid development of drug resistance. The aim of this study was to compare the occurrence of virulence factors produced by S. aureus strains isolated from children in an Iranian referral children's hospital. The presence of genes encoding for the enterotoxins A (sea), B (seb), C (sec), D (sed), TSST-1 (tsst), exfoliative toxin A (eta), and exfoliative toxin B (etb) were detected by Multiplex polymerase chain reaction (PCR) using specific primers. In addition, the standardized Kirby-Bauer disc-diffusion method was performed on Mueller-Hinton agar. In total, 133 S. aureus isolates were obtained from different patients. Of these S. aureus isolates, 64 (48%) were methicillin-resistant S. aureus (MRSA), and all of these tested positive for the mecA gene. Regarding the classical enterotoxin genes, sea gene (40.6%) was the most prevalent followed by seb (19.6%), tsst (12.8%), eta (11.3%), etb (9%), sed (4.5%), and sec (3%). Among methicillin-susceptible S. aureus (MSSA) isolates, seb and tsst were the more prevalent toxins in comparison with MRSA isolates (p  0.05). In our study enterotoxin A was produced by 40.6% of the isolates (48% from MRSA and 33% from MSSA isolates) which was higher than in previous reports. According to our results, strict hygiene and preventative measures during food processing are highly recommended.

Microarray-based analysis of IncA/C plasmid-associated genes from multidrug-resistant Salmonella enterica.

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Lindsey, Rebecca L; Frye, Jonathan G; Fedorka-Cray, Paula J; Meinersmann, Richard J

2011-10-01

In the family Enterobacteriaceae, plasmids have been classified according to 27 incompatibility (Inc) or replicon types that are based on the inability of different plasmids with the same replication mechanism to coexist in the same cell. Certain replicon types such as IncA/C are associated with multidrug resistance (MDR). We developed a microarray that contains 286 unique 70-mer oligonucleotide probes based on sequences from five IncA/C plasmids: pYR1 (Yersinia ruckeri), pPIP1202 (Yersinia pestis), pP99-018 (Photobacterium damselae), pSN254 (Salmonella enterica serovar Newport), and pP91278 (Photobacterium damselae). DNA from 59 Salmonella enterica isolates was hybridized to the microarray and analyzed for the presence or absence of genes. These isolates represented 17 serovars from 14 different animal hosts and from different geographical regions in the United States. Qualitative cluster analysis was performed using CLUSTER 3.0 to group microarray hybridization results. We found that IncA/C plasmids occurred in two lineages distinguished by a major insertion-deletion (indel) region that contains genes encoding mostly hypothetical proteins. The most variable genes were represented by transposon-associated genes as well as four antimicrobial resistance genes (aphA, merP, merA, and aadA). Sixteen mercury resistance genes were identified and highly conserved, suggesting that mercury ion-related exposure is a stronger pressure than anticipated. We used these data to construct a core IncA/C genome and an accessory genome. The results of our studies suggest that the transfer of antimicrobial resistance determinants by transfer of IncA/C plasmids is somewhat less common than exchange within the plasmids orchestrated by transposable elements, such as transposons, integrating and conjugative elements (ICEs), and insertion sequence common regions (ISCRs), and thus pose less opportunity for exchange of antimicrobial resistance.

Diversity and evolution of blaZ from Staphylococcus aureus and coagulase-negative staphylococci

DEFF Research Database (Denmark)

Olsen, John E.; Christensen, Henrik; Aarestrup, Frank Møller

2006-01-01

and sequences retrieved from public databases were compared. A phylogenetic tree showed that blaZ exists in three evolutionary lines: one group was of plasmid origin, one group was of chromosomal origin and one intermediate group. Sixty-nine sequence types were demonstrated. They translated into 11 BlaZ protein...... types. The major types all contained strains of both human and bovine origin, and more than one Staphylococcus species, demonstrating a shared gene pool. In a comparison of S. aureus and CoNS obtained from five Danish cattle herds, the same type of blaZ was only detected in one case. Conclusions...

Interplay of a non-conjugative integrative element and a conjugative plasmid in the spread of antibiotic resistance via suicidal plasmid transfer from an aquaculture Vibrio isolate.

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Nonaka, Lisa; Yamamoto, Tatsuya; Maruyama, Fumito; Hirose, Yuu; Onishi, Yuki; Kobayashi, Takeshi; Suzuki, Satoru; Nomura, Nobuhiko; Masuda, Michiaki; Yano, Hirokazu

2018-01-01

The capture of antimicrobial resistance genes (ARGs) by mobile genetic elements (MGEs) plays a critical role in resistance acquisition for human-associated bacteria. Although aquaculture environments are recognized as important reservoirs of ARGs, intra- and intercellular mobility of MGEs discovered in marine organisms is poorly characterized. Here, we show a new pattern of interspecies ARGs transfer involving a 'non-conjugative' integrative element. To identify active MGEs in a Vibrio ponticus isolate, we conducted whole-genome sequencing of a transconjugant obtained by mating between Escherichia coli and Vibrio ponticus. This revealed integration of a plasmid (designated pSEA1) into the chromosome, consisting of a self-transmissible plasmid backbone of the MOBH group, ARGs, and a 13.8-kb integrative element Tn6283. Molecular genetics analysis suggested a two-step gene transfer model. First, Tn6283 integrates into the recipient chromosome during suicidal plasmid transfer, followed by homologous recombination between the Tn6283 copy in the chromosome and that in the newly transferred pSEA1. Tn6283 is unusual among integrative elements in that it apparently does not encode transfer function and its excision barely generates unoccupied donor sites. Thus, its movement is analogous to the transposition of insertion sequences rather than to that of canonical integrative and conjugative elements. Overall, this study reveals the presence of a previously unrecognized type of MGE in a marine organism, highlighting diversity in the mode of interspecies gene transfer.

Portación y caracterización de Staphylococcus aureus en manipuladores de alimentos Carriage and characterization of Staphylococcus aureus in food handlers

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Graciela B Jordá

2012-06-01

Full Text Available Staphylococcus aureus es una causa de intoxicaciones alimentarias por su capacidad de producir enterotoxinas. Los manipuladores de alimentos que portan S. aureus productores de enterotoxinas pueden provocar intoxicaciones alimentarias. Se estudiaron muestras tomadas de fosas nasales de 88 manipuladores de alimentos en la provincia de Misiones. El 37,5 % de los individuos analizados eran portadores de S. aureus. Mediante técnicas de amplificación (PCR, se detectaron genes que codifican la producción de enterotoxinas en 13 de los 33 aislamientos obtenidos (39,4 % y en el 14,7 % de los manipuladores. De estos aislamientos, 10 portaban el gen sea y 3 el gen sec. El estudio de sensibilidad a los antibióticos mostró un 100 % de sensibilidad a teicoplanina, gentamiclna y rifampicina; 2 aislamientos fueron resistentes a clindamicina y a eritromicina y 4 resultaron resistentes a la meticilina. Estos resultados son un alerta e indicarían la necesidad de desarrollar medidas racionales para reducir el riesgo potencial de intoxicaciones alimentarias.Staphylococcus aureus causes food poisoning due to its ability to produce enterotoxins. Food handlers carrying enterotoxin-producing S. aureus can contaminate food, thus leading to food poisoning. Samples were obtained from 88 food handlers in the Province of Misiones, Argentina. S. aureus was isolated from nasal swaps and PCR amplification was performed for genes encoding staphylococcal enterotoxins. A total of 37.5 % food handlers were positive for S. aureus. Expression of enterotoxin genes was found in 13 of the 33 (39.4 % S. aureus isolates studied, accounting for 14.7 % of food handlers. Gene sea was detected in 10 isolates followed by gene sec in 3 isolates. All isolates were susceptible to teicoplanin, gentamicin and rifampicin. Four isolates were resistant to methicillin whereas 2 isolates were resistant to clindamycin and erythromycin. These results constitute a critical alert and indicate the need

Phage Conversion for β-Lactam Antibiotic Resistance of Staphylococcus aureus from Foods.

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Lee, Young-Duck; Park, Jong-Hyun

2016-02-01

Temperate phages have been suggested to carry virulence factors and other lysogenic conversion genes that play important roles in pathogenicity. In this study, phage TEM123 in wild-type Staphylococcus aureus from food sources was analyzed with respect to its morphology, genome sequence, and antibiotic resistance conversion ability. Phage TEM123 from a mitomycin C-induced lysate of S. aureus was isolated from foods. Morphological analysis under a transmission electron microscope revealed that it belonged to the family Siphoviridae. The genome of phage TEM123 consisted of a double-stranded DNA of 43,786 bp with a G+C content of 34.06%. A bioinformatics analysis of the phage genome identified 43 putative open reading frames (ORFs). ORF1 encoded a protein that was nearly identical to the metallo-β-lactamase enzymes that degrade β-lactam antibiotics. After transduction to S. aureus with phage TEM123, the metallo-β-lactamase gene was confirmed in the transductant by PCR and sequencing analyses. In a β-lactam antibiotic susceptibility test, the transductant was more highly resistant to β-lactam antibiotics than S. aureus S133. Phage TEM123 might play a role in the transfer of β-lactam antibiotic resistance determinants in S. aureus. Therefore, we suggest that the prophage of S. aureus with its exotoxin is a risk factor for food safety in the food chain through lateral gene transfer.

Comparative Sequence Analysis of Plasmids from Lactobacillus delbrueckii and Construction of a Shuttle Cloning Vector▿

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Lee, Ju-Hoon; Halgerson, Jamie S.; Kim, Jeong-Hwan; O'Sullivan, Daniel J.

2007-01-01

While plasmids are very commonly associated with the majority of the lactic acid bacteria, they are only very rarely associated with Lactobacillus delbrueckii, with only four characterized to date. In this study, the complete sequence of a native plasmid, pDOJ1, from a strain of Lactobacillus delbrueckii subsp. bulgaricus was determined. It consisted of a circular DNA molecule of 6,220 bp with a G+C content of 44.6% and a characteristic ori and encoded six open reading frames (ORFs), of which functions could be predicted for three—a mobilization (Mob) protein, a transposase, and a fused primase-helicase replication protein. Comparative analysis of pDOJ1 and the other available L. delbrueckii plasmids (pLBB1, pJBL2, pN42, and pLL1212) revealed a very similar organization and amino acid identities between 85 and 98% for the putative proteins of all six predicted ORFs from pDOJ1, reflecting a common origin for L. delbrueckii plasmids. Analysis of the fused primase-helicase replication gene found a similar fused organization only in the theta replicating group B plasmids from Streptococcus thermophilus. This observation and the ability of the replicon to function in S. thermophilus support the idea that the origin of plasmids in L. delbrueckii was likely from S. thermophilus. This may reflect the close association of these two species in dairy fermentations, particularly yogurt production. As no vector based on plasmid replicons from L. delbrueckii has previously been constructed, an Escherichia coli-L. delbrueckii shuttle cloning vector, pDOJ4, was constructed from pDOJ1, the p15A ori, the chloramphenicol resistance gene of pCI372, and the lacZ polylinker from pUC18. This cloning vector was successfully introduced into E. coli, L. delbrueckii subsp. bulgaricus, S. thermophilus, and Lactococcus lactis. This shuttle cloning vector provides a new tool for molecular analysis of Lactobacillus delbrueckii and other lactic acid bacteria. PMID:17526779

Molecular characterization of a 21.4 kilobase antibiotic resistance plasmid from an α-hemolytic Escherichia coli O108:H- human clinical isolate.

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Fay E Dawes

Full Text Available This study characterizes the 21.4 kilobase plasmid pECTm80 isolated from Escherichia coli strain 80, an α hemolytic human clinical diarrhoeal isolate (serotype O108:H-. DNA sequence analysis of pECTm80 revealed it belonged to incompatibility group X1, and contained plasmid partition and toxin-antitoxin systems, an R6K-like triple origin (ori replication system, genes required for replication regulation, insertion sequences IS1R, ISEc37 and a truncated transposase gene (Tn3-like ΔtnpA of the Tn3 family, and carried a class 2 integron. The class 2 integron of pECTm80 contains an intact cassette array dfrA1-sat2, encoding resistance to trimethoprim and streptothricin, and an aadA1 gene cassette truncated by the insertion of IS1R. The complex plasmid replication system includes α, β and γ origins of replication. Pairwise BLASTn comparison of pECTm80 with plasmid pE001 reveals a conserved plasmid backbone suggestive of a common ancestral lineage. Plasmid pECTm80 is of potential clinical importance, as it carries multiple genes to ensure its stable maintenance through successive bacterial cell divisions and multiple antibiotic resistance genes.

Genetical and functional investigation of fliC genes encoding flagellar serotype H4 in wildtype strains of Escherichia coli and in a laboratory E. coli K-12 strain expressing flagellar antigen type H48

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Schaudinn Christoph

2005-01-01

Full Text Available Abstract Background Serotyping of O-(lipopolysaccharide and H-(flagellar antigens is a wideley used method for identification of pathogenic strains and clones of Escherichia coli. At present, 176 O- and 53 H-antigens are described for E. coli which occur in different combinations in the strains. The flagellar antigen H4 is widely present in E. coli strains of different O-serotypes and pathotypes and we have investigated the genetic relationship between H4 encoding fliC genes by PCR, nucleotide sequencing and expression studies. Results The complete nucleotide sequence of fliC genes present in E. coli reference strains U9-41 (O2:K1:H4 and P12b (O15:H17 was determined and both were found 99.3% (1043 of 1050 nucleotides identical in their coding sequence. A PCR/RFLP protocol was developed for typing of fliC-H4 strains and 88 E. coli strains reacting with H4 antiserum were investigated. Nucleotide sequencing of complete fliC genes of six E. coli strains which were selected based on serum agglutination titers, fliC-PCR genotyping and reference data revealed 96.6 to 100% identity on the amino acid level. The functional expression of flagellin encoded by fliC-H4 from strain U9-41 and from our strain P12b which is an H4 expressing variant type was investigated in the E. coli K-12 strain JM109 which encodes flagellar type H48. The fliC recombinant plasmid carrying JM109 strains reacted with both H4 and H48 specific antisera whereas JM109 reacted only with the H48 antiserum. By immunoelectron microscopy, we could show that the flagella made by the fliC-H4 recombinant plasmid carrying strain are constituted of H48 and H4 flagellins which are co-assembled into functional flagella. Conclusion The flagellar serotype H4 is encoded by closely related fliC genes present in serologically different types of E. coli strainswhich were isolated at different time periods and geographical locations. Our expression studies show for the first time, that flagellins of

Plasmid-mediated AmpC-type beta-lactamase isolated from Klebsiella pneumoniae confers resistance to broad-spectrum beta-lactams, including moxalactam.

Science.gov (United States)

Horii, T; Arakawa, Y; Ohta, M; Ichiyama, S; Wacharotayankun, R; Kato, N

1993-01-01

Klebsiella pneumoniae NU2936 was isolated from a patient and was found to produce a plasmid-encoded beta-lactamase (MOX-1) which conferred resistance to broad spectrum beta-lactams, including moxalactam, flomoxef, ceftizoxime, cefotaxime, and ceftazidime. Resistance could be transferred from K. pneumoniae NU2936 to Escherichia coli CSH2 by conjugation with a transfer frequency of 5 x 10(-7). The structural gene of MOX-1 (blaMOX-1) was cloned and expressed in E. coli HB101. The MIC of moxalactam for E. coli HB101 producing MOX-1 was > 512 micrograms/ml. The apparent molecular mass and pI of this enzyme were calculated to be 38 kDa and 8.9, respectively. Hg2+ and Cu2+ failed to block enzyme activity, and the presence of EDTA in the reaction buffer did not reduce the enzyme activity. However, clavulanate and cloxacillin, serine beta-lactamase inhibitors, inhibited the enzyme activity competitively (Kis = 5.60 and 0.35 microM, respectively). The kinetic study of MOX-1 suggested that it effectively hydrolyzed broad-spectrum beta-lactams. A hybridization study confirmed that blaMOX-1 is encoded on a large resident plasmid (pRMOX1; 180 kb) of strain NU2936. By deletion analysis, the functional region was localized within a 1.2-kb region of the plasmid. By amino acid sequencing, 18 of 33 amino acid residues at the N terminus of MOX-1 were found to be identical to those of Pseudomonas aeruginosa AmpC. These findings suggest that MOX-1 is a plasmid-mediated AmpC-type beta-lactamase that provides enteric bacteria resistance to broad-spectrum beta-lactams, including moxalactam. Images PMID:8517725

Bacillus caldolyticus prs gene encoding phosphoribosyldiphosphate synthase

DEFF Research Database (Denmark)

Krath, Britta N.; Hove-Jensen, Bjarne

1996-01-01

The prs gene, encoding phosphoribosyl-diphosphate (PRPP) synthase, as well as the flanking DNA sequences were cloned and sequenced from the Gram-positive thermophile, Bacillus caldolyticus. Comparison with the homologous sequences from the mesophile, Bacillus subtilis, revealed a gene (gca......D) encoding N-acetylglucosamine-l-phosphate uridyltransferase upstream of prs, and a gene homologous to ctc downstream of prs. cDNA synthesis with a B. caldolyticus gcaD-prs-ctc-specified mRNA as template, followed by amplification utilising the polymerase chain reaction indicated that the three genes are co......-transcribed. Comparison of amino acid sequences revealed a high similarity among PRPP synthases across a wide phylogenetic range. An E. coli strain harbouring the B. caldolyticus prs gene in a multicopy plasmid produced PRPP synthase activity 33-fold over the activity of a haploid B. caldolyticus strain. B. caldolyticus...

Association and Expression of Virulence from Plasmids of the Group B Strain in Pseudomonas syringae pv. eriobotryae

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Tran Dang Khanh

2018-04-01

Full Text Available Pseudomonas syringae pv. eriobotryae causes serious stem canker in loquat (Eriobotrya japonica trees. This study was conducted to determine whether plasmids are involved with its virulence. The strain NAE89, which belonged to the B group, harbored two plasmids at approximately 6.2 and 50 Mdal that caused stem canker and halo leaf spots on loquat plants. Following digestion with BamHI and ligation into the BamHI cloning site of the broad range host cosmid pLAFR3, four DNA fragments at 3.8, 6.6, 12.3, and 22.8 kb were generated. Although the plasmid-encoded virulence gene psvA was undigested with the BamHI, the halo leaf spot gene may be adjacent to the psvA gene was digested. A pLAFR3 cosmid clone was introduced into the non-pathogenic PE0 and NAE89-1 strains by triparental matings and the pathogenicity was recovered. As a result, the pLAFR3 cosmid clone was introduced into the largest size DNA fragment of 22.8 kb and determined to be the causal agent of canker on the stem of the loquat. This study revealed that the psvA gene, previously found in the 50 Mdal plasmid, was also observed in the 22.8 kb DNA fragment.

Characterization of a mouse-adapted Staphylococcus aureus strain.

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Silva Holtfreter

Full Text Available More effective antibiotics and a protective vaccine are desperately needed to combat the 'superbug' Staphylococcus aureus. While in vivo pathogenicity studies routinely involve infection of mice with human S. aureus isolates, recent genetic studies have demonstrated that S. aureus lineages are largely host-specific. The use of such animal-adapted S. aureus strains may therefore be a promising approach for developing more clinically relevant animal infection models. We have isolated a mouse-adapted S. aureus strain (JSNZ which caused a severe outbreak of preputial gland abscesses among male C57BL/6J mice. We aimed to extensively characterize this strain on a genomic level and determine its virulence potential in murine colonization and infection models. JSNZ belongs to the MLST type ST88, rare among human isolates, and lacks an hlb-converting phage encoding human-specific immune evasion factors. Naive mice were found to be more susceptible to nasal and gastrointestinal colonization with JSNZ than with the human-derived Newman strain. Furthermore, naïve mice required antibiotic pre-treatment to become colonized with Newman. In contrast, JSNZ was able to colonize mice in the absence of antibiotic treatment suggesting that this strain can compete with the natural flora for space and nutrients. In a renal abscess model, JSNZ caused more severe disease than Newman with greater weight loss and bacterial burden. In contrast to most other clinical isolates, JSNZ can also be readily genetically modified by phage transduction and electroporation. In conclusion, the mouse-adapted strain JSNZ may represent a valuable tool for studying aspects of mucosal colonization and for screening novel vaccines and therapies directed at preventing colonization.

Characterization of new plasmids from methylotrophic bacteria.

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Brenner, V; Holubová, I; Benada, O; Hubácek, J

1991-07-01

Several tens of methanol-utilizing bacterial strains isolated from soil were screened for the presence of plasmids. From the obligate methylotroph Methylomonas sp. strain R103a plasmid pIH36 (36 kb) was isolated and its restriction map was constructed. In pink-pigmented facultative methylotrophs (PPFM), belonging to the genus Methylobacterium four plasmids were detected: plasmids pIB200 (200 kb) and pIB14 (14 kb) in the strain R15d and plasmids pWU14 (14 kb) and pWU7 (7.8 kb) in the strain M17. Because of the small size and the presence of several unique REN sites (HindIII, EcoRI, NcoI), plasmid pWU7 was chosen for the construction of a vector for cloning in methylotrophs. Cointegrates pKWU7A and pKWU7B were formed between pWU7 and the E. coli plasmid pK19 Kmr, which were checked for conjugative transfer from E. coli into the methylotrophic host.

Hydrodynamic delivery of plasmid DNA encoding human FcγR-Ig dimers blocks immune-complex mediated inflammation in mice.

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Shashidharamurthy, R; Machiah, D; Bozeman, E N; Srivatsan, S; Patel, J; Cho, A; Jacob, J; Selvaraj, P

2012-09-01

Therapeutic use and function of recombinant molecules can be studied by the expression of foreign genes in mice. In this study, we have expressed human Fcγ receptor-Ig fusion molecules (FcγR-Igs) in mice by administering FcγR-Ig plasmid DNAs hydrodynamically and compared their effectiveness with purified molecules in blocking immune-complex (IC)-mediated inflammation in mice. The concentration of hydrodynamically expressed FcγR-Igs (CD16A(F)-Ig, CD32A(R)-Ig and CD32A(H)-Ig) reached a maximum of 130 μg ml(-1) of blood within 24 h after plasmid DNA administration. The in vivo half-life of FcγR-Igs was found to be 9-16 days and western blot analysis showed that the FcγR-Igs were expressed as a homodimer. The hydrodynamically expressed FcγR-Igs blocked 50-80% of IC-mediated inflammation up to 3 days in a reverse passive Arthus reaction model. Comparative analysis with purified molecules showed that hydrodynamically expressed FcγR-Igs are more efficient than purified molecules in blocking IC-mediated inflammation and had a higher half-life. In summary, these results suggest that the administration of a plasmid vector with the FcγR-Ig gene can be used to study the consequences of blocking IC binding to FcγRs during the development of inflammatory diseases. This approach may have potential therapeutic value in treating IC-mediated inflammatory autoimmune diseases such as lupus, arthritis and autoimmune vasculitis.

Characteristics of plasmids in multi-drug-resistant Enterobacteriaceae isolated during prospective surveillance of a newly opened hospital in Iraq.

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Xiao-Zhe Huang

Full Text Available BACKGROUND: Gram-negative multidrug-resistant (MDR bacteria are major causes of nosocomial infections, and antibiotic resistance in these organisms is often plasmid mediated. Data are scarce pertaining to molecular mechanisms of antibiotic resistance in resource constrained areas such as Iraq. METHODOLOGY/PRINCIPAL FINDINGS: In this study, all MDR Enterobacteriaceae (n = 38 and randomly selected non-MDR counterparts (n = 41 isolated from patients, healthcare workers and environmental surfaces in a newly opened hospital in Iraq were investigated to characterize plasmids found in these isolates and determine their contribution to antibiotic resistance. Our results demonstrated that MDR E. coli and K. pneumoniae isolates harbored significantly more (≥ 3 plasmids compared to their non-MDR counterparts, which carried ≤ 2 plasmids (p<0.01. Various large plasmids (~52 to 100 kb from representative isolates were confirmed to contain multiple resistance genes by DNA microarray analysis. Aminoglycoside (acc, aadA, aph, strA/B, and ksgA, β-lactam (bla(TEM1, bla(AMPC, bla(CTX-M-15, bla(OXA-1, bla(VIM-2 and bla(SHV, sulfamethoxazole/trimethoprim (sul/dfr, tetracycline (tet and chloramphenicol (cat resistance genes were detected on these plasmids. Additionally, multiple plasmids carrying multiple antibiotic resistance genes were found in the same host strain. Genetic transfer-associated genes were identified on the plasmids from both MDR and non-MDR isolates. Seven plasmid replicon types (FII, FIA, FIB, B/O, K, I1 and N were detected in the isolates, while globally disseminated IncA/C and IncHI1 plasmids were not detected in these isolates. CONCLUSIONS/SIGNIFICANCE: This is the first report of the characteristics of the plasmids found in Enterobacteriaceae isolated following the opening of a new hospital in Iraq. The information provided here furthers our understanding of the mechanisms of drug resistance in this specific region and their evolutionary

Explanatory chapter: how plasmid preparation kits work.

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Koontz, Laura

2013-01-01

To isolate plasmid DNA from bacteria using a commercial plasmid miniprep kit (if interested, compare this protocol with Isolation of plasmid DNA from bacteria). Copyright © 2013 Elsevier Inc. All rights reserved.

High diversity of plasmids harbouring bla_CMY-2 among clinical Escherichia coli isolates from humans and companion animals in the upper Midwestern USA

DEFF Research Database (Denmark)

Bortolaia, Valeria; Hansen, Katrine Hartung; Nielsen, Christine

2014-01-01

To determine the population structure and genetic relatedness of plasmids encoding CMY-2 β-lactamase in clinical Escherichia coli from humans and companion animals within a defined geographical area....

Screening of the Enterocin-Encoding Genes and Antimicrobial Activity in Enterococcus Species.

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Ogaki, Mayara Baptistucci; Rocha, Katia Real; Terra, MÁrcia Regina; Furlaneto, MÁrcia Cristina; Maia, Luciana Furlaneto

2016-06-28

In the current study, a total of 135 enterococci strains from different sources were screened for the presence of the enterocin-encoding genes entA, entP, entB, entL50A, and entL50B. The enterocin genes were present at different frequencies, with entA occurring the most frequently, followed by entP and entB; entL50A and L50B were not detected. The occurrence of single enterocin genes was higher than the occurrence of multiple enterocin gene combinations. The 80 isolates that harbor at least one enterocin-encoding gene (denoted "Gene(+) strains") were screened for antimicrobial activity. A total of 82.5% of the Gene(+) strains inhibited at least one of the indicator strains, and the isolates harboring multiple enterocin-encoding genes inhibited a larger number of indicator strains than isolates harboring a single gene. The indicator strains that exhibited growth inhibition included Listeria innocua strain CLIP 12612 (ATCC BAA-680), Listeria monocytogenes strain CDC 4555, Enterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, S. aureus ATCC 29213, S. aureus ATCC 6538, Salmonella enteritidis ATCC 13076, Salmonella typhimurium strain UK-1 (ATCC 68169), and Escherichia coli BAC 49LT ETEC. Inhibition due to either bacteriophage lysis or cytolysin activity was excluded. The growth inhibition of antilisterial Gene+ strains was further tested under different culture conditions. Among the culture media formulations, the MRS agar medium supplemented with 2% (w/v) yeast extract was the best solidified medium for enterocin production. Our findings extend the current knowledge of enterocin-producing enterococci, which may have potential applications as biopreservatives in the food industry due to their capability of controlling food spoilage pathogens.

Origin and evolution of European community-acquired methicillin-resistant Staphylococcus aureus.

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Stegger, Marc; Wirth, Thierry; Andersen, Paal S; Skov, Robert L; De Grassi, Anna; Simões, Patricia Martins; Tristan, Anne; Petersen, Andreas; Aziz, Maliha; Kiil, Kristoffer; Cirković, Ivana; Udo, Edet E; del Campo, Rosa; Vuopio-Varkila, Jaana; Ahmad, Norazah; Tokajian, Sima; Peters, Georg; Schaumburg, Frieder; Olsson-Liljequist, Barbro; Givskov, Michael; Driebe, Elizabeth E; Vigh, Henrik E; Shittu, Adebayo; Ramdani-Bougessa, Nadjia; Rasigade, Jean-Philippe; Price, Lance B; Vandenesch, Francois; Larsen, Anders R; Laurent, Frederic

2014-08-26

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) was recognized in Europe and worldwide in the late 1990s. Within a decade, several genetically and geographically distinct CA-MRSA lineages carrying the small SCCmec type IV and V genetic elements and the Panton-Valentine leukocidin (PVL) emerged around the world. In Europe, the predominant CA-MRSA strain belongs to clonal complex 80 (CC80) and is resistant to kanamycin/amikacin and fusidic acid. CC80 was first reported in 1993 but was relatively rare until the late 1990s. It has since been identified throughout North Africa, the Middle East, and Europe, with recent sporadic reports in sub-Saharan Africa. While strongly associated with skin and soft tissue infections, it is rarely found among asymptomatic carriers. Methicillin-sensitive S. aureus (MSSA) CC80 strains are extremely rare except in sub-Saharan Africa. In the current study, we applied whole-genome sequencing to a global collection of both MSSA and MRSA CC80 isolates. Phylogenetic analyses strongly suggest that the European epidemic CA-MRSA lineage is derived from a PVL-positive MSSA ancestor from sub-Saharan Africa. Moreover, the tree topology suggests a single acquisition of both the SCCmec element and a plasmid encoding the fusidic acid resistance determinant. Four canonical SNPs distinguish the derived CA-MRSA lineage and include a nonsynonymous mutation in accessory gene regulator C (agrC). These changes were associated with a star-like expansion into Europe, the Middle East, and North Africa in the early 1990s, including multiple cases of cross-continent imports likely driven by human migrations. With increasing levels of CA-MRSA reported from most parts of the Western world, there is a great interest in understanding the origin and factors associated with the emergence of these epidemic lineages. To trace the origin, evolution, and dissemination pattern of the European CA-MRSA clone (CC80), we sequenced a global collection

Detection of toxic shock toxin (tst gene in Staphylococcus aureus isolated from bovine milk samples

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S. Baniardalan

2017-09-01

Full Text Available Staphylococcus aureus is a major causative pathogen of clinical and subclinical mastitis in dairy cattle all over the world. This agent produces a variety of extracellular toxins and virulence factors in-cluding toxic shock syndrome toxin-1 (TSST-1 which is the major cause of toxic shock syndrome (TSS. In the present study, 76 S. aureus isolates have been obtained from milk samples collected from 7 dairy herds in Hamedan province of Iran. The isolates were identified based on the biochemical and molecular methods using PCR amplification of the femA gene. The staphylococcal isolates were also examined for the presence of TSST-1 (tst encoding gene. This gene was detected in only one S. aureus isolate (1.3%. The results revealed that S. aureus strains causing bovine mastitis may potentially produce staphylococcal toxic shock syndrome toxin-1, indicating that it is very important to follow the presence of TSST-1 producing S. aureus isolates in foodstuffs to protect consumers against the risk of toxic shock syndrome

Detection of an IncA/C plasmid encoding VIM-4 and CMY-4 β-lactamases in Klebsiella oxytoca and Citrobacter koseri from an inpatient in a cardiac rehabilitation unit.

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Caltagirone, Mariasofia; Bitar, Ibrahim; Piazza, Aurora; Spalla, Melissa; Nucleo, Elisabetta; Navarra, Antonella; Migliavacca, Roberta

2015-07-01

A 62-year-old patient was transferred to the cardiac rehabilitation unit of the I.R.C.C.S. Fondazione S. Maugeri after undergoing a heart transplantation at the Acute Care Hospital I.R.C.C.S. S. Matteo of Pavia. On 1 August 2013 and during hospitalization in the rehabilitation unit, Klebsiella oxytoca and Citrobacter koseri clinical isolates were simultaneously recovered from the patient's preputial swab. Both the K. oxytoca and C. koseri strains were carbapenem- resistant by MicroScan System (Beckman Coulter). Carbapenem-resistant K. pneumoniae had previously been reported in the same rehabilitation facility. The aim of the study was to identify the carbapenem resistance mechanisms among the enterobacterial species recovered. Phenotypic screening tests useful to detect the β-lactamases/carbapenemases were performed. Carbapenem MICs were obtained by Etest. AmpC and MBL encoding genes were identified by PCR and sequencing. Conjugation assays and plasmid characterization were performed. Both of the K. oxytoca and C. koseri isolates were multi drug resistant, showing resistance to amoxicillin-clavulanic acid, three generation cephalosporins, ertapenem (K. oxytoca MIC, >32 mg/L; C. koseri MIC, 4 mg/L), imipenem (K. oxytoca MIC, 4 mg/L; C. koseri MIC, 12 mg/L), thrimethoprim sulphamethoxazole and gentamicin. Susceptibility was retained to fluoroquinolones, colistin and tigecycline. Molecular characterization confirmed the co-presence of blaCMY-4 and blaVIM-4 determinants in a 150 Kb transferable plasmid of IncA/C group. This case is the first detection in Italy of the K. oxytoca and C. koseri clinical isolates co-producing the CMY-4 and VIM-4 enzymes.

Alterations in the transcriptome and antibiotic susceptibility of Staphylococcus aureus grown in the presence of diclofenac

Science.gov (United States)

2011-01-01

Background Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) which has been shown to increase the susceptibility of various bacteria to antimicrobials and demonstrated to have broad antimicrobial activity. This study describes transcriptome alterations in S. aureus strain COL grown with diclofenac and characterizes the effects of this NSAID on antibiotic susceptibility in laboratory, clinical and diclofenac reduced-susceptibility (DcRS) S. aureus strains. Methods Transcriptional alterations in response to growth with diclofenac were measured using S. aureus gene expression microarrays and quantitative real-time PCR. Antimicrobial susceptibility was determined by agar diffusion MICs and gradient plate analysis. Ciprofloxacin accumulation was measured by fluorescence spectrophotometry. Results Growth of S. aureus strain COL with 80 μg/ml (0.2 × MIC) of diclofenac resulted in the significant alteration by ≥2-fold of 458 genes. These represented genes encoding proteins for transport and binding, protein and DNA synthesis, and the cell envelope. Notable alterations included the strong down-regulation of antimicrobial efflux pumps including mepRAB and a putative emrAB/qacA-family pump. Diclofenac up-regulated sigB (σB), encoding an alternative sigma factor which has been shown to be important for antimicrobial resistance. Staphylococcus aureus microarray metadatabase (SAMMD) analysis further revealed that 46% of genes differentially-expressed with diclofenac are also σB-regulated. Diclofenac altered S. aureus susceptibility to multiple antibiotics in a strain-dependent manner. Susceptibility increased for ciprofloxacin, ofloxacin and norfloxacin, decreased for oxacillin and vancomycin, and did not change for tetracycline or chloramphenicol. Mutation to DcRS did not affect susceptibility to the above antibiotics. Reduced ciprofloxacin MICs with diclofenac in strain BB255, were not associated with increased drug accumulation. Conclusions The results of

The effect of mutation on Rhodococcus equi virulence plasmid gene expression and mouse virulence.

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Ren, Jun; Prescott, John F

2004-11-15

An 81 kb virulence plasmid containing a pathogenicity island (PI) plays a crucial role in the pathogenesis of Rhodococcus equi pneumonia in foals but its specific function in virulence and regulation of plasmid-encoded virulence genes is unclear. Using a LacZ selection marker developed for R. equi in this study, in combination with an apramycin resistance gene, an efficient two-stage homologous recombination targeted gene mutation procedure was used to mutate three virulence plasmid genes, a LysR regulatory gene homologue (ORF4), a ResD-like two-component response regulator homologue (ORF8), and a gene (ORF10) of unknown function that is highly expressed by R. equi inside macrophages, as well as the chromosomal gene operon, phoPR. Virulence testing by liver clearance after intravenous injection in mice showed that the ORF4 and ORF8 mutants were fully attenuated, that the phoPR mutant was hypervirulent, and that virulence of the ORF10 mutant remained unchanged. A virulence plasmid DNA microarray was used to compare the plasmid gene expression profile of each of the four gene-targeted mutants against the parental R. equi strain. Changes were limited to PI genes and gene induction was observed for all mutants, suggesting that expression of virulence plasmid genes is dominated by a negative regulatory network. The finding of attenuation of ORF4 and ORF8 mutants despite enhanced transcription of vapA suggests that factors other than VapA are important for full expression of virulence. ORF1, a putative Lsr antigen gene, was strongly and similarly induced in all mutants, implying a common regulatory pathway affecting this gene for all four mutated genes. ORF8 is apparently the centre of this common pathway. Two distinct highly correlated gene induction patterns were observed, that of the ORF4 and ORF8 mutants, and that of the ORF10 and phoPR mutants. The gene induction pattern distinguishing these two groups paralleled their virulence in mice.

The Bacillus subtilis Conjugative Plasmid pLS20 Encodes Two Ribbon-Helix-Helix Type Auxiliary Relaxosome Proteins That Are Essential for Conjugation

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Andrés Miguel-Arribas

2017-11-01

Full Text Available Bacterial conjugation is the process by which a conjugative element (CE is transferred horizontally from a donor to a recipient cell via a connecting pore. One of the first steps in the conjugation process is the formation of a nucleoprotein complex at the origin of transfer (oriT, where one of the components of the nucleoprotein complex, the relaxase, introduces a site- and strand specific nick to initiate the transfer of a single DNA strand into the recipient cell. In most cases, the nucleoprotein complex involves, besides the relaxase, one or more additional proteins, named auxiliary proteins, which are encoded by the CE and/or the host. The conjugative plasmid pLS20 replicates in the Gram-positive Firmicute bacterium Bacillus subtilis. We have recently identified the relaxase gene and the oriT of pLS20, which are separated by a region of almost 1 kb. Here we show that this region contains two auxiliary genes that we name aux1LS20 and aux2LS20, and which we show are essential for conjugation. Both Aux1LS20 and Aux2LS20 are predicted to contain a Ribbon-Helix-Helix DNA binding motif near their N-terminus. Analyses of the purified proteins show that Aux1LS20 and Aux2LS20 form tetramers and hexamers in solution, respectively, and that they both bind preferentially to oriTLS20, although with different characteristics and specificities. In silico analyses revealed that genes encoding homologs of Aux1LS20 and/or Aux2LS20 are located upstream of almost 400 relaxase genes of the RelLS20 family (MOBL of relaxases. Thus, Aux1LS20 and Aux2LS20 of pLS20 constitute the founding member of the first two families of auxiliary proteins described for CEs of Gram-positive origin.

The Bacillus subtilis Conjugative Plasmid pLS20 Encodes Two Ribbon-Helix-Helix Type Auxiliary Relaxosome Proteins That Are Essential for Conjugation.

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Miguel-Arribas, Andrés; Hao, Jian-An; Luque-Ortega, Juan R; Ramachandran, Gayetri; Val-Calvo, Jorge; Gago-Córdoba, César; González-Álvarez, Daniel; Abia, David; Alfonso, Carlos; Wu, Ling J; Meijer, Wilfried J J

2017-01-01

Bacterial conjugation is the process by which a conjugative element (CE) is transferred horizontally from a donor to a recipient cell via a connecting pore. One of the first steps in the conjugation process is the formation of a nucleoprotein complex at the origin of transfer ( oriT ), where one of the components of the nucleoprotein complex, the relaxase, introduces a site- and strand specific nick to initiate the transfer of a single DNA strand into the recipient cell. In most cases, the nucleoprotein complex involves, besides the relaxase, one or more additional proteins, named auxiliary proteins, which are encoded by the CE and/or the host. The conjugative plasmid pLS20 replicates in the Gram-positive Firmicute bacterium Bacillus subtilis . We have recently identified the relaxase gene and the oriT of pLS20, which are separated by a region of almost 1 kb. Here we show that this region contains two auxiliary genes that we name aux1 LS20 and aux2 LS20 , and which we show are essential for conjugation. Both Aux1 LS20 and Aux2 LS20 are predicted to contain a Ribbon-Helix-Helix DNA binding motif near their N-terminus. Analyses of the purified proteins show that Aux1 LS20 and Aux2 LS20 form tetramers and hexamers in solution, respectively, and that they both bind preferentially to oriT LS20 , although with different characteristics and specificities. In silico analyses revealed that genes encoding homologs of Aux1 LS20 and/or Aux2 LS20 are located upstream of almost 400 relaxase genes of the Rel LS20 family (MOB L ) of relaxases. Thus, Aux1 LS20 and Aux2 LS20 of pLS20 constitute the founding member of the first two families of auxiliary proteins described for CEs of Gram-positive origin.

Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46.

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Balachandran, Manasi; Giannone, Richard J; Bemis, David A; Kania, Stephen A

2017-01-01

Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A) gene and binds to immunoglobulin (Ig). The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA) enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins with an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models.

Conjugative Plasmid Transfer in Xylella fastidiosa Is Dependent on tra and trb Operon Functions.

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Burbank, Lindsey P; Van Horn, Christopher R

2017-11-01

The insect-transmitted plant pathogen Xylella fastidiosa is capable of efficient horizontal gene transfer (HGT) and recombination. Natural transformation occurs at high rates in X. fastidiosa , but there also is evidence that certain strains of X. fastidiosa carry native plasmids equipped with transfer and mobilization genes, suggesting conjugation as an additional mechanism of HGT in some instances. Two operons, tra and trb , putatively encoding a conjugative type IV secretion system, are found in some but not all X. fastidiosa isolates, often on native plasmids. X. fastidiosa strains that carry the conjugative transfer genes can belong to different subspecies and frequently differ in host ranges. Using X. fastidiosa strain M23 ( X. fastidiosa subsp. fastidiosa ) or Dixon ( X. fastidiosa subsp. multiplex ) as the donor strain and Temecula ( X. fastidiosa subsp. fastidiosa ) as the recipient strain, plasmid transfer was characterized using the mobilizable broad-host-range vector pBBR5pemIK. Transfer of plasmid pBBR5pemIK was observed under in vitro conditions with both donor strains and was dependent on both tra and trb operon functions. A conjugative mechanism likely contributes to gene transfer between diverse strains of X. fastidiosa , possibly facilitating adaptation to new environments or different hosts. IMPORTANCE Xylella fastidiosa is an important plant pathogen worldwide, infecting a wide range of different plant species. The emergence of new diseases caused by X. fastidiosa , or host switching of existing strains, is thought to be primarily due to the high frequency of HGT and recombination in this pathogen. Transfer of plasmids by a conjugative mechanism enables movement of larger amounts of genetic material at one time, compared with other routes of gene transfer such as natural transformation. Establishing the prevalence and functionality of this mechanism in X. fastidiosa contributes to a better understanding of HGT, adaptation, and disease emergence

Identification of IncA/C Plasmid Replication and Maintenance Genes and Development of a Plasmid Multilocus Sequence Typing Scheme.

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Hancock, Steven J; Phan, Minh-Duy; Peters, Kate M; Forde, Brian M; Chong, Teik Min; Yin, Wai-Fong; Chan, Kok-Gan; Paterson, David L; Walsh, Timothy R; Beatson, Scott A; Schembri, Mark A

2017-02-01

Plasmids of incompatibility group A/C (IncA/C) are becoming increasingly prevalent within pathogenic Enterobacteriaceae They are associated with the dissemination of multiple clinically relevant resistance genes, including bla CMY and bla NDM Current typing methods for IncA/C plasmids offer limited resolution. In this study, we present the complete sequence of a bla NDM-1 -positive IncA/C plasmid, pMS6198A, isolated from a multidrug-resistant uropathogenic Escherichia coli strain. Hypersaturated transposon mutagenesis, coupled with transposon-directed insertion site sequencing (TraDIS), was employed to identify conserved genetic elements required for replication and maintenance of pMS6198A. Our analysis of TraDIS data identified roles for the replicon, including repA, a toxin-antitoxin system; two putative partitioning genes, parAB; and a putative gene, 053 Construction of mini-IncA/C plasmids and examination of their stability within E. coli confirmed that the region encompassing 053 contributes to the stable maintenance of IncA/C plasmids. Subsequently, the four major maintenance genes (repA, parAB, and 053) were used to construct a new plasmid multilocus sequence typing (PMLST) scheme for IncA/C plasmids. Application of this scheme to a database of 82 IncA/C plasmids identified 11 unique sequence types (STs), with two dominant STs. The majority of bla NDM -positive plasmids examined (15/17; 88%) fall into ST1, suggesting acquisition and subsequent expansion of this bla NDM -containing plasmid lineage. The IncA/C PMLST scheme represents a standardized tool to identify, track, and analyze the dissemination of important IncA/C plasmid lineages, particularly in the context of epidemiological studies. Copyright © 2017 American Society for Microbiology.

Study of nosocomial isolates of Staphylococcus aureus with special reference to methicillin resistant S. aureus in a tertiary care hospital in Nepal.

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Shrestha, B; Pokhrel, B; Mohapatra, T

2009-06-01

To find out the prevalence of Staphylococcus aureus nosocomial infection and methicillin resistant S. aureus (MRSA), clinical samples from nosocomially infected patients were processed by following standard methodology in microbiology laboratory, Tribhuvan University Teaching Hospital, Kathmandu, Nepal. Of 149 S. aureus isolates, skin infection isolates contributed a major part 72.5% making nosocomial infection by S. aureus most prevalent in skin infection followed by lower respiratory tract infection 11.41% and urinary tract infection 8.7%. Overall MRSA prevalence was 45.0%. MRSA prevalence was 42.6% in skin infection, 82.3% in lower respiratory tract infection and 30.8% in urinary tract infection. MRSA infection was found associated with lower respiratory tract infection only. Highest occurrence of nosocomial infection was observed in female surgical ward, surgical out patient department, orthopedic ward, male surgical ward and maternity ward. MRSA isolation was high from lower respiratory tract of patients admitted in intensive care unit, coronary care unit, Sub-acute intensive care unit, intermediate coronary care unit, neurology ward and post-operative ward. Whereas methicillin sensitive S. aureus (MSSA) occurrence was higher in patients admitted in orthopedic, Surgical out patient department, and female surgical ward. The occurrence of MRSA did not differ with age but MRSA was found associated with male patients and MSSA was associated with female patients. Since MRSA prevalence was high, regular surveillance of MRSA and nosocomial infections should be done and universal precautions to control nosocomial infections should be followed.

Amphibian antimicrobial peptide fallaxin analogue FL9 affects virulence gene expression and DNA replication in Staphylococcus aureus

DEFF Research Database (Denmark)

Gottschalk, Sanne; Gottlieb, Caroline Trebbien; Vestergaard, Martin

2015-01-01

antimicrobials. In the present study, the analogue FL9, based on the amphibian AMP fallaxin, was studied to elucidate its mode of action and antibacterial activity against the human pathogen Staphylococcus aureus. Our data showed that FL9 may have a dual mode of action against S. aureus. At concentrations around...... and at alkaline pH, while it was compromised by acidic pH and exposure to serum. Furthermore, at subinhibitory concentrations of FL9, S. aureus responded by increasing the expression of two major virulence factor genes, namely the regulatory rnaIII and hla, encoding α-haemolysin. In addition, the S. aureus...... the MIC, FL9 bound DNA, inhibited DNA synthesis and induced the SOS DNA damage response, whereas at concentrations above the MIC the interaction between S. aureus and FL9 led to membrane disruption. The antibacterial activity of the peptide was maintained over a wide range of NaCl and MgCl2 concentrations...

Investigation of Virulence Genes by PCR in Stapylococcus aureus Isolates Originated from Subclinical Bovine Mastitis in Turkey

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Murat Karahan, Mehmet Nuri Acik1* and Burhan Cetinkaya

2011-06-01

Full Text Available The aim of the present study was to characterize coagulase (coa positive Staphylococcus aureus strains (n=92 isolated from bovine subclinical mastitis in Turkey by PCR amplification of clumping factor A (clfA and protein A (spa genes. All the coa-positive S. aureus isolates were determined to harbor the genes encoding the IgG binding region (spa-IgG and the X region (spa-X of spa. On the other hand, 84 (91.3% isolates were positive for clfA gene. These three genes displayed size polymorphisms. It was concluded that spa gene polymorphisms for S. aureus, when used together with coa-PCR, can be proposed as good alternatives to conventional methods in typing S. aureus isolates of bovine origin which may provide valuable data for the development of effective control strategies against staphylococcal mastitis. The results of the present study showed that S. aureus isolates responsible for the mastitis cases in Turkey were genetically diverse.

Spread of clonally related Escherichia coli harboring an IncA/C1 plasmid encoding IMP-8 and its recruitment into an unrelated MCR-1-containing isolate.

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Elena, Alan; Cejas, Daniela; Magariños, Francisco; Jewtuchowicz, Virginia; Facente, Andrea; Gutkind, Gabriel; Di Conza, José; Radice, Marcela

2018-04-16

Ten IMP-8-producing Escherichia coli isolates were recovered from the surveillance cultures of a neonatal intensive care unit, of which eight were clonally related. A 168.2-kb- bla IMP-8 plasmid was fully sequenced, and it corresponded to the recently described IncA/C1-ST13. This plasmid was detected in all isolates, even in those no clonally related. One unrelated isolate was also resistant to colistin and positive for mcr-1. This marker was located in a 62.7-kb-IncI2 plasmid, which was also fully sequenced. Copyright © 2018 American Society for Microbiology.

Plasmid-mediated UV-protection in Streptococcus lactis

Energy Technology Data Exchange (ETDEWEB)

Chopin, M.C.; Rouault, A. (Institut National de la Recherche Agronomique, Rennes (France). Lab. de Recherches de Technologie Laitiere); Moillo-Batt, A. (Institut National de la Sante et de la Recherche Medicale (INSERM), Hopital de Pontchaillon, 35 - Rennes (France))

1985-02-01

Streptococcus lactis strain IL594 contains 9 plasmids, designated pIL1 to pIL9. On the basis of protoplast-induced curing experiments the authors showed that derivatives containing pIL7 were resistant to UV-irradiation while derivatives lacking pIL7 were sensitive. The pIL7-determined UV-protection was confirmed by co-transfer of the plasmid and of the character into a plasmid-free derivative of S. lactis IL594. Moreover, prophage induction required higher UV-fluence in this derivative carrying pIL7 than in the plasmid-free strain. This is the first report of a plasmid-mediated UV-protection in group N streptococci.

Plasmid-mediated UV-protection in Streptococcus lactis

International Nuclear Information System (INIS)

Chopin, M.-C.; Rouault, A.

1985-01-01

Streptococcus lactis strain IL594 contains 9 plasmids, designated pIL1 to pIL9. On the basis of protoplast-induced curing experiments the authors showed that derivatives containing pIL7 were resistant to UV-irradiation while derivatives lacking pIL7 were sensitive. The pIL7-determined UV-protection was confirmed by cotransfer of the plasmid and of the character into a plasmid-free derivative of S. lactis IL594. Moreover, prophage induction required higher UV-fluence in this derivative carrying pIL7 than in the plasmid-free strain. This is the first report of a plasmid-mediated UV-protection in group N streptococci. (orig.)

Behavior of IncQ Plasmids in Agrobacterium tumefaciens

NARCIS (Netherlands)

Hille, Jacques; Schilperoort, Rob

1981-01-01

Inc-Q plasmids were introduced into Agrobacterium tumefuciens, by mobilization from Escherichia coli with an Inc-P plasmid, or by transformation with purified plasmid DNA. It was found that they were stably maintained. The presence of an Inc-Q plasmid did not influence tumorigenicity. These results

Phenotypic and genotypic evaluation of fluoroquinolone resistance in clinical isolates of Staphylococcus aureus in Tehran.

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Aligholi, Marzieh; Mirsalehian, Akbar; Halimi, Shahnaz; Imaneini, Hossein; Taherikalani, Morovat; Jabalameli, Fereshteh; Asadollahi, Parisa; Mohajer, Babak; Abdollahi, Alireza; Emaneini, Mohammad

2011-09-01

Fluoroquinolones are broad-spectrum antibiotics widely used in the treatment of bacterial infections such as Staphylococcus aureus isolates. Resistance to these antibiotics is increasing. The occurrence of mutations in the grlA and gyrA loci were evaluated in 69 fluoroquinolone-resistant S. aureus isolates from 2 teaching hospitals of Tehran University of Medical Sciences. Out of the 165 S. aureus isolates, 87 (52.7%) were resistant to methicillin and 69 (41.8%) were resistant to fluoroquinolone. Fluoroquinolone-resistant S. aureus isolates had a mutation at codon 80 in the grlA gene and different mutational combinations in the gyrA gene. These mutational combinations included 45 isolates at codons 84 and 86, 23 isolates at codons 84, 86 and 106 and 1 isolate at codons 84, 86 and 90. Fluoroquinolone-resistant S. aureus isolates were clustered into 33 PFGE types. The findings of this study show that the fluoroquinolone-resistant S. aureus strains isolated in the teaching hospitals in Tehran had multiple mutations in the QRDRs region of both grlA and gyrA genes.

Persistence Mechanisms of Conjugative Plasmids

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Bahl, Martin Iain; Hansen, Lars H.; Sørensen, Søren Johannes

2009-01-01

Are plasmids selfish parasitic DNA molecules or an integrated part of the bacterial genome? This chapter reviews the current understanding of the persistence mechanisms of conjugative plasmids harbored by bacterial cells and populations. The diversity and intricacy of mechanisms affecting the suc...

Dissemination of Methicillin-Resistant Staphylococcus aureus (MRSA), USA300 Sequence Type 8 Lineage in Latin-America

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Reyes, Jinnethe; Rincón, Sandra; Díaz, Lorena; Panesso, Diana; Contreras, Germán A.; Zurita, Jeannete; Carrillo, Carlos; Rizzi, Adele; Guzmán, Manuel; Adachi, Javier; Chowdhury, Shahreen; Murray, Barbara E.; Arias, Cesar A.

2009-01-01

Background Methicillin-resistant Staphylococus aureus (MRSA) is an important nosocomial and community-associated (CA) pathogen. Recently, a variant of the MRSA USA300 clone emerged and disseminated in South-America causing important clinical problems. Methods S. aureus isolates were prospectively collected (2006 to 2008) from 32 tertiary hospitals in Colombia, Ecuador, Peru, and Venezuela. MRSA isolates were subjected to antimicrobial susceptibility testing, pulsed field gel electrophoresis (PFGE), and categorized as healthcare-associated (HA)-like or CA-like clones based on genotypic characteristics and detection of genes encoding the Panton-Valentine leukocidin (PVL) and staphylococcal cassette mec (SCCmec) IV. Additionally, MLST of representative isolates of each major CA-MRSA pulsotype, and detection of USA300-associated toxins and the arcA gene were performed in all isolates categorized as CA-MRSA. Results A total of 1570 S. aureus were included; 651 were MRSA (41%), with the highest rates of MRSA isolation in Peru (62%), and lowest in Venezuela (26%) and 71%, 27%, and 2% were classified as HA-like, CA-like, and non-CA/HA-like clones, respectively. Only 9 MRSA isolates were confirmed to have reduced susceptibility to glycopeptides (GISA phenotype). The most common pulsotype (designated ComA) amongst the CA-like MRSA strains was found in 96% of isolates with the majority (81%) having ≤6 bands difference with the USA300-0114 strain. Representative isolates of this clone were ST8 but, unlike the USA300-0114 strain, they harbored a different SCCmec IV subtype and lacked arcA (an indicator of the arginine catabolic mobile element (ACME)). Conclusion A variant CA-MRSA USA300 clone has now become established in South America and, in some countries, is endemic in hospital settings. PMID:19911971

Plasmids of Raw Milk Cheese Isolate Lactococcus lactis subsp. lactis Biovar diacetylactis DPC3901 Suggest a Plant-Based Origin for the Strain ▿ †

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Fallico, Vincenzo; McAuliffe, Olivia; Fitzgerald, Gerald F.; Ross, R. Paul

2011-01-01

The four-plasmid complement of the raw milk cheese isolate Lactococcus lactis subsp. lactis biovar diacetylactis DPC3901 was sequenced, and some genetic features were functionally analyzed. The complete sequences of pVF18 (18,977 bp), pVF21 (21,739 bp), pVF22 (22,166 bp), and pVF50 (53,876 bp) were obtained. Each plasmid contained genes not previously described for Lactococcus, in addition to genes associated with plant-derived lactococcal strains. Most of the novel genes were found on pVF18 and encoded functions typical of bacteria associated with plants, such as activities of plant cell wall modification (orf11 and orf25). In addition, a predicted high-affinity regulated system for the uptake of cobalt was identified (orf19 to orf21 [orf19-21]), which has a single database homolog on a plant-derived Leuconostoc plasmid and whose functionality was demonstrated following curing of pVF18. pVF21 and pVF22 encode additional metal transporters, which, along with orf19-21 of pVF18, could enhance host ability to uptake growth-limiting amounts of biologically essential ions within the soil. In addition, vast regions from pVF50 and pVF21 share significant homology with the plant-derived lactococcal plasmid pGdh442, which is indicative of extensive horizontal gene transfer and recombination between these plasmids and suggests a common plant niche for their hosts. Phenotypes associated with these regions include glutamate dehydrogenase activity and Na+ and K+ transport. The presence of numerous plant-associated markers in L. lactis DPC3901 suggests a plant origin for the raw milk cheese isolate and provides for the first time the genetic basis to support the concept of the plant-milk transition for Lactococcus strains. PMID:21803914

Staphylococcus aureus Survives with a Minimal Peptidoglycan Synthesis Machine but Sacrifices Virulence and Antibiotic Resistance.

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Patricia Reed

2015-05-01

Full Text Available Many important cellular processes are performed by molecular machines, composed of multiple proteins that physically interact to execute biological functions. An example is the bacterial peptidoglycan (PG synthesis machine, responsible for the synthesis of the main component of the cell wall and the target of many contemporary antibiotics. One approach for the identification of essential components of a cellular machine involves the determination of its minimal protein composition. Staphylococcus aureus is a Gram-positive pathogen, renowned for its resistance to many commonly used antibiotics and prevalence in hospitals. Its genome encodes a low number of proteins with PG synthesis activity (9 proteins, when compared to other model organisms, and is therefore a good model for the study of a minimal PG synthesis machine. We deleted seven of the nine genes encoding PG synthesis enzymes from the S. aureus genome without affecting normal growth or cell morphology, generating a strain capable of PG biosynthesis catalyzed only by two penicillin-binding proteins, PBP1 and the bi-functional PBP2. However, multiple PBPs are important in clinically relevant environments, as bacteria with a minimal PG synthesis machinery became highly susceptible to cell wall-targeting antibiotics, host lytic enzymes and displayed impaired virulence in a Drosophila infection model which is dependent on the presence of specific peptidoglycan receptor proteins, namely PGRP-SA. The fact that S. aureus can grow and divide with only two active PG synthesizing enzymes shows that most of these enzymes are redundant in vitro and identifies the minimal PG synthesis machinery of S. aureus. However a complex molecular machine is important in environments other than in vitro growth as the expendable PG synthesis enzymes play an important role in the pathogenicity and antibiotic resistance of S. aureus.

Phenotypic and Genotypic Characterization of Staphylococcus aureus Strains from Italian Dairy Products

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Stefano Morandi

2009-01-01

Full Text Available Staphylococcus aureus is a known major cause of foodborne illnesses, and milk and dairy products are often contaminated by enterotoxigenic strains of this bacterium. In the present study, 122 S. aureus isolates collected from different dairy products were characterised by phenotypic properties, by the distribution of genes encoding staphylococcal enterotoxins (sea, sec, sed, seg, seh, sei, sej, and sel and by randomly amplified polymorphic DNA PCR (RAPD-PCR. Moreover, strain resistance to vancomycin and methicillin (oxacillin was studied. The differences in the RAPD-PCR profiles obtained with the primers M13 and AP4 revealed the presence of a great genetic heterogeneity among the different S. aureus strains. Using the primer AP4 and M13, eight groups were distinguished by RAPD-PCR cluster analysis, although, except in few cases, it was not possible to correlate the isolates of different animal species (cow or ovine with the presence of se genes. None of the isolates showed resistance to vancomycin or methicillin.

Translocation of integron-associated resistance in a natural system: Acquisition of resistance determinants by Inc P and Inc W Plasmids from Salmonella enterica Typhimurium DT104

DEFF Research Database (Denmark)

Sandvang, Dorthe; Diggle, M.; Platt, D.J.

2002-01-01

to determinate the genetic content. Translocation to R751 and R388 was associated with the loss of the indigenous trimethoprim cassette to both plasmids and also acquisition of sulfonamide resistance by R751 and RP4::Tn7, which indicated movement of the 3' terminus of one or both of the DT104 integrons......Salmonella enterica Typhimurium DT104, 961368, a veterinary field isolate that encodes a chromosomal cluster of resistance genes as well as two integrons, was used to study the mobility of resistance cassettes (aadA2 and pse-1) and nonintegron-associated resistance determinants (chloramphenicol...... and tetracycline). A range of natural plasmids was used as targets for the translocation of resistance. Plasmids that acquired resistance from the DT104 chromosome were segregated by conjugation into Escherichia coli K12. Plasmids R751, R388, and RP4::Tn7 acquired several combinations of resistance determinant...

Health and economic burden of post-partum Staphylococcus aureus breast abscess.

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Branch-Elliman, Westyn; Lee, Grace M; Golen, Toni H; Gold, Howard S; Baldini, Linda M; Wright, Sharon B

2013-01-01

To determine the health and economic burdens of post-partum Staphylococcus aureus breast abscess. We conducted a matched cohort study (N = 216) in a population of pregnant women (N = 32,770) who delivered at our center during the study period from 10/1/03-9/30/10. Data were extracted from hospital databases, or via chart review if unavailable electronically. We compared cases of S. aureus breast abscess to controls matched by delivery date to compare health services utilization and mean attributable medical costs in 2012 United States dollars using Medicare and hospital-based estimates. We also evaluated whether resource utilization and health care costs differed between cases with methicillin-resistant and -susceptible S. aureus isolates. Fifty-four cases of culture-confirmed post-partum S. aureus breast abscess were identified. Breastfeeding cessation (41%), milk fistula (11.1%) and hospital readmission (50%) occurred frequently among case patients. Breast abscess case patients had high rates of health services utilization compared to controls, including high rates of imaging and drainage procedures. The mean attributable cost of post-partum S. aureus breast abscess ranged from $2,340-$4,012, depending on the methods and data sources used. Mean attributable costs were not significantly higher among methicillin-resistant vs. -susceptible S. aureus cases. Post-partum S. aureus breast abscess is associated with worse health and economic outcomes for women and their infants, including high rates of breastfeeding cessation. Future study is needed to determine the optimal treatment and prevention of these infections.

Health and economic burden of post-partum Staphylococcus aureus breast abscess.

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Westyn Branch-Elliman

Full Text Available To determine the health and economic burdens of post-partum Staphylococcus aureus breast abscess.We conducted a matched cohort study (N = 216 in a population of pregnant women (N = 32,770 who delivered at our center during the study period from 10/1/03-9/30/10. Data were extracted from hospital databases, or via chart review if unavailable electronically. We compared cases of S. aureus breast abscess to controls matched by delivery date to compare health services utilization and mean attributable medical costs in 2012 United States dollars using Medicare and hospital-based estimates. We also evaluated whether resource utilization and health care costs differed between cases with methicillin-resistant and -susceptible S. aureus isolates.Fifty-four cases of culture-confirmed post-partum S. aureus breast abscess were identified. Breastfeeding cessation (41%, milk fistula (11.1% and hospital readmission (50% occurred frequently among case patients. Breast abscess case patients had high rates of health services utilization compared to controls, including high rates of imaging and drainage procedures. The mean attributable cost of post-partum S. aureus breast abscess ranged from $2,340-$4,012, depending on the methods and data sources used. Mean attributable costs were not significantly higher among methicillin-resistant vs. -susceptible S. aureus cases.Post-partum S. aureus breast abscess is associated with worse health and economic outcomes for women and their infants, including high rates of breastfeeding cessation. Future study is needed to determine the optimal treatment and prevention of these infections.

Plasmid DNA initiates replication of yellow fever vaccine in vitro and elicits virus-specific immune response in mice.

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Tretyakova, Irina; Nickols, Brian; Hidajat, Rachmat; Jokinen, Jenny; Lukashevich, Igor S; Pushko, Peter

2014-11-01

Yellow fever (YF) causes an acute hemorrhagic fever disease in tropical Africa and Latin America. To develop a novel experimental YF vaccine, we applied iDNA infectious clone technology. The iDNA represents plasmid that encodes the full-length RNA genome of 17D vaccine downstream from a cytomegalovirus (CMV) promoter. The vaccine was designed to transcribe the full-length viral RNA and to launch 17D vaccine virus in vitro and in vivo. Transfection with 10 ng of iDNA plasmid was sufficient to start replication of vaccine virus in vitro. Safety of the parental 17D and iDNA-derived 17D viruses was confirmed in AG129 mice deficient in receptors for IFN-α/β/γ. Finally, direct vaccination of BALB/c mice with a single 20 μg dose of iDNA plasmid resulted in seroconversion and elicitation of virus-specific neutralizing antibodies in animals. We conclude that iDNA immunization approach combines characteristics of DNA and attenuated vaccines and represents a promising vaccination strategy for YF. Copyright © 2014 Elsevier Inc. All rights reserved.

The qacC gene has recently spread between rolling circle plasmids of Staphylococcus, indicative of a novel gene transfer mechanism

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Trudy M. Wassenaar

2016-09-01

Full Text Available Resistance of Staphylococcus species to quaternary ammonium compounds, frequently used as disinfectants and biocides, can be attributed to qac genes. These qac gene products belong to the Small Multidrug Resistant (SMR protein family, and are often encoded by rolling-circle (RC replicating plasmids. Four classes of SMR-type qac gene families have been described in Staphylococcus species: qacC, qacG, qacJ and qacH. Within their class, these genes are highly conserved, but qacC genes are extremely conserved, although they are found in variable plasmid backgrounds. The lower degree of sequence identity of these plasmids compared to the strict nucleotide conservation of their qacC means that this gene has recently spread. In the absence of insertion sequences or other genetic elements explaining the mobility, we sought for an explanation of mobilization by sequence comparison. Publically available sequences of qac genes, their flanking genes and the replication gene that is invariably present in RC-plasmids were compared to reconstruct the evolutionary history of these plasmids and to explain the recent spread of qacC. Here we propose a new model that explains how qacC is mobilized and transferred to acceptor RC-plasmids without assistance of other genes, by means of its location in between the Double Strand replication Origin (DSO and the Single-Strand replication Origin (SSO. The proposed mobilization model of this DSO-qacC-SSO element represents a novel mechanism of gene mobilization in RC-plasmids, which has also been employed by other genes, such as lnuA (conferring lincomycin resistance. The proposed gene mobility has aided to the wide spread of clinically relevant resistance genes in Staphylococcus populations.

Replacement of the folC gene, encoding folylpolyglutamate synthetase-dihydrofolate synthetase in Escherichia coli, with genes mutagenized in vitro.

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Pyne, C; Bognar, A L

1992-03-01

The folylpolyglutamate synthetase-dihydrofolate synthetase gene (folC) in Escherichia coli was deleted from the bacterial chromosome and replaced by a selectable Kmr marker. The deletion strain required a complementing gene expressing folylpolyglutamate synthetase encoded on a plasmid for viability, indicating that folC is an essential gene in E. coli. The complementing folC gene was cloned into the vector pPM103 (pSC101, temperature sensitive for replication), which segregated spontaneously at 42 degrees C in the absence of selection. This complementing plasmid was replaced in the folC deletion strain by compatible pUC plasmids containing folC genes with mutations generated in vitro, producing strains which express only mutant folylpolyglutamate synthetase. Mutant folC genes expressing insufficient enzyme activity could not complement the chromosomal deletion, resulting in retention of the pPM103 plasmid. Some mutant genes expressing low levels of enzyme activity replaced the complementing plasmid, but the strains produced were auxotrophic for products of folate-dependent pathways. The folylpolyglutamate synthetase gene from Lactobacillus casei, which may lack dihydrofolate synthetase activity, replaced the complementing plasmid, but the strain was auxotrophic for all folate end products.

Molecular basis of surface anchored protein A deficiency in the Staphylococcus aureus strain Wood 46.

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Manasi Balachandran

Full Text Available Protein A in Staphylococcus aureus is encoded by the spa (staphylococcal protein A gene and binds to immunoglobulin (Ig. The S. aureus strain Wood 46 has been variously reported as protein A-deficient and/or spa negative and used as a control in animal models of staphylococcal infections. The results of this study indicate that Wood 46 has normal spa expression but transcribes very low levels of the srtA gene which encodes the sortase A (SrtA enzyme. This is consistent with unique mutations in the srtA promoter. In this study, a low level of sortase A explains deficient anchoring of proteins with an LPXTG motif, such as protein A, fibrinogen-binding protein and fibronectin-binding proteins A and B on to the peptidoglycan cell wall. The activity of secreted protein A is an important consideration for use of Wood 46 in functional experiments and animal models.

Plasmid-mediated mineralization of 4-chlorobiphenyl

International Nuclear Information System (INIS)

Shields, M.S.; Hooper, S.W.; Sayler, G.S.

1985-01-01

Strains of Alcaligenes and Acinetobacter spp. were isolated from a mixed culture already proven to be proficient at complete mineralization of monohalogenated biphenyls. These strains were shown to harbor a 35 x 10(6)-dalton plasmid mediating a complete pathway for 4-chlorobiphenyl (4CB) oxidation. Subsequent plasmid curing of these bacteria resulted in the abolishment of the 4CB mineralization phenotype and loss of even early 4CB metabolism by Acinetobacter spp. Reestablishment of the Alcaligenes plasmid, denoted pSS50, in the cured Acinetobacter spp. via filter surface mating resulted in the restoration of 4CB mineralization abilities. 4CB mineralization, however, proved to be an unstable characteristic in some subcultured strains. Such loss was not found to coincide with any detectable alteration in plasmid size. Cultures capable of complete mineralization, as well as those limited to partial metabolism of 4CB, produced 4-chlorobenzoate as a metabolite. Demonstration of mineralization of a purified 14 C-labeled chlorobenzoate showed it to be a true intermediate in 4CB mineralization. Unlike the mineralization capability, the ability to produce a metabolite has proven to be stable on subculture. These results indicate the occurrence of a novel plasmid, or evolved catabolic plasmid, that mediates the complete mineralization of 4CB

Staphylococcus aureus: methicillin-susceptible S. aureus to methicillin-resistant S. aureus and vancomycin-resistant S. aureus.

Science.gov (United States)

Rehm, Susan J; Tice, Alan

2010-09-15

The evolution of methicillin-resistant and vancomycin-resistant Staphylococcus aureus has demanded serious review of antimicrobial use and development of new agents and revised approaches to prevent and overcome drug resistance. Depending on local conditions and patient risk factors, empirical therapy of suspected S. aureus infection may require coverage of drug-resistant organisms with newer agents and novel antibiotic combinations. The question of treatment with inappropriate antibiotics raises grave concerns with regard to methicillin-resistant S. aureus selection, overgrowth, and increased virulence. Several strategies to reduce the nosocomial burden of resistance are suggested, including shortened hospital stays and outpatient parenteral antimicrobial therapy of the most serious infections.

Construction of Biologically Functional Bacterial Plasmids In Vitro

Science.gov (United States)

Cohen, Stanley N.; Chang, Annie C. Y.; Boyer, Herbert W.; Helling, Robert B.

1973-01-01

The construction of new plasmid DNA species by in vitro joining of restriction endonuclease-generated fragments of separate plasmids is described. Newly constructed plasmids that are inserted into Escherichia coli by transformation are shown to be biologically functional replicons that possess genetic properties and nucleotide base sequences from both of the parent DNA molecules. Functional plasmids can be obtained by reassociation of endonuclease-generated fragments of larger replicons, as well as by joining of plasmid DNA molecules of entirely different origins. Images PMID:4594039

Aureusimines in Staphylococcus aureus are not involved in virulence.

Science.gov (United States)

Sun, Fei; Cho, Hoonsik; Jeong, Do-Won; Li, Chunling; He, Chuan; Bae, Taeok

2010-12-29

Recently, dipeptide aureusimines were reported to activate expression of staphylococcal virulence genes, such as alpha-hemolysin, and increase S. aureus virulence. Surprisingly, most of the virulence genes affected by aureusimines form part of the regulon of the SaeRS two component system (TCS), raising the possibility that SaeRS might be directly or indirectly involved in the aureusimine-dependent signaling process. Using HPLC analyses, we confirmed that a transposon mutant of ausA, the gene encoding the aureusimine dipeptide synthesis enzyme, does not produce dipeptides. However, the transposon mutant showed normal hemolysis activity and alpha-hemolysin/SaeP production. Furthermore, the P1 promoter of the sae operon, one of the targets of the SaeRS TCS, showed normal transcription activity. Moreover, in contrast to the original report, the ausA transposon mutant did not exhibit attenuated virulence in an animal infection model. DNA sequencing revealed that the ausA deletion mutant used in the original study has an 83 nt-duplication in saeS. Hemolysis activity of the original mutant was restored by a plasmid carrying the sae operon. A mutant of the sae operon showed elevated resistance to chloramphenicol and erythromycin, two antibiotics widely used during staphylococcal mutagenesis. At 43°C in the presence of erythromycin and aeration, the conditions typically employed for staphylococcal mutagenesis, an saeR transposon mutant grew much faster than a control mutant and the saeR mutant was highly enriched in a mixed culture experiment. Our results show that the previously reported roles of aureusimines in staphylococcal gene regulation and virulence were due to an unintended mutation in saeS, which was likely selected due to elevated resistance of the mutant to environmental stresses. Thus, there is no evidence indicating that the dipeptide aureusimines play a role in sae-mediated virulence factor production or contribute to staphylococcal virulence.

Aureusimines in Staphylococcus aureus are not involved in virulence.

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Fei Sun

2010-12-01

Full Text Available Recently, dipeptide aureusimines were reported to activate expression of staphylococcal virulence genes, such as alpha-hemolysin, and increase S. aureus virulence. Surprisingly, most of the virulence genes affected by aureusimines form part of the regulon of the SaeRS two component system (TCS, raising the possibility that SaeRS might be directly or indirectly involved in the aureusimine-dependent signaling process.Using HPLC analyses, we confirmed that a transposon mutant of ausA, the gene encoding the aureusimine dipeptide synthesis enzyme, does not produce dipeptides. However, the transposon mutant showed normal hemolysis activity and alpha-hemolysin/SaeP production. Furthermore, the P1 promoter of the sae operon, one of the targets of the SaeRS TCS, showed normal transcription activity. Moreover, in contrast to the original report, the ausA transposon mutant did not exhibit attenuated virulence in an animal infection model. DNA sequencing revealed that the ausA deletion mutant used in the original study has an 83 nt-duplication in saeS. Hemolysis activity of the original mutant was restored by a plasmid carrying the sae operon. A mutant of the sae operon showed elevated resistance to chloramphenicol and erythromycin, two antibiotics widely used during staphylococcal mutagenesis. At 43°C in the presence of erythromycin and aeration, the conditions typically employed for staphylococcal mutagenesis, an saeR transposon mutant grew much faster than a control mutant and the saeR mutant was highly enriched in a mixed culture experiment.Our results show that the previously reported roles of aureusimines in staphylococcal gene regulation and virulence were due to an unintended mutation in saeS, which was likely selected due to elevated resistance of the mutant to environmental stresses. Thus, there is no evidence indicating that the dipeptide aureusimines play a role in sae-mediated virulence factor production or contribute to staphylococcal

Elimination of indigenous linear plasmids in Streptomyces hygroscopicus var. jinggangensis and Streptomyces sp. FR008 to increase validamycin A and candicidin productivities.

Science.gov (United States)

Lu, Chenyang; Wu, Hang; Su, Xiurong; Bai, Linquan

2017-05-01

Giant linear plasmids, which replicate independently of the chromosomes, widely exist in actinobacteria. Previous studies mostly focused on the replication and evolution of the linear plasmids or the secondary metabolite gene clusters and the resistance gene clusters therein. However, the relationships of the linear plasmids to the productivities of secondary metabolites have not been studied. In this work, we developed a method to eliminate the indigenous linear plasmid pSHJG1 in Streptomyces hygroscopicus var. jinggangensis, and validamycin A titer increased by 12.5% (from 19.16 ± 1.93 to 21.56 ± 2.25 g/L) in the high-yielding strain TL01 and 43.7% (from 4.67 ± 0.05 to 6.71 ± 0.21 g/L) in the wild-type strain 5008, whereas the cellular growth of the plasmid-cured mutant was reduced. Subsequently, the plasmid-cured mutant was complemented with three structure genes involved in cellular growth in pSHJG1 under the control of a strong PvalA promoter. Among them, the complementation of genes pSHJG1.069 and pSHJG1.072, encoding a putative hydrolase and putative P-loop ATPase, respectively, resulted in the restoration of cellular growth and validamycin A titer. Furthermore, the elimination of indigenous linear plasmid pHZ228 in the candicidin producer Streptomyces sp. FR008 also led to enhanced candicidin production and reduced cellular growth. Because of the wide distribution of indigenous linear plasmids in actinobacteria, the engineering strategy described here could be implemented in a variety of strains for the overproduction of various natural products.

Diversity and homogeneity among small plasmids of Aeromonas salmonicida subsp. salmonicida linked with geographical origin

Directory of Open Access Journals (Sweden)

Sabrina A Attéré

2015-11-01

Full Text Available Furunculosis, which is caused by Aeromonas salmonicida subsp. salmonicida, is a major salmonid disease in fish farms worldwide. Several plasmids found in this bacterium confer phenotypes such drug resistance and virulence. Small plasmids (pAsa1, pAsa2, pAsa3, and pAsal1 related to ColE1- and ColE2-type replicons are usually present in its normal plasmidome. In the present study, with the objective to investigate if these plasmids display particularities related to the origin of the isolates bearing them, a total of 153 isolates, including 78 new and 75 previously described, were analyzed for the presence of small plasmids by PCR and DNA restriction fragment profiling. A geographical dichotomy between Canadian and European isolates for their propensity to do not have pAsa3 or pAsal1 was found. In addition, the genotyping analysis led to the identification of two European isolates harboring an unusual pAsal1. An investigation by next-generation sequencing (NGS of these two isolates shed light on two pAsal1 variants (pAsal1C and pAsal1D. As with pAsal1B, another pAsal1 variant previously described, these two new variants bore a second insertion sequence (ISAS5 in addition to the usual ISAS11. The characterization of these variants suggested that they could predominate over the wild-type pAsal1 in stressful conditions such as growth at temperatures of 25°C and above. To obtain a comprehensive portrait of the mutational pressure on small plasmids, 26 isolates whose DNA had been sequenced by NGS were investigated. pAsa3 and pAsal1 were more prone to mutations than pAsa1 and pAsa2, especially in the mobA gene, which encodes a relaxase and a primase. Lastly, the average copy number of each plasmid per cell was assessed using raw sequencing data. A clear trend with respect to the relative proportion per cell of each plasmid was identified. Our large-scale study revealed a geographical dichotomy in small plasmid repertoire in addition to a clear trend

Prevalence of plasmid-bearing and plasmid-free Chlamydia trachomatis infection among women who visited obstetrics and gynecology clinics in Malaysia.

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Yeow, Tee Cian; Wong, Won Fen; Sabet, Negar Shafiei; Sulaiman, Sofiah; Shahhosseini, Fatemeh; Tan, Grace Min Yi; Movahed, Elaheh; Looi, Chung Yeng; Shankar, Esaki M; Gupta, Rishien; Arulanandam, Bernard P; Hassan, Jamiyah; Abu Bakar, Sazaly

2016-03-18

The 7.5 kb cryptic plasmid of Chlamydia trachomatis has been shown to be a virulence factor in animal models, but its significance in humans still remains unknown. The aim of this study was to investigate the prevalence and potential involvement of the C. trachomatis cryptic plasmid in causing various clinical manifestations; including infertility, reproductive tract disintegrity, menstrual disorder, and polycystic ovarian syndrome (PCOS) among genital C. trachomatis-infected patients. A total of 180 female patients of child bearing age (mean 30.9 years old, IQR:27-35) with gynecological complications and subfertility issues, who visited Obstetrics and Gynecology clinics in Kuala Lumpur, Malaysia were recruited for the study. Prevalence of genital chlamydial infection among these patients was alarmingly high at 51.1% (92/180). Of the 92 chlamydia-infected patients, 93.5% (86/92) were infected with plasmid-bearing (+) C. trachomatis while the remaining 6.5% (6/92) were caused by the plasmid-free (-) variant. Our data showed that genital C. trachomatis infection was associated with infertility issues, inflammation in the reproductive tract (mucopurulent cervicitis or endometriosis), irregular menstrual cycles and polycystic ovarian syndrome (PCOS). However, no statistical significance was detected among patients with plasmid (+) versus plasmid (-) C. trachomatis infection. Interestingly, plasmid (+) C. trachomatis was detected in all patients with PCOS, and the plasmid copy numbers were significantly higher among PCOS patients, relative to non-PCOS patients. Our findings show a high incidence of C. trachomatis infection among women with infertility or gynecological problems in Malaysia. However, due to the low number of plasmid (-) C. trachomatis cases, a significant role of the plasmid in causing virulence in human requires further investigation of a larger cohort.

Short communication: Antimicrobial susceptibility profiling and genotyping of Staphylococcus aureus isolates from bovine mastitis in Poland.

Science.gov (United States)

Jagielski, T; Puacz, E; Lisowski, A; Siedlecki, P; Dudziak, W; Międzobrodzki, J; Krukowski, H

2014-10-01

Staphylococcus aureus is the predominant causative agent of bovine mastitis, a disease that remains a major economic burden for the dairy industry worldwide. In this study, the antimicrobial resistance patterns and the genetic composition of 80 S. aureus mastitis isolates collected from 14 dairy farms in Eastern Poland were determined. Of the 10 antimicrobial agents evaluated, only testing for penicillin G produced drug resistance. As 41% of the S. aureus isolates were penicillin resistant, this drug along with other β-lactamase-sensitive β-lactams, should rather not be considered for the treatment of bovine mastitis caused by S. aureus. Upon genotyping, with a triplex PCR method, a total of 11 distinct PCR types were produced. The population structure of S. aureus isolates was highly clonal, with 1 predominant genotype circulating on each farm. The observed similarities in the genotype composition of S. aureus populations from geographically distant farms underscore the significance of interfarm transmission of S. aureus in Poland. This, in turn, argues for the establishment of a nationwide surveillance program for bovine mastitis due to this pathogen. Copyright © 2014 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Clonal profile, virulence and resistance of Staphylococcus aureus isolated from sheep milk.

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Martins, Katheryne Benini; Faccioli-Martins, Patricia Yoshida; Riboli, Danilo Flávio Moraes; Pereira, Valéria Cataneli; Fernandes, Simone; Oliveira, Aline A; Dantas, Ariane; Zafalon, Luiz Francisco; da Cunha, Maria de Lourdes Ribeiro de Souza

2015-06-01

The objective of this study was to characterize the clonal profile, virulence factors and antimicrobial resistance, particularly oxacillin resistance, of Staphylococcus aureus isolated from sheep milk. Milk samples were collected from all teats for the California Mastitis Test (CMT), somatic cell count, identification of S. aureus, investigation in these strains of genes encoding toxins (sea, seb, sec, sed, tst), biofilm (icaA, icaC, icaD, bap), leukocidin (luk-PV) oxacillin resistance by mecA gene detection and susceptibility testing (12 antibiotics). Messenger RNA expression was evaluated by RT-PCR in isolates carrying toxin and biofilm genes. Biofilm formation was also evaluated phenotypically by adherence to polystyrene plates. The clonal profile of S. aureus was investigated by pulsed-field gel electrophoresis. A total of 473 milk samples were collected from 242 animals on three farms and 20 S. aureus strains were isolated and none carried the mecA gene. The two sec gene-positive isolates and the isolates carrying the tst and luk-PV genes were positive by RT-PCR. Staphylococcus aureus isolated from the three flocks studied showed high susceptibility to the drugs tested and none was biofilm producer, indicating that biofilm formation was not a virulence factor causing infection by these strains. The typing of 17 S. aureus isolates revealed the presence of a common clone on the three farms studied, and the presence and expression of the sec and tst genes in one strain of this clone suggest the possible acquisition of virulence genes by this clone, a fact that is important for animal health and food hygiene.

Clonal profile, virulence and resistance of Staphylococcus aureus isolated from sheep milk

Science.gov (United States)

Martins, Katheryne Benini; Faccioli-Martins, Patricia Yoshida; Riboli, Danilo Flávio Moraes; Pereira, Valéria Cataneli; Fernandes, Simone; Oliveira, Aline A.; Dantas, Ariane; Zafalon, Luiz Francisco; da Cunha, Maria de Lourdes Ribeiro de Souza

2015-01-01

The objective of this study was to characterize the clonal profile, virulence factors and antimicrobial resistance, particularly oxacillin resistance, of Staphylococcus aureus isolated from sheep milk. Milk samples were collected from all teats for the California Mastitis Test (CMT), somatic cell count, identification of S. aureus, investigation in these strains of genes encoding toxins (sea, seb, sec, sed, tst), biofilm (icaA, icaC, icaD, bap), leukocidin (luk-PV) oxacillin resistance by mecA gene detection and susceptibility testing (12 antibiotics). Messenger RNA expression was evaluated by RT-PCR in isolates carrying toxin and biofilm genes. Biofilm formation was also evaluated phenotypically by adherence to polystyrene plates. The clonal profile of S. aureus was investigated by pulsed-field gel electrophoresis. A total of 473 milk samples were collected from 242 animals on three farms and 20 S. aureus strains were isolated and none carried the mecA gene. The two sec gene-positive isolates and the isolates carrying the tst and luk-PV genes were positive by RT-PCR. Staphylococcus aureus isolated from the three flocks studied showed high susceptibility to the drugs tested and none was biofilm producer, indicating that biofilm formation was not a virulence factor causing infection by these strains. The typing of 17 S. aureus isolates revealed the presence of a common clone on the three farms studied, and the presence and expression of the sec and tst genes in one strain of this clone suggest the possible acquisition of virulence genes by this clone, a fact that is important for animal health and food hygiene. PMID:26273271

Plasmid fermentation process for DNA immunization applications.

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Carnes, Aaron E; Williams, James A

2014-01-01

Plasmid DNA for immunization applications must be of the highest purity and quality. The ability of downstream purification to efficiently produce a pure final product is directly influenced by the performance of the upstream fermentation process. While several clinical manufacturing facilities already have validated fermentation processes in place to manufacture plasmid DNA for use in humans, a simple and inexpensive laboratory-scale fermentation process can be valuable for in-house production of plasmid DNA for use in animal efficacy studies. This chapter describes a simple fed-batch fermentation process for producing bacterial cell paste enriched with high-quality plasmid DNA. A constant feeding strategy results in a medium cell density culture with continuously increasing plasmid amplification towards the end of the process. Cell banking and seed culture preparation protocols, which can dramatically influence final product yield and quality, are also described. These protocols are suitable for production of research-grade plasmid DNA at the 100 mg-to-1.5 g scale from a typical 10 L laboratory benchtop fermentor.

Phospholipase C-related catalytically inactive protein participates in the autophagic elimination of Staphylococcus aureus infecting mouse embryonic fibroblasts.

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Kae Harada-Hada

Full Text Available Autophagy is an intrinsic host defense system that recognizes and eliminates invading bacterial pathogens. We have identified microtubule-associated protein 1 light chain 3 (LC3, a hallmark of autophagy, as a binding partner of phospholipase C-related catalytically inactive protein (PRIP that was originally identified as an inositol trisphosphate-binding protein. Here, we investigated the involvement of PRIP in the autophagic elimination of Staphylococcus aureus in infected mouse embryonic fibroblasts (MEFs. We observed significantly more LC3-positive autophagosome-like vacuoles enclosing an increased number of S. aureus cells in PRIP-deficient MEFs than control MEFs, 3 h and 4.5 h post infection, suggesting that S. aureus proliferates in LC3-positive autophagosome-like vacuoles in PRIP-deficient MEFs. We performed autophagic flux analysis using an mRFP-GFP-tagged LC3 plasmid and found that autophagosome maturation is significantly inhibited in PRIP-deficient MEFs. Furthermore, acidification of autophagosomes was significantly inhibited in PRIP-deficient MEFs compared to the wild-type MEFs, as determined by LysoTracker staining and time-lapse image analysis performed using mRFP-GFP-tagged LC3. Taken together, our data show that PRIP is required for the fusion of S. aureus-containing autophagosome-like vacuoles with lysosomes, indicating that PRIP is a novel modulator in the regulation of the innate immune system in non-professional phagocytic host cells.

Identification of point mutations in clinical Staphylococcus aureus strains that produce small-colony variants auxotrophic for menadione.

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Dean, Melissa A; Olsen, Randall J; Long, S Wesley; Rosato, Adriana E; Musser, James M

2014-04-01

Staphylococcus aureus small-colony variants (SCVs) are implicated in chronic and relapsing infections that are difficult to diagnose and treat. Despite many years of study, the underlying molecular mechanisms and virulence effect of the small-colony phenotype remain incompletely understood. We sequenced the genomes of five S. aureus SCV strains recovered from human patients and discovered previously unidentified nonsynonymous point mutations in three genes encoding proteins in the menadione biosynthesis pathway. Analysis of genetic revertants and complementation with wild-type alleles confirmed that these mutations caused the SCV phenotype and decreased virulence for mice.

Antimicrobial susceptibility pattern and plasmid-mediated ...

African Journals Online (AJOL)

negative Staphylococci (CoNS) were isolated from clinical samples and isolates subjected to antibiotic susceptibility testing, plasmid curing and plasmid DNA isolation. Result: The highest percentages isolates were recovered from urine samples and ...

Human cytomegalovirus-encoded miR-US4-1 promotes cell ...

Indian Academy of Sciences (India)

2016-04-05

Apr 5, 2016 ... 3′) for hcmv-miR-US4-1 was designed according to the. miRNA sequence (5′- ... the hcmv-miR-US4-1 hybrid primer and polyA tails were. 184. Yaozhong ... eight hours later, luciferase activities were measured us- ing Dual ..... resolution profiling and analysis of viral and host small RNAs during human ...

Prevalence and molecular characterization of Staphylococcus aureus in commercially available meat over a one-year period in Iowa, USA.

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Thapaliya, Dipendra; Forshey, Brett M; Kadariya, Jhalka; Quick, Megan K; Farina, Sarah; O' Brien, Ashley; Nair, Rajeshwari; Nworie, Amos; Hanson, Blake; Kates, Ashley; Wardyn, Shylo; Smith, Tara C

2017-08-01

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of infectious disease morbidity and mortality. Previous studies have confirmed the presence of S. aureus, including MRSA, on raw meat products. We investigated the prevalence and molecular epidemiology of S. aureus and MRSA in commercially-distributed antibiotic-free and conventional raw meat products (n = 3290) purchased in 8 Iowa retail stores weekly for a period of one year. Isolates were characterized using spa typing, and PCR was used to detect the presence of the Panton-Valentine leukocidin (PVL) and mecA genes. Quantitation of S. aureus on meat products was carried out one week per month. The prevalence of S. aureus on meat samples was 27.8% (913/3290). Compared to antibiotic-free meat samples, higher prevalence of both MRSA and methicillin-susceptible S. aureus (MSSA) were found in conventional meat samples. Among the S. aureus isolates, 18 were PVL-positive (1.9%) and 41 (4.5%) carried mecA. Phenotypic oxacillin resistance was observed for 17.1% (41/239) of the isolates tested, while 23% (55/239) were multi-drug resistant. A total of 132 spa types were detected from 913 contaminated meat samples. Overall, t002 was the most common spa type identified (137; 15.0%). The number of colony-forming units (CFU) per 10 g meat ranged from 2 to 517 (median: 8 CFU per 10 g of meat; mean: 28) with the highest bacterial load observed on turkey samples. These data reinforce the need to consider meat products as potential vehicles of S. aureus transmission from farm into human households, and the potential need for public health intervention programs pre and post-slaughter in meat processing facilities. Copyright © 2017 Elsevier Ltd. All rights reserved.

Methicillin-resistant Staphylococcus aureus nasal colonisation amongst healthcare workers in Kurdistan Region, Iraq.

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Hussein, Nawfal R; Assafi, Mahde S; Ijaz, Tayyaba

2017-06-01

The aim of this study was to determine the prevalence of nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) among healthcare workers (HCWs) compared with non-HCWs at Duhok city, Kurdistan Region, northern Iraq. A total of 182 HCWs with different occupations and working in different hospital units as well as 198 non-HCWs were recruited. Nasal swab samples were collected and were inoculated on mannitol salt agar and incubated at 35°C for 48h. Isolates identified as S. aureus underwent antimicrobial sensitivity testing to oxacillin. MRSA isolates were selected and investigated for presence of the mecA gene. Among the HCWs, 41/182 (22.5%) were carriers of S. aureus compared with 37/198 (18.7%) non-HCWs (P=0.4). Amongst the S. aureus carriers, 25/41 strains (61.0%) isolated from HCWs were MRSA compared with 8/37 strains (21.6%) isolated from non-HCWs (P=0.039). The mean age of MRSA carriers was 35.6±6.7years compared with 30±5.8years for MRSA non-carriers (P=0.0177). The mean working years of MRSA carriers was significantly higher than that of MRSA non-carriers (7.8±5.5years vs. 3.9±5.3years; P=0.04). The prevalence of MRSA was very high amongst HCWs. Regular screening of carriers is required for prevention of nosocomial infections. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

Photorhabdus insect-related (Pir) toxin-like genes in a plasmid of Vibrio parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (AHPND) of shrimp

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Han, Jee Eun; Tang, Kathy F. J.; Tran, Loc H.; Lightner, Donald V.

2016-01-01

The 69 kb plasmid pVPA3-1 was identified in Vibrio parahaemolyticus strain 13-028/A3 that can cause acute hepatopancreatic necrosis disease (AHPND). This disease is responsible for mass mortalities in farmed penaeid shrimp and is referred to as early mortality syndrome (EMS). The plasmid has a GC content of 45.9% with a copy number of 37 per bacterial cell as determined by comparative quantitative PCR analyses. It consists of 92 open reading frames that encode mobilization proteins, replication enzymes, transposases, virulence-associated proteins, and proteins similar to Photorhabdus insect-related (Pir) toxins. In V. parahaemolyticus, these Pir toxin-like proteins are encoded by 2 genes ( pirA- and pirB-like) located within a 3.5 kb fragment flanked with inverted repeats of a transposase-coding sequence (1 kb). The GC content of these 2 genes is only 38.2%, substantially lower than that of the rest of the plasmid, which suggests that these genes were recently acquired. Based on a proteomic analysis, the pirA-like (336 bp) and pirB-like (1317 bp) genes encode for 13 and 50 kDa proteins, respectively. In laboratory cultures of V. parahaemolyticus 13-028/A3, both proteins were secreted into the culture medium. We developed a duplex PCR diagnostic method, with a detection limit of 105 CFU ml−1 and targeting pirA- and pirB-like genes in this strain of V. parahaemolyticus. This PCR protocol can reliably detect AHPND-causing strains of V. parahaemolyticus and does not cross react with non-pathogenic strains or with other species of Vibrio isolated from shrimp ponds. PMID:25667334

Lysine-Grafted MCM-41 Silica as an Antibacterial Biomaterial.

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Villegas, María F; Garcia-Uriostegui, Lorena; Rodríguez, Ofelia; Izquierdo-Barba, Isabel; Salinas, Antonio J; Toriz, Guillermo; Vallet-Regí, María; Delgado, Ezequiel

2017-09-26

This paper proposes a facile strategy for the zwitterionization of bioceramics that is based on the direct incorporation of l-lysine amino acid via the ε-amino group onto mesoporous MCM-41 materials. Fourier transform infrared (FTIR) studies of lysine-grafted MCM-41 (MCM-LYS) simultaneously showed bands at 3080 and 1540 cm -1 and bands at 1625 and 1415 cm -1 corresponding to -NH 3+ /COO - pairs, which demonstrate the incorporation of the amino acid on the material surface keeping its zwitterionic character. Both elemental and thermogravimetric analyses showed that the amount of grafted lysine was 8 wt. % based on the bioceramic total weight. Moreover, MCM-LYS exhibited a reduction of adhesion of S. aureus and E. coli bacteria in 33% and 50%, respectively at physiological pH, as compared with pristine MCM-41. Biofilm studies onto surfaces showed that lysine functionalization elicited a reduction of the area covered by S. aureus biofilm from 42% to only 5% (88%). This research shows a simple and effective approach to chemically modify bioceramics using single amino acids that provides zwitterionic functionality, which is useful to develop new biomaterials that are able to resist bacterial adhesion.

Lysine-Grafted MCM-41 Silica as an Antibacterial Biomaterial

Directory of Open Access Journals (Sweden)

María F. Villegas

2017-09-01

Full Text Available This paper proposes a facile strategy for the zwitterionization of bioceramics that is based on the direct incorporation of l-lysine amino acid via the ε-amino group onto mesoporous MCM-41 materials. Fourier transform infrared (FTIR studies of lysine-grafted MCM-41 (MCM-LYS simultaneously showed bands at 3080 and 1540 cm−1 and bands at 1625 and 1415 cm−1 corresponding to -NH3+/COO− pairs, which demonstrate the incorporation of the amino acid on the material surface keeping its zwitterionic character. Both elemental and thermogravimetric analyses showed that the amount of grafted lysine was 8 wt. % based on the bioceramic total weight. Moreover, MCM-LYS exhibited a reduction of adhesion of S. aureus and E. coli bacteria in 33% and 50%, respectively at physiological pH, as compared with pristine MCM-41. Biofilm studies onto surfaces showed that lysine functionalization elicited a reduction of the area covered by S. aureus biofilm from 42% to only 5% (88%. This research shows a simple and effective approach to chemically modify bioceramics using single amino acids that provides zwitterionic functionality, which is useful to develop new biomaterials that are able to resist bacterial adhesion.

Transcriptome mapping of pAR060302, a blaCMY-2-positive broad-host-range IncA/C plasmid.

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Lang, Kevin S; Danzeisen, Jessica L; Xu, Wayne; Johnson, Timothy J

2012-05-01

The multidrug resistance-encoding plasmids belonging to the IncA/C incompatibility group have recently emerged among Escherichia coli and Salmonella enterica strains in the United States. These plasmids have a unique genetic structure compared to other enterobacterial plasmid types, a broad host range, and a propensity to acquire large numbers of antimicrobial resistance genes via their accessory regions. Using E. coli strain DH5α harboring the prototype IncA/C plasmid pAR060302, we sought to define the baseline transcriptome of IncA/C plasmids under laboratory growth and in the face of selective pressure. The effects of ampicillin, florfenicol, or streptomycin exposure were compared to those on cells left untreated at logarithmic phase using Illumina platform-based RNA sequencing (RNA-Seq). Under growth in Luria-Bertani broth lacking antibiotics, much of the backbone of pAR060302 was transcriptionally inactive, including its putative transfer regions. A few plasmid backbone genes of interest were highly transcribed, including genes of a putative toxin-antitoxin system and an H-NS-like transcriptional regulator. In contrast, numerous genes within the accessory regions of pAR060302 were highly transcribed, including the resistance genes floR, bla(CMY-2), aadA, and aacA. Treatment with ampicillin or streptomycin resulted in no genes being differentially expressed compared to controls lacking antibiotics, suggesting that many of the resistance-associated genes are not differentially expressed due to exposure to these antibiotics. In contrast, florfenicol treatment resulted in the upregulation of floR and numerous chromosomal genes. Overall, the transcriptome mapping of pAR060302 suggests that it mitigates the fitness costs of carrying resistance-associated genes through global regulation with its transcriptional regulators.

Methicillin resistant S. aureus in human and bovine mastitis.

Science.gov (United States)

Holmes, Mark A; Zadoks, Ruth N

2011-12-01

Staphylococcus aureus is a ubiquitous organism that causes a variety of diseases including mastitis in cattle and humans. High-level resistance of S. aureus to β-lactams conferred by a mecA gene encoding a modified penicillin binding protein (PBP2a) was first observed in the early 1960's. These methicillin resistant S. aureus (MRSA) have been responsible for both hospital acquired infections (HA-MRSA) and, more recently, community acquired MRSA (CA-MRSA). A small number of human MRSA mastitis cases and outbreaks in maternity or neonatal units have been reported which are generally the result of CA-MRSA. The establishment of the sequence type 398 (ST398) in farm animals, primarily pigs, in the early 2000's has provided a reservoir of infection for humans and dairy cattle, particularly in continental Europe, described as livestock-associated MRSA (LA-MRSA). Prior to the emergence of ST398 there were sporadic reports of MRSA in bovine milk and cases of mastitis, often caused by strains from human associated lineages. Subsequently, there have been several reports describing bovine udder infections caused by ST-398 MRSA. Recently, another group of LA-MRSA strains was discovered in humans and dairy cattle in Europe. This group carries a divergent mecA gene and includes a number of S. aureus lineages (CC130, ST425, and CC1943) that were hitherto thought to be bovine-specific but are now also found as carriage or clinical isolates in humans. The emergence of MRSA in dairy cattle may be associated with contact with other host species, as in the case of ST398, or with the exchange of genetic material between S. aureus and coagulase negative Staphylococcus species, which are the most common species associated with bovine intramammary infections and commonly carry antimicrobial resistance determinants.

Peptidoglycan architecture can specify division planes in Staphylococcus aureus.

Science.gov (United States)

Turner, Robert D; Ratcliffe, Emma C; Wheeler, Richard; Golestanian, Ramin; Hobbs, Jamie K; Foster, Simon J

2010-06-15

Division in Staphylococci occurs equatorially and on specific sequentially orthogonal planes in three dimensions, resulting, after incomplete cell separation, in the 'bunch of grapes' cluster organization that defines the genus. The shape of Staphylococci is principally maintained by peptidoglycan. In this study, we use Atomic Force Microscopy (AFM) and fluorescence microscopy with vancomycin labelling to examine purified peptidoglycan architecture and its dynamics in Staphylococcus aureus and correlate these with the cell cycle. At the presumptive septum, cells were found to form a large belt of peptidoglycan in the division plane before the centripetal formation of the septal disc; this often had a 'piecrust' texture. After division, the structures remain as orthogonal ribs, encoding the location of past division planes in the cell wall. We propose that this epigenetic information is used to enable S. aureus to divide in sequentially orthogonal planes, explaining how a spherical organism can maintain division plane localization with fidelity over many generations.

ω-Hydroxyemodin Limits Staphylococcus aureus Quorum Sensing-Mediated Pathogenesis and Inflammation

Science.gov (United States)

Daly, Seth M.; Elmore, Bradley O.; Kavanaugh, Jeffrey S.; Triplett, Kathleen D.; Figueroa, Mario; Raja, Huzefa A.; El-Elimat, Tamam; Crosby, Heidi A.; Femling, Jon K.; Cech, Nadja B.; Horswill, Alexander R.; Oberlies, Nicholas H.

2015-01-01

Antibiotic-resistant pathogens are a global health threat. Small molecules that inhibit bacterial virulence have been suggested as alternatives or adjuncts to conventional antibiotics, as they may limit pathogenesis and increase bacterial susceptibility to host killing. Staphylococcus aureus is a major cause of invasive skin and soft tissue infections (SSTIs) in both the hospital and community settings, and it is also becoming increasingly antibiotic resistant. Quorum sensing (QS) mediated by the accessory gene regulator (agr) controls virulence factor production essential for causing SSTIs. We recently identified ω-hydroxyemodin (OHM), a polyhydroxyanthraquinone isolated from solid-phase cultures of Penicillium restrictum, as a suppressor of QS and a compound sought for the further characterization of the mechanism of action. At concentrations that are nontoxic to eukaryotic cells and subinhibitory to bacterial growth, OHM prevented agr signaling by all four S. aureus agr alleles. OHM inhibited QS by direct binding to AgrA, the response regulator encoded by the agr operon, preventing the interaction of AgrA with the agr P2 promoter. Importantly, OHM was efficacious in a mouse model of S. aureus SSTI. Decreased dermonecrosis with OHM treatment was associated with enhanced bacterial clearance and reductions in inflammatory cytokine transcription and expression at the site of infection. Furthermore, OHM treatment enhanced the immune cell killing of S. aureus in vitro in an agr-dependent manner. These data suggest that bacterial disarmament through the suppression of S. aureus QS may bolster the host innate immune response and limit inflammation. PMID:25645827

Purification and Genetic Characterization of Enterocin I from Enterococcus faecium 6T1a, a Novel Antilisterial Plasmid-Encoded Bacteriocin Which Does Not Belong to the Pediocin Family of Bacteriocins

Science.gov (United States)

Floriano, Belén; Ruiz-Barba, José L.; Jiménez-Díaz, Rufino

1998-01-01

Enterocin I (ENTI) is a novel bacteriocin produced by Enterococcus faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation. The bacteriocin is active against many olive spoilage and food-borne gram-positive pathogenic bacteria, including clostridia, propionibacteria, and Listeria monocytogenes. ENTI was purified to homogeneity by ammonium sulfate precipitation, binding to an SP-Sepharose fast-flow column, and phenyl-Sepharose CL-4B and C2/C18 reverse-phase chromatography. The purification procedure resulted in a final yield of 954% and a 170,000-fold increase in specific activity. The primary structure of ENTI was determined by amino acid and nucleotide sequencing. ENTI consists of 44 amino acids and does not show significant sequence similarity with any other previously described bacteriocin. Sequencing of the entI structural gene, which is located on the 23-kb plasmid pEF1 of E. faecium 6T1a, revealed the absence of a leader peptide at the N-terminal region of the gene product. A second open reading frame, ORF2, located downstream of entI, encodes a putative protein that is 72.7% identical to ENTI. entI and ORF2 appear to be cotranscribed, yielding an mRNA of ca. 0.35 kb. A gene encoding immunity to ENTI was not identified. However, curing experiments demonstrated that both enterocin production and immunity are conferred by pEF1. PMID:9835578

The Plasmid Complement of the Cheese Isolate Lactococcus garvieae IPLA 31405 Revealed Adaptation to the Dairy Environment

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Flórez, Ana Belén; Mayo, Baltasar

2015-01-01

Lactococcus garvieae is a lactic acid bacterium found in raw-milk dairy products as well as a range of aquatic and terrestrial environments. The plasmids in L. garvieae have received little attention compared to those of dairy Lactococcus lactis, in which the genes carried by these extrachromosomal elements are considered of adaptive value. The present work reports the sequencing and analysis of the plasmid complement of L. garvieae IPLA 31405, a strain isolated from a traditional, Spanish, starter-free cheese made from raw-milk. It consists of pLG9 and pLG42, of 9,124 and 42,240 nucleotides, respectively. Based on sequence and structural homology in the putative origin of replication (ori) region, pLG9 and pLG42 are predicted to replicate via a theta mechanism. Real-time, quantitative PCR showed the number of copies per chromosome equivalent of pLG9 and pLG42 to be around two and five, respectively. Sequence analysis identified eight complete open reading frames (orfs) in pLG9 and 36 in pLG42; these were organized into functional modules or cassettes containing different numbers of genes. These modules were flanked by complete or interrupted insertion sequence (IS)-like elements. Among the modules of pLG42 was a gene cluster encoding specific components of a phosphoenolpyruvate-phosphotransferase (PEP-PTS) system, including a phospho-β-galacosidase. The cluster showed a complete nucleotide identity respect to that in plasmids of L. lactis. Loss of pLG42 showed this to be involved in lactose assimilation. In the same plasmid, an operon encoding a type I restriction/modification (R/M) system was also identified. The specificity of this R/M system might be broadened by different R/M specificity subunits detected in pLG9 and in the bacterial chromosome. However, challenges of L. garvieae IPLA 31405 against L. lactis phages proved that the R/M system was not involved in phage resistance. Together, these results support the hypothesis that, as in L. lactis, pLG42

Multilocus sequence typing of IncN plasmids

DEFF Research Database (Denmark)

García-Fernández, Aurora; Villa, Laura; Moodley, Arshnee

2011-01-01

that spread and persistence of this particular IncN-carrying blaVIM-1 lineage in Greece. CONCLUSIONS: This study proposes the use of pMLST as a suitable and rapid method for identification of IncN epidemic plasmid lineages. The recent spread of blaCTX-M-1 among humans and animals seems to be associated......OBJECTIVES: Incompatibility group N (IncN) plasmids have been associated with the dissemination of antimicrobial resistance and are a major vehicle for the spread of blaVIM-1 in humans and blaCTX-M-1 in animals. A plasmid multilocus sequence typing (pMLST) scheme was developed for rapid...... in different countries from both animals and humans belonged to ST1, suggesting dissemination of an epidemic plasmid through the food chain. Fifteen of 17 plasmids carrying blaVIM-1 from Klebsiella pneumoniae and Escherichia coli, isolated during a 5year period in Greece were assigned to ST10, suggesting...

Selfish restriction modification genes: resistance of a resident R/M plasmid to displacement by an incompatible plasmid mediated by host killing.

Science.gov (United States)

Naito, Y; Naito, T; Kobayashi, I

1998-01-01

Previous work from this laboratory demonstrated that plasmids carrying a type II restriction-modification gene complex are not easily lost from their bacterial host because plasmid-free segregant cells are killed through chromosome cleavage. Here, we have followed the course of events that takes place when an Escherichia coli rec BC sbcA strain carrying a plasmid coding for the PaeR7I restriction-modification (R/M) gene complex is transformed by a plasmid with an identical origin of replication. The number of transformants that appeared was far fewer than with the restriction-minus (r-) control. Most of the transformants were very small. After prolonged incubation, the number and the size of the colonies increased, but this increase never attained the level of the r- control. Most of the transformed colonies retained the drug-resistance of the resident, r+ m+ plasmid. These results indicate that post-segregational host killing occurs when a plasmid bearing an R/M gene complex is displaced by an incompatible plasmid. Such cell killing eliminates the competitor plasmid along with the host and, thus, would allow persistence of the R/M plasmid in the neighboring, clonal host cells in nature. This phenomenon is reminiscent of mammalian apoptosis and other forms of altruistic cell death strategy against infection. This type of resistance to displacement was also studied in a wild type Escherichia coli strain that was normal for homologous recombination (rec+). A number of differences between the recBC sbcA strain and the rec+ strain were observed and these will be discussed.

Clonal profile, virulence and resistance of Staphylococcus aureus isolated from sheep milk

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Katheryne Benini Martins

2015-06-01

Full Text Available The objective of this study was to characterize the clonal profile, virulence factors and antimicrobial resistance, particularly oxacillin resistance, of Staphylococcus aureus isolated from sheep milk. Milk samples were collected from all teats for the California Mastitis Test (CMT, somatic cell count, identification of S. aureus, investigation in these strains of genes encoding toxins (sea, seb, sec, sed, tst, biofilm (icaA, icaC, icaD, bap, leukocidin (luk-PV oxacillin resistance by mecA gene detection and susceptibility testing (12 antibiotics. Messenger RNA expression was evaluated by RT-PCR in isolates carrying toxin and biofilm genes. Biofilm formation was also evaluated phenotypically by adherence to polystyrene plates. The clonal profile of S. aureus was investigated by pulsed-field gel electrophoresis. A total of 473 milk samples were collected from 242 animals on three farms and 20 S. aureus strains were isolated and none carried the mecA gene. The two sec gene-positive isolates and the isolates carrying the tst and luk-PV genes were positive by RT-PCR. Staphylococcus aureus isolated from the three flocks studied showed high susceptibility to the drugs tested and none was biofilm producer, indicating that biofilm formation was not a virulence factor causing infection by these strains. The typing of 17 S. aureus isolates revealed the presence of a common clone on the three farms studied, and the presence and expression of the sec and tst genes in one strain of this clone suggest the possible acquisition of virulence genes by this clone, a fact that is important for animal health and food hygiene.

Genotyping of Staphylococcus aureus in bovine mastitis and correlation to phenotypic characteristics.

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Artursson, Karin; Söderlund, Robert; Liu, Lihong; Monecke, Stefan; Schelin, Jenny

2016-09-25

Reducing the prevalence of mastitis caused by Staphylococcus aureus (S. aureus) is essential to improve animal health and reduce economic losses for farmers. The clinical outcome of acute mastitis and risk of progression to persistent mastitis can, at least to some extent, be related to genetic variants of the strain causing the infection. In the present study we have used microarrays to investigate the presence of virulence genes in S. aureus isolates from dairy cows with acute clinical mastitis (n=70) and correlated the findings to other genotypic and phenotypic characteristics. Among the most commonly found virulence factors were genes encoding several hemolysin types, leukocidins D and lukM/lukF-P83, clumping factors A and B, fibrinogen binding protein and fibronectin-binding protein A. Some virulence factors e.g. fibronectin-binding protein B and Staphylococcus aureus surface protein G were less common. Genes coding for several staphylococcal enterotoxins and toxic shock syndrome toxin-1 (TSST-1) were commonly found, especially in one major pulsotype. No beta-lactamase genes were found in any common pulsotype, while present in some rare pulsotypes, indicated to be of human origin. Production of TSST-1, enterotoxins, hemolysins and beta-lactamase could all be positively correlated to presence of the corresponding genes. This study reveals a number of genotypic differences and similarities among common and rare pulsotypes of S. aureus from cases of mastitis in Sweden. The results could help the design of diagnostic tools to guide on-farm interventions according to the expected impact on udder health from a specific S. aureus genotype. Copyright © 2016 Elsevier B.V. All rights reserved.

Molecular detection of the carriers of Staphylococcus aureus golden in referred to the Imam Ali Clinic in Shahrekord, Iran

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Maryam Reisi

2014-12-01

Full Text Available Objective: To conduct for the molecular detection of Staphylococcus aureus (S. aureus (Golden staph carriers among patients referred to the Imam Ali Clinic, Shahrekord, Iran. Methods: This sectional-descriptive study was conducted with 200 persons with suspected upper respiratory tract infections, who were referred to the Imam Ali Clinic in Shahrekord, Iran, in 2012. After culturing the nasal swab samples in mannitol salt agar and blood agar, S. aureus colonies were confirmed by biochemical methods. To determine the susceptibility of S. aureus strains isolated, molecular methods were used. Results: Among the 200 investigated samples, 60 cases (30%, comprising 25 men (41.66% and 35 women (58.33%, were found to be S. aureus carriers. Conclusions: The results of the present study showed that the frequency of the S. aureus strain isolated from the nasal swabs of patients with respiratory tract infections admitted to the Imam Ali Clinic in Shahrekord, Iran, was remarkable. Thus, knowing detection of S. aureus carriers, who are at a risk of spreading nosocomial infection among the staff, is vital to control and prevent nosocomial infections.

Increased Age-Dependent Risk of Death Associated With lukF-PV-Positive Staphylococcus aureus Bacteremia

DEFF Research Database (Denmark)

Knudsen, Trine A; Skov, Robert; Petersen, Andreas

2016-01-01

BACKGROUND: Panton-Valentine leucocidin is a Staphylococcus aureus virulence factor encoded by lukF-PV and lukS-PV that is infrequent in S aureus bacteremia (SAB), and, therefore, little is known about risk factors and outcome of lukF-PV/lukS-PV-positive SAB. METHODS: This report is a register......-based nationwide observational cohort study. lukF-PV was detected by polymerase chain reaction. Factors associated with the presence of lukF-PV were assessed by logistic regression analysis. Adjusted 30-day hazard ratios of mortality associated with lukF-PV status were computed by Cox proportional hazards...... regression analysis. RESULTS: Of 9490 SAB cases, 129 were lukF-PV-positive (1.4%), representing 14 different clonal complexes. lukF-PV was associated with younger age, absence of comorbidity, and methicillin-resistant S aureus. In unadjusted analysis, mortality associated with lukF-PV-positive SAB...

A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus*♦

Science.gov (United States)

Laakso, Holly A.; Marolda, Cristina L.; Pinter, Tyler B.; Stillman, Martin J.; Heinrichs, David E.

2016-01-01

Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD–I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis. PMID:26534960

Growth and enterotoxin production of Staphylococcus aureus during the manufacture and ripening of Camembert-type cheeses from raw goats' milk.

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Meyrand, A; Boutrand-Loei, S; Ray-Gueniot, S; Mazuy, C; Gaspard, C E; Jaubert, G; Perrin, G; Lapeyre, C; Vernozy-Rozand, C

1998-09-01

Tests were carried out to determine the effect of manufacturing procedures for a Camembert-type cheese from raw goats' milk on the growth and survival of Staphylococcus aureus organisms added to milk at the start of the process, and to study the possible presence of staphylococcal enterotoxin A in these cheeses. The initial staphylococcal counts were, respectively, 2, 3, 4, 5 and 6 log cfu ml-1. Cheese was prepared following the industrial specifications and ripened for 41 d. Detection of enterotoxins was done by the Vidas SET test and by an indirect double-sandwich ELISA technique using antienterotoxin monoclonal antibodies. Generally, numbers of microbes increased at a similar rate during manufacture in all cheeses until salting. During the ripening period, the aerobic plate count population and Staph. aureus levels remained stable and high. There was an approximately 1 log reduction of Staph. aureus in cheeses made with an initial inoculum of Staph. aureus greater than 10(3) cfu ml-1 at the end of the ripening period (41 d) compared with the count at 22 h. The level of staphylococcal enterotoxin A recovered varied from 1 to 3.2 ng g-1 of cheese made with an initial population of 10(3)-10(6) cfu ml-1. No trace of enterotoxin A was detected in cheeses made with the lowest Staph. aureus inoculum used in this study.

In vitro transfer of methicillin resistance determinants mecA from methicillin resistant Staphylococcus aureus (MRSA) to methicillin susceptible Staphylococcus aureus (MSSA).

Science.gov (United States)

Bitrus, Asinamai Athliamai; Zunita, Zakaria; Bejo, Siti Khairani; Othman, Sarah; Nadzir, Nur Adilah Ahmad

2017-04-04

Staphylococcus aureus more than any other human pathogen is a better model for the study of the adaptive evolution of bacterial resistance to antibiotics, as it has demonstrated a remarkable ability in its response to new antibiotics. This study was designed to investigate the in vitro transfer of mecA gene from methicillin resistant S. aureus to methicillin susceptible S. aureus. The recipient transconjugants were resistant to erythromycin, cefpodoxime and were mecA positive. PCR amplification of mecA after mix culture plating on Luria Bertani agar containing 100 μg/mL showed that 75% of the donor and 58.3% of the recipient transconjugants were mecA positive. Additionally, 61.5% of both the donor cells and recipient transconjugants were mecA positive, while 46.2% and 41.75% of both donor and recipient transconjugants were mecA positive on LB agar containing 50 μg/mL and 30 μg/mL respectively. In this study, the direction of transfer of phenotypic resistance as well as mecA was observed to have occurred from the donor to the recipient strains. This study affirmed the importance of horizontal transfer events in the dissemination of antibiotics resistance among different strains of MRSA.

Mutagenesis in sequence encoding of human factor VII for gene therapy of hemophilia

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B Kazemi

2009-12-01

Full Text Available "nBackground: Current treatment of hemophilia which is one of the most common bleeding disorders, involves replacement therapy using concentrates of FVIII and FIX .However, these concentrates have been associated with viral infections and thromboembolic complications and development of antibodies. "nThe use of recombinant human factor VII (rhFVII is effective  for the treatment of patients with  hemophilia A or B, who develop antibodies ( referred as inhibitors against  replacement therapy , because it induces coagulation independent of FVIII and FIX. However, its short half-life and high cost have limited its use. One potential solution to this problem may be the use of FVIIa gene transfer, which would attain continuing therapeutic levels of expression from a single injection. The aim of this study was to engineer a novel hFVII (human FVII gene containing a cleavage site for the intracellular protease and furin, by PCR mutagenesis "nMethods: The sequence encoding light and heavy chains of hFVII, were amplified by using hFVII/pTZ57R and specific primers, separately. The PCR products were cloned in pTZ57R vector. "nResults and discussion: Cloning was confirmed by restriction analysis or PCR amplification using specific primers and plasmid universal primers. Mutagenesis of sequence encoding light and heavy chain was confirmed by restriction enzyme. "nConclusion: In the present study, it was provided recombinant plasmids based on mutant form of DNA encoding light and heavy chains.  Joining mutant form of DNA encoding light chain with mutant heavy chain led to a new variant of hFVII. This variant can be activated by furin and an increase in the proportion of activated form of FVII. This mutant form of hFVII may be used for gene therapy of hemophilia.

pTC Plasmids from Sulfolobus Species in the Geothermal Area of Tengchong, China: Genomic Conservation and Naturally-Occurring Variations as a Result of Transposition by Mobile Genetic Elements.

Science.gov (United States)

Xiang, Xiaoyu; Huang, Xiaoxing; Wang, Haina; Huang, Li

2015-02-12

Plasmids occur frequently in Archaea. A novel plasmid (denoted pTC1) containing typical conjugation functions has been isolated from Sulfolobus tengchongensis RT8-4, a strain obtained from a hot spring in Tengchong, China, and characterized. The plasmid is a circular double-stranded DNA molecule of 20,417 bp. Among a total of 26 predicted pTC1 ORFs, 23 have homologues in other known Sulfolobus conjugative plasmids (CPs). pTC1 resembles other Sulfolobus CPs in genome architecture, and is most highly conserved in the genomic region encoding conjugation functions. However, attempts to demonstrate experimentally the capacity of the plasmid for conjugational transfer were unsuccessful. A survey revealed that pTC1 and its closely related plasmid variants were widespread in the geothermal area of Tengchong. Variations of the plasmids at the target sites for transposition by an insertion sequence (IS) and a miniature inverted-repeat transposable element (MITE) were readily detected. The IS was efficiently inserted into the pTC1 genome, and the inserted sequence was inactivated and degraded more frequently in an imprecise manner than in a precise manner. These results suggest that the host organism has evolved a strategy to maintain a balance between the insertion and elimination of mobile genetic elements to permit genomic plasticity while inhibiting their fast spreading.

pTC Plasmids from Sulfolobus Species in the Geothermal Area of Tengchong, China: Genomic Conservation and Naturally-Occurring Variations as a Result of Transposition by Mobile Genetic Elements

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Xiaoyu Xiang

2015-02-01

Full Text Available Plasmids occur frequently in Archaea. A novel plasmid (denoted pTC1 containing typical conjugation functions has been isolated from Sulfolobus tengchongensis RT8-4, a strain obtained from a hot spring in Tengchong, China, and characterized. The plasmid is a circular double-stranded DNA molecule of 20,417 bp. Among a total of 26 predicted pTC1 ORFs, 23 have homologues in other known Sulfolobus conjugative plasmids (CPs. pTC1 resembles other Sulfolobus CPs in genome architecture, and is most highly conserved in the genomic region encoding conjugation functions. However, attempts to demonstrate experimentally the capacity of the plasmid for conjugational transfer were unsuccessful. A survey revealed that pTC1 and its closely related plasmid variants were widespread in the geothermal area of Tengchong. Variations of the plasmids at the target sites for transposition by an insertion sequence (IS and a miniature inverted-repeat transposable element (MITE were readily detected. The IS was efficiently inserted into the pTC1 genome, and the inserted sequence was inactivated and degraded more frequently in an imprecise manner than in a precise manner. These results suggest that the host organism has evolved a strategy to maintain a balance between the insertion and elimination of mobile genetic elements to permit genomic plasticity while inhibiting their fast spreading.

Compatibility and entry exclusion of IncA and IncC plasmids revisited: IncA and IncC plasmids are compatible.

Science.gov (United States)

Ambrose, Stephanie J; Harmer, Christopher J; Hall, Ruth M

2018-02-24

In an early study, IncA and IncC plasmids that were reported to be compatible were grouped as the "A-C complex" based on similarities and on strong entry exclusion. However, recently, the term IncA/C has been used frequently to describe plasmids belonging to both of these two groups. Granted that the supporting data was not included in the original reports and that the consensus iteron sequences have since been shown to be essentially identical, we have addressed the question again. The original IncA plasmid, RA1, and the IncC plasmid pRMH760, were introduced into the same cell by transformation, and were found to be maintained stably for over 100 generations in the absence of selection for either plasmid, i.e. they were compatible. We conclude that use of the term IncA/C for this important plasmid group is indeed incorrect and it causes unnecessary confusion. Granted the importance of IncC plasmids in the spread of antibiotic resistance genes, we recommend that use of the misleading terms IncA/C, IncA/C 1 and IncA/C 2 should cease. In addition, RA1 and pRMH760 were shown to each completely prevent entry of the other via conjugative transfer into the cell they reside in. Copyright © 2018 Elsevier Inc. All rights reserved.

A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus.

Science.gov (United States)

Laakso, Holly A; Marolda, Cristina L; Pinter, Tyler B; Stillman, Martin J; Heinrichs, David E

2016-01-01

Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD-I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Ecological and genetic determinants of plasmid distribution in Escherichia coli.

Science.gov (United States)

Medaney, Frances; Ellis, Richard J; Raymond, Ben

2016-11-01

Bacterial plasmids are important carriers of virulence and antibiotic resistance genes. Nevertheless, little is known of the determinants of plasmid distribution in bacterial populations. Here the factors affecting the diversity and distribution of the large plasmids of Escherichia coli were explored in cattle grazing on semi-natural grassland, a set of populations with low frequencies of antibiotic resistance genes. Critically, the population genetic structure of bacterial hosts was chararacterized. This revealed structured E. coli populations with high diversity between sites and individuals but low diversity within cattle hosts. Plasmid profiles, however, varied considerably within the same E. coli genotype. Both ecological and genetic factors affected plasmid distribution: plasmid profiles were affected by site, E. coli diversity, E. coli genotype and the presence of other large plasmids. Notably 3/26 E. coli serotypes accounted for half the observed plasmid-free isolates indicating that within species variation can substantially affect carriage of the major conjugative plasmids. The observed population structure suggest that most of the opportunities for within species plasmid transfer occur between different individuals of the same genotype and support recent experimental work indicating that plasmid-host coevolution, and epistatic interactions on fitness costs are likely to be important in determining occupancy. © 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.

High-level fluoroquinolone resistant Salmonella enterica serovar Kentucky ST198 epidemic clone with IncA/C conjugative plasmid carrying bla(CTX-M-25) gene.

Science.gov (United States)

Wasyl, Dariusz; Kern-Zdanowicz, Izabela; Domańska-Blicharz, Katarzyna; Zając, Magdalena; Hoszowski, Andrzej

2015-01-30

Multidrug resistant Salmonella Kentucky strains have been isolated from turkeys in Poland since 2009. Multiple mutations within chromosomal genes gyrA and parC were responsible for high-level ciprofloxacin resistance. One of the isolates was extended spectrum β-lactamase- (ESBL) positive: the strain 1643/2010 carried a conjugative 167,779 bps plasmid of IncA/C family. The sequence analysis revealed that it carried a blaCTX-M-25 gene and an integron with another β-lactamase encoding gene-blaOXA-21. This is the first known report of a CTX-M-25 encoding gene both in Poland and in Salmonella Kentucky world-wide, as well as in the IncA/C plasmid. Analysis of the integron showed a novel arrangement of gene cassettes-aacA4, aacC-A1 and blaOXA-21 where the latter might result from an intergeneric gene transfer. The study confirmed Salmonella Kentucky population isolated in Poland belongs to global epidemics of high level fluoroquinolone resistant clone ST198 that can carry rare β-lactamase genes. Copyright © 2014 Elsevier B.V. All rights reserved.

Plasmid DNA initiates replication of yellow fever vaccine in vitro and elicits virus-specific immune response in mice

International Nuclear Information System (INIS)

Tretyakova, Irina; Nickols, Brian; Hidajat, Rachmat; Jokinen, Jenny; Lukashevich, Igor S.; Pushko, Peter

2014-01-01

Yellow fever (YF) causes an acute hemorrhagic fever disease in tropical Africa and Latin America. To develop a novel experimental YF vaccine, we applied iDNA infectious clone technology. The iDNA represents plasmid that encodes the full-length RNA genome of 17D vaccine downstream from a cytomegalovirus (CMV) promoter. The vaccine was designed to transcribe the full-length viral RNA and to launch 17D vaccine virus in vitro and in vivo. Transfection with 10 ng of iDNA plasmid was sufficient to start replication of vaccine virus in vitro. Safety of the parental 17D and iDNA-derived 17D viruses was confirmed in AG129 mice deficient in receptors for IFN-α/β/γ. Finally, direct vaccination of BALB/c mice with a single 20 μg dose of iDNA plasmid resulted in seroconversion and elicitation of virus-specific neutralizing antibodies in animals. We conclude that iDNA immunization approach combines characteristics of DNA and attenuated vaccines and represents a promising vaccination strategy for YF. - Highlights: • The iDNA ® platform combines advantages of DNA and live attenuated vaccines. • Yellow fever (YF) 17D vaccine was launched from iDNA plasmid in vitro and in vivo. • Safety of iDNA-generated 17D virus was confirmed in AG129 mice. • BALB/c mice seroconverted after a single-dose vaccination with iDNA. • YF virus-neutralizing response was elicited in iDNA-vaccinated mice

Plasmid DNA initiates replication of yellow fever vaccine in vitro and elicits virus-specific immune response in mice

Energy Technology Data Exchange (ETDEWEB)

Tretyakova, Irina; Nickols, Brian; Hidajat, Rachmat [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States); Jokinen, Jenny; Lukashevich, Igor S. [Department of Pharmacology and Toxicology, School of Medicine, Center for Predictive Medicine and Emerging Infectious Diseases, University of Louisville, Louisville, KY (United States); Pushko, Peter, E-mail: ppushko@medigen-usa.com [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD 21701 (United States)

2014-11-15

Yellow fever (YF) causes an acute hemorrhagic fever disease in tropical Africa and Latin America. To develop a novel experimental YF vaccine, we applied iDNA infectious clone technology. The iDNA represents plasmid that encodes the full-length RNA genome of 17D vaccine downstream from a cytomegalovirus (CMV) promoter. The vaccine was designed to transcribe the full-length viral RNA and to launch 17D vaccine virus in vitro and in vivo. Transfection with 10 ng of iDNA plasmid was sufficient to start replication of vaccine virus in vitro. Safety of the parental 17D and iDNA-derived 17D viruses was confirmed in AG129 mice deficient in receptors for IFN-α/β/γ. Finally, direct vaccination of BALB/c mice with a single 20 μg dose of iDNA plasmid resulted in seroconversion and elicitation of virus-specific neutralizing antibodies in animals. We conclude that iDNA immunization approach combines characteristics of DNA and attenuated vaccines and represents a promising vaccination strategy for YF. - Highlights: • The iDNA{sup ®} platform combines advantages of DNA and live attenuated vaccines. • Yellow fever (YF) 17D vaccine was launched from iDNA plasmid in vitro and in vivo. • Safety of iDNA-generated 17D virus was confirmed in AG129 mice. • BALB/c mice seroconverted after a single-dose vaccination with iDNA. • YF virus-neutralizing response was elicited in iDNA-vaccinated mice.

The extended regulatory networks of SXT/R391 integrative and conjugative elements and IncA/C conjugative plasmids.

Science.gov (United States)

Poulin-Laprade, Dominic; Carraro, Nicolas; Burrus, Vincent

2015-01-01

Nowadays, healthcare systems are challenged by a major worldwide drug resistance crisis caused by the massive and rapid dissemination of antibiotic resistance genes and associated emergence of multidrug resistant pathogenic bacteria, in both clinical and environmental settings. Conjugation is the main driving force of gene transfer among microorganisms. This mechanism of horizontal gene transfer mediates the translocation of large DNA fragments between two bacterial cells in direct contact. Integrative and conjugative elements (ICEs) of the SXT/R391 family (SRIs) and IncA/C conjugative plasmids (ACPs) are responsible for the dissemination of a broad spectrum of antibiotic resistance genes among diverse species of Enterobacteriaceae and Vibrionaceae. The biology, diversity, prevalence and distribution of these two families of conjugative elements have been the subject of extensive studies for the past 15 years. Recently, the transcriptional regulators that govern their dissemination through the expression of ICE- or plasmid-encoded transfer genes have been described. Unrelated repressors control the activation of conjugation by preventing the expression of two related master activator complexes in both types of elements, i.e., SetCD in SXT/R391 ICEs and AcaCD in IncA/C plasmids. Finally, in addition to activating ICE- or plasmid-borne genes, these master activators have been shown to specifically activate phylogenetically unrelated mobilizable genomic islands (MGIs) that also disseminate antibiotic resistance genes and other adaptive traits among a plethora of pathogens such as Vibrio cholerae and Salmonella enterica.

The extended regulatory networks of SXT/R391 integrative and conjugative elements and IncA/C conjugative plasmids.

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Dominic ePoulin-Laprade

2015-08-01

Full Text Available Nowadays, healthcare systems are challenged by a major worldwide drug resistance crisis caused by the massive and rapid dissemination of antibiotic resistance genes and associated emergence of multidrug resistant pathogenic bacteria, in both clinical and environmental settings. Conjugation is the main driving force of gene transfer among microorganisms. This mechanism of horizontal gene transfer mediates the translocation of large DNA fragments between two bacterial cells in direct contact. Integrative and conjugative elements (ICEs of the SXT/R391 family (SRIs and IncA/C conjugative plasmids (ACPs are responsible for the dissemination of a broad spectrum of antibiotic resistance genes among diverse species of Enterobacteriaceae and Vibrionaceae. The biology, diversity, prevalence and distribution of these two families of conjugative elements have been the subject of extensive studies for the past 15 years. Recently, the transcriptional regulators that govern their dissemination through the expression of ICE- or plasmid-encoded transfer genes have been described. Unrelated repressors control the activation of conjugation by preventing the expression of two related master activator complexes in both types of elements, i.e. SetCD in SXT/R391 ICEs and AcaCD in IncA/C plasmids. Finally, in addition to activating ICE- or plasmid-borne genes, these master activators have been shown to specifically activate phylogenetically unrelated mobilizable genomic islands (MGIs that also disseminate antibiotic resistance genes and other adaptive traits among a plethora of pathogens such as Vibrio cholerae and Salmonella enterica.

The characteristics of micrococcus (deinococcus) radiodurans sark plasmids

International Nuclear Information System (INIS)

Sjarief, Sri Hariani; Kikuchi, Masahiro; Watanabe, Hiroshi.

1994-01-01

The characterization of micrococcus (deinococcus) radiodurans sark plasmids. This bacterium has been classified as a new genus deinococcus radiodurans which is resistant to gamma-rays. It can repair itself completely almost all of DNA damages including double strand breaks induced by gamma-rays up to about 5 KGy. To reveal the repair mechanism, several investigations had been done to develop a cloning vector available for the genetic analysis. For this purpose D. radiodurans Sark are to be prepared as a vector by studying the characteristics of its plasmid. Plasmids were isolated by electrophoresis using 0.6% low-melting-temperature agarose in TAE and run for 5.5 hours, followed by the identification. An antibiotic marker was also carried out in this experiment to identify its location in the genetic materials of the cell, beside making a restriction map of the plasmid. Results have shown that D. radiodurans Sark has 4 plasmids (P1, P2, P3, and P4) and the refampicin resistant genes were not found in the plasmid. (authors). 14 refs; 4 figs

Hydrodynamic delivery of plasmid DNA encoding human Fc?R-Ig dimers blocks immune-complex mediated inflammation in mice