Sample records for acriflavine

  1. Spectrofluorometric determination of furosemide in some pharmaceutical product using acriflavine as a reagent

    Qader, Aryan F.; Fakhre, Nabil A.


    Simple, inexpensive, rapid and sensitive determination of furosemide spectrofluorometrically was suggested using acriflavine as a new reagent. The method based on the quantitative quenching effect of furosemide on the native fluorescence of acriflavine in presence of Britton-Robinson buffer medium due to the reaction of furosemide with acriflavine to form an ion associated complex. The decrease of acriflavine fluorescence was observed at 505 nm after excitation at 265 nm. the florescence - concentration plot is rectilinear over the range of 2.0-10 µg/ml with correlation coefficient 0.9991 and detection limit 0.2 µg/ml. No interference was observed from the excipients that are commonly present in pharmaceutical formulations. The proposed method was determination of furosemide in some commercial tablets. The results were compared with that of HPLC method revealed with good agreements and no significant differences in the accuracy and precision.

  2. Cytotoxic effect of acriflavine against clinical isolates of Acanthamoeba spp.

    Polat, Zubeyda Akin; Karakus, Gulderen


    Acanthamoeba keratitis (AK) is a potentially devastating and sight-threatening infection of the cornea caused by the ubiquitous free-living amoebae, Acanthamoeba species. Its eradication is difficult because the amoebas encyst, making it highly resistant to anti-amoebic drugs. Acriflavine neutral (ACF) has been used for treatment of microbial infections for humans and fishes. The aim of our study was to evaluate the time-dependent cytotoxicities of ACF against Acanthamoeba spp. Trophozoites and cysts of three different strains (strain PAT06 Acanthamoeba castellanii, strain 2HH Acanthamoeba hatchetti, and strain 11DS A. hatchetti) of Acanthamoeba spp. were tested. All strains had been isolated from patients suffering from a severe AK. The effects of the ACF with the concentrations ranging from 15 to 500 mg mL(-1) on the cytotoxicity of Acanthamoeba strains were examined. ACF showed a time- and dose-dependent amebicidal action on the trophozoites and cysts. Pat06 (A. castellanii) was the most resistant, while strain 11DS (A. hatchetti) was the most sensitive. As a result, ACF could be concluded as a new agent for the treatment of Acanthamoeba infections. On the other hand, it still needs to be further evaluated by in vivo test systems to confirm the efficiency of its biological effect.

  3. Acriflavine immobilized onto polyethyleneimine-wrapped carbon nanotubes/gold nanoparticles as an eletrochemical sensing platform

    Azadeh Azadbakht; Amirreza Abbasi


    Multi-walled carbon nanotubes wrapped by polyethyleneimine (PEI) and functionalized with a carboxylic acid group (CNT-COOH) were deposited with gold nanoparticles (AuNPs) which has been utilized as a platform to immobilize poly(acriflavine)(PAF) and used as modified electrode (AuNPs/PEI/CNTCOOH/PAF). Electrocatalytic reduction of hydrogen peroxide(H2O2) on the surface of the modified electrode was investigated by cyclic voltammetry and electrochemical impedance spectroscopy (EIS) methods. The cyclic voltammetric results indicated the ability of modified Au electrode to catalyze the reduction of H2O2. AuNPs/PEI/CNT-COOH nanocomposite combined the advantages of PEI, well dispersed CNT-COOH and in situ formed AuNPs, endowed with high stability to the enzyme-free sensor.

  4. Effect of frameshift mutagen acriflavine on control of resistance genes in Acinetobacter baumannii.

    Lopes, B S; Hamouda, A; Findlay, J; Amyes, S G B


    Acinetobacter baumannii is a Gram-negative pathogenic bacterium that often exhibits a multidrug-resistant phenotype causing infections at various sites of the body and increasingly leading to septicaemic shock. This study evaluated the role of acriflavine, a frameshift mutagen, on the movement of insertion sequence ISAba1 in clinical isolates of A. baumannii, with the focus on changes in expression levels of the bla(ADC) and bla(OXA-51-like) genes. Resistance profiles were assessed with consideration of ISAba1 acting as a promoter upstream of the bla(ADC) or bla(OXA-51-like) gene. ISAba1 movement was observed in the acriflavine mutants Ab153M and Ab1225M. Ab153M exhibited an increase in the MIC values of carbapenems and ceftazidime, with ISAba1 gained upstream of the bla(ADC) and bla(OXA-51-like) genes, correlating with an increase in gene expression. Reduced expression of the 17, 23 and 25 kDa outer-membrane proteins (OMPs) was also observed in Ab153M. There was a significant decrease in MIC values of carbapenems with the loss of ISAba1 upstream of the bla(ADC) and bla(OXA-51-like) genes in strain Ab1225M, and a significant decrease in bla(OXA-51-like) gene expression and, to a lesser extent, in bla(ADC) expression. Ab1225M and a serially subcultured Ab1225 strain (Ab1225s) exhibited overexpression of the 17, 23, 25 and 27 kDa OMPs. There was a decrease in MIC values of the carbapenems and piperacillin/tazobactam but not of ceftazidime in Ab1225s, which had ISAba1 upstream of the bla(ADC) and bla(OXA-51-like) genes. A significant decrease in bla(OXA-51-like) expression was observed in Ab1225s, whereas the expression of bla(ADC) was similar to that in the Ab1225 parental strain. The attenuation in this strain may be due to overexpression of OMPs and it is clear that, even if ISAba1 is present upstream of an antibiotic resistance gene, it may not necessarily contribute towards the overexpression of antibiotic resistance genes (bla(OXA-51-like) in Ab

  5. Investigation on the inclusion and toxicity of acriflavine with cyclodextrins: A spectroscopic approach

    Manivannan, C.; Meenakshi Sundaram, K.; Sundararaman, M.; Renganathan, R.


    Acriflavine hydrochloride (AFN) is a prospective drug worn in the eradication of HIV1 infection. The toxicity and adverse side effects renders the potent drug to limits its usage. However, to overcome the dilemma we have aimed to select carriers with great complexation efficiencies in different cyclodextrins (CDs) of varying cavity size. The interaction of AFN with α, β and γ-CDs were investigated using absorption and steady state as well as lifetime measurements. From the obtained data it was found that AFN fits in the cavity of α and β-CDs but unable to form inclusion complex with γ-CD. The effect of quencher molecules during the inclusion phenomena of AFN with CDs was explored via steady state measurements. The nature of binding forces responsible for the inclusion of AFN with CDs was discussed by using thermodynamic parameters. Using Benesi-Hildebrand equation the stoichiometry of AFN with CDs was predominantly found to be 1:1. To get deeper in situ, the in vitro toxicity of AFN and its complexation product were probed by Artemia salina sp. The toxicity of AFN was reduced when complexed with α and β-CDs.

  6. NCBI nr-aa BLAST: CBRC-XTRO-01-0459 [SEVENS

    Full Text Available CBRC-XTRO-01-0459 ref|YP_286542.1| Acriflavin resistance protein [Dechloromonas aro...matica RCB] gb|AAZ48072.1| Acriflavin resistance protein [Dechloromonas aromatica RCB] YP_286542.1 0.0 62% ...

  7. NCBI nr-aa BLAST: CBRC-XTRO-01-0459 [SEVENS

    Full Text Available CBRC-XTRO-01-0459 ref|YP_983051.1| acriflavin resistance protein [Polaromonas naphtha...lenivorans CJ2] gb|ABM38130.1| acriflavin resistance protein [Polaromonas naphthalenivorans CJ2] YP_983051.1 0.0 81% ...

  8. NCBI nr-aa BLAST: CBRC-AGAM-04-0109 [SEVENS

    Full Text Available CBRC-AGAM-04-0109 ref|ZP_02018486.1| acriflavin resistance protein [Methylobacterium extorque...ns PA1] gb|EDN54609.1| acriflavin resistance protein [Methylobacterium extorquens PA1] ZP_02018486.1 2.7 30% ...

  9. NCBI nr-aa BLAST: CBRC-CREM-01-1300 [SEVENS

    Full Text Available CBRC-CREM-01-1300 ref|YP_001267510.1| acriflavin resistance protein [Pseudomonas pu...tida F1] gb|ABQ78326.1| acriflavin resistance protein [Pseudomonas putida F1] YP_001267510.1 0.0 60% ...

  10. NCBI nr-aa BLAST: CBRC-CREM-01-1300 [SEVENS

    Full Text Available CBRC-CREM-01-1300 ref|YP_348253.1| Acriflavin resistance protein [Pseudomonas fluor...escens PfO-1] gb|ABA74263.1| Acriflavin resistance protein [Pseudomonas fluorescens PfO-1] YP_348253.1 0.0 60% ...

  11. NCBI nr-aa BLAST: CBRC-CREM-01-1282 [SEVENS

    Full Text Available CBRC-CREM-01-1282 ref|YP_001268986.1| acriflavin resistance protein [Pseudomonas pu...tida F1] gb|ABQ79802.1| acriflavin resistance protein [Pseudomonas putida F1] YP_001268986.1 1e-175 69% ...

  12. Characterization of putative multidrug resistance transporters of the major facilitator-superfamily expressed in Salmonella Typhi

    Shaheen, Aqsa; Ismat, Fouzia; Iqbal, Mazhar


    conferred resistance to at least ten of the tested antimicrobials: ciprofloxacin, norfloxacin, levofloxacin, kanamycin, streptomycin, gentamycin, nalidixic acid, chloramphenicol, ethidium bromide, and acriflavine, including fluoroquinolone antibiotics, which were drugs of choice to treat S. Typhi infections...

  13. Contact dermatitis presenting as non-healing wound: case report

    Leelavathi M


    Full Text Available Abstract Topical antiseptics are commonly used in the management of minor wounds, burns, and infected skin. These agents are widely used by health professionals and are often self-prescribed by patients as they are easily available over-the-counter. This case illustrates a 73 year old man who presented with a non-healing wound on his right forearm for 4 weeks. The wound started from an insect bite and progressively enlarged with increasing pruritus and burning sensation. Clinically an ill-defined ulcer with surrounding erythema and erosion was noted. There was a yellow crust overlying the center of the ulcer and the periphery was scaly. Further inquiry revealed history of self treatment with a yellow solution to clean his wound for 3 weeks. Patient was provisionally diagnosed to have allergic contact dermatitis secondary to acriflavine. Topical acriflavine was stopped and the ulcer resolved after treatment with non-occlusive saline dressing. Skin patch test which is the gold standard for detection and confirmation of contact dermatitis showed a positive reaction (2+ to acriflavine. Acriflavine is widely used as a topical antiseptic agent in this part of the world. Hence, primary care physicians managing a large variety of poorly healing wounds should consider the possibility of contact allergy in recalcitrant cases, not responding to conventional treatment. Patient education is an important aspect of management as this would help curb the incidence of future contact allergies.

  14. Some Experiments with Respiratory Deficient Mutants of Yeast (Saccharomyces cerevisiae)

    Freeland, P. W.


    Methods are described for the induction and identification of respiratory deficient mutants in yeast. Practical schemes are given to enable students to obtain dose-response information for physical and chemical mutagens such as heat, ultraviolet light, or acriflavine. A simple test for environmental mutagens is described. (Author/MA)

  15. Treatment of trichodiniasis in eel ( Anguilla anguilla ) reared in recirculation systems in Denmark : alternatives to formaldehyde

    Madsen, H.C.K.; Buchmann, Kurt; Mellergaard, Stig


    parasiticidal effect: acriflavin (25 ppm), bithionol (0.1 ppm), chloramine T (50 ppm), Detarox AP(R) (45 ppm), malachite green (1 ppm), raw garlic (200 ppm), potassium permanganate (20 ppm) and Virkon PF(R) vet. (20 ppm). Preliminary screening revealed that the anthelmintic, bithionol, and the decomposable...

  16. Isolation and biotyping of Brucella melitensis from aborted sheep and goat fetuses

    BÜYÜKCANGAZ, Esra; ŞEN, Ayşin; KAHYA, Serpil


    The possible role of Brucella spp. in 65 abortion cases, 55 from sheep and 10 from goats, occurring in the birth seasons of 2004 and 2005 in northwestern Turkey was investigated. Colony morphology, agglutination by acriflavin, H2S production, CO2 requirement, dye sensitivity in thionin, basic fuchsin, growth characteristics in streptomycin, lysis with Tbilisi phage, and agglutination with monospecific A- and M antisera were examined for identification and biotyping. The isolates from 21 of 55...

  17. Activation of cGAS-dependent antiviral responses by DNA intercalating agents.

    Pépin, Geneviève; Nejad, Charlotte; Thomas, Belinda J; Ferrand, Jonathan; McArthur, Kate; Bardin, Philip G; Williams, Bryan R G; Gantier, Michael P


    Acridine dyes, including proflavine and acriflavine, were commonly used as antiseptics before the advent of penicillins in the mid-1940s. While their mode of action on pathogens was originally attributed to their DNA intercalating activity, work in the early 1970s suggested involvement of the host immune responses, characterized by induction of interferon (IFN)-like activities through an unknown mechanism. We demonstrate here that sub-toxic concentrations of a mixture of acriflavine and proflavine instigate a cyclic-GMP-AMP (cGAMP) synthase (cGAS)-dependent type-I IFN antiviral response. This pertains to the capacity of these compounds to induce low level DNA damage and cytoplasmic DNA leakage, resulting in cGAS-dependent cGAMP-like activity. Critically, acriflavine:proflavine pre-treatment of human primary bronchial epithelial cells significantly reduced rhinovirus infection. Collectively, our findings constitute the first evidence that non-toxic DNA binding agents have the capacity to act as indirect agonists of cGAS, to exert potent antiviral effects in mammalian cells. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. Functionally cloned pdrM from Streptococcus pneumoniae encodes a Na(+ coupled multidrug efflux pump.

    Kohei Hashimoto

    Full Text Available Multidrug efflux pumps play an important role as a self-defense system in bacteria. Bacterial multidrug efflux pumps are classified into five families based on structure and coupling energy: resistance-nodulation-cell division (RND, small multidrug resistance (SMR, major facilitator (MF, ATP binding cassette (ABC, and multidrug and toxic compounds extrusion (MATE. We cloned a gene encoding a MATE-type multidrug efflux pump from Streptococcus pneumoniae R6, and designated it pdrM. PdrM showed sequence similarity with NorM from Vibrio parahaemolyticus, YdhE from Escherichia coli, and other bacterial MATE-type multidrug efflux pumps. Heterologous expression of PdrM let to elevated resistance to several antibacterial agents, norfloxacin, acriflavine, and 4',6-diamidino-2-phenylindole (DAPI in E. coli KAM32 cells. PdrM effluxes acriflavine and DAPI in a Na(+- or Li(+-dependent manner. Moreover, Na(+ efflux via PdrM was observed when acriflavine was added to Na(+-loaded cells expressing pdrM. Therefore, we conclude that PdrM is a Na(+/drug antiporter in S. pneumoniae. In addition to pdrM, we found another two genes, spr1756 and spr1877,that met the criteria of MATE-type by searching the S. pneumoniae genome database. However, cloned spr1756 and spr1877 did not elevate the MIC of any of the investigated drugs. mRNA expression of spr1756, spr1877, and pdrM was detected in S. pneumoniae R6 under laboratory growth conditions. Therefore, spr1756 and spr1877 are supposed to play physiological roles in this growth condition, but they may be unrelated to drug resistance.

  19. Photocatalytic degradation of acridine dyes using anatase and rutile TiO2.

    Zubieta, C E; Messina, P V; Schulz, P C


    The adsorption and photodegradation of acridine orange (AO) and acriflavine (AF) dyes on two mesoporous titania crystalline phases, anatase and rutile, were experimentally studied. Anatase and rutile were characterized by nitrogen adsorption, electron scanning and transmission microscopy, and X-ray diffraction. The adsorption capacity of rutile was higher than that of anatase, while the reverse is observed for photodegradation of both dyes. The adsorption of AF on both adsorbents was higher than that of AO, which was related with the smaller size of AF molecules compared with those of AO, therefore the access of AF to the adsorption sites is favored.

  20. There is a baby in the bath water: AcrB contamination is a major problem in membrane-protein crystallization.

    Veesler, David; Blangy, Stéphanie; Cambillau, Christian; Sciara, Giuliano


    In the course of a crystallographic study of the Methanosarcina mazei CorA transporter, the membrane protein was obtained with at least 95% purity and was submitted to crystallization trials. Small crystals (contaminating protein, acriflavine resistance protein B (AcrB), that was present at less than 5% in the protein preparations. AcrB contamination is a major problem when expressing membrane proteins in E. coli since it binds naturally to immobilized metal-ion affinity chromatography (IMAC) resins. Here, the structure is compared with previously deposited AcrB structures and strategies are proposed to avoid this contamination.

  1. Host–guest composite materials of dyes loaded zeolite LTL for antenna applications

    Insuwan, W. [Rajamangala University of Technology Isan Surin Campus, Facculty of Agriculture and Technology, Surin 32000 (Thailand); Jungsuttiwong, S. [Center for Organic Electronic and Alternative Energy, Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Ubon Ratchathani University, Ubon Ratchathani 34190 (Thailand); Rangsriwatananon, K., E-mail: [School of Chemistry, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000 (Thailand)


    This research work directly focuses on a new feasible light harvesting antenna material constructed with Acridine hydrochloride (Ac)/Acriflavine hydrochloride (AF), as donor/acceptor for energy transfer, loaded on a round shape zeolite LTL (K-LTL and H-LTL). The energy transfer was monitored by absorption and fluorescence spectra while the calculated Förster distance (R{sub DA}) and Quenching efficiency (%Q) of Ac/AF on K-LTL and H-LTL varied between 22.0 Å to 19.6 Å and 71.4% to 65.5%, respectively. Also, it was found that the microenvironment of a solid host such as K-LTL and H-LTL has significantly influenced the fluorescence spectra of Ac/AF on H-LTL approximately 50 nm longer than that on K-LTL. - Highlights: • New antenna materials have been performed using dyes loaded on zeolite LTL. • Light emission takes place from acriflavine hydrochloride (AF) due to fluorescence resonance energy transfer (FRET). • The microenvironment of zeolite LTL has significantly influenced the fluorescence spectra.

  2. Functional analysis of Vibrio vulnificus RND efflux pumps homologous to Vibrio cholerae VexAB and VexCD, and to Escherichia coli AcrAB.

    Lee, Seunghwa; Yeom, Ji-Hyun; Seo, Sojin; Lee, Minho; Kim, Sarang; Bae, Jeehyeon; Lee, Kangseok; Hwang, Jihwan


    Resistance-nodulation-division (RND) efflux pumps are associated with multidrug resistance in many gram-negative pathogens. The genome of Vibrio vulnificus encodes 11 putative RND pumps homologous to those of Vibrio cholerae and Escherichia coli. In this study, we analyzed three putative RND efflux pumps, showing homology to V. cholerae VexAB and VexCD and to E. coli AcrAB, for their functional roles in multidrug resistance of V. vulnificus. Deletion of the vexAB homolog resulted in increased susceptibility of V. vulnificus to bile acid, acriflavine, ethidium bromide, and erythromycin, whereas deletion of acrAB homologs rendered V. vulnificus more susceptible to acriflavine only. Deletion of vexCD had no effect on susceptibility of V. vulnificus to these chemicals. Upon exposure to these antibacterial chemicals, expression of tolCV1 and tolCV2, which are putative outer membrane factors of RND efflux pumps, was induced, whereas expression levels of vexAB, vexCD, and acrAB homologs were not significantly changed. Our results show that the V. vulnificus homologs of VexAB largely contributed to in vitro antimicrobial resistance with a broad substrate specificity that was partially redundant with the AcrAB pump homologs.

  3. Characterization of putative multidrug resistance transporters of the major facilitator-superfamily expressed in Salmonella Typhi.

    Shaheen, Aqsa; Ismat, Fouzia; Iqbal, Mazhar; Haque, Abdul; De Zorzi, Rita; Mirza, Osman; Walz, Thomas; Rahman, Moazur


    Multidrug resistance mediated by efflux pumps is a well-known phenomenon in infectious bacteria. Although much work has been carried out to characterize multidrug efflux pumps in Gram-negative and Gram-positive bacteria, such information is still lacking for many deadly pathogens. The aim of this study was to gain insight into the substrate specificity of previously uncharacterized transporters of Salmonella Typhi to identify their role in the development of multidrug resistance. S. Typhi genes encoding putative members of the major facilitator superfamily were cloned and expressed in the drug-hypersensitive Escherichia coli strain KAM42, and tested for transport of 25 antibacterial compounds, including representative antibiotics of various classes, antiseptics, dyes and detergents. Of the 15 tested putative transporters, STY0901, STY2458 and STY4874 exhibited a drug-resistance phenotype. Among these, STY4874 conferred resistance to at least ten of the tested antimicrobials: ciprofloxacin, norfloxacin, levofloxacin, kanamycin, streptomycin, gentamycin, nalidixic acid, chloramphenicol, ethidium bromide, and acriflavine, including fluoroquinolone antibiotics, which were drugs of choice to treat S. Typhi infections. Cell-based functional studies using ethidium bromide and acriflavine showed that STY4874 functions as a H(+)-dependent exporter. These results suggest that STY4874 may be an important drug target, which can now be tested by studying the susceptibility of a STY4874-deficient S. Typhi strain to antimicrobials.

  4. Acute cadmium intoxication: influence of cyproterone acetate on the testis and epididymis of the rat.

    Francavilla, S; Moscardelli, S; Francavilla, F; Casasanta, N; Properzi, G; Martini, M; Santiemma, V


    The changes resulting from treatment with cadmium were studied following the histological changes, the modification of both vascular permeability to vital dyes and of alkaline phosphatase activity in rat testis and epididymis. The testicular extravasation of acriflavine started 90 min following parenteral injection of cadmium and increased thereafter synchronous with an increase in testicular and epididymal weights due to edema. At 14 and 24 hr a striking decrease of interstitial fluorescence and tubular degeneration were noted in testis and caput epididymis due to thrombosis of the microvascular circulation. The barrier noted at 8 hr following cadmium injection. No changes of alkaline phosphatase activity was detected in testicular and epididymal blood vessels after cadmium injection. Previous treatment with cyproterone acetate accelerated the appearance of such alterations. The interstitial nuclear staining with acriflavine appeared in the testis at 1 hr and was diffuse at 90 and 120 min. cyproterone acetate seemed to accelerate the appearance of tubular degeneration at 8 hr after cadmium injection. The changes of the male rat gonad following cadmium treatment were characterized by an increased vascular permeability and generalized thrombosis. An inbalance of androgen stimulation seems to increase the blood vessels susceptibility to cadmium.

  5. Overexpression of patA and patB, which encode ABC transporters, is associated with fluoroquinolone resistance in clinical isolates of Streptococcus pneumoniae.

    Garvey, Mark I; Baylay, Alison J; Wong, Ryan L; Piddock, Laura J V


    Fifty-seven clinical isolates of Streptococcus pneumoniae were divided into four groups based on their susceptibilities to the fluoroquinolones ciprofloxacin and norfloxacin and the dyes ethidium bromide and acriflavine. Comparative reverse transcription-PCR was used to determine the level of expression of the genes patA and patB, which encode putative ABC transporters. Overexpression was observed in 14 of the 15 isolates that were resistant to both fluoroquinolones and dyes and in only 3 of 24 of those resistant to fluoroquinolones only. Isolates overexpressing patA and patB accumulated significantly less of the fluorescent dye Hoechst 33342 than wild-type isolates, suggesting that PatA and PatB are involved in efflux. Inactivation of patA and patB by in vitro mariner mutagenesis conferred hypersusceptibility to ethidium bromide and acriflavine in all isolates tested and lowered the MICs of ciprofloxacin in the patAB-overproducing and/or fluoroquinolone-resistant isolates. These data represent the first observation of overexpression of patA and patB in clinical isolates and show that PatA and PatB play a clinically relevant role in fluoroquinolone resistance.

  6. Maintenance of residual activity of Bt toxin by using natural and synthetic dyes: a novel approach for sustainable mosquito vector control.

    Chandrashekhar, Patil; Rahul, Suryawanshi; Hemant, Borase; Chandrakant, Narkhede; Bipinchandra, Salunke; Satish, Patil


    Mosquito control protein from Bacillus thuringiensis gets inactivated with exposure to sunlight. To address this issue, the potential of synthetic and natural dye was investigated as sunlight protectants. Bt SV2 in absence of dyes when exposed to sunlight showed reduced effectiveness against the fourth instars of mosquito larvae. Whereas acriflavin, congo red and violacein were able to maintain 86.4%, 91.6% and 82.2% mosquito larvicidal efficacy of Bt SV2 against IVth instars larvae of Anopheles stephensi Meigen after exposure to sunlight. Similarly, beetroot dye, acriflavin, congo red and violacein maintained 98.4%, 97.1%, 90.8% and 70.7% larvicidal activities against Aedes aegypti Linnaeus after sunlight exposure. Prodigiosin was found to be the best photo-protectant by simultaneously protecting and enhancing Bt activity by 6.16% and 22.16% against A. stephensi and A. aegypti, respectively. Combination of dyes with Bt formulations can be a good strategy for mosquito control programmes in tropical and sub-tropical regions.

  7. DIFFERENTIATION BETWEEN Staphylococcus aureus, S. intermedius AND S. hyicus USING PHENOTYPICAL TESTS AND PCR

    E. A. GANDRA


    Full Text Available

    The aim of this work was to compare the use of two phenotypical tests (beta-galactosidase and acriflavine sensitivity and PCR for the coa and nuc gene sequences, for the identification of three species of coagulase positive staphylococcus (CPS: S. aureus, S. intermedius and S. hyicus. Sixty five staphylococcus isolates, previously characterized as CPS through the coagulase, thermonuclease and catalase production tests and Gram staining, were identified at species level using the beta-galactosidase production tests and sensitivity to acriflavine (7 g.mL-1. At the same time, the DNA of these isolates was extracted and amplified by PCR, using primers for the coa gene, specific for S. aureus, and for the nuc gene, specific for S. intermedius and for S. hyicus. It was possible to differentiate the three species of Staphylococcus through phenotypical tests as well as through the molecular technique. There was no difference in discriminatory power between the two techniques used, but the reproducibility and reliability of the technique based on PCR make this technique more precise.

  8. Clinical use of a portable dual microscope system for smartphone (Conference Presentation)

    Kurachi, Cristina; Brognara, Gabriel; Gómez-García, Pablo A.; Carbinatto, Fernanda; Silva, Eduardo V.; Lombardi, Wellington; Inada, Natália M.; Bagnato, Vanderlei S.


    Cervical cancer is still one of the most relevant women cancer types, since the 5-year survival rate is of only around 68%. Prevention and early diagnosis are the best strategies to improve cervical cancer prognosis. Conventional diagnosis procedure in Gynecology is mainly based on the macroscopic clinical evaluation, Pap smear cytology, and biopsy, if needed. A portable microscope with dual configuration and its use for diagnosis in Gynecology is investigated. The microscope has interchangeable parts that allow its use for cytopathology smear samples or in situ endoscopic tissue interrogation, both using acriflavine as a nuclei marker. Patients of the Women Ambulatory of the School of Medicine (UNIARA, Araraquara, Brazil) were interrogated during the colposcopy examination. The cervix was initially cleaned using an acetic acid solution, and a 0.05% (wt/vol) acriflavine in saline solution was topically applied at the tissue surface using a cotton swab. Microendoscopy images were taken from clinically normal cervix mucosa and from detected lesions. An image processing is performed to evaluate the cell nuclei morphology and the cytoplasm/nuclei ratio. The Pap smear results and the histology analyses are taken as gold standard for the diagnosis. Preliminary results in 5 patients demonstrated the potential use of our microscope at the clinical setting.


    Maria Aparecida Vasconcelos Paiva Brito


    Full Text Available Os testes de produção de acetoína, determinação da atividade da enzima beta-galactosidase e utilização anaeróbica do manitol em conjunto com a susceptibilidade à acriflavina foram avaliados para diferenciação de amostras de Staphylococcus coagulase-positivas (SCP isoladas de mastite bovina. As amostras foram classificadas no gênero Staphylococcus por meio da sensibilidade a furazolidona, resistência à bacitracina, produção de ácido em aerobiose a partir de glicerol na presença de 0,4mig m-1 de eritromicina e catalase, e foram positivas no teste de coagulase do plasma de coelho em tubos. A susceptibilidade à acriflavina foi testada em placas de ágar Baird Parker e ágar P com 7,0mig m-1 de acriflavina. Como controle dos testes, foram incluídas cinco amostras coagulase-negativas de S. hyicus isoladas de leite bovino e identificadas pelo sistema API Staph e a amostra de S. aureus ATCC 29213. Trinta e oito das 49 amostras de SCP foram identificadas como S. aureus e 11 como S. hyicus, não sendo identificada nenhuma como S. intermedius. O sistema API Staph foi empregado para confirmar a identificação das amostras coagulase-positivas de S. hyicus, sete amostras de S. aureus negativas no teste de produção de acetoína e quatro negativas na fermentação anaeróbica do manitol. Todas as amostras de S. aureus foram resistentes a acriflavina, enquanto as de S. hyicus foram sensíveis. Concluiu-se que a sensibilidade a acriflavina pode ser empregada juntamente com os testes de coagulase e produção de acetoína na diferenciação de SCP isolados de mastite bovina.Production of acetoin, acid production from mannitol under anaerobiosis and beta-galactosidase activity in addition to acriflavin susceptibility were evaluated to differentiate between coagulase-positive strains of Staphylococcus (CPS isolated from bovine mastitis. The strains were classified in the genus Staphylococcus by means of sensitivity to furazolidone, resistance

  10. [Antiseptic efficacy and acceptance of Octenisept computed with common antiseptic mouthwashes].

    Kramer, A; Höppe, H; Krull, B; Pitten, F A; Rosenau, S


    The efficacy of common antiseptic mouth rinses was evaluated in a study with healthy volunteers. Octenisept and Cetylpyridiniumchlorid had a significant stronger impact on the microbial burden of the oral cavity than Chlorhexidin (Corsodyl) immediately and 10 min after the application. Dobendan (contains Cetylpyridiniumchlorid) showed a better antimicrobial effect than Corsodyl immediately after application, after 10 min an increasing loss of efficacy was noted. The antiseptic efficacy of hexetidin-based Gurfix was very similar to the efficacy observed with Corsodyl from the start of the antiseptic treatment until 1 h later. Acriflavine-solution (0.2% m/v) was significantly less efficient after 10, 30 and 60 min compared to Corsodyl. The antimicrobial impact of Fluomint-Lysoform was very similar to the effect of rinsing with dest. sterile water. A subjective assessment of taste and smell of the mouth rinse solutions concludes the evaluation. Further methodological aspects for a common test guideline for antiseptic mouth rinse solutions are discussed.

  11. Colony mutants of compatible nocardiae displaying variations in recombining capacity.

    Brownell, G H; Walsh, R S


    Colonial morphology mutants of Nocardia erythropolis were isolated following ultraviolet (UV) irradiation. The alleles rou-1/smo-1 were located by recombinant analysis and found to be linked to previously mapped characters. On the basis of recombinant class type patterns obtained from various selective characters it was postulated that the rou-1 allele may span a region of unique nucleotides in the Mat-Ce genome. Recombination frequencies of rou-1 and smo-2 bearing mutants of the Mat-Ce mating type were found to differ by over 1000 fold. Attempts to demonstrate that low recombination frequencies produced by the Smo mutants were due to Rec(-) genes were unsuccessful. No increased sensitivity to either UV or X irradiation was observed by the Smo mutants. Acriflavine treatment of either Rou or Smo colony mutants failed to accelerate reversion or to alter the recombining potentials of the mutants.

  12. An epifluorescence microscopy method for direct detection and enumeration of the fungilike marine protists, the thraustochytrids

    Raghukumar, S.; Schaumann, K.

    (Gaertner 19 6 8) and a liquid medium of the following composition at pH 6.5-7.3: glucose, 1.0 g; peptone, 1.0 g; yeast extract, 2.0 g; disodium hydrogen phosphate, Notes 183 0.125 g; ferric chloride, 0.002 g; seawater, 1,000 ml. Cells were harvested....4 x 10” 0.1 x 106 1.7x 108 0.6x lo5 5.0~ lo9 0.9 x 106 1.8 x lOlo Acriflavine (3,6-diamino- 1 O-methyl acri- dinium chloride mixed with 3,6-acridine di- amine) is a cationic acridine dye (Windholz 1983) and has been used as a fluorescent stain...

  13. Molecular mechanisms of DNA repair inhibition by caffeine

    Selby, C.P.; Sancar, A. (Univ. of North Carolina School of Medicine, Chapel Hill (USA))


    Caffeine potentiates the mutagenic and lethal effects of genotoxic agents. It is thought that this is due, at least in some organisms, to inhibition of DNA repair. However, direct evidence for inhibition of repair enzymes has been lacking. Using purified Escherichia coli DNA photolyase and (A)BC excinuclease, we show that the drug inhibits photoreactivation and nucleotide excision repair by two different mechanisms. Caffeine inhibits photoreactivation by interfering with the specific binding of photolyase to damaged DNA, and it inhibits nucleotide excision repair by promoting nonspecific binding of the damage-recognition subunit, UvrA, of (A)BC excinuclease. A number of other intercalators, including acriflavin and ethidium bromide, appear to inhibit the excinuclease by a similar mechanism--that is, by trapping the UvrA subunit in nonproductive complexes on undamaged DNA.

  14. Fiber-optic nonlinear endomicroscopy with focus scanning by using shape memory alloy actuation

    Wu, Yicong; Zhang, Yuying; Xi, Jiefeng; Li, Ming-Jun; Li, Xingde


    A miniature fiber optic endomicroscope with built-in dynamic focus scanning capability is developed for the first time for 3-D two-photon fluorescence (TPF) imaging of biological samples. Fast 2-D lateral beam scanning is realized by resonantly vibrating a double-clad fiber cantilever with a tubular piezoactuator. Slow axial scanning is achieved by moving the distal end of the imaging probe with an extremely compact electrically driven shape memory alloy (SMA). The 10-mm-long SMA allows 150-μm contractions with a driving voltage varying only from 50 to 100 mV. The response of the SMA contraction with the applied voltage is nonlinear, but repeatable and can be accurately calibrated. Depth-resolved imaging of acriflavine-stained biological tissues and unstained white paper with the endomicroscope is performed, and the results demonstrate the feasibility of 3-D nonlinear optical imaging with the SMA-based scanning fiber-optic endomicroscope. PMID:21198147

  15. The biological activity of quaternary ammonium salts (QASs

    Ewa Obłąk


    Full Text Available Quaternary ammonium salts (QASs, especially those of cationic surfactant character, are applied as antibacterial and antifungal disinfectants. QASs affect lipid-enveloped viruses, including human immunodeficiency virus (HIV and hepatitis B virus (HBV, but not non-enveloped viruses. These compounds are extensively used in domestic (as ingredients of shampoos, hair conditioners, agricultural (as fungicides, pesticides, insecticides, healthcare (as medications, and industrial applications (as biocides, fabric softeners, corrosion inhibitors. The extensive use of quaternary ammonium disinfectants in recent years has led to the development of resistance in microorganisms to these drugs. Thus [i]Staphylococcus[/i] [i]aureus[/i] strains contain the plasmid-carrying genes [i]qacA[/i] and [i]qacB [/i]encoding resistance to quaternary ammonium compounds and acriflavine. The membrane proteins QacA and QacB confer multidrug resistance by exporting the compound by the proton motive force which is generated by the transmembrane electrochemical proton gradient.

  16. [The biological activity of quaternary ammonium salts (QASs)].

    Obłak, Ewa; Gamian, Andrzej


    Quaternary ammonium salts (QASs), especially those of cationic surfactant character, are applied as antibacterial and antifungal disinfectants. QASs affect lipid-enveloped viruses, including human immunodeficiency virus (HIV) and hepatitis B virus (HBV), but not non-enveloped viruses. These compounds are extensively used in domestic (as ingredients of shampoos, hair conditioners), agricultural (as fungicides, pesticides, insecticides), healthcare (as medications), and industrial applications (as biocides, fabric softeners, corrosion inhibitors). The extensive use of quaternary ammonium disinfectants in recent years has led to the development of resistance in microorganisms to these drugs. Thus Staphylococcus aureus strains contain the plasmid-carrying genes qacA and qacB encoding resistance to quaternary ammonium compounds and acriflavine. The membrane proteins QacA and QacB confer multidrug resistance by exporting the compound by the proton motive force which is generated by the transmembrane electrochemical proton gradient.

  17. Methylene-blue aided rapid confocal laser endomicroscopy of breast cancer

    Vyas, Khushi; Hughes, Michael; Leff, Daniel Richard; Yang, Guang-Zhong


    Breast conserving surgery allows complete tumor resection while maintaining acceptable cosmesis for patients. Safe and rapid intraoperative margin assessment during the procedure is important to establish the completeness of tumor excision and minimizes the need for reoperation. Confocal laser endomicroscopy has demonstrated promise for real-time intraoperative margin assessment using acriflavine staining, but it is not approved for routine in-human use. We describe a custom high-speed line-scan confocal laser endomicroscopy (LS-CLE) system at 660 nm that enables high-resolution histomorphological imaging of breast tissue stained with methylene-blue, an alternative fluorescent stain for localizing sentinel nodes during breast surgery. Preliminary imaging results on freshly excised human breast tissue specimens are presented, demonstrating the potential of methylene-blue aided rapid LS-CLE to determine the oncological status of surgical margins in-vivo.

  18. Quantitative Segmentation of Fluorescence Microscopy Images of Heterogeneous Tissue: Application to the Detection of Residual Disease in Tumor Margins.

    Jenna L Mueller

    Full Text Available To develop a robust tool for quantitative in situ pathology that allows visualization of heterogeneous tissue morphology and segmentation and quantification of image features.TISSUE EXCISED FROM A GENETICALLY ENGINEERED MOUSE MODEL OF SARCOMA WAS IMAGED USING A SUBCELLULAR RESOLUTION MICROENDOSCOPE AFTER TOPICAL APPLICATION OF A FLUORESCENT ANATOMICAL CONTRAST AGENT: acriflavine. An algorithm based on sparse component analysis (SCA and the circle transform (CT was developed for image segmentation and quantification of distinct tissue types. The accuracy of our approach was quantified through simulations of tumor and muscle images. Specifically, tumor, muscle, and tumor+muscle tissue images were simulated because these tissue types were most commonly observed in sarcoma margins. Simulations were based on tissue characteristics observed in pathology slides. The potential clinical utility of our approach was evaluated by imaging excised margins and the tumor bed in a cohort of mice after surgical resection of sarcoma.Simulation experiments revealed that SCA+CT achieved the lowest errors for larger nuclear sizes and for higher contrast ratios (nuclei intensity/background intensity. For imaging of tumor margins, SCA+CT effectively isolated nuclei from tumor, muscle, adipose, and tumor+muscle tissue types. Differences in density were correctly identified with SCA+CT in a cohort of ex vivo and in vivo images, thus illustrating the diagnostic potential of our approach.The combination of a subcellular-resolution microendoscope, acriflavine staining, and SCA+CT can be used to accurately isolate nuclei and quantify their density in anatomical images of heterogeneous tissue.

  19. Culture of a high-chlorophyll-producing and halotolerant Chlorella vulgaris.

    Nakanishi, Koichi; Deuchi, Keiji


    In order to increase the value of freshwater algae as raw ingredients for health foods and feed for seawater-based farmed fish, we sought to breed high-chlorophyll halotolerant Chlorella with the objective of generating strains with both high chlorophyll concentrations (≥ 5%) and halotolerance (up to 1% NaCl). We used the Chlorella vulgaris K strain in our research institute culture collection and induced mutations with UV irradiation and acriflavine which is known to effect mutations of mitochondrial DNA that are associated with chlorophyll production. Screenings were conducted on seawater-based "For Chlorella spp." (FC) agar medium, and dark-green-colored colonies were visually selected by macroscopic inspection. We obtained a high-chlorophyll halotolerant strain (designated C. vulgaris M-207A7) that had a chlorophyll concentration of 6.7% (d.m.), a level at least three-fold higher than that of K strain. This isolate also exhibited a greater survival rate in seawater that of K strain.

  20. Role of the Mmr efflux pump in drug resistance in Mycobacterium tuberculosis.

    Rodrigues, Liliana; Villellas, Cristina; Bailo, Rebeca; Viveiros, Miguel; Aínsa, José A


    Efflux pumps are membrane proteins capable of actively transporting a broad range of substrates from the cytoplasm to the exterior of the cell. Increased efflux activity in response to drug treatment may be the first step in the development of bacterial drug resistance. Previous studies showed that the efflux pump Mmr was significantly overexpressed in strains exposed to isoniazid. In the work to be described, we constructed mutants lacking or overexpressing Mmr in order to clarify the role of this efflux pump in the development of resistance to isoniazid and other drugs in M. tuberculosis. The mmr knockout mutant showed an increased susceptibility to ethidium bromide, tetraphenylphosphonium, and cetyltrimethylammonium bromide (CTAB). Overexpression of mmr caused a decreased susceptibility to ethidium bromide, acriflavine, and safranin O that was obliterated in the presence of the efflux inhibitors verapamil and carbonyl cyanide m-chlorophenylhydrazone. Isoniazid susceptibility was not affected by the absence or overexpression of mmr. The fluorometric method allowed the detection of a decreased efflux of ethidium bromide in the knockout mutant, whereas the overexpressed strain showed increased efflux of this dye. This increased efflux activity was inhibited in the presence of efflux inhibitors. Under our experimental conditions, we have found that efflux pump Mmr is mainly involved in the susceptibility to quaternary compounds such as ethidium bromide and disinfectants such as CTAB. The contribution of this efflux pump to isoniazid resistance in Mycobacterium tuberculosis still needs to be further elucidated.

  1. Intraoperative fluorescence imaging for personalized brain tumor resection: Current state and future directions

    Evgenii Belykh


    Full Text Available Introduction: Fluorescence-guided surgery is one of the rapidly emerging methods of surgical theranostics. In this review, we summarize current fluorescence techniques used in neurosurgical practice for brain tumor patients, as well as future applications of recent laboratory and translational studies.Methods: Review of the literature.Results: A wide spectrum of fluorophores that have been tested for brain surgery is reviewed. Beginning with a fluorescein sodium application in 1948 by Moore, fluorescence guided brain tumor surgery is either routinely applied in some centers or is under active study in clinical trials. Besides the trinity of commonly used drugs (fluorescein sodium, 5-ALA and ICG, less studied fluorescent stains, such as tetracyclines, cancer-selective alkylphosphocholine analogs, cresyl violet, acridine orange, and acriflavine can be used for rapid tumor detection and pathological tissue examination. Other emerging agents such as activity-based probes and targeted molecular probes that can provide biomolecular specificity for surgical visualization and treatment are reviewed. Furthermore, we review available engineering and optical solutions for fluorescent surgical visualization. Instruments for fluorescent-guided surgery are divided into wide-field imaging systems and hand-held probes. Recent advancements in quantitative fluorescence-guided surgery are discussed.Conclusion: We are standing on the doorstep of the era of marker-assisted tumor management. Innovations in the fields of surgical optics, computer image analysis, and molecular bioengineering are advancing fluorescence-guided tumor resection paradigms, leading to cell-level approaches to visualization and resection of brain tumors.

  2. Gastric Tissue Damage Analysis Generated by Ischemia: Bioimpedance, Confocal Endomicroscopy, and Light Microscopy

    Beltran, Nohra E.; Garcia, Laura E.; Garcia-Lorenzana, Mario


    The gastric mucosa ischemic tissular damage plays an important role in critical care patients' outcome, because it is the first damaged tissue by compensatory mechanism during shock. The aim of the study is to relate bioimpedance changes with tissular damage level generated by ischemia by means of confocal endomicroscopy and light microscopy. Bioimpedance of the gastric mucosa and confocal images were obtained from Wistar male rats during basal and ischemia conditions. They were anesthetized, and stain was applied (fluorescein and/or acriflavine). The impedance spectroscopy catheter was inserted and then confocal endomicroscopy probe. After basal measurements and biopsy, hepatic and gastric arteries clamping induced ischemia. Finally, pyloric antrum tissue was preserved in buffered formaldehyde (10%) for histology processing using light microscopy. Confocal images were equalized, binarized, and boundary defined, and infiltrations were quantified. Impedance and infiltrations increased with ischemia showing significant changes between basal and ischemia conditions (P < 0.01). Light microscopy analysis allows detection of general alterations in cellular and tissular integrity, confirming gastric reactance and confocal images quantification increments obtained during ischemia. PMID:23841094


    M. R. Shakibaie ، A. Khosravan ، A. Frahmand ، S. Zare


    Full Text Available In this research, using mutation in the metal resistant bacteria, the bioremediation of the copper and zinc from copper factory effluents was investigated. Wastewater effluents from flocculation and rolling mill sections of a factory in the city of Kerman were collected and used for further experiments. 20 strains of Pseudomonas spp. were isolated from soil and effluents surrounding factory and identified by microbiological methods. Minimum inhibitory concentrations for copper (Cu and zinc (Zn were determined by agar dilution method. Those strains that exhibited highest minimum inhibitory concentrations values to the metals (5mM were subjected to 400-3200 mg/L concentrations of the three mutagenic agents, acriflavine, acridine orange and ethidium bromide. After determination of subinhibitory concentrations, the minimum inhibitory concentrations values for copper and zinc metal ions were again determined, which showed more than 10 fold increase in minimum inhibitory concentrations value (10 mM for Cu and 20 mM for Zn with P≤0.05. The atomic absorption spectroscopy of dried biomass obtained from resistant strains after exposure to mutagenic agents revealed that strains 13 accumulate the highest amount of intracellular copper (0.35% Cu/mg dried biomass and strain 10 showed highest accumulation of zinc (0.3% Zn/mg dried biomass respectively with P≤0.05. From above results it was concluded that the treatment of industrial waste containing heavy metals by artificially mutated bacteria may be appropriate solution for effluent disposal problems.

  4. Single-step multiresidue determination of ten multiclass veterinary drugs in pork, milk, and eggs using liquid chromatography with tandem mass spectrometry.

    Park, Jin-A; Zhang, Dan; Kim, Dong-Soon; Kim, Seong-Kwan; Cho, Kyeong-Su; Jeong, Dana; Shim, Jae-Han; Kim, Jin-Suk; Abd El-Aty, A M; Shin, Ho-Chul


    A multiclass, multiresidue determination method is reported for the detection of ten veterinary drugs, including scopolamine, metoclopramide, acriflavine, berberine, tripelennamine, diphenhydramine, acrinol, triamcinolone, loperamide, and roxithromycin in pork, milk, and eggs. The method involves a simple extraction using 0.1% formic acid in acetonitrile, followed by defatting with n-hexane, centrifugation, and filtration prior to liquid chromatography with tandem mass spectrometric analysis. As ion suppression and enhancement effects are reported, matrix-matched calibrations are used for quantification, with determination coefficients ≥0.9765. For the majority of the tested analytes, the intra- and interday accuracy (expressed as recovery %) range from 70.6 to 94.6% and from 70.1 to 93.3%, respectively, and the precision (expressed as relative standard deviation) ranges from 0.5 to 19.8% and from 2.8 to 18.4% in all matrices. The limits of quantification range between 0.5 and 10 ng/g. The validated tandem mass spectrometry method is successfully applied to market samples; the target analytes are not detected in any of the tested samples. In terms of accuracy, no extract cleanup is deemed necessary. The developed method is feasible for the simultaneous detection of the tested analytes in pork, milk, and eggs.

  5. Role of novel multidrug efflux pump involved in drug resistance in Klebsiella pneumoniae.

    Vijaya Bharathi Srinivasan

    Full Text Available BACKGROUND: Multidrug resistant Klebsiella pneumoniae have caused major therapeutic problems worldwide due to the emergence of the extended-spectrum β-lactamase producing strains. Although there are >10 major facilitator super family (MFS efflux pumps annotated in the genome sequence of the K. pneumoniae bacillus, apparently less is known about their physiological relevance. PRINCIPAL FINDINGS: Insertional inactivation of kpnGH resulting in increased susceptibility to antibiotics such as azithromycin, ceftazidime, ciprofloxacin, ertapenem, erythromycin, gentamicin, imipenem, ticarcillin, norfloxacin, polymyxin-B, piperacillin, spectinomycin, tobramycin and streptomycin, including dyes and detergents such as ethidium bromide, acriflavine, deoxycholate, sodium dodecyl sulphate, and disinfectants benzalkonium chloride, chlorhexidine and triclosan signifies the wide substrate specificity of the transporter in K. pneumoniae. Growth inactivation and direct fluorimetric efflux assays provide evidence that kpnGH mediates antimicrobial resistance by active extrusion in K. pneumoniae. The kpnGH isogenic mutant displayed decreased tolerance to cell envelope stressors emphasizing its added role in K. pneumoniae physiology. CONCLUSIONS AND SIGNIFICANCE: The MFS efflux pump KpnGH involves in crucial physiological functions besides being an intrinsic resistance determinant in K. pneumoniae.

  6. An ace up their sleeve: a transcriptomic approach exposes the AceI efflux protein of Acinetobacter baumannii and reveals the drug efflux potential hidden in many microbial pathogens

    Karl A Hassan


    Full Text Available The era of antibiotics as a cure-all for bacterial infections appears to be coming to an end. The emergence of multidrug resistance in many hospital-associated pathogens has resulted in superbugs that are effectively untreatable. Multidrug efflux pumps are well known mediators of bacterial drug resistance. Genome sequencing efforts have highlighted an abundance of putative efflux pump genes in bacteria. However, it is not clear how many of these pumps play a role in antimicrobial resistance. Several studies have demonstrated that efflux pump genes that participate in drug resistance are typically under tight regulatory control and expressed only in response to their substrates. Consequently, changes in gene expression following antimicrobial shock treatments may be used to identify efflux pumps that mediate antimicrobial resistance, informing targeted functional analyses of these proteins. Using this approach we have characterised novel efflux pumps in both Gram-negative and Gram-positive bacteria. Notably, we recently applied this strategy to characterise the AceI efflux pump from Acinetobacter. AceI is a prototype for a new family of multidrug efflux proteins that is conserved across many proteobacterial lineages. Different efflux pumps in this family have been shown to confer resistance to biocides including chlorhexidine, dequalinium, benzalkonium, proflavine and/or acriflavine. The discovery of this novel family of multidrug efflux proteins raises the possibility that additional undiscovered intrinsic resistance proteins may be encoded in the core genomes of pathogenic bacteria.

  7. Comparative analysis of the susceptibility to biocides and heavy metals of extended-spectrum β-lactamase-producing Escherichia coli isolates of human and avian origin, Germany.

    Deus, Daniela; Krischek, Carsten; Pfeifer, Yvonne; Sharifi, Ahmad Reza; Fiegen, Ulrike; Reich, Felix; Klein, Guenter; Kehrenberg, Corinna


    A total of 174 extended-spectrum β-lactamase (ESBL)-producing Escherichia coli isolates collected from humans (n=140) and healthy broiler chickens (n = 34) was included in the study. The MIC values of alkyl diaminoethyl glycin hydrochloride, benzethonium chloride, benzalkonium chloride, chlorhexidine, acriflavine, copper sulfate, silver nitrate and zinc chloride were determined by the broth microdilution method. Significant differences in MIC distributions were found between human and avian isolates and between CTX-M-, SHV- and TEM-type ESBL E. coli for chlorhexidine, silver nitrate, zinc chloride and copper sulfate by statistical analysis. Isolates with reduced susceptibility were investigated for the presence and localization of tolerance-mediating genes by PCR analysis and Southern blotting. The genes emrE, mdfA, sugE(c), cueO, copA, zntA and zitB were commonly present in isolates with elevated MICs, while the genes qacE∆1, qacF, qacH, sugE(p), cusC and pcoA, were less prevalent. In several isolates, a plasmid localization of the genes qacE∆1, qacF, qacH and sugE(p) on large plasmids >20 kb was detected.

  8. Combination and monotherapy of Leishmania major infection in BALB/c mice using plant extracts and herbicides

    Judith A. Makwali , Frederick M.E. Wanjala , Josyline C. Kaburi , Johnstone Ingonga , Wabwoba W. Byrum & Christopher O. Anjili


    Full Text Available Background & objectives: Leishmaniasis is a growing health problem in many parts of the world. Efforts to findnew chemotherapeutics for leishmaniasis remain a priority. This study was carried out to determine the effect ofcombination and monotherapies using plant extracts and herbicides on Leishmania major infection in BALB/cmice.Methods: The herbicides and saponin extract were purchased from Sigma. Roots of Plumbago capensis werecollected from Karura forest, Nairobi, Kenya. Plant extractions were done in KEMRI at Center for TraditionalMedicines and Drugs Research.Results: Lesion sizes after infection of BALB/c mice were similar in all the experimental groups till the onset oftherapeutic treatments (p >0.05. At 15 days post-treatment, significant differences (p < 0.05 were discerned inthe lesion sizes of the BALB/c mice in all the mono- and combined-treated groups. However, the combinedtherapies caused total elimination of the parasites from the lesions and significantly reduced parasite burden inliver and spleen compared to the untreated controls at the end of the experiment.Interpretation & conclusion: The results of this study demonstrate that combination therapy using alternativeadministration of saponin, acriflavine, trifluralin and plumbagin is effective in treating L. major infection inmice. In this regard, an investigation into the efficacy of these combined therapies against other Leishmaniastrains should be explored further. Furthermore, studies with these combination therapies should be done onnon-human primates such as the vervet monkey (Cercopithecus aethiops.

  9. Polycyclic aromatic hydrocarbons (PAHs) enriching antibiotic resistance genes (ARGs) in the soils.

    Chen, Baowei; He, Rong; Yuan, Ke; Chen, Enzhong; Lin, Lan; Chen, Xin; Sha, Sha; Zhong, Jianan; Lin, Li; Yang, Lihua; Yang, Ying; Wang, Xiaowei; Zou, Shichun; Luan, Tiangang


    The prevalence of antibiotic resistance genes (ARGs) in modern environment raises an emerging global health concern. In this study, soil samples were collected from three sites in petrochemical plant that represented different pollution levels of polycyclic aromatic hydrocarbons (PAHs). Metagenomic profiling of these soils demonstrated that ARGs in the PAHs-contaminated soils were approximately 15 times more abundant than those in the less-contaminated ones, with Proteobacterial being the preponderant phylum. Resistance profile of ARGs in the PAHs-polluted soils was characterized by the dominance of efflux pump-encoding ARGs associated with aromatic antibiotics (e.g., fluoroquinolones and acriflavine) that accounted for more than 70% of the total ARGs, which was significantly different from representative sources of ARG pollution due to wide use of antibiotics. Most of ARGs enriched in the PAHs-contaminated soils were not carried by plasmids, indicating the low possibilities of them being transferred between bacteria. Significant correlation was observed between the total abundance of ARGs and that of Proteobacteria in the soils. Proteobacteria selected by PAHs led to simultaneously enriching of ARGs carried by them in the soils. Our results suggested that PAHs could serve as one of selective stresses for greatly enriching of ARGs in the human-impacted environment.

  10. Modulation of drug resistance in Staphylococcus aureus by a kaempferol glycoside from Herissantia tiubae (Malvaceae).

    Falcão-Silva, Vivyanne S; Silva, Davi A; Souza, Maria de Fátima V; Siqueira-Junior, José P


    In an ongoing project to evaluate natural compounds isolated from plants from the Brazilian biodiversity as modulators of antibiotic resistance, kaempferol-3-O-beta-d-(6''-E-p-coumaroyl) glucopyranoside (tiliroside), isolated from Herissantia tiubae (Malvaceae) was investigated using the strain SA-1199B of Staphylococcus aureus, which overexpresses the norA gene encoding the NorA efflux protein which extrudes hydrophilic fluorquinolones and some biocides, such as benzalkonium chloride, cetrimide, acriflavine and ethidium bromide. The minimum inhibitory concentrations (MICs) of the antibiotics and biocides were determined by the microdilution assay in the absence and in the presence of sub-inhibitory concentration of tiliroside. Although tiliroside did not display relevant antibacterial activity (MIC = 256 microg/mL), it modulated the activity of antibiotics, i.e. in combination with antibiotics a reduction in the MIC was observed for norfloxacin (16-fold), ciprofloxacin (16-fold), lomefloxacin (four-fold) and ofloxacin (two-fold), and an impressive reduction in the MICs for the biocides (up to 128-fold). The results presented here represent the first report of a kaempferol glycoside as a putative efflux pump inhibitor in bacteria. The present finding indicates that H. tiubae (and broadly Malvaceae) could serve as a source of plant-derived natural products that modulate bacterial resistance, i.e. a source of potential adjuvants of antibiotics.

  11. Curcumin improves hypoxia induced dysfunctions in 3T3-L1 adipocytes by protecting mitochondria and down regulating inflammation.

    Priyanka, Ariyapalli; Anusree, Sasidharan Suseela; Nisha, Vijayakumar Marykutty; Raghu, Kozhiparambil Gopalan


    Obesity induced metabolic syndrome is increasing worldwide at an alarming rate. It is characterized by excessive expansion of white adipose tissue which leads to hypoxia and impairs normal metabolism. Recent studies reveal that hypoxia could be one of the factors for inflammation, insulin resistance and other obesity related complications. There is a high demand for anti-obese phytoceuticals to control and manage the complications resulting from obesity. In this study, we investigated how hypoxia affect the physiological functions of 3T3-L1 adipocytes emphasizing on oxidative stress, inflammation, and mitochondrial functions. We also evaluated the protective role of various doses of curcumin, a well-known dietary antioxidant, on hypoxia induced alterations. The results revealed that hypoxia significantly altered the vital parameters of adipocyte biology like HIF 1α expression (103.47% ↑), lactate, and glycerol release (184.34% and 69.1% ↑, respectively), reactive oxygen species production (432.53% ↑), lipid and protein oxidation (376.6% and 566.6% ↑, respectively), reduction in antioxidant enzymes (superoxide dismutase and catalase) status, secretion of inflammatory markers (TNF α, IL 6, IL 1β, and IFN γ), and mitochondrial functions (mitochondrial mass, membrane potential, permeability transition pore integrity, and superoxide generation). Curcumin substantially protected adipocytes from toxic effects of hypoxia in a dose dependent manner by protecting mitochondria and down regulating inflammation. Acriflavine is used as a positive control. A detailed investigation is required for the development of curcumin as an effective nutraceutical against obesity.

  12. Photoprotection of Bacillus thuringiensis kurstaki from ultraviolet irradiation

    Cohen, E.; Rozen, H.; Joseph, T.; Braun, S.; Margulies, L. (Department of Entomology, Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot (Israel))


    Irradiation of Bacillus thuringiensis var. kurstaki HD1 at 300-350 nm for up to 12 hr using a photochemical reactor results in a rapid loss of its toxicity to larvae of Heliothis armigera. Photoprotection of the toxic component was obtained by adsorption of cationic chromophores such as acriflavin (AF), methyl green, and rhodamine B to B. thuringiensis. AF gave the best photoprotection and a level of 0.42 mmol/g dye absorbed per gram of B. thuringiensis was highly toxic even after 12 hr of ultraviolet (uv) irradiation as compared to the control (77.5 and 5% of insect mortality, respectively). Ultraviolet and Fourier-transform infrared spectroscopic studies indicate molecular interactions between B. thuringiensis and AF. The nature of these interactions and energy or charge transfer as possible mechanisms of photoprotection are discussed. It is speculated that tryptophan residues are essential for the toxic effect of B. thuringiensis. It is suggested that photoprotection is attained as energy is transferred from the excited tryptophan moieties to the chromophore molecules.

  13. Feasibility of confocal endomicroscopy in the diagnosis of pediatric gastrointestinal disorders

    Krishnappa Venkatesh; Marta Cohen; Clair Evans; Peter Delaney; Steven Thomas; Christopher Taylor; Ashraf Abou-Taleb; Ralf Kiesslich; Mike Thomson


    AIM: To evaluate the feasibility and utility of confocal laser endomicroscopy (CLE) in the description of normal gastrointestinal (GI) mucosa and in the diagnosis of GI disorders in children, in comparison to histology. METHODS: Forty-four patients (19 female) median age 10.9 years (range 0.7-16.6 years) with suspected or known GI pathology underwent esophago-gastroduodenoscopy (OGD) ( n = 36) and/or ileocolonoscopy (IC) ( n = 31) with CLE using sodium fluorescein and acriflavine as contrast agents. Histological sections were compared with same site confocal images by two experienced pediatric and GI histopathologists and endoscopists, respectively. RESULTS: Duodenum and ileum were intubated in all but one patient undergoing OGD and IC. The median procedure time was 16.4 min (range 7-25 min) for OGD and 27.9 min (range 15-45 min) for IC. A total of 4798 confocal images were compared with 153 biopsies from the upper GI tract from 36 procedures, and 4661 confocal images were compared with 188 biopsies from the ileocolon from 31 procedures. Confocal images were comparable to conventional histology both in normal and in pathological conditions such as esophagitis, Helicobacter pylori gastritis, celiac disease, inflammatory bowel disease, colonic heterotopia, and graft versus host disease. CONCLUSION: CLE offers the prospect of targeting biopsies to abnormal mucosa, thereby increasing diagnostic yield, reducing the number of biopsies, decreasing the burden on the histopathological services, and reducing costs.

  14. Transcriptomes analysis of Aeromonas molluscorum Av27 cells exposed to tributyltin (TBT): Unravelling the effects from the molecular level to the organism.

    Cruz, Andreia; Rodrigues, Raquel; Pinheiro, Miguel; Mendo, Sónia


    Aeromonas molluscorum Av27 cells were exposed to 0, 5 and 50 μM of TBT and the respective transcriptomes were obtained by pyrosequencing. Gene Ontology revealed that exposure to 5 μM TBT results in a higher number of repressed genes in contrast with 50 μM of TBT, where the number of over-expressed genes is greater. At both TBT concentrations, higher variations in gene expression were found in the functional categories associated with enzymatic activities, transport/binding and oxidation-reduction. A number of proteins are affected by TBT, such as the acriflavin resistance protein, several transcription-related proteins, several Hsps, ABC transporters, CorA and ZntB and other outer membrane efflux proteins, all of these involved in cellular metabolic processes, important to maintain overall cell viability. Using the STRING tool, several proteins with unknown function were related with others involved in degradation processes, such as the pyoverdine chromophore biosynthetic protein, that has been described as playing a role in the Sn-C cleavage of organotins. This approach has allowed a better understanding of the molecular effects of exposure of bacterial cells to TBT. Furthermore it contributes to the knowledge of the functional genomic aspects of bacteria exposed to this pollutant. Furthermore, the transcriptomic data gathered, and now publically available, constitute a valuable resource for comparative genome analysis.

  15. Prevalence of Listeria monocytogenes in the river receiving the effluent of municipal wastewater treatment plant

    Atefeh Taherkhani


    Full Text Available Aims: The objective of this study was to evaluate the prevalence of Listeria spp. in the river water before and after discharge of the effluent of the municipal wastewater treatment plant (WWTP in Isfahan, Iran. Materials and Methods: A total of 66 samples were collected bi-weekly over 4 months from eleven discrete sampling locations in Zayandehrood River, Iran. Three sampling sites were located above the discharge point and five sites were located after the discharge point of WWTP. Samples were also collected from the influent and the effluent of WWTP. Listeria spp. were isolated using a selective enrichment procedure and a subculture onto polymyxin-acriflavine-lithium chloride-ceftazidime-esculin-mannitol Agar. All isolates were subjected to standard biochemical tests. Results: L. monocytogenes was isolated from influent (83%, effluent (50% and (18.5% river water. Listeria spp. was not found before the discharge point in river water. However, L. monocytogenes was isolated in samples collected from 200 m (33%, 500 m (33%, 2 km (16.5%, 5 km (16.5% and 10 km (16.5% downstream from the WWTP. Listeria innocua (9% and Listeria seeligeri (10% were the second most frequently isolated species. Conclusion: During the wastewater treatment, Listeria spp. is not removed completely. L. monocytogenes is widely distributed in the Zayandehrood river. L. monocytogenes released into surface water demonstrates a potential risk for public health. These results indicate the need for appropriate water management in order to reduce human and animal exposure to such pathogens.

  16. Bacteria detection based on the evolution of enzyme-generated volatile organic compounds: determination of Listeria monocytogenes in milk samples.

    Tait, Emma; Perry, John D; Stanforth, Stephen P; Dean, John R


    The rapid detection of Listeria monocytogenes contamination in food is essential to prevent food-borne illness in humans. The aim of this study was to differentiate non-contaminated milk from milk contaminated with L. monocytogenes using enzyme substrates coupled with the analysis of volatile organic compounds (VOCs). The method is based on the activity of β-glucosidase and hippuricase enzymes and the detection of a specific VOC i.e. 2-nitrophenol and 3-fluoroaniline, respectively. VOCs were extracted, separated and detected by headspace-solid phase microextraction coupled to gas chromatography-mass spectrometry (HS-SPME GC-MS). This approach required the inclusion of the selective agent's cycloheximide, nalidixic acid and acriflavine HCl in the growth medium to inhibit interfering bacteria. The VOCs were liberated by L. monocytogenes provided that samples contained at least 1-1.5×10(2) CFU ml(-1) of milk prior to overnight incubation. This approach shows potential for future development as a rapid method for the detection of L. monocytogenes contaminated milk.

  17. Gastric Tissue Damage Analysis Generated by Ischemia: Bioimpedance, Confocal Endomicroscopy, and Light Microscopy

    Nohra E. Beltran


    Full Text Available The gastric mucosa ischemic tissular damage plays an important role in critical care patients’ outcome, because it is the first damaged tissue by compensatory mechanism during shock. The aim of the study is to relate bioimpedance changes with tissular damage level generated by ischemia by means of confocal endomicroscopy and light microscopy. Bioimpedance of the gastric mucosa and confocal images were obtained from Wistar male rats during basal and ischemia conditions. They were anesthetized, and stain was applied (fluorescein and/or acriflavine. The impedance spectroscopy catheter was inserted and then confocal endomicroscopy probe. After basal measurements and biopsy, hepatic and gastric arteries clamping induced ischemia. Finally, pyloric antrum tissue was preserved in buffered formaldehyde (10% for histology processing using light microscopy. Confocal images were equalized, binarized, and boundary defined, and infiltrations were quantified. Impedance and infiltrations increased with ischemia showing significant changes between basal and ischemia conditions (. Light microscopy analysis allows detection of general alterations in cellular and tissular integrity, confirming gastric reactance and confocal images quantification increments obtained during ischemia.

  18. Lignification of developing maize (Zea mays L.) endosperm transfer cells and starchy endosperm cells

    Rocha, Sara; Monjardino, Paulo; Mendonça, Duarte; da Câmara Machado, Artur; Fernandes, Rui; Sampaio, Paula; Salema, Roberto


    Endosperm transfer cells in maize have extensive cell wall ingrowths that play a key role in kernel development. Although the incorporation of lignin would support this process, its presence in these structures has not been reported in previous studies. We used potassium permanganate staining combined with transmission electron microscopy – energy dispersive X-ray spectrometry as well as acriflavine staining combined with confocal laser scanning microscopy to determine whether the most basal endosperm transfer cells (MBETCs) contain lignified cell walls, using starchy endosperm cells for comparison. We investigated the lignin content of ultrathin sections of MBETCs treated with hydrogen peroxide. The lignin content of transfer and starchy cell walls was also determined by the acetyl bromide method. Finally, the relationship between cell wall lignification and MBETC growth/flange ingrowth orientation was evaluated. MBETC walls and ingrowths contained lignin throughout the period of cell growth we monitored. The same was true of the starchy cells, but those underwent an even more extensive growth period than the transfer cells. Both the reticulate and flange ingrowths were also lignified early in development. The significance of the lignification of maize endosperm cell walls is discussed in terms of its impact on cell growth and flange ingrowth orientation. PMID:24688487

  19. Anti-protozoal and anti-bacterial antibiotics that inhibit protein synthesis kill cancer subtypes enriched for stem cell-like properties

    Cuyàs, Elisabet; Martin-Castillo, Begoña; Corominas-Faja, Bruna; Massaguer, Anna; Bosch-Barrera, Joaquim; Menendez, Javier A


    Key players in translational regulation such as ribosomes might represent powerful, but hitherto largely unexplored, targets to eliminate drug-refractory cancer stem cells (CSCs). A recent study by the Lisanti group has documented how puromycin, an old antibiotic derived from Streptomyces alboniger that inhibits ribosomal protein translation, can efficiently suppress CSC states in tumorspheres and monolayer cultures. We have used a closely related approach based on Biolog Phenotype Microarrays (PM), which contain tens of lyophilized antimicrobial drugs, to assess the chemosensitivity profiles of breast cancer cell lines enriched for stem cell-like properties. Antibiotics directly targeting active sites of the ribosome including emetine, puromycin and cycloheximide, inhibitors of ribosome biogenesis such as dactinomycin, ribotoxic stress agents such as daunorubicin, and indirect inhibitors of protein synthesis such as acriflavine, had the largest cytotoxic impact against claudin-low and basal-like breast cancer cells. Thus, biologically aggressive, treatment-resistant breast cancer subtypes enriched for stem cell-like properties exhibit exacerbated chemosensitivities to anti-protozoal and anti-bacterial antibiotics targeting protein synthesis. These results suggest that old/existing microbicides might be repurposed not only as new cancer therapeutics, but also might provide the tools and molecular understanding needed to develop second-generation inhibitors of ribosomal translation to eradicate CSC traits in tumor tissues. PMID:25970790

  20. Anti-protozoal and anti-bacterial antibiotics that inhibit protein synthesis kill cancer subtypes enriched for stem cell-like properties.

    Cuyàs, Elisabet; Martin-Castillo, Begoña; Corominas-Faja, Bruna; Massaguer, Anna; Bosch-Barrera, Joaquim; Menendez, Javier A


    Key players in translational regulation such as ribosomes might represent powerful, but hitherto largely unexplored, targets to eliminate drug-refractory cancer stem cells (CSCs). A recent study by the Lisanti group has documented how puromycin, an old antibiotic derived from Streptomyces alboniger that inhibits ribosomal protein translation, can efficiently suppress CSC states in tumorspheres and monolayer cultures. We have used a closely related approach based on Biolog Phenotype Microarrays (PM), which contain tens of lyophilized antimicrobial drugs, to assess the chemosensitivity profiles of breast cancer cell lines enriched for stem cell-like properties. Antibiotics directly targeting active sites of the ribosome including emetine, puromycin and cycloheximide, inhibitors of ribosome biogenesis such as dactinomycin, ribotoxic stress agents such as daunorubicin, and indirect inhibitors of protein synthesis such as acriflavine, had the largest cytotoxic impact against claudin-low and basal-like breast cancer cells. Thus, biologically aggressive, treatment-resistant breast cancer subtypes enriched for stem cell-like properties exhibit exacerbated chemosensitivities to anti-protozoal and anti-bacterial antibiotics targeting protein synthesis. These results suggest that old/existing microbicides might be repurposed not only as new cancer therapeutics, but also might provide the tools and molecular understanding needed to develop second-generation inhibitors of ribosomal translation to eradicate CSC traits in tumor tissues.

  1. Biochemical and genetic analysis of toxic effect of HOE 15030 in mammalian cells.

    Ishida, R; Nishizawa, M; Kohtani, F; Takahashi, T


    HOE 15030 inhibited the growth of BHK cells at concentrations that did not inhibit their nuclear DNA and RNA syntheses. When BHK cells were cultured in the presence of 30 micrograms/ml of HOE 15030, cells were arrested in the G1 phase after one or two cell divisions. After removal of the drug, cells progressed through the G1 to the S phase. HOE 15030 inhibited the activities of both topoisomerases I and II in vitro. To determine the target molecule of HOE 15030 in cells, we isolated a HOE 15030-resistant (HOEr) mutant of BHK cells. The HOEr cells exhibited cross-resistance to ethidium bromide, acriflavine, and rhodamine 123, and slight cross-resistance to 4'-dimethylepipodophyllotoxin-4-(4,6-O-ethylidine-beta-D-glu copyranoside) (VP-16) and adriamycin, but not to chloramphenicol, oligomycin, novobiocin, colchicine, or vinblastine. The uptake and retention of rhodamine 123 by HOEr cells were lower than those by BHK cells. Mitochondrial DNA synthesis of HOEr cells was more resistant to HOE 15030 and ethidium bromide than that of wild-type cells. These results indicate that the resistance of HOEr cells to drugs is due to reduced uptake or accumulation of the drugs by mitochondria and suggest that the mitochondria are the main target of HOE 15030 in cells.


    Iolanda Aparecida Nunes


    Full Text Available

    It was determined the performance of two selective broths and two agars to isolate Listeria from chicken meat naturally contaminated, acquired in the supermarkets of Goiânia-Goiás, between January and June 1992. Methodology of isolation involved the utilization of two-stage selective enrichment in the University of Vermont and Fraser broths and plating in lithium chloride-phenyletanol-moxalactam agar and the Listeria selective agar base supplemented with cicloheximide, acriflavine and nalidixic acid. Incubation was done at 300C for 48 hours. Cultures of secondary enrichment broth were inoculated in duplicated plates with and without previous treatment with a 0.25% potassium hydroxide solution during one minute, while the plates from primary enrichment broth were inoculated only with treated cultures. Primary and secondary selective broths showed comparable efficiency at the statistic point of view (Z= -1.7393<1.96, identifying 90.28% and 97.22% of all positive samples of this experiment, respectively. Using the lithium chloride-pheniletanol-moxalactam agar and the Listeria selective agar base supplemented with cicloheximide, acriflavine and nalidixic acid were detected 98.61% and 54.17% of the positive samples. These differences were significant at statistic view. The treatment of broths with 0.25% potassium hydroxide showed a little reduction in the number of positive samples detected.

    KEY-WORDS: Listeria; enrichment broth; chicken meat.

    No presente trabalho, avaliou-se o desempenho de dois caldos e dois ágares seletivos para o isolamento de Listeria a partir de carne de frango naturalmente contaminada, adquirida no comércio varejista de Goiânia - GO, no período de janeiro a junho de 1992. A metodologia de isolamento e

  3. Contribution of SecDF to Staphylococcus aureus resistance and expression of virulence factors

    Berger-Bächi Brigitte


    Full Text Available Abstract Background SecDF is an accessory factor of the conserved Sec protein translocation machinery and belongs to the resistance-nodulation-cell division (RND family of multidrug exporters. SecDF has been shown in Escherichia coli and Bacillus subtilis to be involved in the export of proteins. RND proteins can mediate resistance against various substances and might be of relevance in antimicrobial therapy. The role of RND proteins in Staphylococcus aureus has not yet been determined. Results Markerless deletion mutants were constructed to analyze the impact of the so far uncharacterized RND proteins in S. aureus. While the lack of Sa2056 and Sa2339 caused no phenotype regarding growth and resistance, the secDF mutant resulted in a pleiotropic phenotype. The secDF mutant was cold sensitive, but grew normally in rich medium at 37°C. Resistance to beta-lactams, glycopeptides and the RND substrates acriflavine, ethidium bromide and sodium dodecyl sulfate was reduced. The secDF mutant showed an aberrant cell separation and increased spontaneous and Triton X-100 induced autolysis, although the amounts of penicillin-binding proteins in the membrane were unchanged. The impact of secDF deletion on transcription and expression of specific virulence determinants varied: While coagulase transcription and activity were reduced, the opposite was observed for the autolysin Atl. A reduction of the transcription of the cell wall anchored protein A (spa was also found. The accumulation of SpA in the membrane and lowered amounts in the cell wall pointed to an impaired translocation. Conclusions The combination of different effects of secDF deletion on transcription, regulation and translocation lead to impaired cell division, reduced resistance and altered expression of virulence determinants suggesting SecDF to be of major relevance in S. aureus. Thus SecDF could be a potential target for the control and eradication of S. aureus in the future.

  4. High resolution DNA content measurements of mammalian sperm

    Pinkel, D.; Lake, S.; Gledhill, B.L.; Van Dilla, M.A.; Stephenson, D.; Watchmaker, G.


    The high condensation and flat shape of the mammalian sperm nucleus present unique difficulties to flow cytometric measurement of DNA content. Chromatin compactness makes quantitative fluorescent staining for DNA difficult and causes a high index of refraction. The refractive index makes optical measurements sensitive to sperm head orientation. We demonstrate that the optical problems can be overcome using the commercial ICP22 epiillumination flow cytometer (Ortho Instruments, Westwood, MA) or a specially built cell orientating flow cytometer (OFCM). The design and operation of the OFCM are described. Measurements of the angular dependence of fluorescence from acriflavine stained rabbit sperm show that it is capable of orienting flat sperm with a tolerance of +-7/sup 0/. Differences in the angular dependence for the similarly shaped bull and rabbit sperm allow discrimination of these cells. We show that DNA staining with 4-6 diamidino-2-phenylindole (DAPI) or an ethidium bromide mithramycin combination allows resolution of the X and Y populations in mouse sperm. They have also been successful with sperm from the bull, ram, rabbit, and boar. Reliable results with human sperm are not obtained. The accuracy of the staining and measurement techniques are verified by the correct determination of the relative content of these two populations in sperm from normal mice and those with the Cattanach (7 to X) translocation. Among the potential uses of these techniques are measurement of DNA content errors induced in sperm due to mutagen exposure, and assessment of the fractions of X and Y sperm in semen that may have one population artifically enriched.

  5. In vitro activity of older and newer fluoroquinolones against efflux-mediated high-level ciprofloxacin-resistant Streptococcus pneumoniae.

    Daporta, Matilde Trigo; Muñoz Bellido, Juan Luis; Guirao, Genoveva Yagüe; Hernández, Manuel Segovia; García-Rodríguez, José Angel


    The effect of high-level efflux activity on the MICs of fluoroquinolones against Streptococcus pneumoniae in the absence of topoisomerase mutations leading to fluoroquinolones resistance was investigated. A S. pneumoniae ATCC 46619-derived strain with high-level efflux activity was obtained (SP-25A). Both the parent and obtained strains were tested against efflux substrates acriflavine (Acr) and ethidium bromide (EtBr), and against norfloxacin (NFX), ciprofloxacin (CFX), levofloxacin (LFX), moxifloxacin (MFX), trovafloxacin (TVX) and sitafloxacin (SFX), in presence and absence of the efflux pump inhibitor reserpine. gyrA, gyrB, parC and parE QRDR genes were amplified by PCR and sequenced. MICs of NFX and CFX against SP-25A were 64-fold higher than parent strain MICs (256 mg/L versus 4 mg/L and 64 mg/L versus 1mg/L, respectively). MIC of LFX increased from 1 to 4 mg/L and MICs of MFX, TVX and SFX remained virtually unchanged (0.1-0.2 mg/L). MICs of Acr and EtBr against SP-25A were 8- and 16-fold higher than against parent strains. In both cases, reserpine reverted MICs to the parent strain values (1 and 0.2 mg/L). Only parE showed two mutations leading to a Pro(454) --> Ser and Glu(443) changes, which have previously been shown not to lead to significant fluoroquinolones MIC increases. SP-25A showed a significant increase of MICs of the hydrophilic fluoroquinolones, apparently derived only from efflux activity. Efflux activity, at these high levels, can lead to high-level resistance to older hydrophilic fluoroquinolones, but does affect newer fluoroquinolones such as moxifloxacin, trovafloxacin and sitafloxacin.

  6. In vivo subsurface morphological and functional cellular and subcellular imaging of the gastrointestinal tract with confocal mini-microscopy

    Martin Goetz; Beena Memadathil; Stefan Biesterfeld; Constantin Schneider; Sebastian Gregor; Peter R Galle; Markus F Neurath; Ralf Kiesslich


    AIM: To evaluate a newly developed hand-held confocal probe for in vivo microscopic imaging of the complete gastrointestinal tract in rodents.METHODS: A novel rigid confocal probe (diameter 7 mm) was designed with optical features similar to the flexible endomicroscopy system for use in humans using a 488 nm single line laser for fluorophore excitation.Light emission was detected at 505 to 750 nm. The field of view was 475 μm × 475 μm. Optical slice thickness was 7 μm with a lateral resolution of 0.7 μm. Subsurface serial images at different depths (surface to 250 μm)were generated in real time at 1024 × 1024 pixels (0.8 frames/s) by placing the probe onto the tissue in gentle,stable contact. Tissue specimens were sampled for histopathological correlation.RESULTS: The esophagus, stomach, small and large intestine and meso, liver, pancreas and gall bladder were visualised in vivo at high resolution in n = 48 mice.Real time microscopic imaging with the confocal minimicroscopy probe was easy to achieve. The different staining protocols (fluorescein, acriflavine, FITC-labelled dextran and L. esculentum lectin) each highlighted specific aspects of the tissue, and in vivo imaging correlated excellently with conventional histology. In vivo blood flow monitoring added a functional quality to morphologic imaging.CONCLUSION: Confocal microscopy is feasible in vivo allowing the visualisation of the complete GI tract at high resolution even of subsurface tissue structures.The new confocal probe design evaluated in this study is compatible with laparoscopy and significantly expands the field of possible applications to intra-abdominal organs. It allows immediate testing of new in vivo staining and application options and therefore permits rapid transfer from animal studies to clinical use in patients.

  7. The purine-utilizing bacterium Clostridium acidurici 9a: a genome-guided metabolic reconsideration.

    Katrin Hartwich

    Full Text Available Clostridium acidurici is an anaerobic, homoacetogenic bacterium, which is able to use purines such as uric acid as sole carbon, nitrogen, and energy source. Together with the two other known purinolytic clostridia C. cylindrosporum and C. purinilyticum, C. acidurici serves as a model organism for investigation of purine fermentation. Here, we present the first complete sequence and analysis of a genome derived from a purinolytic Clostridium. The genome of C. acidurici 9a consists of one chromosome (3,105,335 bp and one small circular plasmid (2,913 bp. The lack of candidate genes encoding glycine reductase indicates that C. acidurici 9a uses the energetically less favorable glycine-serine-pyruvate pathway for glycine degradation. In accordance with the specialized lifestyle and the corresponding narrow substrate spectrum of C. acidurici 9a, the number of genes involved in carbohydrate transport and metabolism is significantly lower than in other clostridia such as C. acetobutylicum, C. saccharolyticum, and C. beijerinckii. The only amino acid that can be degraded by C. acidurici is glycine but growth on glycine only occurs in the presence of a fermentable purine. Nevertheless, the addition of glycine resulted in increased transcription levels of genes encoding enzymes involved in the glycine-serine-pyruvate pathway such as serine hydroxymethyltransferase and acetate kinase, whereas the transcription levels of formate dehydrogenase-encoding genes decreased. Sugars could not be utilized by C. acidurici but the full genetic repertoire for glycolysis was detected. In addition, genes encoding enzymes that mediate resistance against several antimicrobials and metals were identified. High resistance of C. acidurici towards bacitracin, acriflavine and azaleucine was experimentally confirmed.

  8. An insight into the isolation, enumeration, and molecular detection of Listeria monocytogenes in food

    Law, Jodi Woan-Fei; Ab Mutalib, Nurul-Syakima; Chan, Kok-Gan; Lee, Learn-Han


    Listeria monocytogenes, a foodborne pathogen that can cause listeriosis through the consumption of food contaminated with this pathogen. The ability of L. monocytogenes to survive in extreme conditions and cause food contaminations have become a major concern. Hence, routine microbiological food testing is necessary to prevent food contamination and outbreaks of foodborne illness. This review provides insight into the methods for cultural detection, enumeration, and molecular identification of L. monocytogenes in various food samples. There are a number of enrichment and plating media that can be used for the isolation of L. monocytogenes from food samples. Enrichment media such as buffered Listeria enrichment broth, Fraser broth, and University of Vermont Medium (UVM) Listeria enrichment broth are recommended by regulatory agencies such as Food and Drug Administration-bacteriological and analytical method (FDA-BAM), US Department of Agriculture-Food and Safety (USDA-FSIS), and International Organization for Standardization (ISO). Many plating media are available for the isolation of L. monocytogenes, for instance, polymyxin acriflavin lithium-chloride ceftazidime aesculin mannitol, Oxford, and other chromogenic media. Besides, reference methods like FDA-BAM, ISO 11290 method, and USDA-FSIS method are usually applied for the cultural detection or enumeration of L. monocytogenes. most probable number technique is applied for the enumeration of L. monocytogenes in the case of low level contamination. Molecular methods including polymerase chain reaction, multiplex polymerase chain reaction, real-time/quantitative polymerase chain reaction, nucleic acid sequence-based amplification, loop-mediated isothermal amplification, DNA microarray, and next generation sequencing technology for the detection and identification of L. monocytogenes are discussed in this review. Overall, molecular methods are rapid, sensitive, specific, time- and labor-saving. In future, there are

  9. A novel porous anionic metal–organic framework with pillared double-layer structure for selective adsorption of dyes

    Sheng, Shu-Nan; Han, Yi; Wang, Bin; Zhao, Cui; Yang, Fan; Zhao, Min-Jian; Xie, Ya-Bo, E-mail:; Li, Jian-Rong


    A novel porous anionic metal–organic framework, (Me{sub 2}NH{sub 2}){sub 2}[Zn{sub 2}L{sub 1.5}bpy]·2DMF (BUT-201; H{sub 4}L=4,8-disulfonaphthalene-2,6-dicarboxylic acid; bpy=4,4-bipyridine; DMF=N,N-dimethylformamide), with pillared double-layer structure has been synthesized through the reaction of a sulfonated carboxylic acid ligand and Zn(NO{sub 3}){sub 2}·6H{sub 2}O with 4,4-bipyridine as a co-ligand. It is found that BUT-201 can rapidly adsorb cationic dyes with a smaller size such as Methylene Blue (MB) and Acriflavine Hydrochloride (AH) by substitution of guest (CH{sub 3}){sub 2}NH{sub 2}{sup +}, but has no adsorption towards the cationic dyes with a lager size such as Methylene Violet (MV), the anionic dyes like C. I. Acid Yellow 1 (AY1) and neutral dyes like C. I. Solvent Yellow 7 (SY7), respectively. The results show that the adsorption behavior of BUT-201 relates not only to the charge but also to the size/shape of dyes. Furthermore, the adsorbed dyes can be gradually released in the methanol solution of LiNO{sub 3}. - Graphical abstract: A porous anionic metal–organic framework (BUT-201) can selectively adsorb the cationic dyes by cationic guest molecule substitution, and the adsorbed dyes can be gradually released in the methanol solution of LiNO{sub 3}. - Highlights: • An anionic metal-organic framework (BUT-201) has been synthesized and characterized. • BUT-201 has a three-dimensional (3D) pillared double-layer structure. • BUT-201 can selectively and rapidly adsorb cationic dyes. • The adsorbed dyes can be gradually released in the methanol solution of LiNO{sub 3}.

  10. Anatomy and lignin distribution in reaction phloem fibres of several Japanese hardwoods.

    Nakagawa, Kaori; Yoshinaga, Arata; Takabe, Keiji


    Although tension wood formation and the structure of gelatinous fibres (G-fibres) have been widely investigated, studies of the influence of the reaction phenomenon on phloem fibres have been few and incomplete in comparison with those of xylem wood fibres. This study was undertaken to clarify the influence of stem inclination on phloem fibres using several Japanese hardwood species that produce different G-fibre types in tension wood. Eight hardwood species were inclined at 30-45° at the beginning of April. Specimens were collected in July and December. The cell-wall structure and lignin distribution of phloem fibres on both the tension and opposite sides were compared by light microscopy, ultraviolet microscopy, confocal laser scanning microscopy after staining with acriflavine, and transmission electron microscopy after staining with potassium permanganate. Three types of changes were found in tension-side phloem fibres: (1) increases in the proportion of the syringyl unit in lignin in the S(1) and S(2) layers and compound middle lamella (Cercidiphyllum japonicum), (2) formation of unlignified gelatinous layers (Melia azedarach and Acer rufinerve) and (3) increases in the number of layers (n) in the multi-layered structure of S(1) + S(2) + n (G + L) (Mallotus japonicus). Other species showed no obvious change in cell-wall structure or lignin distribution. Phloem fibres of the tree species examined in our study showed three types of changes in lignin distribution and cell-wall structure. The reaction phenomenon may vary with tree species and may not be closely related to G-fibre type in tension wood.

  11. Supercoiled DNA folded by nonhistone proteins in cultured mouse carcinoma cells.

    Nakane, M; Ide, T; Anzai, K; Ohara, S; Andoh, T


    Upon gentle lysis of exponentially growing mouse carcinoma cells FM3A by sodium dodecyl sulfate, DNA was released as a "DNA-protein complex" in a folded conformation. No histones could be detected in the DNA-protein complex. The proteins bound to DNA were found to be composed of several kinds of nonhistone proteins with a molecular weight range of 50,000 to 60,000; they appear to play a key role in stabilizing and maintaining the compact and folded structure of the complex. Removal of the proteins by Pronase or 2-mercaptoethanol produced a more relaxed structure sedimenting about half as fast as the original complex in a neutral sucrose gradient. DNA in the folded complex is supercoiled, as indicated by the characteristic biphasic response of its sedimentation rate to increasing concentration of various intercalating agents, actinomycin D, ethidium bromide and acriflavine, with which the cells were treated before lysis. Pronase- or 2-mercaptoethanol-treated relaxed DNA still possessed the characteristic of closed-circular structure as judged from its response to intercalating agents. Nicking with gamma-ray or 4NQO broke these superhelical turns and relaxed the folded complex to slower sedimenting forms equivalent to the relaxed DNA obtained on treatment with Pronase or 2-mercaptoethanol. Viscometric observations of DNA-protein complex were consistent with the above results. A tentative model for the structure of this DNA-protein complex is proposed in which supercoiled DNA is folded into loops by several kinds of nonhistone proteins. Autoradiographic examination of the complex appeared to support this model.


    Albenones José de Mesquita


    Full Text Available

    In this paper it was observed the efficiency of enrichment brothes for Listeria (LEB1 and LEB2, associated to the passage of the culture through a 0.25% potassium hidroxide solution, verifying that the secondary enrichment broth (LEB2 and LEB2KOH was better than the primary enrichment broth (LEB1KOH for this purpose. On the other hand, lithium chloride-phenylethanol-moxalactam agar and the selective Listeria agar base, supplemented with cicloheximide, acriflavine and nalidixic acid (LSAIB-CAN showed equivalency, at the statistic point of view (p=0.l442, in relation to the number of positive samples for Listeria spp., although the LSAB-CAN agar had given the greatest number of bacteria isolated from this genus.

    KEY-WORDS: Listeria; enrichment broth; selective agar.

    No presente trabalho verificou-se a eficiência dos caldos de enriquecimento para Listeria (LEB1 e LEB 2 associados à passagem da cultura por uma solução de hidróxido de potássio a 0,25%, constatando-se que o caldo de enriquecimento secundário (LEB2 e LEB2KOH foi superior ao caldo de enriquecimento primário (LEB1KOH. Por outro lado, o ágar cloreto de lítio-feniletanol-moxalactam (LPM e o ágar base seletivo para Listeria, suplementado com cicloheximida, acriflavina e ácido nalidíxico (LSAB-CAN equivaleram-se estatisticamente (p= 0,l442 em relação ao número de amostras positivas para Listeria, embora o ágar LSAB-CAN tenha proporcionado um maior número de isolamentos da bactéria.

    PALAVRAS-CHAVE: Caldo de enriquecimento; ágar seletivo; Listeria.

  13. Viral and immunologic follow up of 4 to 9 years of AIDS treatments by quadruple combinations of virostatics including integrase inhibitors applied in short sequences differing by drug rotation%病毒学和免疫学随访经4-9年四种病毒抑制药包括整合酶抑制剂结合短程药物轮转AIDS治疗

    Georges MATHE; Christian MORETTE; Michel HALARD; Paulo PONTIGGIA; Denise BLANQUET; Francoise HAGE


    AIM: To present the 4 to 9 years (median: 6 years)treatment follow up of 10 HIV1-AIDS patients, 9 at AIDS and 1 at A3 stages. METHODS: We have applied from 1992 to 1994, AZT combined with 2 integrase inhibitors,acriflavine and hydroxy-methyl-ellipticine. We could shift, in 1994, to combinations of 3 drugs including two more retrowanscfiptase inhibitors (RTI), ddI and ddC,and, after 1995, to combinations of 4 drugs including also two other RTI, d4T and 3TC, and 3 protease inhibitors (PI), indinavir, ritonavir, and saquinavir. In 1998, as cobalamine was shown by an in vitro test, to act as integrase inhibitor, vitamin B12 was added in cycles of various lengths. Every three weeks, not only the investigations were repeated, but the virostatics were changed. RESULTS: No grade 2 virostatics toxicity has been registered. The viral loads (VL) decreased according to exponential curves. Their initial parts obeyed first order kinetics. The second parts were and still are asymptotic. The first parts could be rectilinear or sinuous. The sinuosities were associated to cofactors present before treatment (chimerism, UV irradiation,hepatitis C or B and C, brain toxoplasmosis). The asymptotic parts, whose VL were below PCR detectable levels, presented discrete, reversible HIV1 rebounds,associated to other cofactors (such as herpes zoster,herpes 6, CMV, flat condyloma, and influenza).Among immunologic parameters, the monocyte and CTLnumbers increased and presented, during the rapidly decreasing part of VL curve, a significant inverse correlation with it. Neither CT4+ nor suppressor T-cell (STC) numbers presented such correlation. Near 100 %of CTL were CD28+ Later, vitamin B12 applications increased monocyte and CD28+ CTL numbers, and appeared to reinforce VL stabilization. CONCLU-SION: The combinations of inhibitors affecting 3retrovirus targets, retrotranscriptase, integrase, and protease have given to 10 out of 10 AIDS patients survivals varying today between 4 to 9 years, in

  14. Isolation and identification methods of Rothia species in oral cavities.

    Tsuzukibashi, Osamu; Uchibori, Satoshi; Kobayashi, Taira; Umezawa, Koji; Mashimo, Chiho; Nambu, Takayuki; Saito, Masanori; Hashizume-Takizawa, Tomomi; Ochiai, Tomoko


    Rothia dentocariosa and Rothia mucilaginosa which are Gram-positive bacteria are part of the normal flora in the human oral cavity and pharynx. Furthermore, Rothia aeria, which was first isolated from air samples in the Russian space station Mir, is predicted to be an oral inhabitant. Immunocompromised patients are often infected by these organisms, leading to various systemic diseases. The involvement of these organisms in oral infections has attracted little attention, and their distribution in the oral cavity has not been fully clarified because of difficulties in accurately identifying these organisms. A suitable selective medium for oral Rothia species, including R. aeria, is necessary to assess the veritable prevalence of these organisms in the oral cavity. To examine the bacterial population in the oral cavity, a novel selective medium (ORSM) was developed for isolating oral Rothia species in this study. ORSM consists of tryptone, sodium gluconate, Lab-Lemco powder, sodium fluoride, neutral acriflavin, lincomycin, colistin, and agar. The average growth recovery of oral Rothia species on ORSM was 96.7% compared with that on BHI-Y agar. Growth of other representative oral bacteria, i.e. genera Streptococcus, Actinomyces, Neisseria, and Corynebacterium, was remarkably inhibited on the selective medium. PCR primers were designed based on partial sequences of the 16S rDNA genes of oral Rothia species. These primers reacted to each organism and did not react to other non-oral Rothia species or representative oral bacteria. These results indicated that these primers are useful for identifying oral Rothia species. A simple multiplex PCR procedure using these primers was a reliable method of identifying oral Rothia species. The proportion of oral Rothia species in saliva samples collected from 20 subjects was examined by culture method using ORSM. Rothia dentocariosa, Rothia mucilaginosa, and R. aeria accounted for 1.3%, 5.9%, and 0.8% of the total cultivable




    Full Text Available The determination of sex of an individual is an important subject in Forensic Medicine and Criminology and in Civil Law. The determination of sex is necessary in both living and the dead bodies. The expert opinion of the medico legal specialist regarding positive identification of an individual will be required for the following reasons. For the identification of the sex of individual whether living or dead , For deciding cases relating to legitimacy, divorce, paternity, affiliation, marriage, education, im potence, right to disposal of property, and in intersex condition & in cases of concealed sex. Determination of sex will be done by presumptive, probable and certain signs of sex. Sex chromatin is a planoconvex mass of about 1 micron in diameter lying near nuclear membrane - Barr body. In the buccal smear the percentage of the nuclei containing chromatin body ranges from 0 to 4 in males & 20n to 80 in females . In females neutrophil leucocytes contain a small nuclear attachment of drum stick form - David son bo dy in up to 6% of cell . This is absent in males . Exact sex determination can be made by using a single specimen of buccal smear , saliva or hair follicle, by the combined treatment of quinacrinedi hydrochloride staining for Y chromosome which is seen as bright florescent body in the nuclei of male cell & florescent feulgen reaction using acriflavin Schiff reagent for X chromosomes , which is seen as bright yellow spot in the nuclei . The percentage of quanacrine positive bodies ranges from 45 to 80% in male s , and 0 to 4% in females . With feulgen reaction technique florescent bodies are found in 50 to 70 % of cell in females, and 0 to 2% in males. Determination of sex chromatin pattern was done by examination of oral smears technique on 50 males and 50 female s who have attended the Casualty of Government General Hospital, Ananthapuramu., A. P . , in the Dept . of forensic Medicine from 17 - 06 - 2012 to 30 - 12 - 2014. Slides were

  16. Evaluation of techniques for enrichment and isolation of Escherichia coli O157:H7 from artificially contaminated sprouts.

    Weagant, S D; Bound, A J


    Because sprouted seed products are kept wet during and after production, have high levels of nutrients, and a neutral pH, they are subject to the outgrowth of pathogens such as Escherichia coli O157:H7. For these same reasons, these products also contain high levels of heterotrophic organisms and in particular coliform bacteria. Recent outbreaks have focused attention on the need to improve methodology for isolating this pathogen from sprouts. When 40 E. coli O157:H7 strains were grown in pure culture in enterohemorrhagic E. coli enrichment broth (EEB) as prescribed in the U.S. FDA-Bacteriological Analytical Manual (FDA-BAM) and in EEB modified by varying the cefixime concentration, outgrowth for all strains in EEB was inhibited at 0.05 mg/l but for only 2 of 40 strains when the cefixime level was adjusted to 0.0125 mg/l. These two enrichment formulae were compared to modified E. coli broth (mEC), modified Tryptic Soy Broth with 20 mg/l novobiocin (mTSB + N), modified Buffered Peptone Water (mBPW), and mBPW with added 10 mg/l acriflavin, 10 mg/l cefsulodin, and 8 mg/l vancomycin (mBPW + ACV) for isolation of E. coli O157:H7 from sprouts. These comparisons were performed using low-level (0.12 to 0.42 cfu/g) artificially contaminated alfalfa and mixed salad sprouts. After enrichment, two isolation methods were compared for recovery; direct plating to Tellurite-Cefixime Sorbitol MacConkey agar (TCSMAC) and immunomagnetic separation (IMS) (Dynabeads anti-E. coli O157, Dynal, Oslo, Norway) followed by plating to TCSMAC. In addition, an immunoprecipitin detection kit, VIP (BioControl, Bellevue, WA), was evaluated for detection after enrichment. We found that five of the six enrichments were equivalent for detection or recovery while one enrichment (mTSB + N without agitation) was less productive. Incubation for 24 h was more effective in recovering E. coli O157:H7 from sprouts than 6 h for all enrichment broths. Plating after IMS was more productive than direct plating

  17. Reversion to virulence evaluation of a 9R vaccine strain of Salmonella enterica serovar gallinarum in commercial brown layers

    AS Okamoto


    reaction or clinical alteration because of the vaccine. We only managed to re-isolate the vaccine strain in the inocula made from organs of birds in group 1. We confirmed the isolation by means of biochemical tests, serology, and acriflavine agglutination test. All other cultures made from organs or feces, from all the other experimental groups did not show any growth of the vaccine strain or any other Salmonella serovar, suggesting that the vaccinated birds did not shed the SG9R vaccine strain. No bird presented any clinical symptoms or died during the trials, and no gross lesions were observed in the post-mortem examinations. Under the controlled conditions and time-frame of the present experiment, it was possible to conclude that the rough 9R strain of Salmonella Gallinarum present in the vaccine Cevac S. Gallinarum (Ceva Campinas Ltda. - Campinas, SP - Brazil did not revert to virulence.