Prevalence of adhesive genes among uropathogenic Escherichia coli strains isolated from patients with urinary tract infection in Mangalore.

The study was carried out to detect the adhesive genes pap (pyelonephritis associated pili), sfa (S fimbrial adhesin) and afa (afimbrial adhesin) from Escherichia coli strains isolated in patients diagnosed with urinary tract infection (UTI). A total of 23% of the isolates were positive for pap, sfa and afa genes with a prevalence of 60.87% (14/23), 39.1% (9/23) and 39.1% (9/23), respectively. Prevalence of multiple adhesive genes was 8.7% (2/23) for pap and afa, 30.43% (7/23) for pap and sfa. Significant numbers of isolates were positive for Congo red binding (80%) and haemolysin production 60%. The prevalence of multiple adhesive genes indicate the potential to adhere and subsequently cause a systemic infection among UTI patients.

Drug resistance & virulence determinants in clinical isolatesof Enterococcus species.

Background & objectives: Enterococci are the leading cause of nosocomial infections, and are thus a persisting clinical problem globally. We undertook this study to determine the virulence factors and the antibiotic resistance in Enterococcus clinical isolates. Methods: One hundred and fifty Enterococcus isolates obtained from various clinical specimens were speciated biochemically and subjected to antibiotic susceptibility testing using Kirby-Bauer disk diffusion method. Resistance to vancomycin was determined by using agar screen method. Haemolysin and gelatinase productions were detected using 5 per cent sheep blood agar and 12 per cent gelatin agar, respectively. Results: Among the 150 Enterococcus isolates, 84 (56%) were E. faecalis. 51(34%) E. faecium, and 15 (10%) were other Enterococcus spp. Haemolysin production was seen among 123 (82%) isolates while 61 (40.6%) isolates produced gelatinase. Nearly 50 per cent of the isolates showed high level aminoglycoside resistance (HLAR). A total of 13 (8.6%) isolates showed vancomycin resistance, of which 11(7.3%) had an MIC >8 μg/ml. Interpretation & conclusions: Presence of VRE was found to be low among the isolates studied. However, occurrence of VRE along with HLAR calls for regular detection of vancomycin resistance promptly and accurately to recognize VRE colonization and infection. Early detection of VRE and HLAR along with their virulence trait will help in preventing the establishment and spread of multidrug resistant Enterococcus species.

Prevalence of Listeria monocytogenes in Raw Meats Marketed in Bangkok and Characterization of the Isolates by Phenotypic and Molecular Methods.

Listeria monocytogenes causes listeriosis characterized by septicaemia, encephalitis, and abortion or stillbirth. Regular monitoring of its prevalence in food and characterization of its phenotypes and genotypes are necessary for disease surveillance and tracing the epidemic outbreaks. In this study, the prevalence of L. monocytogenes in raw meats marketed in Bangkok was 15.4%. The bacteria isolated from meat were serotyped and genotyped using enterobacterial repetitive intergenic consensus–polymerase chain reaction (ERIC-PCR). Their virulence-associated genes, antimicrobial susceptibility, and ability to invade intestinal epithelial cells were studied. All 22 L. monocytogenes strains isolated from 104 raw meat samples carried virulence-associated genes, such as actA, flaA, hlyA, iap, inlA, inlB, and prfA. These were serotype 4b, suggesting their pathogenic and epidemic potential. These isolates could be classified into six ERIC-PCR groups: A-F. The majority (59.1%) of the isolates belonged to Group A, and three isolates were Group D which was closely related to the Group A. Two isolates each were Group C and E, and one isolate each was group B and F. Although the isolates belonged to the same serotype and genotype and were all equipped with the virulence-associated genes, they showed a different cell invasion capability and antibiotic susceptibility. All the isolates were susceptible to ampicillin, amikacin, chloramphenicol, gentamicin, imipenem, penicillin G, sulphamethoxazole-trimethoprim, and tetracycline. However, one isolate showed only intermediate susceptibility to tetracycline. The data provide the first molecular insight into the L. monocytogenes isolates in Thailand and elucidate a potential risk of people contracting listeriosis.

The role of C-terminus carbohydrate-binding domain of Vibrio cholerae haemolysin/cytolysin in the conversion of the pre-pore β-barrel oligomer to a functional diffusion channel.

Background & objectives: Vibrio cholerae cytolysin/hemolysin (VCC) is a 65 kDa pore-forming toxin (PFT) secreted by O1 El Tor and non-O1 strains. The purified toxin, which contains two C-terminus carbohydrate-binding domains in addition to the cytolytic domain at the core, causes lysis of a wide spectrum of eukaryotic cells at picomolar concentrations, apoptogenesis of intestinal and immune cells and accumulation of fluid in rabbit ligated ileal loop. Therefore, it may potentially complement the action of cholera toxin (CT) in diarrheagenic strains that do not produce CT. We showed earlier that β1-galactosyl-terminated glycoconjugates are strong inhibitors of its pore-forming activity, though carbohydrates are not functional receptors of VCC. Here, we investigate how the 15 kDa C-terminus β-prism lectin domain contributed to pore formation in erthrocytes. Methods: VCC was isolated from the culture supernatant of late log phase grown bacteria and purified to homogeneity by chromatography. The 50 kDa truncated variant was generated by restricted proteolysis. Liposome was prepared by sonication of a suspension of phospholipids and calceine release assay was done by spectrofluorometric monitoring of the released dye trapped in liposome. Formation of β-barrel oligomers in erythrocyte stroma was monitored by scanning electron microscopy. Results: Proteolytic truncation of the C-terminus β-prism lectin domain decreased hemolytic activity of the toxin by ~800-fold without causing a significant change in pore-forming activity toward synthetic lipid vesicles devoid of incorporated glycoproteins/glycolipids. Truncation at the C-terminus did not impair membrane-binding or assembly to the oligomeric pore. Interpretation & conclusions: Our data indicated that the C-terminus domain played a critical role in translocation of the pre-pore oligomeric assembly from the cell surface or lipid-water interface to the hydrocarbon core of the membrane bilayer, signaling the formation of functional diffusion channels.

Culture supernatants from V. cholerae O1 ElTor strains isolated from different geographic areas induce cell vacuolation and cytotoxicity / Cepas de V. cholerae O1 biotipo ElTor aisladas de diferente origen geográfico inducen vacuolización celular y citotoxicidad

OBJECTIVE: To investigate whether the HlyA-induced vacuolating effect is produced by V. cholerae O1 ElTor strains isolated from different geographic origins, including Mexico. MATERIAL AND METHODS: Supernatant-induced haemolysis, vacuolating activity and cytotoxicity in Vero cells were recorded. PCR, RFLP analysis and molecular cloning were performed. RESULTS: All ElTor strains analyzed induced cellular vacuolation. Ribotype 2 strains isolates from the U.S. gulf coast yielded the highest titer of vacuolating activity. Eight of nine strains were haemolytic, while all strains were PCR positive for the hlyA gene. We cloned the hlyA gene from two ElTor strains, a toxigenic (2514-88, ctxAB+) and a non-toxigenic Mexican strain (CM 91-3, ctxAB-). Supernatant from those recombinant E. coli strains induced haemolysis, cell vacuolation and cytotoxicity. RFLP-PCR analysis revealed similarities in the hlyA gene from all strains tested. CONCLUSION: The HlyA-induced vacuolating effect is a widespread phenotype of epidemic V. cholerae O1 ElTor strains.

OBJETIVO: Analizar el efecto vacuolizante de cepas de V. cholerae O1 ElTor aisladas de diferente origen geográfico, incluyendo México. MATERIAL Y MÉTODOS: Se realizaron pruebas de hemolisis, vacuolización y citotoxicidad en células Vero, así como PCR, análisis por RFLP y clonación molecular. RESULTADOS: Todas las cepas indujeron el efecto vacuolizante. Las cepas del ribotipo 2, aisladas de las costas del Golfo en Estados Unidos, presentaron títulos altos de vacuolización. El gen hlyA fue amplificado en las nueve cepas mediante PCR, aunque sólo ocho fueron hemolíticas. Se clonó el gen hlyA de una cepa toxigénica (2514-88, ctxAB+) y de una cepa no toxigénica aislada en México (CM 91-3, ctxAB-). El sobrenadante de las clonas recombinantes indujo hemólisis, efecto vacuolizante y citotoxicidad. El RFLP mostró alta similitud del gen hlyA de las cepas estudiadas. CONCLUSIÓN: El efecto vacuolizante es un fenotipo ampliamente distribuido en cepas epidémicas de V. cholerae O1 biotipo ElTor.