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.

Bacteriological Screening of Water in Mangalore, India.

Introduction: Diarrhoea caused by contaminated water is among the most prevalent waterborne diseases in the developing countries like India. In the interest of public health, water supplies should be tested regularly to confirm their freedom from contamination. Objective: The objectives of the study were to screen different water sources for bacterial contamination, to know the antibiotic susceptibility of the common bacterial isolates and typing of the bacterial isolates by random amplification of polymorphic DNA (RAPD) technique. Place and Duration of the Study: Kasturba Medical College Hospital, Microbiology Laboratory, Mangalore, Karnataka, India between August 2007 and August 2009. Methodology: Water samples (n=324) were analyzed by standard microbiological techniques for bacterial contamination. Isolates were identified biochemically and antibiotic susceptibility testing was done by disc diffusion method. Escherichia coli isolates were typed by RAPD technique. Results: Among the water samples tested, 246 were excellent and 78 were contaminated. Contaminated samples showed the growth of commensal bacteria belonging to the family Enterobacteriaceae along with pathogens like Salmonella spp. and Vibrio spp. Many of the isolates were found to be sensitive and a few were found to be resistant to the antibiotics tested. RAPD typing showed genetic similarity and differences among the E. coli isolates from different water sources. Conclusion: Genetic similarity among isolates of E. coli indicates a common ancestral origin or a common source. Bacterial contamination of water samples with pathogens like, Salmonella spp. and Vibrio spp. as well as the faecal coliform is a concern, as water quality is an index of health and well - being of the society. Degree of contamination observed in this study suggests a need to be vigilant to monitor water quality, in order to prevent enteric diseases.

Streptococcus pseudopneumoniae: an emerging respiratory tract pathogen.

Background & objectives: Streptococcus pseudopneumoniae a member of the Viridans Streptococci, is known to be associated with chronic obstructive pulmonary disease and respiratory tract infections (RTI). Very scanty information is available on the isolation of S. pseudopneumoniae from India. Hence, the present study was an attempt to isolate S. pseudopneumoniae from clinical samples and to study their drug resistance pattern. Methods: Sputum samples (n=150) submitted to the microbiology laboratory for routine culture from patients clinically suspected to have lower respiratory tract infection were inoculated onto sheep blood agar and chocolate agar plates. Alpha haemolytic colonies were identified as S. pseudopneumoniae based on absence of capsule, bile solubility and optochin susceptibility in 5 per cent CO2 and ambient air. Disk diffusion method was used for antibiotic susceptibilily testing. Results: Among the samples screened, 4 per cent showed the growth of only S. pseudopneumoniae. Other pathogens isolated were Streptococcus pneumoniae, Moraxella catarrhalis, Klebsiella spp., Enterococcus spp., Pseudomonas spp., Haemophilus influenzae, Staphylococcus aureus, Candida albicans. All the S. pseudopneumoniae isolates were resistant to erythromycin. Interpretation & conclusions: Our preliminary results showed presence of S. pseudopneumoniae in this part of the country and these were associated with RTI. Currently, most clinical laboratories report optochin susceptible isolates in 5 per cent CO2 as S. pneumoniae and the resistant ones are not further tested for susceptibility in ambient air. As a result, S. pseudopneumoniae may be missed out. Hence, performance of at least two tests, viz. optochin susceptibility with incubation in 5 per cent CO2 and ambient air along with bile solubility is necessary to differentiate S. pneumoniae from S. pseudopneumoniae

Detection of shiga-toxigenic Escherichia coli (STEC) in diarrhoeagenic stool & meat samples in Mangalore, India.

BACKGROUND & OBJECTIVE: Shiga-toxigenic Escherichia coli (STEC) are causative agents of bloody diarrhoea, haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). Humans acquire infections primarily through contaminated beef. In India, STEC has not been implicated as a major cause of diarrhoea. Hence, isolation of STEC from diarrhoeagenic stool samples of patients and beef samples marketed through retail outlets was attempted in Mangalore, India. METHODS: Diarrhoeagenic stool samples (n = 192) and meat samples (n = 103) were screened for STEC, using conventional culture methods and polymerase chain reaction (PCR) from December 2003 to 2006 in the department of Microbiology, Kasturba Medical College, Mangalore. All the E. coli isolates were subjected to antibiotic susceptibility testing and serotyping. RESULTS: Of the 40 eae positive E. coli isolates from meat sample, one was positive for all the STEC genes, namely stx1, stx2, rfb O157 and EHEC hlyA. This isolate belonged to O157 serogroup. Of the 110 eae positive E. coli isolated from stool samples, two were positive for EHEC hlyA and belonged to serogroup O8 and one was positive for bfp gene and found to be of O6 serogroup. Among the 192 stool enrichment broths tested, 160 were positive for eae gene, of which two were EHEC hlyA positive and one was bfp gene positive. Among the 103 meat enrichment cultures, 90 were positive for eae gene and one among them was positive for all the STEC genes. INTERPRETATION & CONCLUSION: Our results showed a low incidence of STEC and high prevalence of eae positive E. coli other than STEC in stool and meat samples. A low positivity was observed for PCR performed directly on stool and meat samples. However, PCR on enrichment cultures gave better results. Since E. coli O157 was isolated and detected by PCR in one of the meat samples, this organism may be of public health significance. A study on a large sample may provide some answer.

Typing of Listeria monocytogenes isolates by random amplification of polymorphic DNA.

BACKGROUND & OBJECTIVES: Listeria monocytogenes is an important food-borne pathogen causing meningitis and septicaemia in newborns and immunocompromised persons, abortion and preterm labour in pregnant women. Though various methods are available for typing L. monocytogenes, RAPD analysis has been used for epidemiological purposes in developed countries due to its greater discriminating ability. However, as there are no published reports from India on the typing of L. monocytogenes by RAPD technique the present study was undertaken to type isolates of L. monocytogenes from clinical, food and veterinary samples. METHODS: Isolates of L. monocytogenes were subjected to RAPD using four decamer random primers R1, R2, R3 and R4. Amplified products were analysed by agarose gel electrophoresis. RESULTS: Eight strains of L. monocytogenes on RAPD analysis generated 4 distinct profiles each with R1 and R4 primers and 3 different profiles with R2 and R3 primers. The isolates from fish, clinical and veterinary samples showed different profiles with respect to each other. Isolate from flat fish (serovar 4) showed a different profile from that of clams (serovar 1). Two isolates from placenta (serovar 1) showed similar profiles and all the isolates from veterinary samples generated similar profiles. INTERPRETATION & CONCLUSION: RAPD analysis in the present study allowed discrimination of isolates among the same serotype but from different sources. Since RAPD is a rapid technique and offers greater discrimination of strains, this method may be used for typing L. monocytogenes in India.