Shigellosis: Epidemiology in India.

Shigellosis is one of the major causes of diarrhoea in India. The accurate estimates of morbidity and mortality due to shigellosis are lacking, though it is endemic in the country and has been reported to cause many outbreaks. The limited information available indicates Shigella to be an important foodborne pathogen in India. S. flexneri is the most common species, S. sonnei and non-agglutinable shigellae seem to be steadily surfacing, while S. dysenteriae has temporarily disappeared from the northern and eastern regions. Antibiotic-resistant strains of different Shigella species and serotypes have emerged all over the world. Especially important is the global emergence of multidrug resistant shigellae, notably the increasing resistance to third generation cephalosporins and fluoroquinolones, and also azithromycin. This calls for a continuous and strong surveillance of antibiotic resistance across the country for periodic updation of the local antibiograms. The prevention of shigellosis is desirable as it will substantially reduce the morbidity associated with diarrhoea in the country. Public health measures like provision of safe water and adequate sanitation are of immense importance to reduce the burden of shigellosis, however, the provision of resources to develop such an infrastructure in India is a complex issue and will take time to resolve. Thus, the scientific thrust should be focused towards development of a safe and affordable multivalent vaccine. This review is focused upon the epidemiology, disease burden and the therapeutic challenges of shigellosis in Indian perspective.

occurrence of blaNDM-1 & absence of blaKPC genes encoding carbapenem resistance in uropathogens from a tertiary care centre from north India.

Background & objectives: Carbapenem resistance mediated by carbapenemases is increasingly being reported worldwide. This study was conducted to know the occurrence of important carbapenem resistance encoding genes in gram-negative bacilli (GNB) causing complicated urinary tract infection (CUTI), and to look at the genetic diversity of these isolates. Methods: The study was carried out on 166 consecutive carbapenem resistant uropathogens (CRU) isolated from cases with CUTI during 2008 and 2012. Carbapenemase production was characterized phenotypically and polymerase chain reaction was used to detect blaVIM, blaIMP, blaKPC, and blaNDM-1. BOX- PCR was done on 80 randomly selected isolates for molecular typing. Results: The blaVIM gene was present in 34 (43.6%), blaIMP in five (6.4%) and none of the isolates from 2008 had blaNDM-1 or blaKPC genes. Among the isolates from 2012, blaNDM-1 gene was present in 47 (53.4%), blaVIM in 19 (24.4%), blaIMP in one (1.1%) and none had blaKPC. There were nine isolates during the two years which had multiple genes encoding carbapenemases; while 66 did not have any of the genes tested. Of the 80 isolates subjected to BOX-PCR, 58 could be used for analysis and showed, presence of multiple clusters of carbapenem resistant isolates and absence of a single dominant clone. Interpretation & conclusions: The blaNDM-1 gene was absent in our isolates obtained during 2008 but was present amongst Enterobacteriaceae isolated in 2012. The blaKPC gene was also not found. Nine isolates obtained during the two years had multiple genes encoding carbapenemases confirming the previous reports of emergence of GNB containing genes encoding multiple carbapenemases. Typing using BOX-PCR indicated that this emergence was not because of clonal expansion of a single strain, and multiple strains were circulating at a single point of time.

Enhanced resistance to fluoroquinolones in laboratory-grown mutants & clinical isolates of Shigella due to synergism between efflux pump expression & mutations in quinolone resistance determining region.

Background & objectives: There is a worldwide emergence of fluoroquinolone resistance in Shigella species. To understand the molecular mechanisms associated with fluoroquinolone resistance, naturally occurring fluoroquinolone-resistant strains and laboratory-induced spontaneous mutants of Shigella spp. were used and the relative contributions of acrAB-tolC efflux pumps, gyrase and topoisomerase target gene mutations towards fluoroquinolone resistance were determined. Methods: Eight Shigella flexneri and six S. dysenteriae clinical isolates were studied. Three consecutive mutants resistant to ciprofloxacin for S. flexneri SFM1 (≥0.25 μg/ml), SFM2 (≥4 μg/ml) and SFM3 (≥32 μg/ml) were selected in 15 steps from susceptible isolates by serial exposure to increasing concentrations of nalidixic acid and ciprofloxacin. Similarly, two mutants for S. dysenteriae SDM1 (≥0.25 μg/ml) and SDM2 (≥4 μg/ml) were selected in eight steps. After PCR amplification sequence analyses of gyrase and topoisomerase target genes were performed. Expression of efflux genes acrA, acrB, acrR and tolC was measured using real-time PCR. Results: Mutations were observed in gyrA Ser83→Leu, Asp87→Asn/Gly, Val196→Ala and in parC Phe93→Val, Ser80→Ile, Asp101→Glu and Asp110→Glu. Overall, acrA and acrB overexpression was associated with fluoroquinolone resistance (p<0.05); while tolC and acrR expression levels did not. Interpretation & conclusions: Fluoroquinolone resistance in Shigella spp. is the end product of either a single or a combination of mutations in QRDRs and/ or efflux activity. Novel polymorphisms were observed at Val196→Ala in gyrA in clinical isolates and Phe93→Val, Asp101→Glu, Asp110→Glu and in parC in majority of laboratory-grown mutants.

Characterization of Shiga-toxigenic Escherichia coli isolated from cases of diarrhoea & haemolytic uremic syndrome in north India.

Background & objectives: Shiga toxin producing Escherichia coli (STEC) is an important zoonotic foodborne pathogen, capable of causing haemorrhagic colitis (HC) and haemolytic uremic syndrome (HUS). As data from India on human infections caused by STEC are limited, this study was carried out for hospital based surveillance for STEC as a causative agent of diarrhoea, bloody diarrhoea and HUS at a tertiary care centre and to study the virulence gene profile and strain relatedness by multi locus variable tandem repeat analysis (MLVA). Methods: A total of 600 stool samples were studied. Stool samples of every fifth patient presenting with non-bloody diarrhoea, all cases of bloody diarrhoea and diarrhoea associated HUS (D+HUS) were collected from October 2009 to September 2011. Stool samples were cultured for STEC and characterization of STEC was done by serogrouping, virulence genes analysis, and MLVA typing. Results: STEC were isolated as a sole pathogen from 11 stool samples [5 of 290 (1.7%) non-blood diarrhoea and 5 of 300 (1.6%) blood diarrhoea cases]. STEC was also isolated from one fatal case of HUS who was an eight month old child. Only six of 11 isolates were positive for stx2 gene, whereas stx1 was present in all 11 isolates. Only one isolate was positive for eae. Other adhesion genes present were iha in five isolates, followed by toxB and efa1 in two each and saa gene in one, isolate. Among the plasmid encoded genes, espP, hly and etpD were each present in one isolate each. In the MLVA typing, diverse profiles were obtained except two untypeable isolates from different patients shared the same MLVA profile. Both these isolates were not epidemiologically linked. Interpretation & conclusions: This study demonstrated that STEC could be a causative agent of diarrhoea, bloody diarrhoea and sporadic HUS. However, further work needs to be done to study and explore the prevalence of these organisms in the food chain in this region.

Hospital acquired urinary tract infection by multidrug-resistant Brevundimonas vesicularis.

Infections caused by Brevundimonas vesicularis, a nonfermenting Gram-negative bacterium, are very rare. Here, we report the first case of multidrug-resistant hospital acquired urinary tract infection by B. vesicularis. Patient was successfully treated with antimicrobial therapy with piperacillin-tazobactam and amikacin.

Evaluation of commercial boric acid containing vials for urine culture: Low risk of contamination and cost effectiveness considerations.

Background: Urine culture is a gold standard in the diagnosis of urinary tract infection. Clean catch midstream urine collection and prompt transportation is essential for appropriate diagnosis. Improper collection and delay in transportation leads to diagnostic dilemma. In developing countries, higher ambient temperatures further complicate the scenario. Here, we have evaluated the role of boric acid as a preservative for urine samples prior to culture in female patients attending outpatient department at our center. Materials and Method: Consecutive 104 urine samples were cultured simultaneously in plain uricol (Control-C) and boric acid containing tubes from Becton Dickinson urine culture kit (Boric acid group-BA). Results: In the real-time evaluation, we found that in almost 57% (59/104) of the urine samples tested, it was more effective in maintaining the number of the organisms as compared to samples in the container without any preservative. Our in vitro study of simulated urine cultures revealed that urine samples could be kept up to 12 h before culture in the preservative without any inhibitory effect of boric acid. Though the use of boric acid kit may marginally increase the initial cost but has indirect effects like preventing delays in treatment and avoidance of false prescription of antibiotics. If the man-hours spent on repeat investigations are also taken into consideration, then the economic cost borne by the laboratory would also decrease manifold with the use of these containers.

Molecular epidemiology of Vibrio cholerae causing outbreaks & sporadic cholera in northern India.

Background & objectives: Several outbreaks of cholera have been reported in Chandigarh region during a span of seven years from 2002-2008. The genetic characteristics of Vibrio cholerae isolates obtained during these outbreaks have not been adequately studied. The aim of this study was to do molecular typing of V. cholerae isolated from the sporadic and outbreak cases by pulsed-field gel electrophoresis (PFGE), Rep-PCR and ribotyping. Methods: Fifty representative isolates of V. cholerae from outbreak as well as sporadic cases were subjected to molecular typing by PFGE, 173 isolates (163 clinical and 10 environmental) were typed by rep-PCR and ribotyping. Ribotyping was done by determination of rRNA restriction pattern of BglI restriction digestion and hybridization with 7.2 kb rRNA probe of pKK3535 plasmid using DIG DNA labelling and detection kit. Universal VC1 primer was used for rep-PCR. Results: PFGE generated 15 pulsotypes, of which four matched the published pulsotypes and there were 11 new pulsotypes. PFGE was the most discriminatory method that could differentiate between isolates belonging to single ribotype. Pulsotype P1 corresponding to known pulsotype H1 was the major pulsotype till 2003. Pulsotype P3 corresponding to known pulsotype L emerged in 2004. The 2007 outbreaks in Punjab and Haryana were caused by P5 though P1 and P3 were isolated from the sporadic cases from the same region. The 2008 outbreak was caused by pulsotypes P6 and P7. Ribotype IV was the most predominant followed by RIII. This ribotype was not isolated after 2003 and ribotype IV became the most predominant 2004 onwards. Of the two unknown ribotypes (UNI and UN2), UNI was more common (27 isolates). Rep-PCR was the least discriminatory and divided all clinical isolates into four major profiles. The dendrogram analysis of PFGE revealed similarity of some clinical isolates with environmental isolates indicating the genetic relatedness. Interpretation & conclusion: Our findings showed that Rep-PCR was least discriminatory method. Ribotyping was a reliable and reproducible method. Ribotype IV was predominant ribotype followed by RIII. A total of 15 pulsotypes were generated and 11 of these were not reported earlier. Genetic relatedness was shown by clinical and environmental isolates which needs to be confirmed in future studies.

High occurrence of blaCMY-1 AmpC lactamase producing Escherichia coli in cases of complicated urinary tract infection (UTI) from a tertiary health care centre in north India.

AmpC beta lactamase producing Gram-negative bacteria have emerged worldwide. It is important to distinguish plasmid mediated AmpC β lactamases from chromosomally mediated enzymes for surveillance, epidemiology and hospital infection control as plasmid mediated genes can spread to other organisms. Occurrence of blaCMY-1 AmpC β-lactamase, a plasmid mediated cephamycinase was studied in 100 consecutive isolates of Escherichia coli from cases of complicated urinary tract infection (UTI). Screening for AmpC production was done by modified Hodge test, three dimensional test and AmpC disk test. All isolates showing a positive result by 2 out of 3 tests were then tested for blaCMY-1 gene by PCR. Fifty nine isolates were positive for AmpC β lactamase production, 56.6 per cent were positive by PCR. Eight out of 13 isolates which were negative by EDTA disk method were positive by PCR, whereas none of the isolates negative by 3D and modified Hodge test was positive by PCR. Among admitted patients urinary catheterisation was the major risk factor followed by obstructive uropathy, three patients developed urosepsis. High occurrence of blaCMY-1 AmpC β-lactamase warrants health care workers to endorse good hospital practices.

Fecal Contamination of Drinking Water Supplies in and around Chandigarh and Correlation with Acute Gastroenteritis.

The study evaluated drinking water from localities in and around Chandigarh for fecal coliforms, V.cholerae and Enterotoxigenic E.coli and correlate with occurrence of acute gastroenteritis occurring from the same region. Drinking water sample were collected from various sources from the defined area. Samples were tested for fecal coliforms and E.coli count by multiple tube method and pathogens by membrane filtration technique. E. coli were screened for heat labile toxin (LT) by the reverse passive agglutination method and heat stable toxin (ST) by ELISA. Stool samples from cases of acute gastroenteritis from the same region and time were collected and processed for V. cholerae, Enterotoxigenic E coli (ETEC) and others like Salmonella, Shigella and Aeromonas spp. Of 364 water samples examined, 116 (31.8%) samples were contaminated with fecal coliforms (58.5% rural, 33.4% semi-urban and 11.1% from urban areas). E. coli were grown from 58 samples. Ninety-two isolates of E. coli were tested for enterotoxins of which 8 and 24 were positive for LT and ST respectively. V. cholerae were isolated from 2 samples during the outbreak investigation. Stored water samples showed a significantly higher level of contamination and most of Enterotoxigenic E. coli were isolated from stored water samples. A total of 780 acute gastroenteritis cases occurred; 445 from semiurban, 265 rural and 70 from urban areas. Out of 189 stool samples submitted, ETEC were the commonest (30%) followed by V. cholerae (19%), Shigellae (8.4%), Salmonellae (2.1%) and Aeromonas (2.6%). ST-ETEC (40/57) were commoner than LT- ETEC(17/57). In the present study, high levels of contamination of drinking water supplies (32.1%) correlated well with cases of acute gastroenteritis. Majority of cases of acute gastroenteritis occurred in the semi-urban area corresponding with high level of contamination (33.4%). The highest level of water contamination was seen in rural areas (58.5%) but the number of acute gastroenteritis cases were lesser (33.9%) as ponds were infrequently used for drinking purpose. Safer household water storage and treatment is recommended to prevent acute gastroenteritis, together with point-of-use water quality monitoring.

Endoscopic jejunal biopsy culture: a simple and effective method to study jejunal microflora.

Background Jejunal fluid culture is the gold standard for assessing jejunal microflora. Aspiration of jejunal fluid is sometime difficult. As the microorganisms rests on the mucosal surface, culture of the mucosal biopsy may be a possible alternative method. Aim To study the role of jejunal mucosal biopsy culture to assess jejunal microflora and to compare it with jejunal fluid culture. Methods Thirty adult subjects with gastroesophageal reflux disease requiring endoscopy underwent enteroscopy. Jejunal fluid aspirate and mucosal biopsy were cultured. The procedure was repeated after omeprazole therapy in 18 patients. Results Forty-eight pairs (30 preomeprazole therapy and 18 postomeprazole therapy) of fluid and mucosal biopsies were cultured. In 45 of the 48 pairs (94%), both the culture of jejunal biopsy and jejunal fluid yielded similar results with respect to the presence (n=27) or absence of growth (n=18). In the remaining 3 pairs, the growth was present either in the biopsy culture (n=2) or in the fluid culture (n=1) only. Among those pairs in which growth was present, microorganisms isolated were identical in 53%, differed by ≤2 organism in 37% and different by >2 organisms in 10%. Ten of the 12 patients who were detected to have small intestinal bacterial overgrowth (SIBO) on fluid culture were also detected to have SIBO on biopsy culture. Sensitivity, specificity, positive and negative predictive value of biopsy culture in diagnosing SIBO was 83.5%, 97.2%, 94.7%, and 91.6%, respectively. Conclusion Culture of unwashed endoscopic jejunal mucosal biopsy is an effective and simple alternative to jejunal fluid culture for assessing jejunal microflora.

Pediatric urinary tract infections in a tertiary care center from north India.

Background & objectives: Paediatric urinary tract infections (UTI) are associated with high morbidity and long term complications like renal scarring, hypertension, and chronic renal failure. A cause of occult febrile illness, they often remain undiagnosed. We studied the clinical and microbiologic profile and antibiotic resistance profile of such infections in paediatric UTI patients at our center. Methods: Clean catch mid-stream urine samples for culture were received from 1974 children aged < 12 yr over a period of 6 months. Quantitative wet mount microscopy and semiquantitative culture on cysteine lactose electrolyte deficient medium were done to diagnose UTI. Isolates were identified by standard biochemical tests and antimicrobial sensitivity was determined. Clinical details including risk factors and underlying illness were noted. Results: Significant bacteriuria was found in 558 children (28.3%). Male gender (25.6%), age < 1 yr (77.5%), vesicoureteric reflux disease (VUR) (19.9%) and posterior urethral valve (PUV) (27.6%) were common risk factors in children suffering from UTI. Pyuria was detected in 53.6 per cent of infections. Common uropathogens isolated were Escherichia coli (47.1%), Klebsiella spp. (15.6%), Enterococcus fecalis (8.7%), members of tribe Proteae (5.9%), Pseudomonas aeruginosa (5.9%) and Candida spp. (5.5%). Against lactose fermenting Enterobacteriaceae, in-vitro resistance was least against amikacin (32.5%), nitrofurantoin (26.7%) and imipenem (3.7%). Among enterococci, vancomycin resistant enterococci constituted 12 per cent of the strains. 93.4 per cent of the UTI detected was nosocomial. Interpretation & conclusion: Paediatric UTI was common in children with male gender, age < 1 yr, and in children suffering from VUR and PUV. Spectrum of pathogens causing paediatric UTI in our center had a preponderance of nosocomial multi-drug resistant pathogens.

Concomitant intestinal parasitism and noncholera vibrio infection.

Concomitant parasitism is not uncommon especially in tropical countries with low socioeconomic status. Here we report an unusual combination of intestinal infection due to Strongyloides stercoralis, Blastomyces hominis and non-cholera Vibrio in a patient suffering from acute gastroenteritis and hypoalbuminemia. Early recognition and accurate treatment of gastrointestinal infections and infestations before the patient develops complications is important.

Intestinal bacterial and parasitic infections among food handlers in a tertiary care hospital of North India.

BACKGROUND: Intestinal infection is still an important public health problem in developing countries like India. Food handlers may be infected by a wide range of enteropathogens and have been implicated in the transmission of many infections to the public in the community and to patients in hospitals. AIM: To study the presence of enteropathogenic bacteria and parasites in food handlers working in the food service establishments of a tertiary care centre in North India. METHOD: The stool samples received from food handlers during January 2001 to December 2006 were examined by wet mount, iodine mount and modified acid fast staining directly and after formol ether concentration technique for parasites. For enteropathogenic bacteria, samples were inoculated onto MacConkey's agar, Deoxycholate Citrate Agar, Xylose Lysine Deoxycholate agar (XLD) as per the WHO protocol. RESULTS: During the years 2001-2006 respectively, 8.75%, 16%, 1.4%, 6.75%, 2.56% and 6.75% food handlers were infected with enteropathogens. The parasitic infections in our study were 1.3 to 7% while the enteropathogenic bacteria were reported from 0% to 13.3% during the years 2001-6. Giardia was the most common parasite while Shigella was the most common bacteria isolated from food handlers. During the year 2001, there was an outbreak of ETEC in the neonatal ICU, Advanced Paediatric Centre, PGIMER, which was traced back to a food handler involved in the preparation of the milk feed. CONCLUSION: Routine screening of food handlers is a valuable tool for prevention of nosocomial food-borne infections.

Occurrence of ESBL & Amp-C beta-lactamases & susceptibility to newer antimicrobial agents in complicated UTI.

BACKGROUND & OBJECTIVE: Production of extended spectrum beta-lactamases (ESBLs) and AmpC beta-lactamases are the most common mechanisms of antimicrobial resistance in Gram negative bacilli. A prospective study was undertaken to know the occurrence of ESBL and AmpC producing strains and their antibiotic susceptibilities to newer agents to guide empirical therapy for complicated urinary tract infections. METHODS: Over a period of five months (January to May 2003), organisms grown in pure culture and in significant numbers from urine sample were identified by standard biochemical tests and antibiotic susceptibility determined by disc diffusion method. Gram-negative bacilli that were resistant to third generation cephalosporins, ciprofloxacin and gentamicin/amikacin were defined as highly drug resistant uropathogens (HDRU). HDRU were further tested for ESBL and AmpC phenotypes. RESULTS: Uropathogens were isolated in significant numbers in 1979 (21.8%) of the total 9,072 samples, of which 438(22.1%) were HDRU. Two hundred and five consecutive HDRU isolates were tested for ESBL production and 36.5 per cent were found to be ESBL producers. The highest positivity was found to be in Klebsiella spp. (51.2%), followed by Escherichia coli (40.2%), Enterobacter aerogenes (33.4%) and Pseudomonas aeruginosa (27.9%). Both ESBL producers and non producers showed a high degree resistance to piperacillin (93.1 and 90.9%), amoxycillin-clavulanic acid (93.4 and 90.9%), aztreonam (79.4 and 78%), cefepime (76.7 and 78%), and ampicillin-sulbactam (76.7 and 70.4%). The most effective antibiotics for ESBL producers were imipenem (8.2% resistance), piperacillin-tazobactam (9.5%) and ceftazidime-clavulanic acid (23.2%). Among ESBL non-producers, piperacillin-tazobactam (31.06%), ceftazidime-clavulanic acid (49.2%) and imipenem (11%) were less effective when compared to ESBL producers. Fifty three piperacillin and piperacillin-tazobactam positive and 20 negative isolates were further tested for AmpC production and found that all 53 positive isolates were also positive by for AmpC beta-lactamase. INTERPRETATION & CONCLUSION: Overall, 22.1 per cent of our isolates were highly drug resistant, and ESBL producers could explain only 36.5 per cent of HDRU in our study. Therefore, we assume that AmpC beta-lactamases are more important in our setting. Based on our finding a test using discs containing piperacillin and piperacillin-tazobactam ( PtPc) disc at a distance of 20 mm would act as a useful screening procedure for AmpC production as AmpC beta-lactamase producers are more susceptible to tazobactam as compared to clavulanic acid.

Modification of small bowel microflora in chronic alcoholics with alcoholic liver disease.

The bacterial flora of the gastrointestinal tract plays an essential role in human physiology. Our aim was to study the pattern of change in bacterial microflora in the small intestines of patients with chronic alcoholic liver disease (ALD). Patients of ALD made up the test group and nonalcoholics served as controls. Duodenal (D2) biopsies were obtained by upper gastrointestinal endoscopy and processed immediately for microbiological analysis. Marked qualitative and quantitative alterations of small intestinal microflora was documented in chronic alcoholics. There was increased bacterial growth of both gram-positive cocci and gram-negative bacilli in the ALD group.