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.

Role of 30 kDa antigen of enteric bacterial pathogens as a possible arthritogenic factor in post-dysenteric reactive arthritis.

Background: Reactive arthritis (ReA)/Reiter’s syndrome (RS) may be caused as a sequel of infections caused by enteric bacterial pathogens, although the mechanisms through, which different pathogens cause similar disease are not clear. Aim: This study was done to look for the presence and role of any common bacterial antigen among the pathogens isolated from such patients. Materials and Methods: A total of 51 patients of ReA and 75 controls (three groups of 25 subjects each: Group 1: Patients who did not develop arthritic complications within 3 months after bacillary dysentery/diarrhea; Group 2: Patients with other arthritic diseases and Group 3: Normal healthy subjects) were included. The isolated enteric pathogens were tested to detect the immunodominant antigens. Results and Conclusions: A common 30 kDa antigen was found to be specifi cally present among seven arthritogenic enteric bacterial strains belonging to three genera, Salmonella, Shigella and Hafnia. Post-dysenteric ReA patients’ sera show higher levels of immunoglobulin G, immunoglobulin M and immunoglobulin A antibodies against this antigen as compared to the controls. Lymphocytes of ReA patients recognize this antigen, proliferate and produce interleukin-2 in response to this antigen more than the lymphocytes of controls. 30 kDa antigen may be a common arthritogenic factor associated with postdysenteric ReA/RS. The association of Hafnia alvei with post-dysenteric ReA is described for the fi rst time. Four cases of mycobacterial ReA had an association with this antigen, suggesting that the arthritogenic antigen of mycobacteria and enteric bacteria may be of a similar nature.

Mycoplasma genitalium: An emerging sexually transmitted pathogen.

Mycoplasma genitalium is a member of genital mycoplasmas, which is emerging as an important causative agent of sexually transmitted infections both in males and females. The advent of polymerase chain reaction and other molecular methods have made studies on M. genitalium more feasible, which is otherwise a difficult organism to isolate. Besides Chlamydia trachomatis, M. genitalium is now an important and established cause of non gonococcal urethritis (NGU) in men, more so in persistent and recurrent NGU. Multiple studies have also shown a positive association of M. genitalium with mucopurulent cervicitis and vaginal discharge in females as well. The evidences for M. genitalium pelvic inflammatory diseases and infertility are quite convincing and indicate that this organism has potential to cause ascending infection. Lack of clear association with M. genitalium has been reported for bacterial vaginosis and adverse pregnancy outcomes. Diagnosis of M. genitalium infections is performed exclusively using nucleic acid amplification tests (NAATs), owing to poor or slow growth of bacterium in culture. Although there are no guidelines available regarding treatment, macrolide group of antimicrobials appear to be more effective than tetracyclines. The present review provides an overview of the epidemiology, pathogenesis, clinical presentation and management of sexually transmitted infections due to M. genitalium.

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.

Characterization of low molecular weight antimicrobial peptide from human female reproductive tract.

Background & objectives: The mechanisms that protect female upper genital tract from ascending infection by microbes present in vagina are only partially understood. It is expected that epithelial cells in mucosal surfaces and their secretions directly interfere with microbial colonization and invasion. This study was aimed to demonstrate the expression of 2 kDa antimicrobial peptide which was identified and purified from female genital tract tissues using chromatographic techniques. Methods: Low molecular weight proteins were isolated from human female reproductive tract tissues obtained from premenopausal women. Antimicrobial activity of these LMW proteins was assessed against different reproductive tract pathogens viz., Neisseria gonorrhoeae, Group B streptococcus, Gardnerella vaginalis, Escherechia coli and Candida albicans. The expression of these peptides were also documented in reproductive tract tissues with the help of hyperimmune sera raised against the rabbits. The purified peptide was characterized by N-terminal sequencing. Results: Immunohistochemical and immunofluorescence studies demonstrated that 2 kDa peptide was expressed in the stratified squamous epithelial cells of the ectocervix while it was absent in columnar epithelial cells of upper genital tract. Upregulation of the expression of this peptide was observed in patients of chronic non-specific cervicitis and acute on chronic cervicitis. This purified antimicrobial peptide also showed broad spectrum antimicrobial activity against different reproductive tract pathogens. Interpretation & conclusions: Considering the emerging bacterial resistance against conventional antibiotics, isolation and understanding of the expression of antimicrobial peptides from female reproductive tissue extracts may provide some leads towards the development of strategies for the treatment of reproductive tract infections.

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.

Efficacy of an in-house polymerase chain reaction assay for rapid diagnosis of Mycobacterium tuberculosis in patients with tubercular lymphadenitis: Comparison with fine needle aspiration cytology and conventional techniques.

Introduction: Tubercular lymphadenitis (TB-L) is the most common manifestation of extrapulmonary tuberculosis. Excisional biopsy with histopathological examination, Ziehl-Neelsen staining (ZNS) and culture and fine needle aspiration (FNA) cytology, although useful in the diagnosis of TB-L, cannot diagnose a substantial proportion of cases. We investigated the role of an in-house polymerase chain reaction (PCR) assay targeting the IS6110 gene from the FNA material in the diagnosis of the disease. Materials and Methods: The clinical profile of 150 patients with lymphadenopathy was noted and the fine needle aspirate was collected. After cytological processing, ZNS and culture on Lowenstein-Jensen media, mycobacterial DNA was isolated from the residual aspirate material and IS6110 gene PCR was performed. Results of cytology, ZNS, culture and IS6110 gene PCR were compared. Results: There were 49 confirmed patients of TB-L based on laboratory parameters (either culture isolation of Mycobacterium tuberculosis or any two of cytology, ZNS, PCR positive) and clinical response to therapy. Sensitivity and specificity of FNA was 89.8% and 96%, of ZNS was 40.8% and 99%, of culture was 40.8% and 100% and of IS6110 gene PCR test was 100% and 92.1%. Conclusion: IS6110 PCR can be considered a valuable adjunct to cytology, ZNS and culture techniques in the diagnosis of TB-L.

Detection of TEM & SHV genes in Escherichia coli & Klebsiella pneumoniae isolates in a tertiary care hospital from India.

Background & objectives: Extended spectrum beta lactamases (ESBLs) have been observed in virtually all the species of family Enterobacteriaceae. The enzymes are predominantly plasmid mediated and are derived from broad-spectrum beta lactamase TEM-1, TEM-2 or SHV-1 by a limited number of mutations. This study was undertaken to characterize ESBL producers among Escherichia coli and Klebsiella pneumoniae by PCR-RFLP, which were initially screened by phenotypic method. Methods: A total of 100 isolates of each species (E. coli and K. pneumoniae) were screened for ESBL production. PCR analysis for b-lactamase genes of the family TEM and SHV was also carried out. PCR products of TEM and SHV genes were subjected to digest with three different restriction enzymes. The digested products were run on 1.5 per cent agarose gel, stained and examined for DNA bands. Results: PCR carried out on plasmid DNA alone detected 30 per cent ESBL positive isolates using TEM primer and 38 per cent using SHV primer, whereas PCR for both plasmid and chromosomal DNA showed 56 per cent positivity for TEM and 60 per cent positivity for SHV. Interpretation & conclusion: RFLP yielded homogeneous band pattern, suggesting that there may be a point source or a common evolutionary origin for all the ESBL isolates.

Plasmid profile of ESBL producing Gram-negative bacteria and correlation with susceptibility to β-lactam drugs.

Background: Members of family Enterobacteriaceae can acquire resistance to extended spectrum beta lactams by a number of mechanisms; most important being the plasmid encoded extended spectrum beta lactamase (ESBL) and AmpC beta lactamase. This study has been designed to look for the presence of plasmids and their correlation with drug resistance. Methods: ESBL production was studied in different gram-negative bacteria and susceptibility testing of ESBL positive isolates was done for various beta lactams, cephalosporins and other commonly used drugs against them. Plasmid DNA isolation of all the ESBL positive strains was done by alkalilysis method. Finally the presence of plasmid was correlated with susceptibility to beta lactam drugs. Results: E. coli, K. pneumoniae, Enterobacter aerogenes and A. anitratus harbored multiple plasmids. One plasmid (M.W greater than 21,226 bp) was unanimously present in all the isolates. Conclusion: There is a strong correlation between the number of plasmids harbored by an isolate and resistance to various drugs tested.

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.