Background & objectives: Among cell surface proteins, biofilm-associated protein (Bap) promotes biofilm development in Staphylococcus aureus strains. The aim of this study was to investigate proteinase-mediated biofilm dispersion in different isolates of S. aureus. Methods: Biofilm assay was done in 96-well microtitre plate to evaluate the effect of proteinase K on biofilms of bovine mastitis S. Aureus isolates. Extracellular polymeric substances were extracted and evaluated for their composition (protein, polysaccharides and extracellular DNA), before and after the proteinase K treatment. Results: Biofilm assay showed that 2 μg/ml proteinase K significantly inhibited biofilm development in bap-positive S. aureus V329 as well as other S. aureus isolates (SA7, SA10, SA33, SA352), but not in bap-mutant M556 and SA392 (a weak biofilm-producing strain). Proteinase K treatment on S. aureus planktonic cells showed that there was no inhibition of planktonic growth up to 32 μg/ml of proteinase K. Proteinase K treatment on 24 h old preformed biofilms showed an enhanced dispersion of bap-positive V329 and SA7, SA10, SA33 and SA352 biofilms; however, proteinase K did not affect the bap-mutant S. aureus M556 and SA392 biofilms. Biofilm compositions study before and after proteinase K treatment indicated that Bap might also be involved in eDNA retention in the biofilm matrix that aids in biofilm stability. When proteinase K was used in combination with antibiotics, a synergistic effect in antibiotic efficacy was observed against all biofilm-forming S. aureus isolates. Interpretation & conclusions: Proteinase K inhibited biofilms growth in S. aureus bovine mastitis isolates but did not affect their planktonic growth. An enhanced dispersion of preformed S. aureus biofilms was observed on proteinase K treatment. Proteinase K treatment with antibiotics showed a synergistic effect against S. aureus biofilms. The study suggests that dispersing S. aureus by protease can be of use while devising strategies againstS. aureus biofilms.

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Background & objectives: The emergence and rapid spread of carbapenem resistance mediated by metallo-beta-lactamase (MBL) in Pseudomonas aeruginosa is of major concern due to limited therapeutic options. This study was aimed at detecting the presence of MBL and its association with integrons in imipenem-resistant P. aeruginosa isolates and to determine their genetic relatedness. Methods: A total of 213 P. aeruginosa isolates were collected from two tertiary care centres and tested against anti-pseudomonal antibiotics by antimicrobial susceptibility testing, followed by the detection of MBL production by combined disk method. Minimum inhibitory concentration (MIC) of meropenem was determined by E-test. Multiplex polymerase chain reaction (PCR) was performed for the detection of bla_SPM, bla_IMP, bla_VIM, bla_NDM, bla_GIM and bla_SIM. PCR was carried out to characterize the variable region of class 1 integron. Transcongujation assay was carried out for the confirmation of plasmid-mediated resistance. Enterobacterial repetitive intergenic consensus sequence (ERIC)-PCR was performed for determining the genetic relatedness among P. aeruginosa isolates. Results: Of the 213 P. aeruginosa isolates, 22 (10%) were found to be carbapenem resistant and these were from pus 18 (82%), urine 2 (9%), sputum 1 (5%) and tracheal wash 1 (5%). Among 22 isolates, 18 (81.8%) were found to be MBL producers by phenotypic method and MIC range of meropenem was 8 to >32 μg/ml. PCR amplification showed that 20 (91%) isolates carried any one of the MBL genes tested: bla_VIM and bla_NDM in seven (32%) and six (27%) isolates, respectively; bla_VIM and bla_NDMin three (14%); bla_IMP and bla_NDM in two (9%); bla_VIM and bla_IMP in one (5%) isolate. The bla_VIM, bla_IMP and bla_NDM were found to co-exist in one isolate. None of the isolates were positive for bla_SPM, bla_SIM and bla_GIM. All 22 isolates carried class I integron. Of the 20 MBL-positive isolates, transconjugants were obtained for 15 isolates. ERIC-PCR analysis showed all isolates to be clonally independent. Interpretation & conclusions: Our results showed 10.3 per cent of carbapenem resistance among P. aeruginosa isolates, and the coexistence of MBL-encoding genes among P. aeruginosa mediated by class I integron.

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Background & objectives: Several outbreaks of acute encephalitis syndrome (AES) have been reported in Alappuzha district, Kerala State, India, in the past. The aetiology of these outbreaks was either inconclusive or concluded as probable Japanese encephalitis virus (JEV) infection based on clinical presentation. The role of West Nile virus (WNV) in AES outbreaks was also determined. However, the extent of WNV infection has not been studied in this region previously. A population-based cross-sectional serosurvey study was undertaken to determine the seroprevalence of JEV and WNV in Alappuzha district. Methods: A total of 30 clusters were identified from 12 blocks and five municipalities as per the probability proportional to size sampling method. A total of 1125 samples were collected from all age groups. A microneutralization assay was performed to estimate the prevalence of JEV and WNV neutralizing antibodies in the sample population. Results: Of 1125 serum samples tested, 235 [21.5%, 95% confidence interval (CI): 15.2-27.8%] and 179 (15.9%, 95% CI: 9.6-22.3%) were positive for neutralizing antibodies against WNV and JEV, respectively. In addition, 411 (34.5%, 95% CI: 26.7-42.2%) were positive for cross-reactive antibodies against flaviviruses. Interpretation & conclusions: The study showed the seroprevalence of WNV and JEV antibodies in the surveyed area and the WNV seroprevalence was greater than JEV. It is necessary to create awareness in public and adopt suitable policy to control these diseases.

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Background & objectives: Genital Chlamydia trachomatis (CT) infections are one of the most prevalent sexually transmitted infections across the world. In pregnant women, if not detected and treated early, these may result in poor pregnancy outcomes and complications. The present study was aimed to screen CT infections from first void urine (FVU) samples of asymptomatic pregnant women using molecular methods. The secondary objective was to evaluate cost-effectiveness in pooling FVU samples for their diagnostic application. Methods: FVU samples were collected from 1000 asymptomatic pregnant women over a period of three years. Pooling was done by including five specimens in one pool in the amount of 10 μl and subjected to polymerase chain reaction (PCR) and further confirmed by direct fluorescent antibody assay (DFA). Results: The age of study participants ranged from 18 to 43 yr with the median±standard deviation of 26±3.84 yr. Majority of positive participants were younger than 25 years. A total of 200 pools were prepared and 20 of these were PCR positive. When individual specimen in 20 positive pools was tested, 20 PCR-positive specimens were identified from 19 pools, of which 16 were positive by DFA. Thus, CT was detected in 1.6 per cent asymptomatic pregnant women in India and pooling strategy resulted in 70 per cent reduction in a number of tests performed. Interpretation & conclusions: Our study detected C. trachomatis infection in 1.6 per cent asymptomatic pregnant women, and pooling of FVU specimens for PCR testing was found to be a cost-saving strategy in comparison to testing individual samples. Further evaluation and studies on the bigger sample size are warranted to validate these results.

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Background & objectives: Antimicrobial resistance in Neisseria gonorrhoeae, the causative agent of gonorrhoea, is a subject of worldwide attention. The present study was undertaken to examine the rates of ciprofloxacin resistance, to correlate mutations in gyrA and parC genes with the level of resistance and to look for a variation in mutation pattern, if any, in isolates from across the country. Methods: A total of 113 isolates of N. gonorrhoeae collected from sexually transmitted infection patients in six centres during November 2010 to October 2013 were investigated. Minimum inhibitory concentration (MIC) determination was done by E-test and results interpreted as per Calibrated Dichotomous Sensitivity criteria. DNA sequence analysis of gyrA and parC genes was done. Results: Of the 113 isolates, only three (2.6%) were susceptible whereas eight (7.07%) were less susceptible, 32 [28.3%, 95% confidence interval (CI): 20.4-37.6%] resistant (MIC 1-3 μg/ml) and 70 (61.9%, 95% CI: 52.2-70.7%) exhibited high-level resistance (HLR) (MIC ≥4 μg/ml) to ciprofloxacin. A S91F substitution in gyrA gene was demonstrated in all ciprofloxacin non-susceptible isolates. All resistant and HLR isolates had a double mutation in gyrA gene. However, only 5.7 per cent of HLR isolates showed double mutations in parC gene. One isolate (MIC 32 μg/ml) had a previously undescribed G85D substitution in the parC gene. Interpretation & conclusions: A S91F substitution in gyrA gene was seen in all non-susceptible isolates of N. gonorrhoeae. It may be used as a marker for ciprofloxacin resistance for molecular surveillance approaches to complement the culture-based methods.

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Background & objectives: Aeromonas species have been reported to cause various illnesses in humans such as wound infections, septicaemia, peritonitis and pneumonia. Their role in causation of cholera-like illness is also being increasingly recognized. This retrospective study was done to know the presence of Aeromonas as a cause of acute diarrhoea in a tertiary care hospital and to find the common species of Aeromonas causing diarrhoea and their antibiotic susceptibility patterns. Methods: Fifty isolates of Aeromonas were obtained over a period of 15 yr from 2000 to 2014 from patients of suspected acute gastroenteritis resembling cholera. Biotyping was done for 35 of these isolates available in culture collection, based on a panel of 13 biochemical reactions. Antibiogram was put up for all of these isolates by disk diffusion methods and interpreted according to the Clinical and Laboratory Standards Institute guidelines. Results: Of the 50 patients of Aeromonas-related acute gastroenteritis, 13 (26%) had typical features of cholera with rice water stools and severe dehydration. Eight patients (16%) had dysentery-like picture. One patient died of severe dehydration and septicaemia. The most common species were found to be Aeromonas caviae (34%) followed by Aeromonas veronii biovar veronii (29%), Aeromonas veronii biovar sobria (26%) and Aeromonas hydrophila (9%). All tested isolates were uniformly susceptible to cefepime, amikacin, azithromycin and meropenem; 14 per cent were susceptible to amoxicillin, 32 per cent to nalidixic acid, 60 per cent to co-trimoxazole, 54 per cent to ciprofloxacin, 60 per cent to ofloxacin, 74 per cent to chloramphenicol, 76 per cent to ceftriaxone, 74 per cent to cefotaxime, 88 per cent to gentamicin and 86 per cent to furoxone. Interpretation & conclusions: Aeromonas is an important, often neglected pathogen capable of causing a variety of gastrointestinal tract symptoms such as acute diarrhoea and dysentery and may even mimic cholera. It is, therefore, pertinent to recognize this pathogen as an important agent in the causation of severe diarrhoea.

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Background & objectives: Outbreaks of infection due to Salmonella enterica servovar Typhi (S. Typhi) are a great threat to public health. A rapid molecular typing method to characterize strains implicated in an outbreak is critical in implementing appropriate control measures. This study was done to demonstrate the power of a PCR-based method to provide rapid insights into the genetic relatedness amongst the Salmonella isolates implicated in a suspected typhoid fever outbreak. Methods: Forty two S. Typhi isolates originating from three geographically distinct areas, with one area suspected to have a single-source outbreak were included in the study. The genetic fingerprint of all isolates was generated using enterobacterial repetitive intergenic consensus sequence based-PCR (ERIC-PCR). The antimicrobial susceptibility profiles were also evaluated. Results: ERIC-PCR was found to be rapid and reproducible with a discriminatory index of 0.766. The dendrogram constructed based on ERIC-PCR fingerprinting revealed the existence of 12 distinct genotypes. The location suspected to have an outbreak displayed two genotypes amongst the 24 isolates. The other two locations (18 isolates) displayed genetic heterogeneity. The clonality of the outbreak isolates from the time-matched control isolates was established. The observed antimicrobial susceptibility profiles did not have any discriminatory power to subtype the isolates compared to the genetic fingerprints. Interpretation & conclusions: Our study demonstrated the discriminatory power and value of ERIC-PCR in the typing of S. Typhi isolates and providing valuable epidemiological insights.

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Background & objectives: The prevalence of multidrug-resistant (MDR) Escherichia coli isolates producing β-lactamase enzyme is a growing problem across the globe. Strain typing is an epidemiologically important tool not only for detecting the cross transmission of nosocomial pathogens but also for determining the source of infection. The present study was conducted to understand the clonal relationship among various β-lactamase-producing MDR E. coli isolates using enterobacterial repetitive intergenic consensus (ERIC) polymerase chain reaction (PCR). Methods: A total of 41 MDR E. coli isolates were randomly collected from various clinical samples and processed. Isolated organisms were tested for antibiotics resistance pattern. Phenotypic detection of metallo β-lactamases (MBL) was carried out by the imipenem-ethylenediaminetetraacetic acid disc diffusion/double-disc synergy test. AmpC enzyme production was tested by a modified three-dimensional extract test. Results: Almost all isolates were found sensitive to colistin. A high percentage of drug resistance was observed in these isolates against ceftazidime (100%), cefotaxime (100%), cefepime (100%), ofloxacin (97.56%), amoxicillin/clavulanic acid (97.56%) and norfloxacin (85.36%). Of the 41 isolates, ESBL producers were found to be predominant, i.e., 22 (53.65%), followed by AmpC (6, 14.63%) and MBL (5, 12.19%). Interpretation & conclusions: At 60 per cent similarity cut-off value, the dendrogram analysis showed that there were a total of 14 unique clusters of ERIC (CL-1 - CL-14) within the 41 E. coli isolates, which revealed the genetic diversity existing between them.

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Background & objectives: β-lactamases play a predominant role in drug-resistance amongst Enterobacteriaceae. Presence of genes on transferable plasmids encoding these enzymes favours their dissemination across species and genera within and outside geographical boundaries. This study was aimed to understand the presence of β-lactamases and transferable plasmids in clinical isolates of Klebsiella spp. which can contribute to the spread of resistance determinants. Methods: A total of 41 clinical isolates of Klebsiella spp., collected from a tertiary care centre in Kerala, India, were checked for antibiotic sensitivity and the presence of plasmids. The ability to produce extended-spectrum β-lactamases (ESBLs) and metallo-β-lactamases (MBLs) was screened for and confirmed in 29 plasmid-harbouring isolates. bla_NDM-1-specific primers were used for polymerase chain reaction amplification with plasmid DNA as template to determine episomal prevalence of this gene and its sequence-based phylogeny employing similar sequences from GenBank. Plasmid replicon typing was also carried out to determine the presence of transferable plasmids. Results: Our results showed a high degree of multidrug-resistant (MDR) pathogens with ESBL production confirmed in 52 per cent, MBL in 31 per cent and co-production of both enzymes in seven per cent of the plasmid-bearing isolates. Plasmid DNA from 14 per cent of the isolates produced bla_NDM-1-specific amplicons which showed sequence homology with those from bacteria of different genera and geographical areas. The predominant replicon type was found to be that of conjugative plasmids belonging to the incompatibility group - IncFII_K. Interpretation & conclusions: This study provides insight into the predominance of various β-lactamases and potent gene-disseminating agents in Klebsiella spp. and emphasizes the need for constant surveillance of these pathogens to determine appropriate treatment strategies.

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Background & objectives: Typhoid fever is a major cause of morbidity and mortality in the developing countries including India. Resistance to multiple antimicrobial agents is an emerging global problem that has serious impact on the treatment of disease. There are many factors associated with the emergence of resistance. Most important of them is the acquisition and further transmission and spread of resistance markers among various bacterial species. Therefore, we conducted this study to characterize the resistance plasmids in terms of their transferability and stability among Salmonella enterica serovar Typhi. Methods: Six multidrug-resistant S. Typhi isolates were evaluated for the stability and transfer of resistance markers. The resistance plasmids were also checked for the presence of RepHI1A replicon. Results: All resistance markers were found to be transferred to the recipient through conjugation and transformation, except for nalidixic acid. None of the resistance plasmid was found to harbour RepHI1A replicon and therefore, did not belong to incompatibility group IncHI1. Resistance markers were found to be highly stable in all the isolates during serial passages and storage as stab cultures at different temperatures for different time periods. Interpretation & conclusions: Resistance markers for chloramphenicol, ampicillin, streptomycin and trimethoprim were transferred through conjugation as well as transformation whereas that for nalidixic acid was not transferred in any of the isolates. Markers for chloramphenicol and streptomycin resistance were found to be most stable during various storage conditions. Presence of small-sized non-IncHI1 resistance plasmids is a matter of concern due to their capability to exist inside the host, thereby increasing the possibility of their transmission and spread among S. Typhi and other bacterial species.

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Background & objectives: Shigatoxic Escherichia coli (STEC) recovered from dairy animals of Kolkata, India, harboured the putative virulence genes; however, the animals did not exhibit clinical symptoms. Similarly, human isolates in this locality also showed variations in degree of symptoms. Hence, this study was designed to know the presence of recognized gene(s) in the locus of enterocyte effacement (LEE) pathogenicity island in these STEC isolates and functional status of the cardinal gene (eae) related to pathogenicity. Methods: Genes were characterized using polymerase chain reaction (PCR) assays, and functional status of cardinal gene (eae) was evaluated by fluorescent actin staining (FAS) assay. Variation in eae gene was determined by intimin PCR. Results: Cattle STEC isolates carried 22 genes in LEE pathogenicity island in different frequencies ranging from 5.63 to 47.88 per cent of the isolates. In human isolates, the genes namely ler, escRSTU, orf 2, esc C, esc V, orf 3 and tir that are associated with secretory function, were found to be absent and rest of the genes were present in lower frequency. Further, the cardinal gene (eae) responsible for initiation of pathogenesis was in a very low frequency in human (n=2; 10.5%) and cattle (n=11; 15.5%) isolates. None of theseeae⁺ STEC isolates from human and cattle revealed positivity in FAS assay. Interpretation & conclusions: Majority of human STEC isolates lacked the cardinal virulence gene (eae), and genes for secretory function that are essential for facilitating pathogenesis. This may partially be attributed to low occurrence of STEC in human clinical diarrhoea in this area. Although a few isolates (11 of 71) from cattle had eae gene, they did not express phenotypically. This could be one of the reasons for not appearing of clinical symptoms in the hosts.

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