Krupali V. Poharkar

Biofilm-Forming Abilities of Listeria monocytogenes Serotypes Isolated from Different Sources.

A total of 98 previously characterized and serotyped L. monocytogenes strains, comprising 32 of 1/2a; 20 of 1/2b and 46 of 4b serotype, from clinical and food sources were studied for their capability to form a biofilm. The microtiter plate assay revealed 62 (63.26%) strains as weak, 27 (27.55%) strains as moderate, and 9 (9.18%) strains as strong biofilm formers. Among the strong biofilm formers, 6 strains were of serotype 1/2a and 3 strains were of serotype 1/2b. None of the strain from 4b serotype exhibited strong biofilm formation. No firm correlation (p = 0.015) was noticed between any serotype and respective biofilm formation ability. Electron microscopic studies showed that strong biofilm forming isolates could synthesize a biofilm within 24 h on surfaces important in food industries such as stainless steel, ceramic tiles, high-density polyethylene plastics, polyvinyl chloride pipes, and glass. Cell enumeration of strong, moderate, and weak biofilm was performed to determine if the number of cells correlated with the biofilm-forming capabilities of the isolates. Strong, moderate, and weak biofilm showed 570±127× 103 cells/cm2, 33±26× 103 cells/cm2, 5±3× 103 cells/cm2, respectively, indicating that the number of cells was directly proportional to the strength of the biofilm. The hydrophobicity index (HI) analysis revealed higher hydrophobicity with an increased biofilm formation. Fatty acid methyl esterase analysis revealed the amount of certain fatty acids such as iso-C15:0, anteiso-C15:0, and anteiso-C17:0 fatty acids correlated with the biofilm-forming capability of L. monocytogenes. This study showed that different strains of L. monocytogenes form biofilm of different intensities which did not completely correlate with their serotype; however, it correlated with the number of cells, hydrophobicity, and amount of certain fatty acids.

Characterization and biofilm forming ability of diarrhoeagenic enteroaggregative Escherichia coli isolates recovered from human infants and young animals

Enteroaggregative Escherichia coli (EAEC) is an important pathotype that causes infection in humans and animals. EAEC isolates (n=86) recovered from diarrhoeal cases in human infants (37) and young animals (49) were characterized as typical and/or atypical EAEC strains employing PCR for virulence associated genes (cvd432, aaiA, astA, pilS, irp2, ecp, pic, aggR, aafA, aggA, and agg3A). Besides, biofilm formation ability of human and animal EAEC isolates was assessed using microtiter plate assay. In addition, the transcriptional profile of biofilm associated genes (fis and ecp) was also evaluated and correlated with biofilm formation assay for few selected EAEC isolates of human and animal origins. Overall, a diverse virulence gene profile was observed for the EAEC isolates of human and animal origins as none of the EAEC isolates revealed the presence of all the genes that were targeted. Nine typical EAEC isolates were identified (6 from humans and 3 from animals) while, the majority of the isolates were atypical EAEC strains. Isolation and identification of three typical EAEC isolates from animals (canines) appears to be the first report globally. Further, based on the observations of the biofilm formation assay, the study suggested that human EAEC isolates in particular were comparatively more biofilm producers than that of the animal EAEC isolates. The fis gene was highly expressed in majority of typical EAEC isolates and the ecp gene in atypical EAEC isolates.

Presence of a widely disseminated Listeria monocytogenes serotype 4b clone in India.

Details about the members of the Indian Listeria Consortium are provided in the Supplementary Data. Emerging Microbes and Infections (2016) 5, e55; doi:10.1038/emi.2016.55; published online 8 June 2016

Multi-Virulence-Locus Sequence Typing of 4b Listeria monocytogenes Isolates Obtained from Different Sources in India over a 10-Year Period

Listeria monocytogenes is an emerging foodborne pathogen responsible for listeriosis. The incidence of listeriosis has increased during the last 2 decades due to the increase in consumption of ready-to-eat foods and change in food consumption habits. Outbreaks and sporadic cases of listeriosis have been reported in developed countries. These reports have helped determine the safety practices needed to control listeriosis. Although L. monocytogenes has been reported from humans, animals, and a variety of foods in India, limited data exist with respect to prevalence and distribution of L. monocytogenes in the Indian subcontinent. The Indian Listeria Culture Collection Centre in Goa maintains all of the isolates received for subtyping and molecular characterization. Of the listerial isolate collection maintained by this center, three fourths of the isolates are of 4b serotype, while the number of other serotypes is very low. Therefore, we screened L. monocytogenes serotype 4b isolates to determine their relevance to previously defined epidemics and/or outbreaks using multi-virulence-locus sequence typing (MVLST). A total of 25 isolates in serogroup 4b of L. monocytogenes were randomly selected from a repository of 156 L. monocytogenes 4b isolates obtained from different sources in India over a period of 10 years. MVLST sequence types (virulence types, VTs) were compared to known epidemic clones and other known isolates in the L. monocytogenes MVLST database. The 25 isolates were grouped into three clusters. Cluster I comprised 21 isolates including animal (n=9), human (n=4), and food (n=8), which matched Epidemic Clone I (ECI, VT20). Three isolates-two from animal and one from food-formed a cluster while a single animal isolate was placed into two novel VTs (VT98 and VT99), respectively. Based on these findings, it can be inferred that ECI has been isolated from a variety of sources and places and has persisted in India for at least 10 years.

16S rRNA PCR followed by restriction endonuclease digestion: A rapid approach for genus level identification of important enteric bacterial pathogens

The study describes a rapid approach for detection of common enteric bacterial pathogens, which involves partial amplification of the 16S rRNA gene by PCR using a colony from selective medium followed by restriction enzyme (RE) digestion using the EcoRI, HindIII and SalI enzymes. On the basis of RE digestion analysis different genera namely, Escherichia, Salmonella, Shigella, Vibrio, Campylobacter, Arcobacter, Yesinia and Listeria were differentiated.

A multiplex PCR for detection of Listeria monocytogenes and its lineages.

A novel multiplex PCR assay was developed to identify genus Listeria, and discriminate Listeria monocytogenes and its major lineages (LI, LII, LIII). This assay is a rapid and inexpensive subtyping method for screening and characterization of L. monocytogenes.

Prevalence, Serogroups, Shiga-toxin Genes and Pulsed Field Gel Electrophoresis Analyses of Escherichia coli Isolated from Bovine Milk

Shiga toxin-producing Escherichia coli (STEC) including non-O157 strains have been linked to outbreaks and sporadic cases of illness worldwide. A total of 647 milk samples were collected at different levels of collection and processing (udder, milking utensils, milk collection centres and receiving dock) within West Coast region of India. The milk samples were screened for the presence of E. coli and further tested for the Shiga-toxin (stx) genes by PCR. The isolates were characterized for their serogroups and XbaI digestion patterns of total DNA separated by pulsed-field gel electrophoresis (PFGE). A total of 77 (11.90xa0%) isolates were confirmed as having E. coli. The serogroups reported were O4, O60, O112, O56, O159, O120, O2, O83, O88, O95, O141, O21, O25, O80, O140, O97, O24, O166, O146, O51, O169, O147, O103, O18, O100, O15, O69, O43, O7, O3, O45, O124, O110, O84, and O114. Out of the 77 E. coli isolates, 25 (32.46xa0%) could be classified as Shiga-toxigenic based on PCR results. Of these 11, 3 and 11 isolates were positive for stx1, stx2, and both stx1 and stx2, respectively. PFGE profiles indicated genetic diversity of E. coli strains. Much variation was observed among isolates recovered at different levels of collection. Further research is needed to uncover unique characteristics and resistance of non-O157 STEC strains.

Genetic diversity, virulence potential and antimicrobial susceptibility of Listeria monocytogenes recovered from different sources in India

Listeria monocytogenes isolates (n = 36) recovered from human and animal clinical cases and foods from different geographical regions of India were characterized using multiplex PCR-based serotyping, pulsed field gel electrophoresis (PFGE), in vitro and in vivo pathogenicity tests and antibiogram profiling. Multiplex PCR-based serotyping distributed L. monocytogenes isolates into 3 serogroups, of which 91.67% belonged to 4b, 4d, 4e serogroup, followed by 5.56% to 1/2a, 3a and 2.78% to 1/2b, 3b serogroups. PFGE analysis using ApaI and AscI restriction enzymes revealed 17 pulsotypes among 36 L. monocytogenes isolates with 6 major clusters having similar fingerprint profile within their cluster and 11 unique fingerprint profiles. Interestingly, PFGE analysis inferred that foods of animal origin could be a significant source of infection for spread of listeriosis among human populations. Furthermore, on comparison of in vitro and in vivo pathogenicity tests, an overall good correlation was observed between hemolytic titer assay and chick embryo inoculation test as most of the isolates with a hemolytic titer of ≥ 16 were found to be lethal to chick embryo. All the isolates were found to be susceptible to tested antimicrobials except for one animal isolate which showed resistance towards co-trimoxazole.

Prevalence and genetic profiles of Escherichia coli from mangroves and mangrove associated foods off Goa, India.

A total of 120 samples comprising of water (45), sediment (45) and mangrove originated food (30) collected from mangrove ecosystems of Goa were screened for Escherichia coli employing ISO-16654 method. Seventy-one (59.16%) samples were positive for E. coli. The E. coli isolates were further characterized by serotyping, virulence gene profiling and pulsed field gel electrophoresis (PFGE). Water and sediment samples were analyzed for physico-chemical parameters. The serotypes reported were O1, O10, O13, O17, O36, O41, O50, O68, O105, O116, O141, O148, O159, O162 and rough types while, 23 strains could not be typed. The stx1 and stx2 genes were detected in 33(46.47%) and 16(22.53%) isolates, respectively. The XbaI restriction digestion patterns of the stx positive strains were diverse. Interestingly, few strains isolated from diarrheal patients and from water, sediment and food from mangrove sources were genetically similar. The study showed that the mangrove ecosystem could be a potential reservoir for pathogenic E. coli.

Genetic diversity and antibiogram profile of diarrhoeagenic Escherichia coli pathotypes isolated from human, animal, foods and associated environmental sources

Introduction Infectious diarrhoea particularly due to pathogenic bacteria is a major health problem in developing countries, including India. Despite significant reports of diarrhoeagenic Escherichia coli (DEC) pathotypes around the globe, studies which address genetic relatedness, antibiogram profile and their correlation with respect to their isolation from different sources are sparse. The present study determines isolation and identification of DEC pathotypes from different sources, their genetic characterisation, antibiogram profile and their correlation if any. Materials and methods A total of 336 samples comprising diarrhoeic stool samples from infants (n=103), young animal (n=106), foods (n=68) and associated environmental sources (n=59) were collected from Bareilly region of India. All the samples were screened by using standard microbiological methods for the detection of E. coli. The identified E. coli were then confirmed as DEC pathotypes using polymerase chain reaction–based assays. Those DEC pathotypes identified as Enteroaggregative E. coli (EAEC) were further confirmed using HEp-2 adherence assay. All the isolated DEC pathotypes were studied for their genetic diversity using pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing was performed by using disc diffusion method as per Clinical Laboratory Standards Institute guidelines. Results and discussion Of the four DEC pathotypes investigated, EAEC was found to be the predominant pathogen with an isolation rate of 16.5% from infants, 17.9% from young animals, 16.2% from foods and 3.4% from the associated environmental sources. These EAEC isolates, on further characterisation, revealed predominance of ‘atypical’ EAEC, with an isolation rate of 10.7% from infants, 15.1% from young animals, 16.2% from foods, and 3.4% from the associated environmental sources. On PFGE analysis, discrimination was evident within DEC pathotypes as 52 unique pulsotypes were observed for 59 recovered DEC pathotypes. However, a few EAEC isolates were found to be clonal (clusters A, B, C, D, F, G, and H) irrespective of their source of isolation, suggests sharing and/or circulation among different sources. Further, a high antibiotic resistance pattern was observed among isolated DEC pathotypes as almost 86.4% of isolates were found to be resistant against ≥3 tested drugs.