Listeria goaensis sp. nov.

Two Listeria-like isolates obtained from mangrove swamps in Goa, India were characterized using polyphasic combinations of phenotypic, chemotaxonomic and whole-genome sequence (WGS)-based approaches. The isolates presented as short, non-spore-forming, Gram-positive rods, that were non-motile, oxidase-negative, catalase-positive and exhibited α-haemolysis on 5 % sheep- and horse-blood agar plates. The 16S rRNA gene sequences exhibited 93.7-99.7 % nucleotide identity to other Listeria species and had less than 92 % nucleotide identity to species of closely related genera, indicating that the isolates are de facto members of the genus Listeria. Their overall fatty acid composition resembled that of other Listeria species, with quantitative differences in iso C15 : 0, anteiso C15 : 0, iso C16 : 0, C16 : 0, iso C17 : 0 and anteiso C17 : 0 fatty acid profiles. Phylogeny based on 406 core coding DNA sequences grouped these two isolates in a monophyletic clade within the genus Listeria. WGS-based average nucleotide identity and in silico DNA-DNA hybridization values were lower than the recommended cut-off values of 95 and 70 %, respectively, to the other Listeria species, indicating that they are founding members of a novel Listeria species. We suggest the name Listeriagoaensis sp. nov. be created and the type strain is ILCC801T (=KCTC 33909;=DSM 29886;=MCC 3285).

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.

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.

Mangrove Ecosystems: An Adopted Habitat for Pathogenic Salmonella spp.

Mangroves are affected by industrial and anthropogenic factors. Although mangroves have been widely studied, investigations of pathogens that may affect public health significance are largely lacking even while incidences of diseases linked with the consumption of mangrove-associated food have increased. A total of 150 samples of water, sediment, and biota were collected from ten mangrove ecosystems in Goa, India. Total viable counts of pathogens such as E. coli, Listeria, Salmonella, and Vibrio spp. ranged from 1.25 to 3.9 × 10(3) cfu/ mL, which were above the relevant standards. Salmonella counts were the highest at 3.1 to 3.9 × 10(3)cfu/mL, with a prevalence of 40%. Considering its high prevalence, the virulence of Salmonella spp. was studied. The invA gene was detected in 35% of the Salmonella isolates by polymerase chain reaction (PCR). The findings suggested that pathogens adapt to this habitat, resulting in contamination of the indigenous fauna.

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.

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.

Evaluation of a PCR targeting fimbrial subunit gene (fimA) for rapid and reliable detection of Enteroaggregative Escherichia coli recovered from human and animal diarrhoeal cases.

The present study describes the utility of a chromosomal associated fimbrial subunit gene (fimA) for screening 'typical' as well as 'atypical' Enteroaggregative Escherichia coli (EAEC) by PCR and its possible role as a promising molecular marker for rapid detection of 'typical' and 'atypical' EAEC pathotype.

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.