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

Biofilm formation and growth ability of <i>L</i>. <i>monocytogenes</i> strains of different serotypes.

<p><b>(a)</b> OD₅₉₅ of the biofilm after staining with crystal violet, and growth turbidity of the strains belonging to serotype 1/2a; <b>(b)</b> OD₅₉₅ of the biofilm after staining with crystal violet, and growth turbidity of the strains belonging to serotype 1/2b; <b>(c)</b>. OD₅₉₅ of the biofilm after staining with crystal violet, and growth turbidity of the strains belonging to serotype 4b.</p

Biofilm-Forming Abilities of <i>Listeria monocytogenes</i> Serotypes Isolated from Different Sources

<div><p>A total of 98 previously characterized and serotyped <i>L</i>. <i>monocytogenes</i> 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 <i>L</i>. <i>monocytogenes</i>. This study showed that different strains of <i>L</i>. <i>monocytogenes</i> 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.</p></div

Strong, moderate, and weak biofilm-forming capabilities of <i>L</i>. <i>monocytogenes</i> isolates from different sources analyzed by microtiter plate assay.

<p>Strong, moderate, and weak biofilm-forming capabilities of <i>L</i>. <i>monocytogenes</i> isolates from different sources analyzed by microtiter plate assay.</p

SEM of <i>Listeria monocytogenes</i> ILCC306 on different industrially important surfaces.

<p><b>(a)</b><i>L</i>. <i>monocytogenes</i> ILCC306 on PVC pipe after 24 h. Multilayered and mat-like biofilms were observed; <b>(b)</b><i>L</i>. <i>monocytogenes</i> ILCC306 on ceramic tiles after 24 h. The cells were found to be aggregated all over the surfaces of ceramic tiles; <b>(c)</b><i>L</i>. <i>monocytogenes</i> ILCC306 on stainless steel (SS304) after 24 h. Biofilm aggregated near suture; <b>(d)</b><i>L</i>. <i>monocytogenes</i> ILCC306 on stainless steel suture (artificially made) after 24 h; <b>(e)</b><i>L</i>. <i>monocytogenes</i> ILCC306 on HDPE plastic after 24 h. The circled area shows biofilm rooted in the sutures; bacterial growth can be seeninside the sutures and aggregates formation toward the surfaces.</p

Fatty acid profile of the strong, moderate, and weak biofilm-forming isolates as analyzed by the FAME analysis.

<p>Fatty acid profile of the strong, moderate, and weak biofilm-forming isolates as analyzed by the FAME analysis.</p

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 201