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Isolation of Human Pathogen Escherichia Albertii from Faeces of Seals (Leptonychotes Weddelli) in James Ross Island, Antarctica

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Isolation of Human Pathogen Escherichia Albertii from Faeces of Seals (Leptonychotes Weddelli) in James Ross Island, Antarctica CZECH POLAR REPORTS 3 (2): 173-183, 2013 Isolation of human pathogen Escherichia albertii from faeces of seals (Leptonychotes weddelli) in James Ross Island, Antarctica Ivo Sedláček1*, Linda Grillová2, Eva Kroupová1, Jitka Černohlávková1, David Šmajs2 1Czech Collection of Microorganisms, Department of Experimental Biology, Faculty of Science, Masaryk University, Tvrdého 14, 602 00 Brno, Czech Republic 2Department of Biology, Faculty of Medicine, Masaryk University, Kamenice 5, 602 00 Brno, Czech Republic Abstract A set of nine gram-negative fermenting rods biochemically identified as Escherichia coli was isolated from faeces of seals. These bacteria were characterized by phenotypic classification, 16S rDNA sequence analyses, automated ribotyping, study of whole-cell protein profiles by SDS-PAGE and finally by bacteriocin production. The results of our polyphasic taxonomic study supported the recognition of P4652, P4653 and P4740 isolates as true members of Escherichia albertii species – probably a major enteric human pathogen. To our best knowledge, this is the first evidence showing that E. albertii produces bacteriocin, colicin D. Obtained data unambiguously showed incon- venience of commercial identification systems to distinguish both Escherichia species due to missing data of E. albertii in the commercial databases. The results of Escherichia isolates taxonomy suggest seals as a novel source of human and animal pathogen, E. albertii in the Antarctic region. Key words: identification, bacteriocin, biotyping, ribotyping, SDS-PAGE The GenBank accession numbers for the 16S rRNA gene sequences of Escherichia albertii P4652 (CCM 8505) and P4653 are KF891381 and KF891382, respectively. DOI: 10.5817/CPR2013-2-18 ——— Received November 22, 2013, accepted December 12, 2013. *Corresponding author: Ivo Sedláček <[email protected]> Acknowledgements: This work was supported by the Operational Programme Education for Competitiveness project CEB (CZ.1.07/2.3.00/20.0183). The authors would also like to thank to the scientific infrastructure of the Czech Antarctic Station ‘‘J.G. Mendel’’ and its crew for their support (CzechPolar, project LM2010009). 173 I. SEDLÁČEK et al. Introduction Antarctica is the specific cold envi- enteropathogenic (EPEC) E. coli (Ooka et ronment area far from human activities al. 2012). This species is not only a human that exhibit totally natural ecosystem. pathogen but was found in wild and do- Analyses of microbiota from animals in- mestic birds worldwide, both healthy and habiting such cold environments reveal death birds (Oaks et al. 2010, Oh et al. their biodiversity and specific relation- 2011). The bird and human E. albertii iso- ships. Enteric bacteria in the gut of lates are heterogeneous and do not segre- animals may entail positive (e.g. probiotics gate on the basis of host, geographic ori- such as lactobacilli) but also detrimental gin, or disease status. Strains of E. albertii (e.g. Salmonella infections) consequences. possessed intimin (eae) and cytolethal dis- Regulation of the intestinal flora depends tending toxin (cdtB) genes as putative on complex interactions between many virulence factors (Oaks et al. 2010). Re- factors determined by the host environ- cently, the E. albertii was found as the ment, e.g. secretion of gastric acid, in- causative agent of human gastroenteritis testinal motility, local immunity, structure outbreak (Konno et al. 2012, Ooka et al. of the inner gut and mucous layers, as well 2013). as diet (Potti et al. 2002). The genus Escherichia spp., as well as many Escherichia, one of the most important bacterial species, produce antimicrobial a- enteric bacteria, is composed of five valid gents called bacteriocins (comprise coli- species, including the type species Esche- cins, microcins). Production of bacterio- richia coli and four less frequently en- cins is considered to be an important countered members: Escherichia herman- feature of interactions between microbial nii, Escherichia vulneris, Escherichia fer- populations (Cascales et al. 2007). Al- gusonii and Escherichia albertii (Euzéby though there is an evidence of the role of 1997). However, the current taxonomic some bacteriocins in bacterial virulence position of Escherichia spp. is little (Šmajs et al. 2010), in probiotic phenotype complicated. Comparative sequence analy- of E. coli strains (Šmajs et al. 2008), and sis between the genes for 16S rRNA of E. in E. coli colonization of gastrointestinal coli, E. vulneris, and E. hermannii placed tract (Gillor et al. 2009), the exact role of E. coli and E. vulneris together in a tightly bacteriocin production on bacterial popu- related cluster with shigellae, and E. her- lation level is unclear. New informa- mannii between Salmonella spp. and Ci- tion about the frequency of bacteriocins trobacter freundii (Cilia et al. 1996). and their molecular types help understand Recently, based on phylogenetic data, Eza- a bacteriocin role in bacterial populations ki (2010) suggested that only two species (Šmajs et al. 2012). of genus Escherichia should be valid, In this study, we have classified nine E. coli and E. albertii. strains of the genus Escherichia isolated Species E. albertii was described ten from faeces of seals (Leptonychotes wed- years ago by Huys and co-workers (2003) delli) in James Ross Island, Antarctica, in as the pathogen associated with diarrheal the austral summer of 2012-13. These disease in Bangladesh children. After- isolates were analysed on the basis of wards, E. albertii was found as probable e- phenotypic, genetic and phylogenetic data merging major human enteric pathogen be- to the species level and both E. coli and cause many strains might have been mis- E. albertii were proved. identified as enterohemorrhagic (EHEC) or 174 ESCHERICHIA ALBERTII FROM SEALS Material and Methods Bacterial strains The study was carried out in austral 21 to 49 days. The strains, originating summer of 2012-13 in James Ross Island, from different seals (Table 1), were iso- Antarctica (63.778338°S, 57.780993°W) lated on Columbia blood agar (Bio-Rad, and the culture-dependent technique was Czech Republic) by primo-cultivation of used to characterize the diversity of enteric samples maintained in E-Swabs (Dispolab, bacteria in faeces of seals (L. weddelli). A Czech Republic ). Subcultures, based on group of nine Escherichia strains was distinct colony differences, were cultivated isolated from faecal samples of randomly again on Columbia agar (Bio-Rad, Czech discovered seals in the Lachman cape in Republic) supplemented with 7% sheep the frame of cultivable faecal bacteria blood and after incubation at 30°C for 24 communities study project. The samples hours were used for further experiments. A were taken from seal faeces and/or rectum selected representative strain P4652 was with sterile swab/transport tube system E- deposited in the Czech Collection of Swabs (Dispolab, Czech Republic), kept at Microorganisms (http:\\www.sci.muni.cz\ 4°C and brought to the Czech Collection ccm) as Escherichia albertii CCM 8505. of Microorganisms, Masaryk University, Reference strains E. albertii CCM 7160T Czech Republic where they were plated and E. coli CCM 4225 and CCM 5172T and analysed. The time from sampling to were used for ribotyping and SDS-PAGE initial culture in the laboratory was from experiments. Isolate Source Isolate Source No: No: Escherichia albertii Escherichia coli P4652 fresh seal faeces (L. weddelli) P4656 seal faeces on iceberg (unknown species) P4653 fresh seal faeces (L. weddelli) P4657 seal faeces on iceberg (unknown species) P4740 fresh seal faeces (L. weddelli) P4664 fresh seal faeces (L. weddelli) P4665 fresh seal faeces (L. weddelli) P4722 smear from seal rectum (L. weddelli) P4733 fresh seal faeces (L. weddelli) Table 1. Source of isolation (Lachman Cape, James Ross Island, Antarctica). Phenotyping The phenotypic characteristics of the gas from glucose, motility, growth at 5°C isolates were initially performed using two and 37°C, beta glucuronidase, hydrolysis commercial identification kits ENTERO- of gelatine etc.) were additionally carried test 24 (Erba Lachema, Czech Republic) out according to Abbott et al. (2003) to and Biolog Identification System, GP2 confirm assignment of isolates into the Micro Plate (Biolog, USA) according to genus Escherichia and to supplement test the manufacturer’s instructions. Further- results with a few key tests missing in more, some physiological and biochemical identification kits. tube or plate tests (e.g. catalase, oxidase, 175 I. SEDLÁČEK et al. Bacteriocins Bacteriocin production was tested in (1.5%, w/v, solid agar) and a top layer seven strains identified biochemically as (0.7%, w/v, soft agar). As a relatively E. coli (P4652, P4653, P4656, P4657, unenriched medium, a Difco™ nutrient P4664, P4665 and P4733) in parallel using broth (Difco Laboratories, USA) 8 g l-1, four different agar plates containing TY NaCl 5 g l-1, was used for 1.5% (w/v) agar agar, nutrient agar, TY agar supplemented plates. For induction of colicin production, with mitomycin C, and TY agar supple- the base agar layer was supplemented with mented with trypsin (Šmajs et al. 2010). 0.01% (w/v) mitomycin C. To test pro- The rich TY medium consisted of yeast tease sensitivity of the inhibitive agents, extract (Hi-Media) 5 g l-1, tryptone (Hi- 0.1% (w/v) trypsin was added to the base Media) 8 g l-1, and sodium chloride 5 g l-1; layer of agar. the TY agar consisted of a base layer Detection of bacteriocin producers, identification of bacteriocin types and
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