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Environmental Isolates of Citrobacter

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Environmental Isolates of Citrobacter Epidemiol. Infect. (2003), 130, 179–186. f 2003 Cambridge University Press DOI: 10.1017/S0950268802008117 Printed in the United Kingdom Environmental isolates of Citrobacter braakii that agglutinate with Escherichia coli O157 antiserum but do not possess the genes responsible for the biosynthesis of O157 somatic antigen A. KHAN 1,R.K.NANDI1,S.C.DAS2, T. RAMAMURTHY1, J. KHANAM3, T. SHIMIZU 4, S. YAMASAKI5, S.K. BHATTACHARYA1,W.CHAICUMPA6, 7 1,8 Y. TAKEDA AND G. BALAKRISH NAIR * 1 National Institute of Cholera and Enteric Diseases, Calcutta, India 2 Indian Veterinary Research Institute, Belgachia, Calcutta, India 3 Jadavpur University, Jadavpur, Calcutta, India 4 Institute of Basic Medical Sciences, University of Tsukuba, Japan 5 Department of Veterinary Sciences, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Japan 6 Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand 7 Faculty of Human Life Sciences, Jissen Women’s University, Tokyo 181-8510, Japan 8 International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) Dhaka, Bangladesh (Accepted 31 October 2002) SUMMARY While searching for Escherichia coli O157 in the aquatic environment of Calcutta using an immunodetection procedure, we fortuitously detected five strains of Citrobacter braakii, which cross-reacted with the commercially available O157 polyvalent antiserum. The five C. braakii isolates gave positive results when a sensitive dot-ELISA was performed with E. coli O157 monoclonal antibody. Further, the O157 monoclonal antibody recognized the bands of proteinase K treated whole cells of lipopolysaccharide of all the C. braakii isolates. Apart from weak reactions with two or three of the DNA probes, all the C. braakii strains did not hybridize with the other probes spanning the minimum region required for O157 O-antigen biosynthesis. These strains did not possess any of the virulence genes that are commonly found in the Shiga toxin-producing E. coli (STEC) specially the serotype O157:H7. Therefore, it appears that the serological cross-reaction between C. braakii and E. coli O157 antiserum is based on structural mimicry between the O-polysaccharide of C. braakii and E. coli O157. INTRODUCTION result have gained extensive publicity due to recent outbreaks [2]. The clinical manifestations of consum- Escherichia coli is a genetically and phenotypically ing foods contaminated with E. coli O157:H7 range diverse species with serotypes normally being ident- from haemorrhagic colitis (HC) to life-threatening ified by their combination of ‘O’ and ‘H’ (and some- systemic complications, including haemolytic uraemic times ‘K’) antigens [1]. Some E. coli, especially strains syndrome (HUS) and thrombotic thrombocytopenic belonging to the serotype O157:H7, are capable of purpura (TTP). Surveillance data indicate that the causing severe and fatal illness in humans and as a epidemiology of enterohaemorrhagic E. coli (EHEC) O157 infection is evolving. The geographic range of occurrence of the organism is growing and there is * Author for correspondence: Laboratory Sciences Division, International Centre for Diarrhoeal Disease Research, Bangladesh evidence of changing patterns of disease transmission (ICDDR,B), Mohakhali, Dhaka-1212, Bangladesh. [3]. Vehicles of Shiga toxin-producing E. coli (STEC) Downloaded from https://www.cambridge.org/core. IP address: 170.106.40.40, on 30 Sep 2021 at 05:13:23, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268802008117 180 A. Khan and others infection continue to be identified, and the massive day, the colonies from the plates (which contained water-borne outbreak, points to environmental con- around 300 colonies) were transferred on to a Hybond taminations as major risk factors [4]. nylon membrane (Amersham, USA). The membranes Though only few strains belonging to the E. coli were subjected to screening for E. coli O157 by an O157 serotype have been isolated in India from spor- antigen based screening method using O157 polyclonal adic cases of diarrhoea [5], these strains have not been antibody (Difco). The colonies on the Hybond mem- characterized in detail and the origin of these isolates brane were denatured by adding 1% SDS and then remain uncertain. In this context, we initiated a com- kept at 70 xC in a water bath. The unbound area of prehensive study to understand the prevalence of E. the membrane was blocked using 3% (w/v) bovine coli O157:H7 and other STEC in Calcutta, India. Our serum albumin phosphate buffered saline (BSA-PBS) study showed STEC strains were present in bovine solution containing 0.002% sodium azide. The mem- sources and in beef samples in Calcutta but were in- brane was then washed with PBS solution and in- frequently isolated from human diarrhoea cases (1.4 cubated with appropriately diluted (1:100) O157 and 0.7% from watery and bloody diarrhoea cases, antiserum (Difco). This was followed by washing the respectively) and therefore were not construed as an membrane with PBS and adding of anti-rabbit per- important enteric pathogen among humans in this part oxidase conjugate (Sigma, USA). The O157 colonies of the world [6]. Further, in this survey, we could not appeared as dark spots when the colour-developing isolate E. coli O157:H7 strains but isolated a variety of reagent, 3,3k-diaminobe iodine (Sigma) was added. STEC isolates belonging to diverse serogroups. In this A human O157:H7 strain harbouring stx1 and stx2 study, we extend our previous investigation to search genes VTEC3 and a K12 E. coli strain of DH5a were for O157 strains of E. coli in the aquatic environs of used as positive and negative controls, respectively. Calcutta using an antibody-based method in an effort Motility of the positive colonies in the antigen-based to understand if there are any reservoirs of the O157 screening method was tested using motility test me- strains of E. coli in the environment. dium (BBL, MD, USA). For E. coli O157 antigen determination, slide agglutination test was performed using commercially available O157 (polyvalent) and H MATERIALS AND METHODS (H2, H4–7, H9, H10–12, H16, H18–21, H27, H28, The present study was conducted in the eastern part H34, H40–42, H45 and H51) antisera (Denka Seiken of the city of Calcutta (longitude 88x 20k E, latitude Co., Japan). For O157 antigen detection, a highly 22x 33k N). Water samples were examined from the sensitive and specific dot-ELISA assay (SDM, Thai- following sites: (i) two fresh-water ponds, located in land) based on monoclonal antibody (Mab) to lipo- the eastern part of the city in Tangra, used by the polysaccharide (LPS) of E. coli O157 was applied population residing in its vicinity for different purposes according to the manufacturer’s instruction. In brief, including washing clothes and bathing, (ii) four fresh- environmental isolates were inoculated in 3 ml LB water ponds located in Topsia and (iii) a fresh-water broth (Difco) and incubated for 6 h at 37 xC tem- lake used by humans but which does not receive any perature under shaking condition. After incubation, industrial effluents. 500 ml of each sample were taken and boiled for The study was carried out over a period of 2 months 20 min. Three microlitres of the boiled sample were from January to February 2001. Samples were col- taken and spotted on the nitrocellulose membrane lected twice every month from all the sites. After col- provided by the manufacturer and air-dried for 5 min. lecting the samples, 100 ml of water samples was After that the membrane was covered with a blocking prefiltered using a filter paper (Grade 3; size 90 mm; solution for 10 min, the membrane was washed three thickness 0.39; Whatman, USA) and then passed times with Buffer A solution and incubated with the through a 0.22 mm membrane (Millipore Corporation, Mab for 30 min. After incubation, the membrane Bedford, MA, USA). The membrane was aseptically was washed three times with Buffer A solution. This transferred to 10 ml Luria Broth (LB) (Difco, USA) was followed by addition of rabbit antimouse im- and incubated at 37 xC overnight with agitation. After munoglobin-biotin conjugate (Dako, Japan) (1:2000 incubation, the enriched broths were serially diluted in dilution), incubated for 20 min and membrane washed 10 ml PBS (pH 7). A 100 ml volume of each dilution twice with Buffer A and once with Buffer B solutions. was spread on Luria Agar (Difco) plates in duplicate Subsequently, the membrane was incubated with a followed by overnight incubation at 37 xC. On the next substrate solution (1:2 dilution) and left in the dark for Downloaded from https://www.cambridge.org/core. IP address: 170.106.40.40, on 30 Sep 2021 at 05:13:23, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0950268802008117 Environmental isolates of Citrobacter braakii 181 5 min. O157 LPS antigen positive samples appeared as as positive and negative control strains, respectively. purplish-blue spots while negative samples appeared Immunochemical detection of O157 antigen in the LPS as spots of brown or other non-specific colour or as preparation of the environmental strains was carried clear areas. out by immunoblotting experiments. LPS separated by The biochemical characterization and identification SDS–PAGE were blotted on to Trans-Blot, Transfer of the environmental isolates, which reacted with the Medium (Bio-Rad) by use of Semi dry Transfer Cell O157 antiserum, were performed using a commer- (Bio-Rad). Following the transfer, the blotted mem- cially available API-20E (BioMe´rieux, France) identifi- brane was sequentially treated with monoclonal anti- cation system, which uses 23 miniaturized biochemical O157 serum, followed by appropriately diluted alka- tests. Interpretation of the results was done by refer- line phosphatase-labelled conjugate and developed ring to the identification table and matching with the with colour-developing solution.
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