Shiga Toxin

The outbreak isolate LB226692 and the historic isolates 01-09591 (HUSEC041; serotype O104:H4) were selected for whole genome sequencing. LB226692 originates from a HUS patient from Paderborn (Germany), who was associated with the current outbreak. The stool from this patient was received by the German National Consulting Laboratory for HUS at the Institute of Hygiene, University of Münster, on May 23. LB226692 was isolated on May 24, 2011, using stool enrichment in GN broth (Hajna) followed by plating the enriched cultures on extended spectrum beta-lactamase (ESBL) agar (chromID ESBL, bioMérieux, Nürtingen, Germany) and cefixime-tellurite sorbitol MacConkey agar (CT-SMAC, Oxoid, Wesel, Germany). Analyses of the isolate using PCR on May 24, 2011 detected a stx₂-positive (99% homologous to stx2 sequence of prototypic Shiga toxin- producing E. coli O157:H7 strain EDL933 with one synonymous nucleotide change in each subunit gene) [16] , eae-negative EHEC, that was one day later subtyped as gnd O104, fliC H4 and multilocus sequence typing (MLST) sequence type (ST) 678 [32] (link)-[34] (link). The strain 01-09591 (gnd O104, fliC H4, ST678) was isolated from an HUS patient in Germany in 2001 and is part of the HUSEC collection [9] (link).

The Infectious Diseases and Beliaghata General Hospital (ID&BGH), in Kolkata, a 770 bedded hospital, provides treatment for about 20,000 to 25,000 hospitalized patients with acute diarrhoea annually. In the present systematic active surveillance, every fifth patient with diarrhoea or dysentery without other associated illness on two randomly selected days of the week was enrolled as study subjects from cases admitted at the ID&BGH. This study was conducted between November 2007 and October 2009. The dehydration status of each diarrhoea case was classified as no, some or severe dehydration according to WHO guidelines. The clinical, demographic and laboratory data was checked manually and entered into pre-designed data entry proforma developed in visual basic with inbuilt entry validation checking facilitated programme in structure query language (SQL) server by dual entry method by trained data entry professionals. Data was randomly checked and matched to derive consistency and validity for analysis. The edited data was exported and a final analysis was performed using the SPSS.17.0 software (SPSS Inc., Chicago, IL, USA).
This study was approved by the duly constituted Institutional Ethics Committee (IEC). As per the recommendation of IEC, individual informed consent was obtained from each patient enrolled in this study and confidentiality was maintained. Faecal specimens were collected in McCartney bottles using sterile catheters or as rectal swabs in Cary Blair medium and were examined within 2 hrs for 24 enteric pathogens comprising bacterial, viral and parasitic pathogens using a combination of conventional, immunological and molecular methods (Fig. 6). PCR targeting ompW and toxR were performed for the species confirmation of V. cholerae and V. fluvialis, respectively [31 (link),32 (link)]. Confirmed strains of V. parahaemolyticus, Shigella spp and Salmonella spp were serotyped using commercially available antisera (Denka Seiken, Tokyo, Japan, BioRad, Marnes-la-Coquette, France). V. cholerae strains were serotyped using antisera prepared in NICED. Representative strains of V. cholerae O1 were examined by MAMA-PCR to determine the type of cholera toxin B subunit gene (ctxB) [33 (link)]. Three different lactose-fermenting colonies isolated from each sample were picked from MacConkey agar plate and included in the multiplex PCR assay for the detection of different DEC that include enterotoxigenic E. coli (ETEC, inclusive of both heat-labile and heat-stable enterotoxin producers), enteropathogenic E. coli (typical and atypical EPEC) and enteroaggregative E. coli (EAEC) [34 (link)]. Simplex PCR was also performed for the detection of enteroinvasive E. coli (EIEC) and Shiga toxin-producing E. coli (STEC) [35 (link),36 (link)].
Antimicrobial susceptibility testing was performed by disk diffusion (Kirby- Bauer method) using commercially available disks (Becton Dickinson Co., Sparks, MD, USA) with interpretation stipulated by the Clinical and Laboratory Standard Institute [37 ]. Two hundred and thirty representative (one third from the total number of strains) V. cholerae O1 strains covering all the months and all the Shigella strains were included in the testing. Rotavirus was detected by polyacrylamide gel electrophoresis and silver staining [38 (link)]. Norovirus [Group I and II (NVGI and NVGII)], Sapovirus and Astrovirus were detected by RT-PCR using random primers for reverse transcription and specific primers for polymerase chain reaction [24 (link),39 (link)]. Different viruses were detected according to the appropriate amplicon sizes observed in agarose gels stained with ethidium bromide. Adenovirus was detected by the commercially available RotaAdeno VIKIA kit (biomereux, France), which is a qualitative test-based on immunochromatography in lateral flow format [40 (link)]. For detection of enteric parasites, faecal samples were processed separately for microscopic and molecular analysis. For microscopic analysis, the samples were first concentrated using formalin ethyl acetate concentration method [41 ] and an aliquot of each sample was preserved in 10% formalin and stored at 4°C for subsequent use. Aliquots of fresh stool specimens were also preserved at -80°C for ELISA and PCR assays. All the faecal samples were screened using a highly sensitive antigen capture ELISA (Tech Lab, Blacksburg, USA) and PCR for the detection of Giardia lamblia, Cryptosporidium parvum and Entamoeba histolytica. Faecal samples were processed by microscopy using iodine wet mount staining and trichome staining procedure for Blastocystis hominis [42 ].
Using the surveillance data, an estimate of the total number of cases specific for each pathogen in two consecutive years was extrapolated. From the monthly enrolled cases, the isolation rate of different pathogens was calculated for that particular month. An estimate of total number of cases with particular pathogen for a particular month was then extrapolated by multiplying the total admitted cases with particular isolation rate of the pathogenic with an assumption that similar isolation rate would be among non-enrolled cases. In this way, pathogen-specific total number of yearly estimated cases was calculated.
The risk age group was also explored for predominant enteric pathogens such as V. cholerae O1, Rotavirus, shigellae and G. lamblia by Multinomial Logistic Regression (MLR) analysis [43 (link),44 (link)]. This analysis helps to determine the likelihood age of the patient associated with any enteric pathogen. The age groups were classified into 8 categories viz. <1 year, 1-2 years, >2-5 years, >5-14 years, >14-30 years, >30-45 years, >45-60 years and >60 years and were coded from 1 to 8, respectively. Infection caused by an enteric pathogen was coded as '1' for the pathogen present and '2' for its absence. The extreme values of the classified age group was fixed as a reference category.

Strains were cultured from the feces of infants or children who had acute MSD or who were recruited as healthy controls; tEPEC strains had been identified as described [16 (link)]. Individual colonies were shipped from countries of origin to Baltimore, Maryland, USA and verified by PCR, using primers listed in S1 Table as previously described [9 (link)] for bfpA and escV to identify tEPEC, and stxA1 and stxA2 to identify and exclude Shiga-toxin producing E. coli. PCR using the same conditions and primers also listed in S1 Table was used to detect nleG clusters in a larger collection of 63 NSI and 56 AI unmatched tEPEC and atypical EPEC (aEPEC, bfpA-/escV+/stx-) GEMS isolates.

Each sample (10 g) was enriched at a 1:10 ratio in Enterobacteriaceae enrichment (EE) broth (Becton, Dickinson, Heidelberg, Germany) for 24 h at 37 °C. One loopful of each of the enrichment cultures was cultured on sheep blood agar (Difco™ Columbia Blood Agar Base EH; Becton Dickinson AG, Allschwil, Switzerland) using the streak-plate method. The resulting colonies were suspended in 2 ml 0.85% NaCl. Samples were then screened by real-time PCR for stx1 and stx2 using the Assurance GDS^® for Shiga Toxin Genes (Bio Control Systems, Bellevue, WA, USA).

In this study, we used six serovars of S. enterica, including Enteritidis, Typhimurium, Newport, Heidelberg, Kentucky, and Gallinarum. Out of these six serovars, S. Typhimurium (ATCC LT2) and S. Enteritidis (ATCC13076) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and S. Newport, S. Heidelberg, S. Kentucky, and S. Gallinarum were isolated from animal farms and characterized previously in our laboratory [17 (link)]. In addition, a Shiga toxin-producing enterohemorrhagic Escherichia coli (E. coli) O157: H7 EDL933 (ATCC 700927) was also used (Table 1). All strains were previously preserved in 40% glycerol (v/v) at −80 °C and revived on Luria–Bertani (LB) agar (Becton, Dickinson and Co., Sparks, MD, USA) through incubation at 37 °C overnight.