Isolation of non-0157 shiga toxin-producing escherichia coli from cattle and goats and detection of their virulence genes

Shigatoxin-producing Escherichia coli (STEC) have emerged as the important food borne pathogen since after the first documented outbreak of O157:H7 STEC in 1982. Clinical symptoms range widely from uncomplicated diarrhea to life threatening hemolytic uremic syndrome (HUS). Shigatoxin 1 and Shigatoxin 2 (Stx1and Stx2) play the major role in causing diseases in humans. Genes coding for attaching and effacing lesion (eae)and enterohemolysin production (ehlyA) also contribute to the potency of STEC in causing the disease. Many of the STEC outbreaks were linked to the consumption of raw or undercooked meat contaminated with STEC. Farm animals, particularly cattle, sheep and goats, serve as major reservoir hosts for human STEC transmission. Although serogroup E. coli O157 is reported as a major strain in causing STEC infections, other serogroups have also been reported in many outbreaks worldwide and they are categorized as non-O157 STEC. Serogroups O8, O26, O91, O103, O111, O113, O128 and O145 are considered as the most common among non-O157 STEC in causing severe human infections. Unlike O157, which can be differentiated easily from other E. colidue to its late fermentation of sorbitol, there is no culture method to differentiate all non-O157 STEC strains from commensal E. coli. It was reported that enterohemolysin (E-Hly) productionis a characteristic feature of many of the human pathogenic non-O157 STEC strains. Studies showed that E-Hly production could be seen on special blood agar made with washed sheep blood erythrocytes. The common E.coliisolation media such as MacConkey agar or Sorbitol MacConkey (SMAC) agar were also suggested for isolation. In Malaysia, both O157 and non-O157 STEC have been reported from hospitalized patients, market beef and meat products. However, report on non-O157 STEC in farm animals is lacking. The aims of the present study were to investigate the presence of non-O157 STEC in cattle and goats, to determine a reliable culture based isolation method for non-O157 STEC for use in routine laboratory diagnosis and to detect the virulence genes of non-O157 STEC isolates. Recto-anal swab samples (RAMS) were collected from 144 cattle from seven farms and 87 goats from four farms during a six-month period. The samples were cultured on two agar media, namely Sorbitol MacConkey (SMAC) agar and washed sheep blood agar (WSBA) and identifedas E. coli using biochemical tests. ConfirmedE. coli isolates were serogrouped against seven monovalent ‘O’ antisera namely, O8, O26, O91, O103, O111, O128 and O145. Colorless colonies from SMAC agar and E-Hly positive colonies from WSBA were tested for O157. Each seropositive isolate was investigated for the presence of Shigatoxin 1 and 2 (Stx1 and Stx2) using Duopath Verotoxins test kit (DV test, Merck, Germany). Isolates from 19 of 144 (13%) cattle and seven of 87 (8%) goats were positive in serogrouping. Using DV test, non-O157 STEC were identified from 71% of cattle farms and 75% of goat farms. Non-O157 STEC serogroups O8, O103 and O128 were isolated from 6.9% of cattle and serogroups O8 and O128 from 4.6% of goats. All non-O157 STEC produced Stx1 alone except one isolate from goat that produced both Stx1 and Stx2. Ninety three percent (93%) of total non-O157 STEC were isolated from SMAC agar and 77% from WSBA. Serogroup O157 were also isolated from two cattle at one farm but failed to produce Stx1/Stx2 on DV test. With multiplex PCR (m-PCR),58% of seropositve isolates from cattle and 71% from goats were found to harbor stx1 with or without other virulence genes.stx2was detected in 46% and 25% of non-O157 STEC isolates from cattle and goats respectively. eaeAwas also found in 46% of cattle and 25% of goat isolates. All non-O157 STEC isolates from goats and 60% non-O157 STEC isolates from cattle were positive for ehlyA. The most common patterns of virulence genes combinations were stx1+ eaeA + ehlyA for cattle and stx1 + ehlyA for goats. O157 isolates were also detected by m-PCR and found to be positive for stx2, eaeA and ehlyA. The present study highlighted the occurrence of common zoonotic serogroups of non-O157 in cattle and goats in Malaysia. Serogroups included in this study were considered as most common human pathogenic strains of non-O157 STEC worldwide. Isolation rates onSMAC agar and WSBA were not statistically significant although SMAC agar gave higher isolation rate.WSBA failed to isolate E-Hly negative strains of non-O157 STEC. This could be due to not all STEC produce E-Hly and all E-Hly producing E. coli are not STEC. There was a strong agreement between DV test and m-PCR in detecting the presence of Shigatoxin. However, isolates that showed stx2 positive in m-PCR failed to produce Stx2 on DV test. It could be probably that Stx2 produced by those strains were below the detection limit of DV test or the strains failed to produce Stx2 although they possessed stx2. In the present study, 93% of non-O157 STEC from cattle and goats possessed more than one virulence genes with 40% harboringstx2 and eaeA which are associated with disease severity in humans. Thus it is of public health significance. For future studies, sampling areas should be extended to other parts of Malaysia to determine the nationwide prevalence of non-O157 STEC. The number of common non-O157 STECserogroups should also be increased as there are more than 100 different serotypes of non-O157 STEC reported worldwide.

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