Molecular characterization of virulence determinants in Escherichia coli pathotypes isolated from abattoirs

Abstract

Escherichia coli are Gram negative organisms that live as normal flora in the gastrointestinal tract of humans and warm blooded animals. Despite the fact that these organisms were previously considered to be non-pathogenic a number of species are currently known to cause diseases to their hosts worldwide. Infections caused by E. coli are usually associated with the consumption of contaminated food products especially those that are of animal origin. Meat products particularly beef have been implicated as causative agents of food borne disease outbreaks as well as sporadic cases of infections such as diarrhoea caused by E. coli strains in many countries worldwide. Diarrhoeagenic E. coli pathotypes (DEP) are classified into different pathological groups depending on their virulence characteristics and comprise enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), diffusely adherent E. coli (DAEC) and enteroinvasive E. coli (EIEC). These E. coli strains can easily be transmitted to meat if standard operating procedures as well as proper hygiene are not implemented in abattoirs. The main aim of the study was to characterize E. coli pathotypes isolated from beef carcasses in selected abattoirs in North West Province, South Africa. In the present study, a total of 196 swab samples were collected from beef carcasses and analysed using Eosin Methylene Blue agar (EMBA). A total of 291 presumptive E. coli isolates that were metallic sheen based on macroscopic morphologies were obtained from 152 samples. The cellular morphology of presumptive E. coli isolates was determined by Gram-staining and Gram negative rods were subjected to both preliminary (oxidase, carbohydrate metabolising and citrate utilization) and confirmatory (uidA specific PCR analysis) tests. Bacterial 16S rRNA genes were also amplified through PCR as an internal contro l. Confirmed E. coli isolates were further subjected to PCR analysis designed to amplify the stx1 , stx2, eaeA and hlyA virulence gene determinants. The E. coli isolates were further analysed for combinations of the various viru lence genes detected per isolate and a summary of data indicating the different genotypes obtained for iso lates from the different sampling sites was generated. Generally, a large proportion 152 (77 .6%) of the samples were positive for E. coli based on macroscopic morphologies. In addition, large proportions (80.3% to 80.7%) of the samples from Kareesp ruit and Potchefstroom were potentially contaminated with E. coli strains. All the isolates from Kareespruit and Potchefstroom were Gram negative rod- shaped bacteria that were also oxidase negative. In add ition, these isolates also fermented sugars in the TSI medium but did not produce hydrogen sulphide gas. Results for the citrate utilization test indicated that 86%, 57 .3% and 40% of the isolates from Kareespruit, Potchefstroom and Zeerust respectively were citrate negative. Bacterial 16S rRNA gene fragments were successfu lly amplified for all the 291 (100%) isolates. Out of the 29 1 isolates, a large proportion 256 (88 %) possessed the uidA gene which is specific for E. coli. The proportion of isolates that possessed the uidA gene was higher among the isolates from Kareespruit 95 (95%) and Potchefstroom 88 (91.7%) than those from Zeerust 73 (76.8%). Sixteen major genotypes designated G1 to G16 were identified in the study and large proportions (70.7% to 77.7%) of the isolates screened possessed the stx ,, stx2 and eaeA genes respectively. Despite this the stx, was the most freque ntly identified virulence determinant among isolates from Kareespruit 79 (83 .2%) and Zeerust 61 (83.6%) while the stx2 gene was commonly detected 60 (82.2%) among isolates from Zeerust. A large proportion 49 (67 .1 %) of the E. coli isolates from Zee rust also possessed the hlyA gene. On the contrary, eaeA gene that codes for intimin was frequently detected 72 (75 .8%) among isolates from Kareespruit. There was no major difference in the number of isolates obtained in this study that possessed the stx1, stx2 and eaeA genes and only 3 (1.2%) harboured both stx,, and stx2 sh iga toxin genes as shown in genotype (G6). In addition, only 1 (0.4%) isolate that is classified as genotype (G12) harboured all these three genes. Despite this, the number of isolates that possessed the stx1 gene, i.e. 199 (77.7%), was higher than those with the stx2 gene in this study, 6 (2 .3%) of the E. coli isolates screened harboured all the four virulence gene determinants investigated. Only 16 (6 .3%) of the E. coli isolates possessed the stx, and stx2 genes respectively in combin ation with the two accessory viru lence gene determinants (eaeA and hlyA) as indicated in genotypes G14, G15 and G16. ERIC-PCR analysis of the 256 E. coli isolates revealed between· 1 and 17 polymorphic bands per isolate. Isolates produced band sizes that ranged from 100 bp to 1 kb. A large band of approximately 2 kb as well as a smaller band of 400 bp were very common in most of the isolates typed.