Molecular diagnostic and epidemiological investigations of Shiga toxin-producing Escherichia coli and Campylobacter jejuni in dairy cattle.

Abstract

Foodborne illness is a significant public health concern, contributing to 25 to 30% of gastroenteritis. Among bacterial causes, Shiga toxin-producing Escherichia coli (STEC) is an important enteric pathogens that causes bloody diarrhea, hemorrhagic colitis, or hemolytic uremic syndrome which occasionally leads to death at high rate. E.coli O157, the most notorious of the STEC strain, and hundreds of STEC non-O157 strains are associated with both endemic and epidemic infection. Campylobacter is also recognized as the leading cause of diarrheal disease especially in developed countries. In order to minimize human infections, rapid detection and characterization, especially for the virulence potentials of STEC and Campylobacter jejuni are needed. Cattle are a natural reservoir of STEC and have recently been recognized as a major source of C. jejuni contamination. In this study, molecular diagnostic and epidemiological investigations were performed for the STEC and C. jejuni strains isolated from cattle farm samples, a primary source of infection. First, loop-mediated isothermal amplification (LAMP) assay, the newest and the most advanced method for amplification of nucleic acid, was developed for rapid and sensitive detection of STEC and C. jejuni. Next, the prevalence and virulence potentials of STEC in cattle were investigated. Finally, the influence of microbiota on shedding STEC or C. jejuni was investigated. The first study as discussed about the development of real-time LAMP assay for detection of C. jejuni targeting hipO. The developed LAMP assay was specific (100% inclusivity and exclusivity for 84 C. jejuni and 41 non-C. jejuni strains, respectively), sensitive (detection limit of 100 fg/μl), and quantifiable (R2 = 0.9133). When applied the LAMP assay on all C. jejuni strains (n = 51) isolated from cattle farm during 2012 to 2013, the hipO gene was successfully amplified within 30 min (mean = 10.8 min) demonstrating the accuracy and rapidity. In addition, the LAMP assay was applied to the enriched broth cultures of the naturally-contaminated cattle farms samples. In the comparison between LAMP and PCR, the higher sensitivity of the LAMP assay (84.4% vs. 35.5%) was observed in boiled DNA samples, indicating less susceptibility of LAMP assay to the existence of inhibitors in sample material. The detection of this organism in cattle and their environment is important for the control of C. jejuni transmission and the prevention of Campylobacteriosis. However, isolation of C. jejuni using culture method is difficult due to its high oxygen sensitivity and difficulty in reading typical phenotypes on media. The use of LAMP assay together with culture method would enhance identifying and screening of C. jejuni in cattle farm samples, and play an important role in the prevention of C. jejuni contamination in the food chain, thereby reducing the future risk of human Campylobacteriosis. The second study was also focused on the development of LAMP for detection of STEC targeting stx genes. Whilst LAMP assay was accepted as a novel nucleic acid amplification method, the application of LAMP assay to several genes was limited due to formation of cauliflower-like amplified product. In this study, multiplex LAMP assay was developed targeting stx1 and stx2 genes of STEC. The mLAMP was able to distinguish two target genes based on different Tm values (85.03 ± 0.54°C for stx1 and 87.47 ± 0.35°C for stx2). The mLAMP was highly specific (100% inclusivity and exclusivity), sensitive (with a detection limit as low as 10 fg/μL), and quantifiable (R2 = 0.9313). In addition, the mLAMP assay was able to type shiga toxin types of a total of 12 (12/253

4.7%) and 17 (17/253

6.7%) STEC O157, and 11 (11/236

4.7%) STEC non-O157 strains, which were isolated from cattle farm samples by conventional selective culture, immunomagnetic separation, and PCR-based culture methods, respectively. Due to lack of characteristic phenotype of STEC non-O157, it is hard to isolate STEC non-O157 by culture method, thus a molecular based detection method is often accompanied. Application of mLAMP assay would be helpful not only to detect STEC but also to identify its shiga toxin type simultaneously. Furthermore, the high detection rate of specific genes from enriched broth samples indicates the potential utility of this assay as a screening method for detecting STEC in cattle farm samples. The third study was discussed about the prevalence and the virulence potentials of STEC in cattle farm samples. In total, 63 STEC were isolated from 496 cattle farm samples, and temperature and rainfall affected STEC prevalence (p < 0.001). The O157 serogroup was most prevalent, followed by O108, O8, O84, O15, and O119. In the stx variant test, high prevalence of stx2a and stx2c (known to be associated with high STEC virulence) were observed, and stx2g, a bovine STEC variant, was detected in STEC O15 and O109. Additionally, stx1c was detected in eae-positive STEC, suggesting genetic dynamics among the virulence genes in the STEC isolates. STEC non-O157 strains were resistant to tetracycline (7.9%), ampicillin (6.4%), and cefotaxime (1.6%), while STEC O157 was susceptible to all tested antimicrobials, except cefotaxime. The antimicrobial resistance genes, blaTEM (17.5%), tetB (6.3%), and tetC (4.8%), were only detected in STEC non-O157, whereas tetE (54.0%) was detected in STEC O157. AmpC was detected in all STEC isolates. Clustering was performed based on the virulence gene profiles, which grouped STEC O84, O108, O111, and O157 together as potentially pathogenic STEC strains. Finally, PFGE suggested the presence of a prototype STEC that continues to evolve by genetic mutation and causes within- and between-farm transmission within the Gyeonggi province. The final study was dealt with the bovine gut microbiota in relation to the presence of the foodborne zoonotic pathogens, STEC and C. jejuni. Recent studies have focused on the shedding lebel of fooborne pathogens. The host shedding pathogens at high levels called high-shedder or super-shedder, which is thought to be related to the high prevalence or incidence of the pathogens. In this study, the fecal microbiota of dairy cattle (n = 24) was investigated using next-generation sequencing to identify the microbial impact on shedding STEC or C. jejuni. The core microbiota (9 phyla, 13 classes, 18 orders, 47 families, 148 genera, and 261 species) was identified, which covered 80.0~100.0% of the fecal microbial community. The presence of STEC had a minor effect on alpha-diversity and relative abundance (RA) of taxa, identifying 2 and 3 genera that were significantly higher and lower, respectively, in STEC shedding cattle. On the other hand, a high diversity index was observed in C. jejuni-positive samples. In addition, C. jejuni-positive cattle had a higher RA of Bacteroidetes (p = 0.035) and a lower RA of Firmicutes (p = 0.035) compared to C. jejuni-negative cattle. In genus level, the RA of 6 and 3 genera were significantly higher and lower, respectively, in cattle shedding C. jejuni. While diverse microbial communities were observed between cattle shedding foodborne pathogens and non-shedding cattle, these differences had a minor influence on the overall microbial community. Rapid, and sensitive detection of STEC and C. jejuni at cattle farm is especially important in terms of the initial stage of the food chain, which prevent further contamination along the food production line. With the use of developed LAMP assay in this study, effective control and prevention would be possible. In addition, considerable numbers of STEC non-O157 were isolated from cattle farms, and the virulence and antimicrobial resistance features were different between the STEC O157 and non-O157 strains. STEC from cattle with virulence or antimicrobial resistance genes might represent a threat to public health and therefore, continual surveillance of both STEC O157 and non-O157 would be beneficial for controlling and preventing STEC-related illness. Finally, diverse microbial community was observed in cattle groups shedding STEC or C. jejuni, implying the interaction between indigenous bacteria and foodborne pathoges. While several factors are known to be associated with bacterial colonization, the underlying microbial factors have not been clarified. Microbiota is thought to be one of the important animal factor causing super-shedding, because microbiota is closely reltated to survival and colonization of STEC and C. jejuni as a niche for bacterial pathogens. These finding would provide fundamental information on bacterial ecology in cattle feces, and would be useful to develop strategies for controlling bacterial shedding.