Development of three multiplex-PCR assays for virulence profiling of different iron acquisition systems in Escherichia coli

Background and Objectives: Escherichia coli is responsible for various enteric and extraintestinal infections in animals and humans. Iron as an essential nutrient, has a proven role in pathogenicity of E. coli. Pathogenic E. coli benefits of having complicated systems for iron acquisition but our current knowledge is limited because of complexity of these systems. In the present study, three multiplex-PCR assays were developed to screen nine different virulence genes related to diverse iron acquisition systems in E. coli. Materials and Methods: The multiplex-PCR systems were designed and optimized in three panels. Each panel includes a triplex-PCR cocktail. The panels are as follow: panel 1: iroN, iutA and fecA; panel 2: fyuA, sitA and irp2; and panel 3: iucD, chuA and tonB. A total of 39 pathogenic E. coli was screened according to the designed multiplex-PCR. Results: In total, the top three frequent genes were tonB (100%), fecA (66.6%) and sitA (58.9%). With the exception of fecA and tonB, comparing the prevalence of genes among different origin of isolates (human, cattle, poultry and pigeon) showed significant associations (P < 0.05). Moreover, the iroN, sitA and iucD genes were significantly prevalent (P < 0.05) among members of extraintestinal pathogenic E. coli in comparison with the group of diarrheagenic E. coli. Conclusion: The current multiplex-PCR assays could be a valuable, rapid and economic tool to investigate diverse iron acquisition systems in E. coli for more precise virulence typing of pathogenic or commensal strains.

Virulence typing and antibiotic susceptibility profiling of thermophilic Campylobacters isolated from poultry, animal, and human species

Background and Aim: Campylobacteriosis finds its place among the four important global foodborne illnesses. The disease, though self-limiting, needs antibacterial therapy in extraintestinal complications. Therefore, the present study was designed to estimate the prevalence of thermophilic Campylobacters in poultry, animals, and humans of the Kumaon region of Uttarakhand. Materials and Methods: A total of 609 samples comprising of poultry ceca (n=116), poultry droppings (n=203), and feces of pigs (n=71), cattle (n=61), sheep (n=19), goat (n=17), human beings (n=88), and laboratory animals (n=34) (rats, rabbits, and guinea pigs) were collected. The thermophilic Campylobacters, Campylobacter jejuni and Campylobacter coli were confirmed using multiplex polymerase chain reaction. The isolates were also screened for the presence of virulence genes, and their antibiotic susceptibility testing was done against eight antibiotics. Results: An overall prevalence of 6.24% was revealed with highest from poultry ceca (15.52%), followed by poultry droppings (5.91%), cattle feces (4.92%), human stools (3.40%), and pig feces (2.82%). The virulence genes, namely cadF, flaA, virB11, and pldA, were present in 38 (100%), 37 (97.37%), 7 (18.42%), and 14 (36.84%) isolates, respectively. All the isolates were resistant to nalidixic acid, while all were sensitive to erythromycin and co-trimoxazole. Conclusion: It was concluded that the animals and humans in the region harbored the thermophilic Campylobacters which may contribute to the human illness. Resistance shown among the isolates may complicate the antimicrobial therapy.

Risk determinants for the development of typical haemolytic uremic syndrome in Belgium and proposition of a new virulence typing algorithm for Shiga toxin-producing Escherichia coli

In Belgium, it is mandatory to report Shiga toxin-producing Escherichia coli (STEC) infections to the health inspection authorities. To facilitate the decision making regarding infection control measures, information about the risk factors for the development of the haemolytic uremic syndrome (HUS) can be helpful. We performed statistical analyses on a dataset of 411 Belgian STEC strains. Demographic and clinical patient characteristics as well as phenotypical and genotypical STEC strain characteristics were taken into account. Multivariate logistic regression models indicated that age categories ⩽5, 6–12 and ⩾75; the stx2 gene; and the eae gene were significant HUS development risk determinants. The stx2a subtype had the highest risk (OR 29.6, 95% CI 7.0–125.1), while all stx1 subtypes encompassed a significant lower risk (OR 0.3, 95% CI 0.1–0.5). Presence of the stx1 gene without stx2 encompassed a lower risk than the combined presence of stx1 and stx2, or stx2 solely. Based on these results, we propose a new virulence typing algorithm that will enable the National Reference Centre to provide the physicians and health inspection authorities with a risk classification for the development of HUS. We believe this will contribute to a more efficient STEC infection control management in Belgium.

Frequency of virulence genes and antibiotic resistances inEnterococcusspp. isolates from wastewater and feces of domesticated mammals and birds, and wildlife

Enterococci are gastrointestinal tract residents and also an important cause of nosocomial infections. To understand which species, virulence determinants, and antibiotic resistances are prevalent in enterococci shed by various hosts groups, a total of 1460 strains isolated from 144 fecal samples obtained from wastewater, domesticated mammals and birds, and wildlife were characterized. Identification of isolates to the species level showed that Enterococcus faecalis was dominant in domesticated mammals and birds and wildlife feces, whereas Enterococcus faecium was dominant among wastewater isolates, and that no single Enterococcus species could be associated with a specific host group. The frequency of 12 virulence determinants was evaluated among isolates, but no single virulence determinant could be associated with a specific host group. Resistance to 12 antibiotics was evaluated among isolates, and it was shown that the highest frequency of resistance at breakpoint concentration was found in domesticated mammals and birds (P ≤ 0.05 for 4 antibiotics). Our results suggests that (1) species identification and virulence typing of Enterococcus spp. isolates are not useful for the identification of the host groups responsible for fecal contamination of water by microbial source tracking and that (2) antibiotic use for clinical, veterinary, or animal husbandry practices is promoting resistance.