A total of 112 Escherichia coli O149:K91 strains isolated from pigs with diarrhea in Quebec, Canada, between 1978 and 2000 were characterized for their genotypic antimicrobial resistance profiles. Tests for resistance to 10 antimicrobial agents were conducted. Resistance to tetracycline and sulfonamides was found to be the most frequent, but resistance to cefotaxime and ceftiofur was absent. An increase in the number of isolates resistant to at least three antimicrobials was observed over time. The distribution of 28 resistance genes covering six antimicrobial families (beta-lactams, aminoglycosides, phenicols, tetracycline, trimethoprim, and sulfonamides) was assessed by colony hybridization. Significant differences in the distributions of tetracycline [tet(A), tet(B), tet(C)], trimethoprim (dhfrI, dhfrV, dhfrXIII), and sulfonamide (sulI, sulII) resistance genes were observed during the study period (

One approach to the accurate determination of the pathogenic potential (pathotype) of isolated Escherichia coli strains would be through a complete assessment of each strain for the presence of all known E. coli virulence factors. To accomplish this, an E. coli virulence factor DNA microarray composed of 105 DNA PCR amplicons printed on glass slides and arranged in eight subarrays corresponding to different E. coli pathotypes was developed. Fluorescently labeled genomic DNAs from E. coli strains representing known pathotypes were initially hybridized to the virulence gene microarrays for both chip optimization and validation. Hybridization pattern analysis with clinical isolates permitted a rapid assessment of their virulence attributes and determination of the pathogenic group to which they belonged. Virulence factors belonging to two different pathotypes were detected in one human E. coli isolate (strain H87-5406). The microarray was also tested for its ability to distinguish among phylogenetic groups of genes by using gene probes derived from the attaching-and-effacing locus (espA, espB, tir). After hybridization with these probes, we were able to distinguish E. coli strains harboring espA, espB, and tir sequences closely related to the gene sequences of an enterohemorrhagic strain (EDL933), a human enteropathogenic strain (E2348/69), or an animal enteropathogenic strain (RDEC-1). Our results show that the virulence factor microarray is a powerful tool for diagnosis-based studies and that the concept is useful for both gene quantitation and subtyping. Additionally, the multitude of virulence genes present on the microarray should greatly facilitate the detection of virulence genes acquired by horizontal transfer and the identification of emerging pathotypes.Escherichia coli is a normal inhabitant of the intestinal tract of humans and warm-blooded animals. Although usually harmless, various E. coli strains have acquired genetic determinants (virulence genes) rendering them pathogenic for both humans and animals. These pathogens are responsible for three main types of clinical infections: (i) enteric and diarrheal diseases, (ii) urinary tract infections, and (iii) sepsis and meningitis. On the basis of their distinct virulence properties and the clinical symptoms of the host, pathogenic E. coli strains are divided into numerous categories or pathotypes. The diarrheagenic E. coli strains include (i) enterotoxigenic E. coli (ETEC) strains, which are associated with traveler's diarrhea and porcine and bovine diarrhea; (ii) enteropathogenic E. coli (EPEC) strains, which cause diarrhea in children and animals; (iii) enterohemorrhagic E. coli (EHEC) strains, which are associated with hemorrhagic colitis and hemolytic-uremic syndrome in humans; (iv) enteroaggregative E. coli (EAEC) strains, which are associated with persistent diarrhea in humans; and (v) enteroinvasive E. coli (EIEC) strains, which are involved in invasive intestinal infections, watery diarrhea, and dysentery in humans and animals (71). Extraintestina...

Extraintestinal pathogenic Escherichia coli (ExPEC) isolates collected from different infected animals and from human patients with extraintestinal infections in 2001 were characterized for their phenotypic and genotypic antimicrobial resistance profiles, genotypes, and key virulence factors. Among the 10 antimicrobial agents tested, resistance to ampicillin, tetracycline, and sulfonamides was most frequent. Multiresistant strains were found in both the animal and the human groups of isolates. Resistance gene distribution was assessed by colony hybridization. Similar antibiotic resistance patterns could be observed in the animal and the human isolates. Although some resistance genes, such as bla TEM , sulI, and sulII, were equally represented in the animal and human ExPEC isolates, differences in the distributions of tetracycline [tet(D)], chloramphenicol (catI, catIII, and floR), and trimethoprim (dhfrI, dhfrV, dhfrVII, and dhfrXIII) resistance genes were observed between the animal and the human isolates. Approximately one-third of the ExPEC isolates possessed a class 1 integron. The four major different variable regions of the class 1 integron contained aminoglycoside (aadA1, aadA2, aadA5, and aadA6) and/or trimethoprim (dhfrIb, dhfrXII, and dhfrXVII) resistance genes. The ExPEC strains belonged to different phylogenetic groups, depending on their host origin. Strains isolated from animal tissues belonged to either a commensal group (group A or B1) or a virulent group (group B2 or D), while the majority of the human isolates belonged to a virulent group (group B2 or D). Although the limited number of isolates evaluated in the present study prevents firm epidemiological conclusions from being made, on a more global scale, these data demonstrate that extraintestinal isolates of E. coli can possess relatively distinct intra-and intergroup resistance gene profiles, with animal isolates presenting a more heterogeneous group than human isolates.

Escherichia coli is generally described as a commensal species with occasional pathogenic strains. Due to technological limitations, there is currently little information concerning the prevalence of pathogenic E. coli strains in the environment. For the first time, using a DNA microarray capable of detecting all currently described virulence genes and commonly found antimicrobial resistance genes, a survey of environmental E. coli isolates from recreational waters was carried out. A high proportion (29%) of 308 isolates from a beach site in the Great Lakes carried a pathotype set of virulence-related genes, and 14% carried antimicrobial resistance genes, findings consistent with a potential risk for public health. The results also showed that another 8% of the isolates had unusual virulence gene combinations that would be missed by conventional screening. This new application of a DNA microarray to environmental waters will likely have an important impact on public health, epidemiology, and microbial ecology in the future.

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid, highly accurate, and cost-effective method for routine identification of a wide range of microorganisms. We carried out a side by side comparative evaluation of the performance of Bruker Biotyper versus VITEK MS for identification of a large and diverse collection of microorganisms. Most difficult and/or unusual microorganisms, as well as commonly encountered microorganisms were selected, including Gram-positive and negative bacteria, mycobacteria, actinomycetes, yeasts and filamentous fungi. Six hundred forty two strains representing 159 genera and 441 species from clinical specimens previously identified at the Laboratoire de santé publique du Québec (LSPQ) by reference methods were retrospectively chosen for the study. They included 254 Gram-positive bacteria, 167 Gram-negative bacteria, 109 mycobacteria and aerobic actinomycetes and 112 yeasts and moulds. MALDI-TOF MS analyses were performed on both systems according to the manufacturer’s instructions. Of the 642 strains tested, the name of the genus and / or species of 572 strains were referenced in the Bruker database while 406 were present in the VITEK MS IVD database. The Biotyper correctly identified 494 (86.4%) of the strains, while the VITEK MS correctly identified 362 (92.3%) of the strains (excluding 14 mycobacteria that were not tested). Of the 70 strains not present in the Bruker database at the species level, the Biotyper correctly identified 10 (14.3%) to the genus level and 2 (2.9%) to the complex/group level. For 52 (74.2%) strains, we obtained no identification, and an incorrect identification was given for 6 (8.6%) strains. Of the 178 strains not present in the VITEK MS IVD database at the species level (excluding 71 untested mycobacteria and actinomycetes), the VITEK MS correctly identified 12 (6.8%) of the strains each to the genus and to the complex/group level. For 97 (54.5%) strains, no identification was given and for 69 (38.7%) strains, an incorrect identification was obtained. Our study demonstrates that both systems gave a high level (above 85%) of correct identification for a wide range of microorganisms. However, VITEK MS gave more misidentification when the microorganism analysed was not present in the database, compared to Bruker Biotyper. This should be taken into account when this technology is used alone for microorganism identification in a public health laboratory, where isolates received are often difficult to identify and/or unusual microorganisms.

An oligonucleotide microarray detecting 189 Escherichia coli virulence genes or markers and 30 antimicrobial resistance genes was designed and validated using DNA from known reference strains. This microarray was confirmed to be a powerful diagnostic tool for monitoring emerging E. coli pathotypes and antimicrobial resistance, as well as for environmental, epidemiological, and phylogenetic studies including the evaluation of genome plasticity.

Salmonella enterica serovar Heidelberg is the second most frequently occurring serovar in Quebec and the third-most prevalent in Canada. Given that conventional pulsed-field gel electrophoresis (PFGE) subtyping for common Salmonella serovars, such as S. Heidelberg, yields identical subtypes for the majority of isolates recovered, public health laboratories are desperate for new subtyping tools to resolve highly clonal S. Heidelberg strains involved in outbreak events. As PFGE was unable to discriminate isolates from three epidemiologically distinct outbreaks in Quebec, this study was conducted to evaluate whole-genome sequencing (WGS) and phylogenetic analysis as an alternative to conventional subtyping tools. Genomes of 46 isolates from 3 Quebec outbreaks (2012, 2013, and 2014) supported by strong epidemiological evidence were sequenced and analyzed using a highquality core genome single-nucleotide variant (hqSNV) bioinformatics approach (SNV phylogenomics [SNVphyl] pipeline). Outbreaks were indistinguishable by conventional PFGE subtyping, exhibiting the same PFGE pattern (SHEXAI.0001/ SHEBNI.0001). Phylogenetic analysis based on hqSNVs extracted from WGS separated the outbreak isolates into three distinct groups, 100% concordant with the epidemiological data. The minimum and maximum number of hqSNVs between isolates from the same outbreak was 0 and 4, respectively, while >59 hqSNVs were measured between 2 previously indistinguishable outbreaks having the same PFGE and phage type, thus corroborating their distinction as separate unrelated outbreaks. This study demonstrates that despite the previously reported high clonality of this serovar, the WGS-based hqSNV approach is a superior typing method, capable of resolving events that were previously indistinguishable using classic subtyping tools. Nontyphoidal Salmonella enterica strains are important bacterial agents of salmonellosis in humans and animals (1) and represent up to 125,000 cases annually of foodborne gastroenteric disease arising from sporadic and outbreak events in Canada (2). More than 2,500 Salmonella enterica serovars have been described, but only a few have been associated with cases of human illness (3, 4). Salmonella Heidelberg ranks third and fourth among serovars causing human illness in Canada (5) and the United States (6), respectively, and is commonly detected in retail meat samples and food animals. While the majority of Salmonella infections are mild and self-limiting, S. Heidelberg can cause more severe diseases, including septicemia, myocarditis, extraintestinal infections, and death (7,8).Pulsed-field gel electrophoresis (PFGE) is the gold standard method used by Canadian public health laboratories for the molecular typing of S. Heidelberg, following standardized procedures set out by the PulseNet Canada guidelines. A well-recognized limitation of this classic typing method is that strains bearing highly common PFGE patterns occasionally render PFGE ineffective at detecting foodborne outbreaks from background sporadic cases, thus li...

Campylobacter fetus is a venereal pathogen of cattle and sheep, and an opportunistic human pathogen. It is often assumed that C. fetus infection occurs in humans as a zoonosis through food chain transmission. Here we show that mammalian C. fetus consists of distinct evolutionary lineages, primarily associated with either human or bovine hosts. We use whole-genome phylogenetics on 182 strains from 17 countries to provide evidence that C. fetus may have originated in humans around 10,500 years ago and may have “jumped” into cattle during the livestock domestication period. We detect C. fetus genomes in 8% of healthy human fecal metagenomes, where the human-associated lineages are the dominant type (78%). Thus, our work suggests that C. fetus is an unappreciated human intestinal pathobiont likely spread by human to human transmission. This genome-based evolutionary framework will facilitate C. fetus epidemiology research and the development of improved molecular diagnostics and prevention schemes for this neglected pathogen.