Michel Cloutier

Distribution of selected virulence genes and antibiotic resistance in Enterococcus species isolated from the South Nation River drainage basin, Ontario, Canada.

Aims:  Isolate and characterize water enterococci from the South Nation River drainage basin, an area dominated by agriculture.

Identification, characterization and description of Arcobacter faecis sp nov., isolated from a human waste septic tank

A study on the taxonomic classification of Arcobacter species was performed on the cultures isolated from various fecal sources where an Arcobacter strain AF1078(T) from human waste septic tank near Ottawa, Ontario, Canada was characterized using a polyphasic approach. Genetic investigations including 16S rRNA, atpA, cpn60, gyrA, gyrB and rpoB gene sequences of strain AF1078(T) are unique in comparison with other arcobacters. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the strain is most closely related to Arcobacter lanthieri and Arcobacter cibarius. Analyses of atpA, cpn60, gyrA, gyrB and rpoB gene sequences suggested that strain AF1078(T) formed a phylogenetic lineage independent of other species in the genus. Whole-genome sequence, DNA-DNA hybridization, fatty acid profile and phenotypic analysis further supported the conclusion that strain AF1078(T) represents a novel Arcobacter species, for which the name Arcobacter faecis sp. nov. is proposed, with type strain AF1078(T) (=LMG 28519(T); CCUG 66484(T)).

Detection of virulence, antibiotic resistance and toxin (VAT) genes in Campylobacter species using newly developed multiplex PCR assays.

Campylobacter species are one of the leading causes of bacterial gastroenteritis in humans worldwide. This twofold study was sought to: i) develop and optimize four single-tube multiplex PCR (mPCR) assays for the detection of six virulence (ciaB, dnaJ, flaA, flaB, pldA and racR), three toxin (cdtA, cdtB and cdtC) and one antibiotic resistance tet(O) genes in thermophilic Campylobacter spp. and ii) apply and evaluate the developed mPCR assays by testing 470 previously identified C. jejuni, C. coli and C. lari isolates from agricultural water. In each mPCR assay, a combination of two or three sets of primer pairs for virulence, antibiotic resistance and toxin (VAT) genes was used and optimized. Assay 1 was developed for the detection of dnaJ, racR and cdtC genes with expected amplification sizes of 720, 584 and 182bp. Assay 2 generated PCR amplicons for tet(O) and cdtA genes of 559 and 370bp. Assay 3 amplified cdtB ciaB, and pldA genes with PCR amplicon sizes of 620, 527 and 385bp. Assay 4 was optimized for flaA and flaB genes that generated PCR amplicons of 855 and 260bp. The primer pairs and optimized PCR protocols did not show interference and/or cross-amplification with each other and generated the expected size of amplification products for each target VAT gene for the C. jejuni ATCC 33291 reference strain. Overall, all ten target VAT genes were detected at a variable frequency in tested isolates of thermophilic Campylobacter spp. where cdtC, flaB, ciaB, cdtB, cdtA and pldA were commonly detected compared to the flaA, racR, dnaJ and tet(O) genes which were detected with less frequency. The developed mPCR assays are simple, rapid, reliable and sensitive tools for simultaneously assessing potential pathogenicity and antibiotic resistance profiling in thermophilic Campylobacter spp. The mPCR assays will be useful in diagnostic and analytical settings for routine screening of VAT characteristics of Campylobacter spp. as well as being applicable in epidemiological studies by providing information that could be related to the risk of human infection.

Development and evaluation of multiplex PCR assays for rapid detection of virulence-associated genes in Arcobacter species.

As the pathogenicity of Arcobacter species might be associated with various virulence factors, this study was aimed to develop and optimize three single-tube multiplex PCR (mPCR) assays that can efficiently detect multiple virulence-associated genes (VAGs) in Arcobacter spp. including the Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii, respectively. The recognized target virulence factors used in the study were fibronectin binding protein (cj1349), filamentous hemagglutinin (hecA), hemolysin activation protein (hecB), hemolysin (tlyA), integral membrane protein virulence factor (mviN), invasin (ciaB), outer membrane protein (irgA) and phospholipase (pldA). Identical results were obtained between singleplex PCR and mPCR assays and no cross- and/or non-specific amplification products were obtained when tested against other closely related bacterial species. The sensitivities of these three mPCR assays were ranging from 1ngμL(-1) to 100ngμL(-1) DNA. The developed assays with combinations of duplex or triplex PCR primer pairs of VAGs were further evaluated and validated by applying them to isolates of the A. butzleri, A. cryaerophilus and A. skirrowii recovered from fecal samples of human and animal origins. The findings revealed that the distribution of the ciaB (90%), mviN (70%), tlyA (50%) and pldA (45%) genes among these target species was significantly higher than the hecA (16%), hecB (10%) and each of irgA and cj1349 (6%) genes, respectively. The newly developed mPCR assays can be used as rapid technique and useful markers for the detection, prevalence and profiling of VAGs in the Arcobacter spp. Moreover, these assays can easily be performed with a high throughput to give a presumptive identification of the causal pathogen in epidemiological investigation of human infections.

Draft Genome Sequences of Three Arcobacter Strains of Pig and Dairy Cattle Manure Origin

ABSTRACT The genus Arcobacter has been associated with human illness and fecal contamination by humans and animals. Here, we announce the draft genome sequences of three strains of Arcobacter species cultured from pig and dairy cattle manure tanks. This information will assist in the characterization of features related to host specificities and identify potential pathogenic health risks to humans and animals.

Draft Genome Sequence of Arcobacter cibarius Strain LMG21996T, Isolated from Broiler Carcasses

ABSTRACT The draft genome sequence of Arcobacter cibarius strain LMG21996T, isolated from chicken carcasses, is reported here. The draft genome consists of 2.2 Mbp, with a 27.12% G+C content. A total of 2,179 protein-coding genes, 46 tRNA genes, and 15 rRNAs have been identified and annotated.

Draft genome sequences of two arcobacter strains isolated from human feces.

ABSTRACT Arcobacter species are members of the family Campylobacteraceae and are considered emerging enteropathogens and potential zoonotic agents. Here, we report the draft genome sequences of two Arcobacter strains isolated from human feces in an effort to provide further genetic resources for understanding the pathogenic dynamics and diversity of this important genus.

Enhanced Single-tube Multiplex PCR Assay for Detection and Identification of Six Arcobacter Species

A single‐tube multiplex PCR (mPCR) assay was developed for rapid, sensitive and simultaneous detection and identification of six Arcobacter species including two new species, A. lanthieri and A. faecis, along with A. butzleri, A. cibarius, A. cryaerophilus and A. skirrowii on the basis of differences in the lengths of their PCR products. Previously designed monoplex, mPCR and RFLP assays do not detect or differentiate A. faecis and A. lanthieri from other closely related known Arcobacter spp.

Quantitative real-time PCR-based assessment of tile drainage management influences on bacterial pathogens in tile drainage and groundwater

This study compared the impact of controlled tile drainage (CD) and freely draining (FD) systems on the prevalence and quantitative real-time PCR-based enumeration of four major pathogens including Arcobacter butzleri, Campylobacter jejuni, Campylobacter coli, and Helicobacter pylori in tile- and groundwater following a fall liquid swine manure (LSM) application on clay loam field plots. Although the prevalence of all target pathogens were detected in CD and FD systems, the loads of A. butzleri, C. jejuni, and C. coli were significantly lower in CD tile-water (p<0.05), in relation to FD tile-water. However, concentrations of A. butzleri were significantly greater in CD than FD tile-water (p<0.05). In shallow groundwater (1.2m depth), concentrations of A. butzleri, C. coli, and H. pylori showed no significant difference between CD and FD plots, while C. jejuni concentrations were significantly higher in FD plots (p<0.05). No impact of CD on the H. pylori was observed since quantitative detection in tile- and groundwater was scarce. Although speculative, H. pylori occurrence may have been related to the application of municipal biosolids four years prior to the LSM experiment. Overall, CD can be used to help minimize off-field export of pathogens into surface waters following manure applications to land, thereby reducing waterborne pathogen exposure risks to humans.