Simon Lévesque

Macrolide Resistance in Campylobacter jejuni and Campylobacter coli: Molecular Mechanism and Stability of the Resistance Phenotype

ABSTRACT A collection of 23 macrolide-resistant Campylobacter isolates from different geographic areas was investigated to determine the mechanism and stability of macrolide resistance. The isolates were identified as Campylobacter jejuni or Campylobacter coli based on the results of the hippurate biochemical test in addition to five PCR-based genotypic methods. Three point mutations at two positions within the peptidyl transferase region in domain V of the 23S rRNA gene were identified. About 78% of the resistant isolates exhibited an A→G transition at Escherichia coli equivalent base 2059 of the 23S rRNA gene. The isolates possessing this mutation showed a wide range of erythromycin and clarithromycin MICs. Thus, this mutation may incur a greater probability of treatment failure in populations infected by resistant Campylobacter isolates. Another macrolide-associated mutation (A→C transversion), at E. coli equivalent base 2058, was detected in about 13% of the isolates. An A→G transition at a position cognate with E. coli 23S rRNA base 2058, which is homologous to the A2142G mutation commonly described in Helicobacter pylori, was also identified in one of the C. jejuni isolates examined. In the majority of C. jejuni isolates, the mutations in the 23S rRNA gene were homozygous except in two cases where the mutation was found in two of the three copies of the target gene. Natural transformation demonstrated the transfer of the macrolide resistance phenotype from a resistant Campylobacter isolate to a susceptible Campylobacter isolate. Growth rates of the resulting transformants containing A-2058→C or A-2059→G mutations were similar to that of the parental isolate. The erythromycin resistance of six of seven representative isolates was found to be stable after successive subculturing in the absence of erythromycin selection pressure regardless of the resistance level, the position of the mutation, or the number of the mutated copies of the target gene. One C. jejuni isolate showing an A-2058→G mutation, however, reverted to erythromycin and clarithromycin susceptibility after 55 subcultures on erythromycin-free medium. Investigation of ribosomal proteins L4 and L22 by sequence analysis in five representative isolates of C. jejuni and C. coli demonstrated no significant macrolide resistance-associated alterations in either the L4 or the L22 protein that might explain either macrolide resistance or enhancement of the resistance level.

Multilocus Sequence Typing of Campylobacter jejuni Isolates from Humans, Chickens, Raw Milk, and Environmental Water in Quebec, Canada

ABSTRACT Molecular strain typing is essential for deciphering the epidemiology of Campylobacter jejuni infections. We applied two different methods, multilocus sequence typing (MLST) and analysis of the flaA short variable repeat (SVR), to 289 isolates (163 human, 56 chicken, 34 raw milk, and 36 environmental water isolates) collected in the province of Québec, Canada, over 3 years; in addition, the analysis included the pulsed-field gel electrophoresis (PFGE) typing results for a subset of 131 isolates studied previously. MLST defined 96 sequence types (STs) and 20 clonal complexes (CCs), including 49 STs (73 isolates, 25%) and 39 alleles not previously documented in an international database. The frequency of new STs was significantly higher among water isolates than among isolates from other sources (18/36 [50%] and 55/253 [22%], respectively; P < 0.001). Nine of the 10 most prevalent CCs included isolates from humans and at least one other source; five CCs comprised exclusively or mostly human and chicken isolates. However, water and milk were the predominant nonhuman sources among the remaining CCs, suggesting that sporadic C. jejuni infections in humans may frequently arise from sources other than chickens. All three typing systems were discriminatory (discriminatory index > 0.9). Among 131 isolates analyzed by PFGE, each of the 20 types represented by two or more isolates corresponded to a single CC. In contrast, among the 14 most prevalent types detected by analysis of the flaA SVR (5 to 27 isolates each), 8 (57%) included isolates that represented multiple different CCs. The basis for these discordant results was uncertain. Antimicrobial resistance was randomly distributed among the CCs and appeared to be more closely related to the source of an isolate than its genotype. Although MLST is labor-intensive and expensive, it remains the single best method for the genotyping of C. jejuni isolates and deciphering the epidemiologic relationships among isolates.

Campylobacteriosis in urban versus rural areas: a case-case study integrated with molecular typing to validate risk factors and to attribute sources of infection.

Campylobacter infection is a leading cause of bacterial gastroenteritis worldwide, and most clinical cases appear as isolated, sporadic infections for which the source is rarely apparent. From July 2005 to December 2007 we conducted a prospective case-case study of sporadic, domestically-acquired Campylobacter enteritis in rural versus urban areas and a prevalence study of Campylobacter in animal and environmental sources in the Eastern Townships, Quebec. Isolates were typed using Multilocus Sequence Typing (MLST) to reinforce the case-case findings and to assign a source probability estimate for each human isolate. The risk of human campylobacteriosis was 1.89-fold higher in rural than urban areas. Unconditional multivariate logistic regression analysis identified two independent risk factors associated with human Campylobacter infections acquired in rural area: occupational exposure to animals (OR = 10.6, 95% CI: 1.2–91, p = 0.032), and household water coming from a private well (OR = 8.3, 95% CI: 3.4–20.4, p<0.0001). A total of 851 C. jejuni isolates (178 human, 257 chicken, 87 bovine, 266 water, 63 wild bird) were typed using MLST. Among human isolates, the incidence rates of clonal complexes (CC) CC-21, CC-45, and CC-61 were higher in rural than urban areas. MLST-based source attribution analysis indicated that 64.5% of human C. jejuni isolates were attributable to chicken, followed by cattle (25.8%), water (7.4%), and wild birds (2.3%). Chicken was the attributable source for the majority of cases, independent of residential area, sex and age. The increased incidence in rural compared to urban areas was associated with occupational exposure to animals, particularly cattle among those aged 15–34 years, and with consumption of private well water. Both bovine and water exposure appeared to contribute to the seasonal variation in campylobacteriosis. These results provide a basis for developing public education and preventive programs targeting the risk factors identified.

A Side by Side Comparison of Bruker Biotyper and VITEK MS: Utility of MALDI-TOF MS Technology for Microorganism Identification in a Public Health Reference Laboratory

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.

Genomic characterization of a large outbreak of Legionella pneumophila serogroup 1 strains in Quebec City, 2012.

During the summer of 2012, a major Legionella pneumophila serogroup 1 outbreak occurred in Quebec City, Canada, which caused 182 declared cases of Legionnaires disease and included 13 fatalities. Legionella pneumophila serogroup 1 isolates from 23 patients as well as from 32 cooling towers located in the vicinity of the outbreak were recovered for analysis. In addition, 6 isolates from the 1996 Quebec City outbreak and 4 isolates from patients unrelated to both outbreaks were added to allow comparison. We characterized the isolates using pulsed-field gel electrophoresis, sequence-based typing, and whole genome sequencing. The comparison of patients-isolated strains to cooling tower isolates allowed the identification of the tower that was the source of the outbreak. Legionella pneumophila strain Quebec 2012 was identified as a ST-62 by sequence-based typing methodology. Two new Legionellaceae plasmids were found only in the epidemic strain. The LVH type IV secretion system was found in the 2012 outbreak isolates but not in the ones from the 1996 outbreak and only in half of the contemporary human isolates. The epidemic strains replicated more efficiently and were more cytotoxic to human macrophages than the environmental strains tested. At least four Icm/Dot effectors in the epidemic strains were absent in the environmental strains suggesting that some effectors could impact the intracellular replication in human macrophages. Sequence-based typing and pulsed-field gel electrophoresis combined with whole genome sequencing allowed the identification and the analysis of the causative strain including its likely environmental source.

High-Resolution Melting System to Perform Multilocus Sequence Typing of Campylobacter jejuni

Multi-locus sequence typing (MLST) has emerged as the state-of-the-art method for resolving bacterial population genetics but it is expensive and time consuming. We evaluated the potential of high resolution melting (HRM) to identify known MLST alleles of Campylobacter jejuni at reduced cost and time. Each MLST locus was amplified in two or three sub fragments, which were analyzed by HRM. The approach was investigated using 47 C. jejuni isolates, previously characterized by classical MLST, representing isolates from diverse environmental, animal and clinical sources and including the six most prevalent sequence types (ST) and the most frequent alleles. HRM was then applied to a validation set of 84 additional C. jejuni isolates from chickens; 92% of the alleles were resolved in 35 hours of laboratory time and the cost of reagents per isolate was

Ciprofloxacin-resistant Shigella sonnei among men who have sex with men, Canada, 2010.

20 compared with

Thermotolerant Coliforms Are Not a Good Surrogate for Campylobacter spp. in Environmental Water

100 for sequence-based typing. HRM has the potential to complement sequence-based methods for resolving SNPs and to facilitate a wide range of genotyping studies.

vanA-containing Enterococcus faecium susceptible to vancomycin and teicoplanin because of major nucleotide deletions in Tn1546

In 2010, we observed isolates with matching pulsed-field gel electrophoresis patterns from 13 cases of ciprofloxacin-resistant Shigella sonnei in Montréal. We report on the emergence of this resistance type and a study of resistance mechanisms. The investigation suggested local transmission among men who have sex with men associated with sex venues.

Comparison of antimicrobial resistance of Campylobacter jejuni isolated from humans, chickens, raw milk, and environmental water in Québec

ABSTRACT This study aimed to assess the importance of quantitatively detecting Campylobacter spp. in environmental surface water. The prevalence and the quantity of Campylobacter spp., thermotolerant coliforms, and Escherichia coli in 2,471 samples collected weekly, over a 2-year period, from 13 rivers and 12 streams in the Eastern Townships, Québec, Canada, were determined. Overall, 1,071 (43%), 1,481 (60%), and 1,463 (59%) samples were positive for Campylobacter spp., thermotolerant coliforms, and E. coli, respectively. There were weak correlations between the weekly distributions of Campylobacter spp. and thermotolerant coliforms (Spearmans ρ coefficient = 0.27; P = 0.008) and between the quantitative levels of the two classes of organisms (Kendall tau-b correlation coefficient = 0.233; P < 0.0001). Well water samples from the Eastern Townships were also tested. Five (10%) of 53 samples from private surface wells were positive for Campylobacter jejuni, of which only 2 were positive for thermotolerant coliforms. These findings suggest that microbial monitoring of raw water by using only fecal indicator organisms is not sufficient for assessing the occurrence or the load of thermophilic Campylobacter spp. Insights into the role of environmental water as sources for sporadic Campylobacter infection will require genus-specific monitoring techniques.