Influence of Conserved and Hypervariable Genetic Markers on Genotyping Circulating Strains of Neisseria gonorrhoeae

Presently there is no vaccine against Neisseria gonorrhoeae and therefore accurate information on gonococcal transmission plays a crucial role for interventions designed to limit the spread of infections caused by this microorganism. We evaluated the impact of two different categories of genetic markers, (i) concatenated sequences of 10 housekeeping genes and (ii) hypervariable porB DNA sequences, on the genetic relatedness and subsequently on genotyping analysis of this human pathogen. Eighty gonococcal isolates from Canada, China, the US, Argentina, Venezuela and Chile, collected over different times, were analyzed. Our results show that the choice of genetic marker had a profound effect on the interpretation of genotyping results associated with N. gonorrhoeae. The concatenated sequences of the housekeeping genes preserved the genetic relatedness of closely related isolates, enabling detection of the predominant strains circulating within a community (Saskatchewan, Canada) over an extended period of time. In contrast, a genetic marker based on antigen gene, porB, may lead to a failure to detect these predominant circulating strains. Based on the analysis of the DNA sequences of the 10 housekeeping genes, we identified two major clonal complexes, CC33 and CC22, which comprised STs from China, and Argentina as well as two STs from Canada. Several minor clonal complexes were observed among isolates from Saskatchewan. eBURST analysis suggested that the majority of the tested gonococcal isolates from Saskatchewan, Canada were endemic, with only a couple of genotypes introduced

Pertactin-negative Bordetella pertussis strains in Canada: characterization of a dozen isolates based on a survey of 224 samples collected in different parts of the country over the last 20 years

SummaryObjectiveTo detect and characterize pertactin-negative Bordetella pertussis in Canada, especially for isolates collected in recent years.MethodsA total of 224 isolates from the years 1994–2013 were screened by Western immuno-blot for expression of pertactin. Pertactin-negative isolates were characterized by serotyping, pulsed-field gel electrophoresis (PFGE), and genotyping of their pertactin, fimbriae 3, pertussis toxin subunit 1, and pertussis toxin gene promoter region, as well as the complete sequence of the pertactin gene.ResultsTwelve isolates were pertactin-negative, giving an overall prevalence of 5.4%. However, no such isolate was found prior to 2011 and 17.8% of 62 isolates examined in 2012 were pertactin-negative. Ten pertactin-negative isolates contained a significant mutation in their pertactin (prn) genes. IS481 was found in the prn genes of eight isolates, while a single point mutation occurred either in the coding region (resulting in a premature stop codon) or in the promoter region (preventing gene transcription) in two other isolates. PFGE analysis also showed multiple profiles suggesting that several independent genetic events might have led to the emergence of these pertactin-negative strains rather than expansion of a single clone.ConclusionsAs reported elsewhere, pertactin-negative B. pertussis has emerged in Canada in recent years, notably in 2012. This coincided with an increase in pertussis activity in Canada. A further systematic study with a larger geographical representative sample is required to determine how these vaccine-negative strains may contribute to the overall changing epidemiology of pertussis in Canada

Ceftiofur Resistance in Salmonella enterica Serovar Heidelberg from Chicken Meat and Humans, Canada

Use of this drug in chickens may limit effectiveness of cephalosporins in treating human infections

Rapid Genotyping of Hepatitis C Virus by Primer-Specific Extension Analysis

Quick and accurate genotyping of hepatitis C virus (HCV) is becoming increasingly important for clinical management of chronic infection and as an epidemiological marker. Furthermore, the incidence of HCV infection with mixed genotypes has clinical significance that is not addressed by most genotyping methods. We have developed a fluorescence-based genotyping assay called primer-specific extension analysis (PSEA) for the most prevalent HCV genotypes and have demonstrated the capacity of PSEA-HCV for detecting mixed-genotype HCV infections. PSEA-HCV detects genotype-specific sequence differences in the 5′ untranslated region of HCV in products amplified by the COBAS AMPLICOR HCV Test, v2.0. Simulated mixed HCV infection of plasma with RNase-resistant RNA controls demonstrates that PSEA-HCV can detect as many as five genotypes in one specimen. Furthermore, in dual-genotype simulations, PSEA-HCV can unequivocally detect both genotypes, with one genotype representing only 3.1% of the mixture (313/10,000 IU in starting plasma). Compared to INNO-LiPA HCV II, both assays determined the same genotype for 191/199 (96%) patient specimens (175 subtype and 16 genotype-only identifications). Following the initial evaluation, PSEA-HCV was used routinely to genotype HCV from patient specimens submitted to our laboratory (n = 312). Seventeen (5.4%) mixed infections were identified. The distribution of single-infection HCV genotypes in our population was 60.9% type 1 (n = 190), 12.8% type 2 (n = 40), 20.2% type 3 (n = 63), 0.3% type 4 (n = 1), and 0.3% other (n = 1). In conclusion, PSEA-HCV provides an inexpensive, high-throughput screening tool for rapid genotyping of HCV while reliably identifying mixed HCV infections

Development of a Triplex Real-Time PCR Assay for Detection of Panton-Valentine Leukocidin Toxin Genes in Clinical Isolates of Methicillin-Resistant Staphylococcus aureus

Community-associated methicillin-resistant Staphylococcus aureus harboring Panton-Valentine leukocidin (PVL) genes is an emerging pathogen. A novel real-time PCR assay for identification of MRSA isolates containing PVL was developed. The PVL assay was used in a triplex format allowing simultaneous amplification of mecA, nuc, and PVL genes in 614 clinical isolates. This assay facilitates the rapid identification of PVL-positive isolates of MRSA

Phylogenetic analysis of emergent Streptococcus pneumoniae serotype 22F causing invasive pneumococcal disease using whole genome sequencing.

Since implementation of the 13-valent polyvalent conjugate vaccine (PCV13) in Canada during 2010, the proportion of PCV13 serotypes causing invasive pneumococcal disease (IPD) has declined from 55% (n = 1492) in 2010 to 31% (n = 764) in 2014. A concurrent increase of non-PCV13 serotypes has occurred and 22F has become the most prevalent serotype in Canada increasing from 7% (n = 183) to 11% (n = 283). Core single nucleotide variant phylogenetic analysis was performed on 137 Streptococcus pneumoniae serotype 22F isolates collected across Canada from 2005-2015. Six phylogenetic lineages (n = 117) were identified among a serotype 22F/ST433 clonal complex (CC), including a recently expanding erythromycin-resistant clone. Erythromycin-resistance was observed in 25 isolates possessing ermB, mef or a 23S rRNA A2061G point mutation; 2 penicillin-resistant isolates had recombinant pbp1a, pbp2a and/or pbp2x; 3 tetracycline-resistant isolates contained tetM; and 1 isolate was multidrug-resistant. Virulence factor analysis indicated a high level of homogeneity among the 22F/ST433 clonal complex strains. A group of 6 phylogenetic outlier strains had differing MLST, antimicrobial resistance and molecular profiles suggestive of capsule switching events. While capsule switch events among S. pneumoniae serotype 22F has been observed, increasing prevalence of S. pneumoniae serotype 22F can be attributed to an evolving homogenous clone expanding nationally through local transmission events

Epidemic of Invasive Pneumococcal Disease, Western Canada, 2005–2009

A single clone of Streptococcus pneumoniae serotype 5 caused this epidemic