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Mass spectrometry-based phenotypic H-antigen typing (MS-H) combined with whole-genome-sequencing-based genetic identification of H antigens, O antigens, and toxins (WGS-HOT) was used to type 60 clinical Escherichia coli isolates, 43 of which were previously identified as nonmotile, H type undetermined, or O rough by serotyping or having shown discordant MS-H and serotyping results. Whole-genome sequencing confirmed that MS-H was able to provide more accurate data regarding H antigen expression than serotyping. Further, enhanced and more confident O antigen identification resulted from gene cluster based typing in combination with conventional typing based on the gene pair comprising wzx and wzy and that comprising wzm and wzt . The O antigen was identified in 94.6% of the isolates when the two genetic O typing approaches (gene pair and gene cluster) were used in conjunction, in comparison to 78.6% when the gene pair database was used alone. In addition, 98.2% of the isolates showed the existence of genes for various toxins and/or virulence factors, among which verotoxins (Shiga toxin 1 and/or Shiga toxin 2) were 100% concordant with conventional PCR based testing results. With more applications of mass spectrometry and whole-genome sequencing in clinical microbiology laboratories, this combined phenotypic and genetic typing platform (MS-H plus WGS-HOT) should be ideal for pathogenic E. coli typing.

We developed a real-time PCR assay to detect single nucleotide polymorphisms associated with ciprofloxacin resistance in specimens submitted for nucleic acid amplification testing (NAAT). All three single nucleotide polymorphism (SNP) targets produced high sensitivity and specificity values. The presence of ≥2 SNPs was sufficient to predict ciprofloxacin resistance in an organism.

A population-based study combining (i) antimicrobial, (ii) genetic, and (iii) virulence analyses with molecular evolutionary analyses revealed segregative characteristics distinguishing human clinical and bovine Escherichia coli O157 strains from western Canada. Human ( n = 50) and bovine ( n = 50) strains of E. coli O157 were collected from Saskatchewan and Manitoba in 2006 and were analyzed by using the six-marker lineage-specific polymorphism assay (LSPA6), antimicrobial susceptibility analysis, the colicin assay, plasmid and virulence profiling including the eae , ehxA , espA , iha , stx 1 , stx 2 , stx 2c , stx 2d , stx 2d-activatable , stx 2e , and stx 2f virulence-associated genes, and structure analyses. Multivariate logistic regression and Fisher's exact test strongly suggested that antimicrobial susceptibility was the most distinctive characteristic ( P = 0.00487) associated with human strains. Among all genetic, virulence, and antimicrobial determinants, resistance to tetracycline ( P < 0.000) and to sulfisoxazole ( P < 0.009) were the most strongly associated segregative characteristics of bovine E. coli O157 strains. Among 11 virulence-associated genes, stx 2c showed the strongest association with E. coli O157 strains of bovine origin. LSPA6 genotyping showed the dominance of the lineage I genotype among clinical (90%) and bovine (70%) strains, indicating the importance of lineage I in O157 epidemiology and ecology. Population structure analysis revealed that the more-diverse bovine strains came from a unique group of strains characterized by a high degree of antimicrobial resistance and high frequencies of lineage II genotypes and stx 2c variants. These findings imply that antimicrobial resistance generated among bovine strains of E. coli O157 has a large impact on the population of this human pathogen.

Fifty-two Haemophilus influenzae isolates from patients with invasive disease in the province of Manitoba, Canada, were examined for serotype, biotype, genotype, and antibiotic susceptibility. Half of the 52 isolates were found to be serotype a, and 38.5% (20 isolates) were found to be nonserotypeable (NST). There were only three serotype b strains and one each for serotypes c, d, and f. All 26 serotype a isolates belonged to biotype II and demonstrated identical or highly similar DNA fingerprints by pulsed-field gel electrophoresis. An analysis of these isolates by multilocus sequence typing showed that they belong to the clonal complex ST-23. While 69% (18 of 26) of the serotype a cases were found in males, only 9 (45%) of the 20 patients with NST isolates were males. Twenty (77%) of the 26 serotype a isolates were from patients who were ≤24 months old. Twelve (63%) of the NST isolates were from adult or adolescent patients. In contrast to the clonal nature of serotype a isolates, the 20 NST isolates were found to belong to 18 different sequence types. Most of these 18 different sequence types were unrelated to each other, with the exception of 7 sequence types grouped into three clonal groups. Two (6.25%) out of 32 serotypeable isolates (1 serotype a and 1 serotype b) and 6 (30%) of 20 NST isolates were resistant to ampicillin due to β-lactamase production. These results suggest a change in the epidemiology of H. influenzae disease, with the majority of invasive H. influenzae isolates being associated with serotype a and NST strains.

Recently, acquisition of methicillin-resistant Staphylococcus aureus (MRSA) has been increasingly seen in community settings. Beginning in 1995, we have routinely conducted pulsed-field gel electrophoresis (PFGE) of MRSA isolates received at Cadham Provincial Laboratory (CPL) in Manitoba, Canada. Our diverse collection of isolates coupled with molecular subtype information allowed us to assess the extent to which MRSA isolates in general were associated with community acquisition and whether specific PFGE types were more likely to be found in community settings. Forty percent of the MRSA isolates in our analysis were designated community associated (CA), with two of the six most common PFGE types showing a greater likelihood to be CA-MRSA. Overall, CA-MRSA were more likely to show multiple sensitivity to antibiotics and to be associated with younger age groups. Mapping of specific CA-MRSA types over successive 5-year periods showed rapid temporal shifts in prevalence in different parts of the province.

The kdsA and kdsB genes from Chlamydia trachomatis encoding 3-deoxy-D-manno-octulosonate (KDO)-8-phosphate synthetase and CMP-KDO synthetase were identified by functional complementation of temperature-sensitive Salmonella typhimurium mutants, homology to known KDO-8-phosphate synthetase and CMP-KDO synthetase proteins, and in vitro enzyme activity. The kdsA gene was transcribed as part of a polycistronic mRNA with two downstream open reading frames (ORFs). One of these ORFs appeared to encode a membrane-anchored protein, while the second encoded a protein showing homology to the ATP-binding component of periplasmic binding protein-dependent ABC transporters. Transcription of kdsA and kdsB in C. trachomatis was evident within 4 h of initiation of the C. trachomatis infection process and continued throughout the chlamydial life cycle.

The vanE operon was characterized from Enterococcus faecalis N00-410 (MIC of vancomycin = 24 μg/ml). The organization of the vanE operon was identical to that of the vanC1 operon from Enterococcus gallinarum , with protein identities ranging from 46 to 63%. An open reading frame located downstream of the vanE operon showed significant homology to a number of integrase genes, all of which are located downstream of the chromosomal GMP synthase gene guaA .

Multilocus sequence typing of 31 stx -carrying Escherichia coli O26:H11 strains isolated in Canada between 1999 and 2003 revealed a high degree of genetic relatedness at 10 loci, suggesting either that this is a clonal serotype (similar to O157:H7) or that additional genetic loci need to be examined.

We conducted a comparative evaluation of the Chlamydiazyme (Abbott Laboratories), PACE 2 (Gen-Probe), and AMP-CT (Gen-Probe) assays for the detection of Chlamydia trachomatis in endocervical samples. Specimens from 787 females were included in the study. The sensitivities of the PACE 2 and Chlamydiazyme assays in comparison to the results of the AMP-CT assay were 79.3 and 63.4%, respectively. The specificities of the Chlamydiazyme and PACE 2 assays were 100%. All of the positive specimens detected in this study were positive by the AMP-CT assay. On the basis of the final results of the comparison, the prevalence of C. trachomatis in the population was 10.4%. Retesting of specimens whose results were in the intermediate zone by the PACE 2 assay by a probe competition assay identified some additional true-positive specimens. Amplification assay testing of such specimens did not significantly increase the yield. The majority of specimens which tested positive by the AMP-CT assay only were not in the intermediate zone by the PACE 2 assay. We were unable to identify demographic or clinical factors which could predict those individuals who tested positive by amplified tests but not by nonamplified tests. The Gen-Probe PACE 2 assay proved to be superior to the Chlamydiazyme assay for the screening and diagnosis of C. trachomatis infections in female endocervical specimens.