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ABSTRACTA curated Web-based user-friendly sequence typing tool based on antimicrobial resistance determinants in Neisseria gonorrhoeaewas developed and is publicly accessible (https://ngstar.canada.ca). The N. gonorrhoeaeSequence Typing for Antimicrobial Resistance (NG-STAR) molecular typing scheme uses the DNA sequences of 7 genes (penA, mtrR, porB, ponA, gyrA, parC, and 23S rRNA) associated with resistance to ß-lactam antimicrobials, macrolides, or fluoroquinolones. NG-STAR uses the entire penAsequence, combining the historical nomenclature for penAtypes I to XXXVIII with novel nucleotide sequence designations; the full mtrRsequence and a portion of its promoter region; portions of ponA, porB, gyrA, and parC; and 23S rRNA sequences. NG-STAR grouped 768 isolates into 139 sequence types (STs) (n= 660) consisting of 29 clonal complexes (CCs) having a maximum of a single-locus variation, and 76 NG-STAR STs (n= 109) were identified as unrelated singletons. NG-STAR had a high Simpson's diversity index value of 96.5% (95% confidence interval [CI] = 0.959 to 0.969). The most common STs were NG-STAR ST-90 (n= 100; 13.0%), ST-42 and ST-91 (n= 45; 5.9%), ST-64 (n= 44; 5.72%), and ST-139 (n= 42; 5.5%). Decreased susceptibility to azithromycin was associated with NG-STAR ST-58, ST-61, ST-64, ST-79, ST-91, and ST-139 (n= 156; 92.3%); decreased susceptibility to cephalosporins was associated with NG-STAR ST-90, ST-91, and ST-97 (n= 162; 94.2%); and ciprofloxacin resistance was associated with NG-STAR ST-26, ST-90, ST-91, ST-97, ST-150, and ST-158 (n= 196; 98.0%). All isolates of NG-STAR ST-42, ST-43, ST-63, ST-81, and ST-160 (n= 106) were susceptible to all four antimicrobials. The standardization of nomenclature associated with antimicrobial resistance determinants through an internationally available database will facilitate the monitoring of the global dissemination of antimicrobial-resistant N. gonorrhoeaestrains.

ABSTRACTThe incidence of antimicrobial-resistant Neisseria gonorrhoeaecontinues to rise in Canada; however, antimicrobial resistance data are lacking for approximately 70% of gonorrhea infections that are diagnosed directly from clinical specimens by nucleic acid amplification tests (NAATs). We developed a molecular assay for surveillance use to detect mutations in genes associated with decreased susceptibility to cephalosporins that can be applied to both culture isolates and clinical samples. Real-time PCR assays were developed to detect single nucleotide polymorphisms (SNPs) in ponA, mtrR, penA, porB, and one N. gonorrhoeae-specific marker (porA). We tested the real-time PCR assay with 252 gonococcal isolates, 50 nongonococcal isolates, 24 N. gonorrhoeae-negative NAAT specimens, and 34 N. gonorrhoeae-positive NAAT specimens. Twenty-four of the N. gonorrhoeae-positive NAAT specimens had matched culture isolates. Assay results were confirmed by comparison with whole-genome sequencing data. For 252 N. gonorrhoeaestrains, the agreement between the DNA sequence and real-time PCR was 100% for porA, ponA, and penA, 99.6% for mtrR, and 95.2% for porB. The presence of =2 SNPs correlated with decreased susceptibility to ceftriaxone (sensitivities of >98%) and cefixime (sensitivities of >96%). Of 24 NAAT specimens with matched cultures, the agreement between the DNA sequence and real-time PCR was 100% for porB, 95.8% for ponAand mtrR, and 91.7% for penA. We demonstrated the utility of a real-time PCR assay for sensitive detection of known markers for the decreased susceptibility to cephalosporins in N. gonorrhoeae. Preliminary results with clinical NAAT specimens were also promising, as they correlated well with bacterial culture results.

ABSTRACTA total of 142 cefoxitin-resistant Escherichia coliisolates from water sources were collected across Canada. Multidrug resistance was observed in 65/142 (45.8%) isolates. The blaCMY-2gene was identified in 110/142 (77.5%) isolates. Sequencing of the chromosomal ampCpromoter region showed mutations from the wild type, previously shown to hyperproduce AmpC. CMY-2-producing plasmids predominantly belonged to replicon groups I1-Iγ, A/C, and K/B. The majority of the E. coliisolates belonged to the nonvirulent phylogenetic groups A and B1.

ABSTRACTA panel of 24 methicillin-resistant Staphylococcus aureusstrains was distributed to 15 laboratories in Canada to evaluate their in-house pulsed-field gel electrophoresis (PFGE) protocols and interpretation criteria. Attempts to compare fingerprint images using computer-aided analysis were not successful due to variability in individual laboratory PFGE protocols. In addition, individual site interpretation of the fingerprint patterns was inadequate, as 7 of 13 sites (54%) made at least one error in interpreting the fingerprints from the panel. A 2-day standardized PFGE protocol (culture to gel image) was developed and distributed to all of the sites. Each site was requested to use the standardized protocol on five strains from the original panel. Thirteen sites submitted gel images for comparisons. The protocol demonstrated excellent reproducibility and allowed interlaboratory comparisons with Molecular Analyst DST software (Bio-Rad) and 1.5% band tolerance.

ABSTRACTWe 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.

We have recently identified ferritin as a cellular protein particle whose synthesis is stimulated in mouse or human cells infected by the picornavirus Mengo. Immunoprecipitation of the particle from infected murine L929 cells showed a 4- and 6-fold increase in the intracellular concentrations of H and L apoferritin subunits, respectively. This differential expression altered the H/L subunit ratio from 3.0 in uninfected cells to 2.2 in Mengo virus-infected cells. The induction is not due to an increase in transcription of the apoferritin L and H genes, nor is it due to an increase in stability of the apoferritin mRNAs. At the level of translation, the iron regulatory protein (IRP) remained intact, with similar amounts being detected in uninfected and infected cells. The Mengo virus RNA genome does not compete with the iron regulatory element (IRE) for the binding of IRP, and sequence analysis confirmed that there are no IREs in the virus RNA. The IRE binding activity of IRP in infected cells decreased approximately 30% compared with uninfected cells. The decrease in binding activity could be overcome by the addition of Desferal (deferoxamine mesylate; CIBA) an intracellular iron chelator, which suggests that virus infection causes an increase in intracellular free iron. Electron paramagnetic resonance (EPR) studies have confirmed the increase in free iron in Mengo virus infected cells. The permeability of cells for iron does not change in virus infected cells, suggesting that the induction of ferritin by Mengo virus is due to a change in the form of intracellular iron from a bound to a free state.

Fusion plasmids with lacZ under the control of the katE (encoding catalase or hydroperoxidase HPII) and katF (encoding a sigma factor-like protein required for katE expression) promoters were constructed. Expression from both katE and katF promoters was low in rich medium but elevated in poor medium during log-phase growth. Furthermore, the slowdown in growth as cells entered the stationary phase in rich medium, a result of carbon source depletion, was associated with an increase in katE and katF expression. A simple reduction in the carbon source level as the cells entered the stationary phase was not responsible for the increase in expression, because transferring the culture to a medium with no glucose did not induce expression from either promoter. Spent rich medium from stationary-phase cells was capable of inducing expression, as were simple aromatic acids such as benzoate, o-hydroxybenzoate, and p-aminobenzoate added to new medium. Anaerobiosis did not cause an increase in expression, nor did it significantly change the pattern of expression. Regardless of the medium, katF expression was always turned on before or coincidently with katE expression; in the presence of benzoate katF was fully induced, whereas katE was only partially induced, suggesting that a factor in addition to KatF protein was involved in katE expression. During prolonged aerobic incubation, cells lacking katF died off more rapidly than did cells lacking either katE or katG.

A transcriptional fusion of katF to the lacZ gene was expressed at increasingly higher levels throughout the exponential phase, but a translational fusion was expressed at low levels during exponential-phase growth and was induced 160-fold during the transition to stationary phase, implicating a posttranscriptional mechanism in the regulation of KatF synthesis. Mutational analyses suggested that the initiation codon of katF is the second ATG in the previously identified open reading frame.

A 3,466-bp nucleotide sequence containing the katE gene of Escherichia coli has been determined. An open reading frame of 2,259 bp was found and was preceded by a potential ribosome-binding site. The predicted N-terminal sequence agreed with the sequence determined by direct amino acid sequencing, and the predicted direction of transcription was confirmed by expression of the gene cloned in both directions behind a T7 promoter. The start site of transcription was determined to be 127 bp upstream from the start of the open reading frame, and a potential RNA polymerase-binding site similar to a sequence preceding the xthA gene, which is also controlled by the KatF protein, was identified. The predicted sequence of the 753-amino-acid protein was compared with known sequences of other catalases, revealing significant similarity to the shorter catalases, including the residues in the putative active site and residues involved in heme binding.

The gene katG, encoding catalase HPI of Escherichia coli, was sequenced, predicting a 726-amino-acid protein. The sequence was confirmed by identification of potential regulatory elements and amino acid sequencing of peptides. HPI shows no homology to other catalases. The distances between katG, metF, and ppc were defined.

ABSTRACTThe vanEoperon was characterized from Enterococcus faecalisN00-410 (MIC of vancomycin = 24 μg/ml). The organization of the vanEoperon was identical to that of the vanC1operon from Enterococcus gallinarum, with protein identities ranging from 46 to 63%. An open reading frame located downstream of the vanEoperon showed significant homology to a number of integrase genes, all of which are located downstream of the chromosomal GMP synthase gene guaA.

ABSTRACTA single vanD-containing Enterococcus faeciumstrain (N97-330) was isolated in Canada. ThevanD-containing region was cloned and sequenced. Although the proteins have more than 96% identity to a previously described vanDregion in BM4339, thevanSDgene contains a frameshift mutation that leads to a predicted truncated protein. Furthermore, sequence analysis of the ddlgene revealed the presence of an IS982-like element (ISEfm1) which interrupted the d-Ala–d-Ala ligase. This suggested the constitutive expression of the vanDoperon, which was confirmed. Pulsed-field gel electrophoresis fingerprinting demonstrated that BM4339 was not related to N97-330 (>15 band differences). Both strains contained multiple copies of the IS982-like element.