New Delhi Metallo-Beta-Lactamase (NDM)-5 in Uropathogenic Klebsiella pneumoniae in a Tertiary Care Hospital in Jamaica.

We have investigated the prevalence of carbapenemase-producing uropathogens at the University Hospital of the West Indies, Jamaica. From 64 unique urine samples collected between January and March 2020, only 2 closely related Klebsiella pneumoniae (ST11, 14 SNPs of difference; no clear epidemiological links found between patients) were carbapenemase-producers. By whole-genome sequencing (WGS), blaNDM-5 was found on ~46 kb, IncX3 plasmid. These findings highlight the necessity for continuous surveillance of these pathogens in Jamaica. IMPORTANCE As the problem of antibiotic resistance continues to be a global problem, we hope to be able to shed further insight into what is happening within the Caribbean, from which there has been a paucity of data. The ability to appropriately tackle the problem of resistance requires surveillance from all territories, including resource limited settings. In this paper, we look at a mechanism of resistance that renders some critical antibiotics useless, including carbapenems, cephalosporins, and penicillin.

Prevalence and distribution of ampc beta-lactamase producing escherichia coli and klebsiella pneumoniae isolates obtained from urine samples at a tertiary care hospital in the caribbean.

Introduction: The aim of this study was to investigate the prevalence and distribution of AmpC beta-lactamases (BLs) in uropathogens (E. coli and K. pneumoniae) at the University Hospital of the West Indies Jamaica (UHWI). Method: De-duplicated consecutive urine samples, collected from January to March 2020 at the UHWI, were analyzed. Screening and phenotypic confirmatory tests were conducted using resistance to cefoxitin and the Disc Approximation Test (DAT) respectively, for isolates of interest. Multiplex PCR was performed on cefoxitin resistant (CR) isolates for the detection of bla CIT, bla MOX, bla FOX, bla ACC, and bla DHA genes. Whole genome sequencing (WGS) was used to further detect AmpC BL genes in PCR negative isolates with indeterminate phenotypic results. Results: Sixty-four Gram negative isolates were obtained from 61 patients (55% female), aged 18 months to 88 years old. At least 35% (26) had complicated urinary tract infections. Only 7 out of 64 isolates were E. coli or K. pneumoniae, had antibiograms suggestive of possible AmpC BL production and were CR. DATs confirmed AmpC BL in two of these (1 K. pneumoniae; 1 E. coli), one tested negative (E. coli) and four had inconclusive results (K. pneumoniae). PCR detected bla DHA and bla CIT in two CR isolates. WGS further detected bla CMY-42 in one isolate. The prevalence of screened CR isolates with AmpC BL is 57.14% (4 of 7), representing 6.25% of the sample. AmpC BL producers tested had 100% susceptibility to meropenem and nitrofurantoin. Conclusion: AmpC BL prevalence among E. coli and K. pneumoniae, common urinary pathogens, in the studied isolates is low. Although cefoxitin screening is helpful, phenotypic screening using the DAT can yield indeterminate results best clarified by molecular testing.

Emergence of KPC-31, a KPC-3 Variant Associated with Ceftazidime-Avibactam Resistance, in an Extensively Drug-Resistant ST235 Pseudomonas aeruginosa Clinical Isolate.

A ceftazidime-avibactam-resistant KPC-producing Pseudomonas aeruginosa strain was isolated in Argentina from a tracheal aspirate. The patient was treated with ceftazidime-avibactam in combination with other agents for 130 days. Whole-genome sequencing of P. aeruginosa identified a D179Y substitution in the Ω loop of KPC-3, corresponding to KPC-31, integrated at the chromosome. The strain belonged to the sequence type 235/O11 (ST235/O11) high-risk clone. Evaluation of carbapenemase detection assays most used by clinical laboratories failed to identify the isolate as a KPC producer.

Performance Characteristics of Next-Generation Sequencing for the Detection of Antimicrobial Resistance Determinants in Escherichia coli Genomes and Metagenomes.

Short-read sequencing can provide detection of multiple genomic determinants of antimicrobial resistance from single bacterial genomes and metagenomic samples. Despite its increasing application in human, animal, and environmental microbiology, including human clinical trials, the performance of short-read Illumina sequencing for antimicrobial resistance gene (ARG) detection, including resistance-conferring single nucleotide polymorphisms (SNPs), has not been systematically characterized. Using paired-end 2 × 150 bp (base pair) Illumina sequencing and an assembly-based method for ARG prediction, we determined sensitivity, positive predictive value (PPV), and sequencing depths required for ARG detection in an Escherichia coli isolate of sequence type (ST) 38 spiked into a synthetic microbial community at varying abundances. Approximately 300,000 reads or 15× genome coverage was sufficient to detect ARGs in E. coli ST38, with comparable sensitivity and PPV to ~100× genome coverage. Using metagenome assembly of mixed microbial communities, ARG detection at E. coli relative abundances of 1% would require assembly of approximately 30 million reads to achieve 15× target coverage. The minimum sequencing depths were validated using public data sets of 948 E. coli genomes and 10 metagenomic rectal swab samples. A read-based approach using k-mer alignment (KMA) for ARG prediction did not substantially improve minimum sequencing depths for ARG detection compared to assembly of the E. coli ST38 genome or the combined metagenomic samples. Analysis of sequencing depths from recent studies assessing ARG content in metagenomic samples demonstrated that sequencing depths had a median estimated detection frequency of 84% (interquartile range: 30%-92%) for a relative abundance of 1%. IMPORTANCE Systematically determining Illumina sequencing performance characteristics for detection of ARGs in metagenomic samples is essential to inform study design and appraisal of human, animal, and environmental metagenomic antimicrobial resistance studies. In this study, we quantified the performance characteristics of ARG detection in E. coli genomes and metagenomes and established a benchmark of ~15× coverage for ARG detection for E. coli in metagenomes. We demonstrate that for low relative abundances, sequencing depths of ~30 million reads or more may be required for adequate sensitivity for many applications.

Dissemination of Verona Integron-encoded Metallo-ß-lactamase among clinical and environmental Enterobacteriaceae isolates in Ontario, Canada.

Surveillance data from Southern Ontario show that a majority of Verona Integron-encoded Metallo-ß-lactamase (VIM)-producing Enterobacteriaceae are locally acquired. To better understand the local epidemiology, we analysed clinical and environmental blaVIM-positive Enterobacteriaceae from the area. Clinical samples were collected within the Toronto Invasive Bacterial Diseases Network (2010-2016); environmental water samples were collected in 2015. We gathered patient information on place of residence and hospital admissions prior to the diagnosis. Patients with and without plausible source of acquisition were compared regarding risk exposures. Microbiological isolates underwent whole-genome sequencing (WGS); blaVIM carrying plasmids were characterized. We identified 15 patients, thereof 11 with blaVIM-1-positive Enterobacter hormaechei within two genetic clusters based on WGS. Whereas no obvious epidemiologic link was identified among cluster I patients, those in cluster II were connected to a hospital outbreak. Except for patients with probable acquisition abroad, we did not identify any further risk exposures. Two blaVIM-1-positive E. hormaechei from environmental waters matched with the clinical clusters; plasmid sequencing suggested a common ancestor plasmid for the two clusters. These data show that both clonal spread and horizontal gene transfer are drivers of the dissemination of blaVIM-1-carrying Enterobacter hormaechei in hospitals and the aquatic environment in Southern Ontario, Canada.

Molecular characteristics of mcr-1-carrying plasmids and new mcr-1 variant recovered from polyclonal clinical Escherichia coli from Argentina and Canada.

We have characterized nine mcr-1-harboring plasmids from clinical Escherichia coli isolates previously described in Argentina and Canada. Three of these plasmids carried a mcr-1-variant called here mcr-1.5. All these E. coli isolates were not clonally related and were recovered in different years and locations. However, their mcr-1-harboring plasmids showed high identity among them and to others characterized in other countries, which strongly suggests that this plasmid-type is playing an important role in spreading this mechanism of resistance to polymyxins.

Mycoplasma genitalium in Toronto, Ont: Estimates of prevalence and macrolide resistance.

OBJECTIVE: To estimate the prevalence of Mycoplasma genitalium in Toronto, Ont; detect mutations associated with macrolide and fluoroquinolone resistance; and describe treatment outcomes. DESIGN: Prospective, cross-sectional study. SETTING: A sexual health clinic in Toronto. PARTICIPANTS: A consecutive sample of men and women attending the sexual health clinic between September 1, 2013, and December 20, 2013. INTERVENTIONS: Participants underwent testing for M genitalium, along with standard sexually transmitted infection screening. All samples that had positive results for M genitalium were tested for mutations associated with resistance to macrolides and fluoroquinolones. Mycoplasma genitalium treatment was based on resistance profile and verified with a test of cure. MAIN OUTCOME MEASURES: Positive results for M genitalium and antibiotic resistance.

Population structure and antimicrobial resistance of invasive serotype IV group B Streptococcus, Toronto, Ontario, Canada.

We recently showed that 37/600 (6.2%) invasive infections with group B Streptococcus (GBS) in Toronto, Ontario, Canada, were caused by serotype IV strains. We report a relatively high level of genetic diversity in 37 invasive strains of this emerging GBS serotype. Multilocus sequence typing identified 6 sequence types (STs) that belonged to 3 clonal complexes. Most isolates were ST-459 (19/37, 51%) and ST-452 (11/37, 30%), but we also identified ST-291, ST-3, ST-196, and a novel ST-682. We detected further diversity by performing whole-genome single-nucleotide polymorphism analysis and found evidence of recombination events contributing to variation in some serotype IV GBS strains. We also evaluated antimicrobial drug resistance and found that ST-459 strains were resistant to clindamycin and erythromycin, whereas strains of other STs were, for the most part, susceptible to these antimicrobial drugs.

Azithromycin resistance is coevolving with reduced susceptibility to cephalosporins in Neisseria gonorrhoeae in Ontario, Canada.

Azithromycin (AZM) is routinely recommended as a component of dual therapy for gonorrhea in combination with third-generation cephalosporins (3GC). In this study, we examined the prevalence of AZM-resistant (AZM(r)) Neisseria gonorrhoeae from July 2010 to February 2013, assessed the rate of concurrent cephalosporin resistance under the current treatment recommendations, and analyzed the clonal distribution of AZM(r) isolates in Ontario, Canada. Nineteen AZM(r) clinical isolates (one per patient; MIC, ≥2 µg/ml) were included in the study. Susceptibility profiles of these isolates to 11 antibiotics, molecular typing, characterization of macrolide resistance mechanisms, and penicillin-binding protein 2 (PBP2) patterns were determined for all the isolates. Two groups were defined based on AZM(r) level; group A isolates displayed high-level resistance (MIC, ≥2,048 µg/ml) due to mutations (A2143G) in the four copies of the 23S rRNA rrl gene, and group B isolates had moderate resistance to AZM (MICs, 2 to 8 µg/ml, C2599T mutation in the rrl gene), with a subgroup belonging to sequence type 3158 (ST3158) (n = 8), which also showed reduced susceptibility to 3GC (MICs, 0.12 to 0.25 µg/ml, PBP2 pattern XXXIV). This AZM(r) phenotype was not observed in previous provincial surveillance in 2008 (the ST3158 clone was found, with AZM MICs of 0.25 to 0.5 µg/ml associated with mtrR mutations). We hypothesized that the AZM mutant prevention concentration (MPC) in the ST3158 subpopulation we found in 2008 was higher than the MPC in wild-type isolates (AZM MIC, ≤0.031 µg/ml), increasing the chances of additional selection of AZM(r) mutations. Full AZM resistance is now emerging in this clone together with reduced susceptibility to 3GC, threatening the future efficacy of these antibiotics as therapeutic options for treatment of gonorrhea.

Determination of In Vitro Activities of Solithromycin at Different pHs and Its Intracellular Activity against Clinical Isolates of Neisseria gonorrhoeae from a Laboratory Collection.

We evaluated the activity of solithromycin against 196 clinical gonococcal isolates collected at the Public Health Ontario Laboratories, Toronto, Canada, including isolates with different levels of azithromycin resistance, as well as the role of pH in MIC determinations using pH-adjusted agar plates (pH range, 5.6 to 7.6). In vitro invasion assays were performed using monolayers of HeLa epithelial cells and clinical gonococci displaying different azithromycin MICs; infected cultures were treated with solithromycin, and its intracellular activity was determined by CFU assays after 3 and 20 h of exposure. Solithromycin displayed a MIC50 and MIC90 of 0.0625 and 0.125 µg/ml, respectively, making its activity at least 4-fold higher than that of azithromycin. Clinical isolates with elevated MICs for azithromycin (MICs of ≥2,048 µg/ml and 4 to 8 µg/ml) showed solithromycin MIC values of 8 and 0.5 µg/ml, respectively. In contrast to azithromycin, solithromycin MICs were not significantly affected by acidic pHs, suggesting more stability at lower pH. Moreover, when intracellular Neisseria gonorrhoeae isolates were incubated with solithromycin at 4 times, 1 times, and one-fourth of the MIC, the exposure to solithromycin resulted in the progressive loss of viability of most isolates over time. The intracellular activity of solithromycin, combined with the low MICs to this agent, indicates that it may be an attractive option for gonorrhea treatment if clinical trials in development reveal that this drug can be used safely in adult indications, especially when multidrug-resistant clinical isolates are now emerging.

Neisseria gonorrhoeae treatment failure and susceptibility to cefixime in Toronto, Canada.

IMPORTANCE: Although cephalosporins are the cornerstone of treatment of Neisseria gonorrhoeae infections, cefixime is the only oral antimicrobial option. Increased minimum inhibitory concentrations (MICs) to cefixime have been identified worldwide and have been associated with reports of clinical failure. OBJECTIVE: To assess the risk of clinical treatment failure of N. gonorrhoeae infections associated with the use of cefixime. DESIGN, SETTING, AND POPULATION: A retrospective cohort study of culture-positive N. gonorrhoeae infections at a single sexual health clinic in Toronto, Canada, that routinely performs test of cure. The cohort comprised N. gonorrhoeae culture-positive individuals identified between May 1, 2010, and April 30, 2011, treated with cefixime as recommended by Public Health Agency of Canada guidelines. MAIN OUTCOME MEASURES: Cefixime treatment failure, defined as the repeat isolation of N. gonorrhoeae at the test-of-cure visit identical to the pretreatment isolate by molecular typing and explicit denial of reexposure. RESULTS: There were 291 N. gonorrhoeae culture-positive individuals identified. Of 133 who returned for test of cure, 13 were culture positive; 9 patients were determined to have experienced cefixime treatment failure, involving urethral (n = 4), pharyngeal (n = 2), and rectal (n = 3) sites. The overall rate of clinical treatment failure among those who had a test of cure was 6.77% (95% CI, 3.14%-12.45%; 9/133). The rate of clinical failure associated with a cefixime MIC of 0.12 µg/mL or greater was 25.0% (95% CI, 10.69%-44.87%; 7/28) compared with 1.90% (95% CI, 0.23%-6.71%; 2/105) of infections with cefixime MICs less than 0.12 µg/mL, with a relative risk of 13.13 (95% CI, 2.88-59.72; P < .001). CONCLUSION AND RELEVANCE: The rate of clinical failure following treatment of N. gonorrhoeae infections with cefixime was relatively high at a Toronto clinic and was associated with elevated MICs.

MupB, a new high-level mupirocin resistance mechanism in Staphylococcus aureus.

Mupirocin is a topical antibiotic used for the treatment of skin infections and the eradication of methicillin-resistant Staphylococcus aureus carriage. It inhibits bacterial protein synthesis by interfering with isoleucyl-tRNA synthetase activity. High-level mupirocin resistance (MIC of ≥ 512 µg/ml) is mediated by the expression of mupA (ileS2), which encodes an alternate isoleucyl-tRNA synthetase. In this study, we describe high-level mupirocin resistance mediated by a novel locus, mupB. The mupB gene (3,102 bp) shares 65.5% sequence identity with mupA but only 45.5% identity with ileS. The deduced MupB protein shares 58.1% identity (72.3% similarity) and 25.4% identity (41.8% similarity) with MupA and IleS, respectively. Despite this limited homology, MupB contains conserved motifs found in class I tRNA synthetases. Attempts to transfer high-level mupirocin resistance via conjugation or transformation (using plasmid extracts from an mupB-containing strain) were unsuccessful. However, by cloning the mupB gene into a shuttle vector, it was possible to transfer the resistance phenotype to susceptible S. aureus by electroporation, proving that mupB was responsible for the high-level mupirocin resistance. Further studies need to be done to determine the prevalence of mupB and to understand risk factors and outcomes associated with resistance mediated by this gene.

Comparison of the FXG™: RESP (Asp+) real-time PCR assay with direct immunofluorescence and calcofluor white staining for the detection of Pneumocystis jirovecii in respiratory specimens.

We compared the FXG™: RESP (Asp +) real-time PCR assay (Myconostica Ltd) with two microscopic staining methods (direct immunofluorescence [IFA] and calcofluor white) for the detection of Pneumocystis jirovecii in 411 respiratory specimens submitted for P. jirovecii examination. We considered the specimen to be microscopically positive if the organism could be visualized through the use of either IFA or calcofluor white. A second, published real-time PCR assay targeting the cdc2 gene of P. jirovecii was used to adjudicate those specimens that were microscopically negative but Myconostica PCR positive. The Myconostica PCR positive samples were deemed to be true positives if they were concordant with microscopically positive results or if they were positive by the second PCR assay. As a result, the Myconostica PCR assay was found to be more sensitive than the two microscopy methods in detecting P. jirovecii (10.5% true positivity rate by PCR, 8.0% by immunofluorescence, and 7.1% by calcofluor white). The Myconostica PCR assay showed 93.5% sensitivity, 95.1% specificity, 70.5% positive predictive value, and 99.1% negative predictive value. Its high negative predictive value suggests a role of the Myconostica PCR assay in ruling out Pneumocystis pneumonia.

Rapid identification of Cryptococcus neoformans var. grubii, C. neoformans var. neoformans, and C. gattii by use of rapid biochemical tests, differential media, and DNA sequencing.

Rapid identification of Cryptococcus neoformans var. grubii, Cryptococcus neoformans var. neoformans, and Cryptococcus gattii is imperative for facilitation of prompt treatment of cryptococcosis and for understanding the epidemiology of the disease. Our purpose was to evaluate a test algorithm incorporating commercial rapid biochemical tests, differential media, and DNA sequence analysis that will allow us to differentiate these taxa rapidly and accurately. We assessed 147 type, reference, and clinical isolates, including 6 other Cryptococcus spp. (10 isolates) and 14 other yeast species (24 isolates), using a 4-hour urea broth test (Remel), a 24-hour urea broth test (Becton Dickinson), a 4-hour caffeic acid disk test (Hardy Diagnostics and Remel), 40- to 44-hour growth assessment on l-canavanine glycine bromothymol blue (CGB) agar, and intergenic spacer (IGS) sequence analysis. All 123 Cryptococcus isolates hydrolyzed urea, along with 7 isolates of Rhodotorula and Trichosporon. Eighty-five of 86 C. neoformans (99%) and 26 of 27 C. gattii (96%) isolates had positive caffeic acid results, unlike the other cryptococci (0/10) and yeast species (0/24). Together, these two tests positively identified virtually all C. neoformans/C. gattii isolates (98%) within 4 h. CGB agar or IGS sequencing further differentiated these isolates within 48 h. On CGB, 25 of 27 (93%) C. gattii strains induced a blue color change, in contrast to 0 of 86 C. neoformans isolates. Neighbor-joining cluster analysis of IGS sequences differentiated C. neoformans var. grubii, C. neoformans var. neoformans, and C. gattii. Based on these results, we describe a rapid identification algorithm for use in a microbiology laboratory to distinguish clinically relevant Cryptococcus spp.

Distribution of antiseptic resistance genes qacA, qacB, and smr in methicillin-resistant Staphylococcus aureus isolated in Toronto, Canada, from 2005 to 2009.

Decreased susceptibility to chlorhexidine gluconate (CHDN) in methicillin-resistant Staphylococcus aureus (MRSA) is associated with the qacA, qacB, and smr genes, encoding efflux pumps. A total of 334 MRSA isolates were collected from two Canadian intensive care units between 2005 and 2009. We identified the qacAB genes in 7 strains (2%; 2 qacA genes and 5 qacB genes) and the smr gene in 23 (7%) strains. CHDN minimal bactericidal concentrations were slightly higher for strains harboring smr genes.

Comparative evaluation of a chromogenic agar medium, the modified Hodge test, and a battery of meropenem-inhibitor discs for detection of carbapenemase activity in Enterobacteriaceae.

Three phenotypic methods (modified Hodge test, chromogenic agar, and meropenem discs combined with specific inhibitors) used for the detection of carbapenemase activity were tested on a panel of characterized Enterobacteriaceae expressing various ß-lactamase mechanisms. Overall, the meropenem-plus-inhibitor approach was more sensitive and specific than the other methods, despite its limitation of being unable to detect class D carbapenemases.

Analytical and clinical validation of novel real-time reverse transcriptase-polymerase chain reaction assays for the clinical detection of swine-origin H1N1 influenza viruses.

During the early stages of the 2009/2010 swine-origin H1N1 influenza A (S-OIV H1N1 FluA) outbreak, the development and validation of sensitive and specific detection methods were a priority for rapid and accurate diagnosis. Between May and June 2009, 2 real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) assays targeting the hemagglutinin and neuraminidase genes of the S-OIV H1N1 FluA virus were developed. These assays are highly specific, showing no cross-reactivity against a panel of respiratory viruses and can differentiate S-OIV H1N1 from seasonal FluA viruses. Analytical sensitivities of the 2 assays were found to be 10(-1) tissue culture infectious dose, 50%/ml. Clinical testing showed 99.2% sensitivity and 94.6-98.1% specificity. A large prospective analysis showed that 94.8-95.5% of S-OIV positive specimens were negative by seasonal H1/H3 subtyping. The large-scale validation data presented in this report indicate that these novel assays provide an accurate and efficient method for the rapid detection of S-OIV H1N1 FluA viruses.