Use of genome sequencing to resolve differences in gradient diffusion and agar dilution antimicrobial susceptibility testing performance of Neisseria gonorrhoeae isolates in Alberta, Canada.

Agar dilution is the gold standard method for phenotypic antimicrobial susceptibility testing (AST) for Neisseria gonorrhoeae. However, this method is laborious and requires expertise, so laboratories that perform N. gonorrhoeae AST may choose alternative methods such as disk diffusion and gradient diffusion. In this study, we retrospectively compare the performance of gradient diffusion to agar dilution for 2,394 unique N. gonorrhoeae isolates identified in Alberta from 2017 to 2020 against azithromycin, cefixime, ceftriaxone, ciprofloxacin, penicillin, and tetracycline. Genome sequencing was utilized to resolve discrepancies between AST methods, detect antimicrobial resistance markers, and identify trends between error rates and sequence types (STs) of isolates. Over 90% of N. gonorrhoeae isolates were susceptible to azithromycin, cefixime, and ceftriaxone, whereas decreased susceptibility was observed for ciprofloxacin, penicillin, and tetracycline. Categorical (CA) and essential agreement (EA) was poorest between the two methods for penicillin (CA: 86.02%; EA: 77.69%) and tetracycline (CA: 47.22%; EA: 55.96%); however, the low CA was primarily attributed to minor errors. Antimicrobial agents with errors outside of acceptable limits included azithromycin (very major error: 18.42%; major error: 7.73%) and tetracycline (very major error: 6.17%). Genome sequencing on a subset of isolates resolved 30.3% of the azithromycin major errors and confirmed the azithromycin or tetracycline very major errors. Significant associations between certain STs and error types for azithromycin and tetracycline were also identified. Overall, gradient diffusion compared well to agar dilution for cefixime, ceftriaxone, and ciprofloxacin, and genome sequencing was identified as a useful tool to arbitrate discrepant susceptibility testing results between gradient diffusion and agar dilution for N. gonorrhoeae.

A conceptual framework for nomenclatural stability and validity of medically important fungi: a proposed global consensus guideline for fungal name changes supported by ABP, ASM, CLSI, ECMM, ESCMID-EFISG, EUCAST-AFST, FDLC, IDSA, ISHAM, MMSA, and MSGERC.

The rapid pace of name changes of medically important fungi is creating challenges for clinical laboratories and clinicians involved in patient care. We describe two sources of name change which have different drivers, at the species versus the genus level. Some suggestions are made here to reduce the number of name changes. We urge taxonomists to provide diagnostic markers of taxonomic novelties. Given the instability of phylogenetic trees due to variable taxon sampling, we advocate to maintain genera at the largest possible size. Reporting of identified species in complexes or series should where possible comprise both the name of the overarching species and that of the molecular sibling, often cryptic species. Because the use of different names for the same species will be unavoidable for many years to come, an open access online database of the names of all medically important fungi, with proper nomenclatural designation and synonymy, is essential. We further recommend that while taxonomic discovery continues, the adaptation of new name changes by clinical laboratories and clinicians be reviewed routinely by a standing committee for validation and stability over time, with reference to an open access database, wherein reasons for changes are listed in a transparent way.

The Ins and Outs of Susceptibility Testing for New ß-Lactam/ß-Lactamase Inhibitor Combinations for Gram-Negative Organisms.

Ceftazidime-avibactam, meropenem-vaborbactam, and imipenem-relebactam are among the newest ß-lactam/ß-lactamase inhibitors (BL/BLIs) introduced to the North American antibiotic market. All have broad Gram-negative activity, including against certain carbapenemases. Despite this, susceptibility testing is warranted due to variable activity against certain ß-lactamases (e.g., oxacillinases) and the presence of acquired resistance mechanisms in some isolates. Here, we discuss what we know about these new antimicrobial agents and how to navigate implementation of susceptibility testing and reporting of these agents in clinical laboratories.

Prevalence and Characterization of the Cefazolin Inoculum Effect in North American Methicillin-Susceptible Staphylococcus aureus Isolates.

Antistaphylococcal penicillins and cefazolin remain the primary treatments for infections with methicillin-susceptible Staphylococcus aureus (MSSA). The cefazolin inoculum effect (CzIE) causes the cefazolin MIC to be elevated in proportion to the number of bacteria in the inoculum. The objective of this multicenter study was to evaluate the prevalence of the CzIE in North American MSSA isolates. Clinical MSSA isolates from six microbiology laboratories in the United States and one microbiology laboratory in Canada were screened for the CzIE by broth microdilution at a standard inoculum (~5 × 105 CFU/mL) and a high inoculum (~5 × 107 CFU/mL). Genome sequencing was performed to further characterize the MSSA isolates. The CzIE was present in 57/305 (18.6%) MSSA isolates, ranging from 0% to 27.9% across study sites. More of the CzIE-positive isolates (29.8%) had standard inoculum cefazolin MICs of 1.0 µg/mL than the CzIE-negative isolates did (3.2%) (P < 0.0001). Conversely, more CzIE-negative isolates (39.5%) had standard inoculum MICs of 0.25 µg/mL than the CzIE positive isolates did (5.3%) (P < 0.0001). The most common BlaZ ß-lactamase types found in the CzIE-positive strains were type C (53.7%) and type A (44.4%). ST8 and ST30 were the most common sequence types among CzIE-positive isolates and correlated with BlaZ type C and A, respectively. The CzIE was present in up to a quarter of clinical MSSA isolates from North American clinical laboratories. Further studies to determine the impact of the presence of the CzIE on clinical outcomes are needed.

Four genomic clades of Candida auris identified in Canada, 2012-2019.

Candida auris is an emerging yeast that is associated with antifungal resistance and healthcare-associated outbreaks. From 2012 to 2019, there were 24 known cases of C. auris colonization or infection in Canada. Isolates were from axilla/groin (n = 6), ear (n = 5), blood (n = 4), toe (n = 2), and a variety of other sites (n = 7). Canadian isolates belonged to the four main genomic clades: Clade I (formerly called South Asian clade, n = 12), Clade II (East Asian, n = 3), Clade III (African, n = 4), and Clade IV (South American, n = 5). Isolates within each clade were clonal; however, whole genome sequencing may be helpful in identifying clusters within healthcare facilities. LAY SUMMARY: The fungal pathogen Candida auris has caused many hospital outbreaks and is often multidrug resistant. All four major strains of C. auris were identified in Canada from 2012 to 2019. Genomic epidemiology may be useful for identifying and reducing transmission of C. auris within hospitals.

Association between gut colonization of vancomycin-resistant enterococci and liver transplant outcomes.

BACKGROUND: Vancomycin-resistant enterococci (VRE) colonization is common in liver transplant recipients and has been associated with worse posttransplant outcomes. METHODS: We conducted a retrospective cohort study at the University of Alberta Hospital including patients who underwent a liver transplant between September 2014 and December 2017. RESULTS: Of 343 patients, 68 (19.8%) had pretransplant VRE colonization and 27 (27/275, 9.8%) acquired VRE posttransplant, 67% were males and the median age was 56.5 years. VRE colonized patients at baseline had higher MELD scores and required longer posttransplant hospitalization. VRE colonization was associated with increased risk of early acute kidney injury (AKI) (64% vs. 52%, p = .044), clinically significant bacterial/fungal infection (29% vs. 17%, p = .012) and invasive VRE infection (5% vs. 1%, p = .017). Mortality at 2 years was 13% in VRE-colonized versus 7% in noncolonized (p = .085). On multivariate analysis, VRE colonization increased the risk of posttransplant AKI (HR 1.504, 95% CI: 1.077-2.100, p = .017) and clinically significant bacterial or fungal infection at 6 months (HR 2.038, 95% CI: 1.222-3.399, p = .006), and was associated with nonsignificant trend toward increased risk of mortality at 2 years posttransplant (HR 1.974 95% CI 0.890-4.378; p = .094). CONCLUSIONS: VRE colonization in liver transplant patients is associated with increased risk of early AKI, clinically significant infections, and a trend toward increased mortality at 2 years.

High Rates of Influenza-Associated Invasive Pulmonary Aspergillosis May Not Be Universal: A Retrospective Cohort Study from Alberta, Canada.

From 2014-2019, invasive pulmonary aspergillosis complicated 7.2% (0-23.1% in different influenza seasons) of cases of influenza-associated respiratory failure in Edmonton, Alberta. Disease outcomes ranged from survival without therapy to death despite antifungals. Clinician vigilance, longitudinal local surveillance, and refined criteria to identify patients requiring therapy are needed.

Evaluation of Surrogate Tests for the Presence of mecA-Mediated Methicillin Resistance in Staphylococcus capitis, Staphylococcus haemolyticus, Staphylococcus hominis, and Staphylococcus warneri.

Testing of staphylococci other than Staphylococcus aureus (SOSA) for mecA-mediated resistance is challenging. Isolates of Staphylococcus capitis, Staphylococcus haemolyticus, Staphylococcus hominis, and Staphylococcus warneri were evaluated by cefoxitin and oxacillin broth microdilution (BMD), disk diffusion (DD), and PBP2a immunoassay, and the results were compared to mecA PCR results. No phenotypic susceptibility test correlated well with PCR results across all species, although the PBP2a immunoassay yielded 100% correlation. Oxacillin BMD testing by current Clinical and Laboratory Standards Institute (CLSI) SOSA breakpoints led to 2.1% very major errors (VMEs) and 7.1% major errors (ME). Adjusting this breakpoint up by a dilution (susceptible, ≤0.5 µg/ml; resistant, ≥1.0 µg/ml) led to 2.8% VMEs and 0.3% MEs. Among species evaluated, S. haemolyticus had unacceptable VMEs with this new breakpoint (6.4%), as did S. hominis (4.0%). MEs were acceptable by this new breakpoint, ranging from 0 to 1.2%. Oxacillin DD yielded high ME rates (20.7 to 21.7%) using CLSI or European Committee on Antimicrobial Susceptibility Testing breakpoints. VMEs ranged from 0 to 5.3%. Cefoxitin BMD led to 4.9% VMEs and 1.6% MEs. Cefoxitin DD performed best when interpreted with the CLSI SOSA breakpoint, with 1.0% VMEs and 2.9% MEs. This study led CLSI to adjust the oxacillin MIC breakpoints for SOSA. Laboratories should be aware that no individual phenotypic test correlates well across all species of SOSA with mecA PCR results. Molecular testing for mecA or evaluation for PBP2a is the preferred approach.

Brucella Exposure Risk Events in 10 Clinical Laboratories, New York City, USA, 2015 to 2017.

From 2015 to 2017, 11 confirmed brucellosis cases were reported in New York City, leading to 10 Brucella exposure risk events (Brucella events) in 7 clinical laboratories (CLs). Most patients had traveled to countries where brucellosis is endemic and presented with histories and findings consistent with brucellosis. CLs were not notified that specimens might yield a hazardous organism, as the clinicians did not consider brucellosis until they were notified that bacteremia with Brucella was suspected. In 3 Brucella events, the CLs did not suspect that slow-growing, small Gram-negative bacteria might be harmful. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), which has a limited capacity to identify biological threat agents (BTAs), was used during 4 Brucella events, which accounted for 84% of exposures. In 3 of these incidents, initial staining of liquid media showed Gram-positive rods or cocci, including some cocci in chains, suggesting streptococci. Over 200 occupational exposures occurred when the unknown isolates were manipulated and/or tested on open benches, including by procedures that could generate infectious aerosols. During 3 Brucella events, the CLs examined and/or manipulated isolates in a biological safety cabinet (BSC); in each CL, the CL had previously isolated Brucella Centers for Disease Control and Prevention recommendations to prevent laboratory-acquired brucellosis (LAB) were followed; no seroconversions or LAB cases occurred. Laboratory assessments were conducted after the Brucella events to identify facility-specific risks and mitigations. With increasing MALDI-TOF MS use, CLs are well-advised to adhere strictly to safe work practices, such as handling and manipulating all slow-growing organisms in BSCs and not using MALDI-TOF MS for identification until BTAs have been ruled out.

A Complete Genome Screening Program of Clinical Methicillin-Resistant Staphylococcus aureus Isolates Identifies the Origin and Progression of a Neonatal Intensive Care Unit Outbreak.

Whole-genome sequencing (WGS) of Staphylococcus aureus is increasingly used as part of infection prevention practices. In this study, we established a long-read technology-based WGS screening program of all first-episode methicillin-resistant Staphylococcus aureus (MRSA) blood infections at a major urban hospital. A survey of 132 MRSA genomes assembled from long reads enabled detailed characterization of an outbreak lasting several months of a CC5/ST105/USA100 clone among 18 infants in a neonatal intensive care unit (NICU). Available hospital-wide genome surveillance data traced the origins of the outbreak to three patients admitted to adult wards during a 4-month period preceding the NICU outbreak. The pattern of changes among complete outbreak genomes provided full spatiotemporal resolution of its progression, which was characterized by multiple subtransmissions and likely precipitated by equipment sharing between adults and infants. Compared to other hospital strains, the outbreak strain carried distinct mutations and accessory genetic elements that impacted genes with roles in metabolism, resistance, and persistence. This included a DNA recognition domain recombination in the hsdS gene of a type I restriction modification system that altered DNA methylation. Transcriptome sequencing (RNA-Seq) profiling showed that the (epi)genetic changes in the outbreak clone attenuated agr gene expression and upregulated genes involved in stress response and biofilm formation. Overall, our findings demonstrate the utility of long-read sequencing for hospital surveillance and for characterizing accessory genomic elements that may impact MRSA virulence and persistence.

Identification of a novel metallo-ß-lactamase, CAM-1, in clinical Pseudomonas aeruginosa isolates from Canada.

OBJECTIVES: To identify the ß-lactamase responsible for the positive detection of carbapenemase production in four clinical isolates of Pseudomonas aeruginosa that were negative by PCR for KPC, OXA-48, NDM, VIM, IMP, GES and NMC/IMI carbapenemase genes. METHODS: WGS using short-read and long-read methods was used to characterize the isolates. Bioinformatic analysis was used to identify the potential gene encoding a carbapenemase. Cloning, antimicrobial susceptibility testing and biochemical and phenotypic characterization were used to determine metallo-enzyme activity. Single-nucleotide variant (SNV) typing was used to determine strain relatedness. Conjugation experiments were used to determine transmissibility of the novel carbapenemase-encoding gene. RESULTS: WGS analysis revealed a novel class B ß-lactamase gene, blaCAM-1 (Central Alberta Metallo-ß-lactamase), located in a 73 kb integrative element, named IMEPaCAM-1, in the chromosome of four clinical isolates of P. aeruginosa. The cloned blaCAM-1 gene conferred carbapenem resistance to Escherichia coli TOP10. The four isolates, which were all closely related, were from three patients, all of whom spent time in the same hospital in 2008 and/or 2009. IMEPaCAM-1 could not be transferred by conjugation. CONCLUSIONS: A novel metallo-enzyme, CAM-1, is encoded on an integrative element, IMEPaCAM-1, located in the chromosome of clinical isolates of P. aeruginosa. No additional isolates harbouring CAM-1 have been identified in Alberta since 2007.

Species distribution and antifungal susceptibility of invasive Candida isolates from Canadian hospitals: results of the CANWARD 2011-16 study.

OBJECTIVES: Understanding the epidemiology of invasive Candida infections is essential to patient management decisions and antifungal stewardship practices. This study characterized the species distribution and antifungal susceptibilities of prospectively collected isolates of Candida species causing bloodstream infections (BSIs) in patients admitted to tertiary care hospitals located in 14 cities across 8 of the 10 Canadian provinces between 2011 and 2016. METHODS: Antifungal susceptibility testing was performed by broth microdilution using CLSI methods, breakpoints and epidemiological cut-off values. DNA sequencing of fks loci was performed on all echinocandin-non-susceptible isolates. RESULTS: Candida albicans (49.6%), Candida glabrata (20.8%) and Candida parapsilosis complex (12.0%) were the most common species out of 1882 isolates associated with BSIs. Candida tropicalis (5.2%), Candida krusei (4.3%), Candida dubliniensis (4.1%), Candida lusitaniae (1.4%) and Candida guilliermondii (1.1%) were less frequently isolated. Between 2011 and 2016, the proportion of C. albicans significantly decreased from 60.9% to 42.1% (P < 0.0001) while that of C. glabrata significantly increased from 16.4% to 22.4% (P = 0.023). C. albicans (n = 934), C. glabrata (n = 392) and C. parapsilosis complex (n = 225) exhibited 0.6%, 1.0% and 4.9% resistance to fluconazole and 0.1%, 2.5% and 0% resistance to micafungin, respectively. Mutations in fks hot-spot regions were confirmed in all nine micafungin non-susceptible C. glabrata. CONCLUSIONS: Antifungal resistance in contemporary isolates of Candida causing BSIs in Canada is uncommon. However, the proportion of C. glabrata isolates has increased and echinocandin resistance in this species has emerged. Ongoing surveillance of local hospital epidemiology and appropriate antifungal stewardship practices are necessary to preserve the utility of available antifungal agents.

The use of genome sequencing to investigate an outbreak of hospital-acquired mucormycosis in transplant patients.

We report three cases of hospital-acquired mucormycosis in heart and lung transplant patients over a 6-month period. Traditional epidemiological investigation tools were used to look for a common link between patients to explain the outbreak. Genome sequencing of each fungal strain was used to supplement the investigation. By disproving a close genetic link between infecting strains of mucormycosis, we were able to conclude the outbreak investigation. Genome sequencing is a novel tool that can be used in addition to traditional epidemiologic investigations to help determine linkage of patients during outbreak investigations.

CLSI Methods Development and Standardization Working Group Best Practices for Evaluation of Antimicrobial Susceptibility Tests.

Effective evaluations of antimicrobial susceptibility tests (ASTs) require robust study design. The Clinical and Laboratory Standards Institute (CLSI) Subcommittee on Antimicrobial Susceptibility Testing has recognized that many published studies reporting the performance of commercial ASTs (cASTs) suffer from major design and/or analysis flaws, rendering the results difficult or impossible to interpret. This minireview outlines the current consensus of the Methods Development and Standardization Working Group of the CLSI Subcommittee on Antimicrobial Susceptibility Testing regarding best practices for systematic evaluation of the performance of an AST, including the analysis and presentation of essential data intended for publication.

First Report of Wohlfahrtiimonas chitiniclastica Isolation from a Patient with Cellulitis in the United States.

We report the first documented isolation of Wohlfahrtiimonas chitiniclastica from a human in the United States. Initially misidentified as Acinetobacter lwoffii by Vitek-2, the isolate was subsequently identified as W. chitiniclastica by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing. While the clinical significance of the isolate in this case is unclear, it highlights the superior performance of MALDI-TOF MS for bacterial identification.