WGS of a cluster of MDR Shigella sonnei utilizing Oxford Nanopore R10.4.1 long-read sequencing.

OBJECTIVES: To utilize long-read nanopore sequencing (R10.4.1 flowcells) for WGS of a cluster of MDR Shigella sonnei, specifically characterizing genetic predictors of antimicrobial resistance (AMR). METHODS: WGS was performed on S. sonnei isolates identified from stool and blood between September 2021 and October 2022. Bacterial DNA from clinical isolates was extracted on the MagNA Pure 24 and sequenced on the GridION utilizing R10.4.1 flowcells. Phenotypic antimicrobial susceptibility testing was interpreted based on CLSI breakpoints. Sequencing data were processed with BugSeq, and AMR was assessed with BugSplit and ResFinder. RESULTS: Fifty-six isolates were sequenced, including 53 related to the cluster of cases. All cluster isolates were identified as S. sonnei by sequencing, with global genotype 3.6.1.1.2 (CipR.MSM5), MLST 152 and PopPUNK cluster 3. Core genome MLST (cgMLST, examining 2513 loci) and reference-based MLST (refMLST, examining 4091 loci) both confirmed the clonality of the isolates. Cluster isolates were resistant to ampicillin (blaTEM-1), trimethoprim/sulfamethoxazole (dfA1, dfrA17; sul1, sul2), azithromycin (ermB, mphA) and ciprofloxacin (gyrA S83L, gyrA D87G, parC S80I). No genomic predictors of resistance to carbapenems were identified. CONCLUSIONS: WGS with R10.4.1 enabled rapid sequencing and identification of an MDR S. sonnei community cluster. Genetic predictors of AMR were concordant with phenotypic antimicrobial susceptibility testing.

Evaluating the accuracy of the MBT Lipid Xtract Kit for assessing colistin resistance in comparison to broth microdilution.

Colistin resistance testing methods such as broth microdilution (BMD) are time-consuming and labour intensive for clinical laboratories. MBT Lipid Xtract Kit on MALDI Biotyper Sirius System (Bruker, Billerica, MA, USA) utilizes lipidomic analysis to identify specific cell wall modifications associated with colistin resistance. We compared MBT to BMD (ComASP Colistin, Liofilchem) across 36 Gram-negative isolates (non-resistant MIC ≤2 µg ml-1, resistant MIC ≥4 µg ml-1). All samples were tested twice on MBT with discrepant results repeated before assessing categorical agreement between MBT and BMD. 44.4% (16/36) of isolates were colistin resistant via BMD. MBT Lipid Xtract had 80.6% agreement (29/36) with BMD, with 5/7 discrepancies corrected to match upon repeat testing. There was 100% agreement for Escherichia coli isolates (n=16). The whole-genome sequencing was completed on the two discrepant Klebsiella pneumoniae isolates, with variants within colistin resistance-associated loci identified (MIC 0.5 µg ml-1: arnC S30T, pmrB T246A, lapB N212T, lpxM S253G, crrB Q287K and MIC >16 µg ml-1: arnC S30T, pmrB R90insRN, pmrB T246A, pmrA E57G, lpxM S253G). Further evaluation, particularly for non-E. coli, of MBT is required prior to implementation in clinical laboratories.

Melting curve analysis reveals false-positive norovirus detection in a molecular syndromic panel.

BACKGROUND: Molecular syndromic panels can improve rapidity of results and ease clinical laboratory workflow, although caution has been raised for potential false-positive results. Upon implementation of a new panel for infectious diarrhea (BioFire® FilmArray® Gastrointestinal [GI] Panel, bioMérieux) in our clinical laboratory, a higher than expected number of stool samples with norovirus were detected. OBJECTIVES: The goal of this study was to investigate positive percent agreement and the false-positive rate of norovirus detected by the multiplex BioFire GI panel compared to a singleplex commercial assay. STUDY DESIGN: From October 2023 to January 2024, all prospective stool samples with a positive norovirus result by BioFire had melting curves reviewed manually using the BioFire FilmArray Torch System. Stool samples further underwent testing by a supplementary real-time RT-PCR assay (Xpert® Norovirus, Cepheid) for comparative analysis. RESULTS: Of the 50 stool samples with norovirus detected by BioFire, 18 (36 %) tested negative by Xpert (deemed "false-positives"). Furthermore, melting curve analysis revealed nearly all of these samples had atypical melting curve morphologies for the "Noro-1" target on BioFire (16/18, 89 %), which was statistically significant (Odds Ratio 173.2, 95 % CI [22.2, 5326.9], p < 0.0001). Stool samples with multiple pathogens detected by BioFire including norovirus were not more likely to produce false-positive norovirus results (Odds Ratio 1, 95 % CI [0.3, 3.3], p = 1). CONCLUSIONS: Although not described in the manufacturer's Instructions for Use, we propose routine manual review of melting curves for the BioFire GI panel prior to reporting, to mitigate potential false-positive norovirus results.

Matrix-assisted laser desorption/ionization time-of-flight MS for the accurate identification of Burkholderia cepacia complex and Burkholderia gladioli in the clinical microbiology laboratory.

Introduction. Burkholderia cepacia complex (Bcc) bacteria, currently consisting of 23 closely related species, and Burkholderia gladioli, can cause serious and difficult-to-treat infections in people with cystic fibrosis. Identifying Burkholderia bacteria to the species level is considered important for understanding epidemiology and infection control, and predicting clinical outcomes. Matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF) is a rapid method recently introduced in clinical laboratories for bacterial species-level identification. However, reports on the ability of MALDI-TOF to accurately identify Bcc to the species level are mixed.Aim. The aim of this project was to evaluate the accuracy of MALDI-TOF using the Biotyper and VITEK MS systems in identifying isolates from 22 different Bcc species and B. gladioli compared to recA gene sequencing, which is considered the current gold standard for Bcc.Methodology. To capture maximum intra-species variation, phylogenetic trees were constructed from concatenated multi-locus sequence typing alleles and clustered with a novel k-medoids approach. One hundred isolates representing 22 Bcc species, plus B. gladioli, were assessed for bacterial identifications using the two MALDI-TOF systems.Results. At the genus level, 100 and 97.0 % of isolates were confidently identified as Burkholderia by the Biotyper and VITEK MS systems, respectively; moreover, 26.0 and 67.0 % of the isolates were correctly identified to the species level, respectively. In many, but not all, cases of species misidentification or failed identification, a representative library for that species was lacking.Conclusion. Currently available MALDI-TOF systems frequently do not accurately identify Bcc bacteria to the species level.

Reduction in hospital-associated methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus with daily chlorhexidine gluconate bathing for medical inpatients.

BACKGROUND: Daily bathing with chlorhexidine gluconate (CHG) is increasingly used in intensive care units to prevent hospital-associated infections, but limited evidence exists for noncritical care settings. METHODS: A prospective crossover study was conducted on 4 medical inpatient units in an urban, academic Canadian hospital from May 1, 2014-August 10, 2015. Intervention units used CHG over a 7-month period, including a 1-month wash-in phase, while control units used nonmedicated soap and water bathing. Rates of hospital-associated methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE) colonization or infection were the primary end point. Hospital-associated S. aureus were investigated for CHG resistance with a qacA/B and smr polymerase chain reaction (PCR) and agar dilution. RESULTS: Compliance with daily CHG bathing was 58%. Hospital-associated MRSA and VRE was decreased by 55% (5.1 vs 11.4 cases per 10,000 inpatient days, P = .04) and 36% (23.2 vs 36.0 cases per 10,000 inpatient days, P = .03), respectively, compared with control cohorts. There was no significant difference in rates of hospital-associated Clostridium difficile. Chlorhexidine resistance testing identified 1 isolate with an elevated minimum inhibitory concentration (8 µg/mL), but it was PCR negative. CONCLUSIONS: This prospective pragmatic study to assess daily bathing for CHG on inpatient medical units was effective in reducing hospital-associated MRSA and VRE. A critical component of CHG bathing on medical units is sustained and appropriate application, which can be a challenge to accurately assess and needs to be considered before systematic implementation.