Journal of Clinical Microbiology最新文献

Whole genome sequencing is an essential cornerstone of pathogen surveillance and outbreak detection. Established sequencing technologies are currently being challenged by Oxford Nanopore Technologies (ONT), which offers an accessible and cost-effective alternative enabling gap-free assemblies of chromosomes and plasmids. Limited accuracy has hindered its use for investigating pathogen transmission, but recent technology updates have brought significant improvements. To evaluate its readiness for outbreak detection, we selected 78 Listeria monocytogenes isolates from diverse lineages or known epidemiological clusters for sequencing with ONT's V14 Rapid Barcoding Kit and R10.4.1 flow cells. The most accurate of several tested workflows generated assemblies with a median of one error (SNP or indel) per assembly. For 66 isolates, the cgMLST profiles from ONT-only assemblies were identical to those generated from Illumina data. Eight assemblies were of lower quality, with more than 20 erroneous sites each, primarily caused by methylations at the GAAGAC motif (5'-GAAG6mAC-3'/5'-GT4mCTTC-3'). This led to inaccurate clustering, failing to group isolates from a persistence-associated clone that carried the responsible restriction-modification system. Out of 50 methylation motifs detected among the 78 isolates, only the GAAGAC motif was linked to substantially increased error rates. Our study shows that most L. monocytogenes genomes assembled from ONT-only data are suitable for high-resolution genotyping, but further improvements of chemistries or basecallers are required for reliable routine use in outbreak and food safety investigations.

Repeat blood cultures are common in children after an initial positive culture. However, in contrast to adults, there are little data to help guide clinicians when a repeat culture is necessary to assess for persistent bacteremia. This study identifies factors associated with persistent bloodstream infections (BSI) in children to inform diagnostic stewardship. This cross-sectional study of children less than 18 years with at least one positive blood culture over a 5-year period utilized a generalized linear equation model to predict patient and microbial factors associated with persistent BSI defined as a positive blood culture with the same organism >48 hours after the index culture. Four hundred and five patients had 502 positive blood cultures yielding 556 organisms. Sixty-seven (13.2%) cultures were persistently positive. Anaerobic organisms (0/37) and Streptococcus species (0/104) were never recovered from repeat cultures. Staphylococcus aureus (OR 9.45, CI 5.15-17.35) and yeast (OR 78.18, CI 9.45-646.6) were statistically associated with persistent BSI. Patients with prior positive cultures (OR 1.44, CI 1.12-1.84) or a central venous catheter (OR 2.20, 95% CI 1.04-3.92) were also at risk for persistence. Immune dysfunction and elevated inflammatory markers at the time of the index blood culture were not significantly associated with persistence. Yeast or S. aureus were associated with persistent BSI, while anaerobes and Streptococcus species were never persistent. Patient characteristics at the time of blood draw did not predict persistence other than having previous positive blood cultures or a central venous catheter. These data can inform when repeat blood cultures have clinical value and reduce the risk of unnecessary blood draws in children.

Importance: We identify factors associated with bloodstream infection persistence in children. Our findings can help guide blood culture stewardship efforts in pediatric patients, especially in light of blood culture supply shortages.

Neisseria meningitidis (Nm) and Neisseria gonorrhoeae (Ng) are human pathogens that sometimes occupy the same anatomical niche. Ng, the causative agent of gonorrhea, infects 87 million individuals annually worldwide and is an urgent threat due to increasing drug resistance. Ng is a pathogen of the urogenital tract and may infect the oropharyngeal or rectal site, often asymptomatically. Conversely, Nm is an opportunistic pathogen. While often a commensal in the oropharyngeal tract, it is also the leading cause of bacterial meningitis with 1.2 million cases globally, causing significant morbidity and mortality. Horizontal gene transfer (HGT) is likely to occur between Ng and Nm due to their shared anatomical niches and genetic similarity, which poses challenges for accurate detection and treatment. Routine surveillance through the Gonococcal Isolate Surveillance Project and Strengthening the U.S. Response to Resistant Gonorrhea detected six concerning urogenital Neisseria isolates with contradicting species identification in Milwaukee (MIL). While all six isolates were positive for Ng using nucleic acid amplification testing (NAAT) and matrix-assisted laser desorption/ionization time of flight identified the isolates as Ng, two biochemical tests, Gonochek-II and API NH, classified them as Nm. To address this discrepancy, we performed whole-genome sequencing (WGS) using Illumina MiSeq on all isolates and employed various bioinformatics tools. Species detection analysis using BMScan, which uses WGS data, identified all isolates as Ng. Furthermore, Kraken revealed over 98% of WGS reads mapped to the Ng genome and <1% to Nm. Recombination analysis identified putative HGT in all MIL isolates within the γ-glutamyl transpeptidase (ggt) gene, a key component in the biochemical tests used to differentiate between Nm and Ng. Further analysis identified Nm as the source of HGT event. Specifically, the active Nm ggt gene replaced the Ng pseudogenes, ggt1 and ggt2. Together, this study demonstrates that closely related Neisseria species sharing a niche underwent HGT, which led to the misidentification of species following biochemical testing. Importantly, NAAT accurately detected Ng. The misidentification highlights the importance of using WGS to continually evaluate diagnostic or bacterial identification tests.

The emergence of metallo-β-lactamase (MBL)-producing Enterobacterales presents unique clinical treatment challenges. Recently developed β-lactam/ β-lactamase inhibitor combination agents, while effective against other carbapenemase-producing organisms, are notably ineffective against MBL producers. While MBLs do not hydrolyze monobactams (aztreonam), many MBL-producing organisms are resistant to aztreonam through alternate mechanisms, leaving cefiderocol as the sole monotherapy treatment option recommended for MBL producers. Recent guidelines for the treatment of MBL-harboring organisms have added combination therapy with aztreonam and ceftazidime-avibactam, using ceftazidime-avibactam as a source of the β-lactamase inhibitor avibactam. Current laboratory testing options for the combination of aztreonam-avibactam are limited to broth microdilution (BMD) and broth disk elution (BDE) methods, which are not practical in most clinical laboratories. In this study, we evaluated the performance of aztreonam/avibactam gradient strips on 103 MBL-producing Enterobacterales patient isolates as well as an additional 31 isolates from the CDC AR Bank. All MBL Enterobacterales patient isolates included in this study harbored a New Delhi metallo-β-lactamase (bla_NDM) gene. Essential agreement of gradient strip minimal inhibitory concentrations (MICs) for patient isolates compared to BMD was 93.2%. While there are no established breakpoints for aztreonam-avibactam, category agreement (CA) for patient isolates was 97.1% when using the CLSI aztreonam breakpoints. There were no major or very major errors observed. There were three minor errors. Precision for aztreonam-avibactam gradient strip diffusion was 100%. These data demonstrate that the use of gradient strip diffusion for aztreonam-avibactam MIC determination in MBL-producing Enterobacterales is a viable option for clinical laboratories.