Whole-genome sequencing of carbapenem-resistant Enterobacterales isolates in southeast Louisiana reveals persistent genetic clusters spanning multiple locations.

BACKGROUND: We investigated 51 g-negative carbapenem-resistant Enterobacterales (CRE) isolates collected from 22 patients over a five-year period from six health care institutions in the Ochsner Health network in southeast Louisiana. METHODS: Short genomic reads were generated using Illumina sequencing and assembled for each isolate. Isolates were classified as Enterobacter spp. (n = 20), Klebsiella spp. (n = 30), and Escherichia coli (n = 1) and grouped into 19 different multi-locus sequence types (MLST). Species and patient-specific core genomes were constructed representing ∼50% of the chromosomal genome. RESULTS: We identified two sets of patients with genetically related infections; in both cases, the related isolates were collected > 6 months apart, and in one case, the isolates were collected in different locations. On the other hand, we identified four sets of patients with isolates of the same species collected within 21 days from the same location; however, none had genetically related infections. Genes associated with resistance to carbapenem drugs (blaKPC and/or blaCTX-M-15) were found in 76% of the isolates. We found three blaKPC variants (blaKPC-2, blaKPC-3, and blaKPC-4) associated with four different Enterobacter MLST variants, and two blaKPC variants (blaKPC-2, blaKPC-3) associated with seven different Klebsiella MLST variants. CONCLUSIONS: Molecular surveillance is increasingly becoming a powerful tool to understand bacterial spread in both community and clinical settings. This study provides evidence that genetically related infections in clinical settings do not necessarily reflect temporal associations, and vice versa. Our results also highlight the regional genomic and resistance diversity within related bacterial lineages.

Comparing antimicrobial resistant genes and phenotypes across multiple sequencing platforms and assays for Enterobacterales clinical isolates.

INTRODUCTION: Whole genome sequencing (WGS) of bacterial isolates can be used to identify antimicrobial resistance (AMR) genes. Previous studies have shown that genotype-based AMR has variable accuracy for predicting carbapenem resistance in carbapenem-resistant Enterobacterales (CRE); however, the majority of these studies used short-read platforms (e.g. Illumina) to generate sequence data. In this study, our objective was to determine whether Oxford Nanopore Technologies (ONT) long-read WGS would improve detection of carbapenem AMR genes with respect to short-read only WGS for nine clinical CRE samples. We measured the minimum inhibitory breakpoint (MIC) using two phenotype assays (MicroScan and ETEST) for six antibiotics, including two carbapenems (meropenem and ertapenem) and four non-carbapenems (gentamicin, ciprofloxacin, cefepime, and trimethoprim/sulfamethoxazole). We generated short-read data using the Illumina NextSeq and long-read data using the ONT MinION. Four assembly methods were compared: ONT-only assembly; ONT-only assembly plus short-read polish; ONT + short-read hybrid assembly plus short-read polish; short-read only assembly. RESULTS: Consistent with previous studies, our results suggest that the hybrid assembly produced the highest quality results as measured by gene completeness and contig circularization. However, ONT-only methods had minimal impact on the detection of AMR genes and plasmids compared to short-read methods, although, notably, differences in gene copy number differed between methods. All four assembly methods showed identical presence/absence of the blaKPC-2 carbapenemase gene for all samples. The two phenotype assays showed 100% concordant results for the non-carbapenems, but only 65% concordance for the two carbapenems. The presence/absence of AMR genes was 100% concordant with AMR phenotypes for all four non-carbapenem drugs, although only 22%-50% sensitivity for the carbapenems. CONCLUSIONS: Overall, these findings suggest that the lack of complete correspondence between CRE AMR genotype and phenotype for carbapenems, while concerning, is independent of sequencing platform/assembly method.

SARS-CoV-2 genomic surveillance as an evidence-based infection control approach in an offshore petroleum employee population.

BACKGROUND: Industrial hygienists (IH) in the oil and gas business instituted an extraordinary number of safety protocols to limit spread of SARS-CoV-2 onto offshore platforms in the Gulf of Mexico. We used genomic surveillance to provide actionable information concerning the efficacy of their efforts. METHODS: Over 6 months, employees at a single company were serology and PCR tested during a 1-5 day predeployment quarantine and when postdeployment symptoms were reported. From each positive test (n = 49), SARS-CoV-2 genomes were sequenced. Phylogenetic analysis was used to investigate the epidemiology of transmissions. RESULTS: Genomic surveillance confirmed 2 viral strains were infecting 18 offshore workers. Genomic data combined with epidemiological data suggested that a change in quarantine protocols contributed to these outbreaks. A pre-deployment outbreak involved a WHO variant of interest (Theta) that had infected 4 international workers. Two additional predeployment clusters of infections were identified. CONCLUSIONS: Our findings support that IH quarantine/testing protocols limited viral transmissions, halted offshore outbreaks, and stopped the spread of a variant of interest. The study demonstrates how genomic data can be used to understand viral transmission dynamics in employee populations and evaluate safety protocols in the offshore oil and gas industry.