Alison Laufer Halpin

Background: Wastewater surveillance at healthcare facilities for carbapenem-resistant (CR) bacteria harboring carbapenemase genes could result in earlier detection of circulating or emerging antimicrobial resistance (AR) threats. However, knowledge gaps exist regarding the CR bacterial community and carbapenemase genes present in facility wastewater and plumbing, which need to be addressed. Methods: Wastewater effluent samples from three skilled nursing facilities (SNFs) (Georgia, USA) were collected weekly over six months. Selective chromogenic media were examined for their performance in screening the samples for targeted CR bacteria. Culture-dependent and culture-independent methods (ie, PCR) were applied to enriched wastewater samples (n = 73) to screen isolates and pooled samples for the most common carbapenemase genes circulating in the United States (blaKPC, blaNDM, blaVIM, blaOXA-48-like, and blaIMP), and method sensitivity was compared. Whole genome sequencing (WGS) was used to identify all AR genes present in isolates. Results: Bacteria (n = 209 isolates) were identified using MALDI-TOF and included >20 different genera. Clinically relevant (Acinetobacter baumannii complex, Enterobacter cloacae complex, Enterococcus faecium, Stenotrophomonas spp.) and environmental/opportunistic (Comamonas spp., Pandoraea spp., Pseudomonas putida group) CR bacteria were identified in each of the three SNFs. In Facility A and C, only blaKPC was detected in 9 (26.5%) of 34 and 14 (11.4%) of 123 recovered isolates, respectively. There were no PCR-positive results in Facility B. WGS confirmed the presence or absence of all genes screened by PCR (100% concordance). Conclusions: These data provide a proof-of-concept and insights into CR bacteria present in healthcare facility wastewater using culture, PCR, and sequencing methods.

Wastewater surveillance at healthcare facilities for carbapenem-resistant (CR) bacteria harboring carbapenemase genes could result in earlier detection of circulating or emerging antimicrobial resistance (AR) threats. However, knowledge gaps exist regarding the CR bacterial community and carbapenemase genes present in facility wastewater and plumbing, which need to be addressed. Wastewater effluent samples from three skilled nursing facilities (SNFs) (Georgia, USA) were collected weekly over six months each. Selective chromogenic media were examined for their performance in screening the samples for targeted CR bacteria. Culture-dependent and culture-independent methods (i.e., PCR) were applied to enriched wastewater samples (n=73) to screen isolates and pooled samples for the most common carbapenemase genes circulating in the United States ( bla _KPC , bla _NDM , bla _VIM , bla _OXA-48-like , and bla _IMP ), and method sensitivity was compared. Whole genome sequencing (WGS) was used to identify all AR genes present in isolates. Bacteria (n=209 isolates) were identified using MALDI-TOF and included >20 different genera. Clinically relevant ( Acinetobacter baumannii complex, Enterobacter cloacae complex, Enterococcus faecium, Stenotrophomonas spp.) and environmental/opportunistic ( Comamonas spp., Pandoraea spp ., Pseudomonas putida group) CR bacteria were identified in each of the three SNFs. In Facility A and C, only bla _KPC was detected in 9 (26.5%) of 34 and 14 (11.4%) of 123 recovered isolates, respectively. There were no PCR-positive results in Facility B. WGS confirmed the presence or absence of all genes screened for by PCR (100% concordance). These data provide a proof of concept and insights into CR bacteria present in healthcare facility wastewater using culture, PCR, and sequencing methods.

During an outbreak investigation of infections following apheresis platelet transfusion, a potentially novel species in the Acinetobacter calcoaceticus-baumannii complex was identified. Here, we report the hybrid Nanopore-Illumina assembly resulting in a complete circular genome sequence of a strain of this species isolated from apheresis platelet product collected during the investigation.

Through their ability to hydrolyze carbapenems, Ambler class D beta-lactamases endanger patients by limiting the clinical efficacy of beta-lactam antimicrobials. Further, plasmid-mediated transmission can increase mobility of carbapenemase genes between bacteria and facilitate their spread between patients. In the United States and elsewhere, the plasmid-mediated Ambler class D carbapenemase genes bla _OXA-23-like , bla _{OXA-24/40-like} , and bla _OXA-58-like are commonly associated with Acinetobacter species and have rarely been reported outside of this genus. However, multiple recent international reports indicate detection of Enterobacterales isolates carrying Acinetobacter -associated class D carbapenemase genes. This evaluation aimed to provide insight into whether Enterobacterales harboring these class D genes may be circulating undetected in the United States, thereby signaling a need for adapting testing strategies to prioritize the detection of these potentially emerging public health threats. We analyzed whole-genome sequencing data generated through multiple Centers for Disease Control and Prevention (CDC) activities, including testing conducted across the Antimicrobial Resistance Laboratory Network, to determine whether any Enterobacterales isolates sequenced from 2017–2022 harbored Acinetobacter -associated class D carbapenemase genes. Among ∼10,000 predominantly carbapenem-resistant Enterobacterales isolates, we identified only a single Enterobacterales isolate harboring an Acinetobacter -associated class D gene – a Klebsiella pneumoniae isolate harboring a bla _OXA-23-like gene. Our findings suggest that bla _OXA-23-like , bla _{OXA-24/40-like} , and bla _OXA-58-like genes are rare among Enterobacterales isolates sequenced through CDC public health activities in the United States and do not warrant changes to current testing priorities at this time.

Bacterial pathogenicity has traditionally focused on gene-level content with experimentally confirmed functional properties. Hence, significant inferences are made based on similarity to known pathotypes and DNA-based genomic subtyping for risk. Herein, we achieved de novo prediction of human virulence in Klebsiella pneumoniae by expanding known virulence genes with spatially proximal gene discoveries linked by functional domain architectures across all prokaryotes. This approach identified gene ontology functions not typically associated with virulence sensu stricto. By leveraging machine learning models with these expanded discoveries, public genomes were assessed for virulence prediction using categorizations derived from isolation sources captured in available metadata. Performance for de novo strain-level virulence prediction achieved 0.81 F1-Score. Virulence predictions using expanded “discovered” functional genetic content were superior to that restricted to extant virulence database content. Additionally, this approach highlighted the incongruence in relying on traditional phylogenetic subtyping for categorical inferences. Our approach represents an improved deconstruction of genome-scale datasets for functional predictions and risk assessment intended to advance public health surveillance of emerging pathogens.

Enterococcus faecalis and Enterococcus faecium are frequent causes of healthcare-associated infections. Antimicrobial-resistant enterococci pose a serious public health threat, particularly vancomycin-resistant enterococci (VRE), for which treatment options are limited. The Centers for Disease Control and Prevention’s Division of Healthcare Quality Promotion Sentinel Surveillance system conducted surveillance from 2018 to 2019 to evaluate antimicrobial susceptibility profiles and molecular epidemiology of 205 E. faecalis and 180 E. faecium clinical isolates collected from nine geographically diverse sites in the United States. Whole genome sequencing revealed diverse genetic lineages, with no single sequence type accounting for more than 15% of E. faecalis or E. faecium . Phylogenetic analysis distinguished E. faecium from 19 E. lactis (previously known as E. faecium clade B). Resistance to vancomycin was 78.3% among E. faecium , 7.8% among E. faecalis , and did not occur among E. lactis isolates. Resistance to daptomycin and linezolid was rare: E. faecium (5.6%, 0.6%, respectively), E. faecalis (2%, 2%), and E. lactis (5.3%, 0%). All VRE harbored the vanA gene. Three of the seven isolates that were not susceptible to linezolid harbored optrA , one chromosomally located and two on linear plasmids that shared a conserved backbone with other multidrug-resistant conjugative linear plasmids. One of these isolates contained optrA and vanA co-localized on the linear plasmid. By screening all enterococci, 20% of E. faecium were predicted to harbor linear plasmids, whereas none were predicted among E. faecalis or E. lactis . Continued surveillance is needed to assess the future emergence and spread of antimicrobial resistance by linear plasmids and other mechanisms. IMPORTANCE: This work confirms prior reports of E. faecium showing higher levels of resistance to more antibiotics than E. faecalis and identifies that diverse sequence types are contributing to enterococcal infections in the United States. All VRE harbored the vanA gene. We present the first report of the linezolid resistance gene optrA on linear plasmids in the United States, one of which co-carried a vanA cassette. Additional studies integrating epidemiological, antimicrobial susceptibility, and genomic methods to characterize mechanisms of resistance, including the role of linear plasmids, will be critical to understanding the changing landscape of enterococci in the United States.