Objectives Linezolid is an important therapeutic option for the treatment of infections caused by VRE. Linezolid is a synthetic antimicrobial and resistance to this antimicrobial agent remains relatively rare. As a result, data on the comparative genomics of linezolid resistance determinants in Enterococcus faecium are relatively sparse. Methods To address this knowledge gap in E. faecium, we deployed phenotypic antibiotic susceptibility testing and Illumina WGS on hospital surface (environmental) and clinical isolates from the USA and Pakistan. Results We found complete concordance between isolate source country and mechanism of linezolid resistance, with all the US isolates possessing a 23S rRNA gene mutation and the Pakistan isolates harbouring two to three acquired antibiotic resistance genes. These resistance genes include the recently elucidated efflux-pump genes optrA and poxtA and a novel cfr-like variant. Although there was no difference in the linezolid MIC between the US and Pakistan isolates, there was a significant difference in the geometric mean of the MIC between the Pakistan isolates that had two versus three of the acquired antibiotic resistance genes. In five of the Pakistan E. faecium that possessed all three of the resistance genes, we found no difference in the local genetic context of poxtA and the cfr-like gene, but we identified different genetic contexts surrounding optrA. Conclusions These results demonstrate that E. faecium from different geographical regions employ alternative strategies to counter selective pressure of increasing clinical linezolid use.

Bacterial bloodstream infections (BSIs) resulting in late-onset sepsis affect up to half of extremely preterm infants and have substantial morbidity and mortality. Bacterial species associated with BSIs in neonatal intensive care units (NICUs) commonly colonize the preterm infant gut microbiome. Accordingly, we hypothesized that the gut microbiome is a reservoir of BSI-causing pathogenic strains that increase in abundance before BSI onset. We analyzed 550 previously published fecal metagenomes from 115 hospitalized neonates and found that recent ampicillin, gentamicin, or vancomycin exposure was associated with increased abundance of Enterobacteriaceae and Enterococcaceae in infant guts. We then performed shotgun metagenomic sequencing on 462 longitudinal fecal samples from 19 preterm infants (cases) with BSI and 37 non-BSI controls, along with whole-genome sequencing of the BSI isolates. Infants with BSI caused by Enterobacteriaceae were more likely than infants with BSI caused by other organisms to have had ampicillin, gentamicin, or vancomycin exposure in the 10 days before BSI. Relative to controls, gut microbiomes of cases had increased relative abundance of the BSI-causing species and clustered by Bray-Curtis dissimilarity according to BSI pathogen. We demonstrated that 11 of 19 (58%) of gut microbiomes before BSI, and 15 of 19 (79%) of gut microbiomes at any time, harbored the BSI isolate with fewer than 20 genomic substitutions. Last, BSI strains from the Enterobacteriaceae and Enterococcaceae families were detected in multiple infants, indicating BSI-strain transmission. Our findings support future studies to evaluate BSI risk prediction strategies based on gut microbiome abundance in hospitalized preterm infants.

Background Premature infants frequently receive antibiotics, which diminishes gut microbial diversity and increases susceptibility to infections by antibiotic resistant pathogens. Neonates with decreased gut microbiota diversity, termed dysbiotic, have dysregulated immune systems marked by increased concentrations of circulating activated T cells and decreased concentrations of circulating neutrophils and dendritic cells. We hypothesize that antibiotics (1) enrich for pathogens within the gut, 2) promote a systemic, proinflammatory host response, and 3) cause death in an antibiotic- and microbiome-specific manner in a gnotobiotic model of preterm gut microbiota disruption. Methods We colonized germ free (GF) dams with stools from preterm infants. Mouse pups acquire this neonatal microbiota, and at 10 days of life (DOL), we treat them with clinically-relevant doses of antibiotics subcutaneously for 3 days. We determined serum concentrations of antibiotics in 10 DOL pups using tandem mass spectrometry to achieve approximate pharmacokinetics as observed in the neonatal intensive care unit (NICU). We ascertained phylogenetic composition using metagenomic shotgun sequencing of individual pup fecal samples longitudinally. We performed flow cytometry on peripheral blood and gut permeability assays to determine the local and peripheral immune response. Results We found adding probenecid prolonged the half-life of ampicillin and meropenem allowing for an approximation of serum levels observed in the NICU with an every 8 hour dosing regimen. Using two representative microbiomes from human neonates (hereafter referred to as microbiota A or B), we show that 95% of pups given microbiota A survive versus 54% given microbiota B after meropenem/probenecid treatment (Fig. 1A; p<0.01; n= 18–42 mice in 3–6 independent experiments). Conversely, only 28% of microbiota-A humanized pups survive during ampicillin/probenecid treatment (Fig. 1; p<0.0001). Ampicillin-resistant Klebsiella species and E. coli dominated the gut of microbiota A-humanized pups who succumbed during ampicillin/probenecid treatment whereas Enterococci dominated the gut of microbiota B-humanized pups who died during treatment. To test the reproducibility of this phenotype, we colonized mice with 2 additional preterm neonatal microbiomes with similar compositions to microbiota A and B (D and C, respectively). We found that microbiota-C humanized pups were similarly dominated by Enterococcus faecalis resulting in 42% mortality during meropenem/probenecid treatment (Fig. 1). Pups colonized with microbiota B had decreased circulating granulocytes, B cells, and CD8+ T cells at sacrifice after treatment compared to microbiota A-humanized pups. We next assessed gut permeability after antibiotic treatment by measuring 4kDa FITC-Dextran in mouse serum after oral gavage. Microbiota-A humanized pups treated with ampicillin/probenecid and microbiota B-humanized pups treated with meropenem/probenecid had elevated serum levels of FITC-Dextran (p<0.05 relative to vehicle control, one way ANOVA), indicative of increased gut permeability. Conclusions Our model of preterm microbiota perturbation by antibiotics demonstrates increased gut permeability, proinflammatory immune response, and death dependent on the microbiota-antibiotic combination. Our transgenerational humanized-microbiota mouse model can be utilized to determine antibiotic by microbiota perturbation and examine risks of late onset sepsis from specific antimicrobial administration. This research can lead to a personalized medicine approach of antibiotic treatment in the NICU to limit antibiotic side effects and mortality.