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The environmental microbiome harbors a vast repertoire of antibiotic resistance genes (ARGs) which can serve as evolutionary predecessors for ARGs found in pathogenic bacteria, or can be directly mobilized to pathogens in the presence of selection pressures. Thus, ARGs from benign environmental bacteria are an important resource for understanding clinically relevant resistance. Here, we conduct a comprehensive functional analysis of the Antibiotic_NAT family of aminoglycoside acetyltransferases. We determined a pan-family antibiogram of 21 Antibiotic_NAT enzymes, including 8 derived from clinical isolates and 13 from environmental metagenomic samples. We find that environment-derived representatives confer high-level, broad-spectrum resistance, including against the atypical aminoglycoside apramycin, and that a metagenome-derived gene likely is ancestral to an aac(3) gene found in clinical isolates. Through crystallographic analysis, we rationalize the molecular basis for diversification of substrate specificity across the family. This work provides critical data on the molecular mechanism underpinning resistance to established and emergent aminoglycoside antibiotics and broadens our understanding of ARGs in the environment.

Bacterial pathogens that infect patients also contaminate hospital surfaces. These contaminants impact hospital infection control and epidemiology, prompting quantitative examination of their transmission dynamics. Here we investigate spatiotemporal and phylogenetic relationships of multidrug resistant (MDR) bacteria on intensive care unit surfaces from two hospitals in the United States (US) and Pakistan collected over one year. MDR bacteria isolated from 3.3% and 86.7% of US and Pakistani surfaces, respectively, include common nosocomial pathogens, rare opportunistic pathogens, and novel taxa. Common nosocomial isolates are dominated by single lineages of different clones, are phenotypically MDR, and have high resistance gene burdens. Many resistance genes (e.g., blaNDM, blaOXA carbapenamases), are shared by multiple species and flanked by mobilization elements. We identify Acinetobacter baumannii and Enterococcus faecium co-association on multiple surfaces, and demonstrate these species establish synergistic biofilms in vitro. Our results highlight substantial MDR pathogen burdens in hospital built-environments, provide evidence for spatiotemporal-dependent transmission, and demonstrate potential mechanisms for multi-species surface persistence., The authors investigate the burden and spatiotemporal dynamics of multidrug-resistant bacteria on intensive care unit surfaces from two hospitals in the US and Pakistan over a year. They find Acinetobacter baumannii and Enterococcus faecium co-association, likely due to synergistic biofilm formation.

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.

In agricultural settings, microbes and antimicrobial resistance genes (ARGs) have the potential to be transferred across diverse environments and ecosystems. The consequences of these microbial transfers are unclear and understudied. On dairy farms, the storage of cow manure in manure pits and subsequent application to field soil as a fertilizer may facilitate the spread of the mammalian gut microbiome and its associated ARGs to the environment. To determine the extent of both taxonomic and resistance similarity during these transitions, we collected fresh manure, manure from pits, and field soil across 15 different dairy farms for three consecutive seasons. We used a combination of shotgun metagenomic sequencing and functional metagenomics to quantitatively interrogate taxonomic and ARG compositional variation on farms. We found that as the microbiome transitions from fresh dairy cow manure to manure pits, microbial taxonomic compositions and resistance profiles experience distinct restructuring, including decreases in alpha diversity and shifts in specific ARG abundances that potentially correspond to fresh manure going from a gut-structured community to an environment-structured community. Further, we did not find evidence of shared microbial community or a transfer of ARGs between manure and field soil microbiomes. Our results suggest that fresh manure experiences a compositional change in manure pits during storage and that the storage of manure in manure pits does not result in a depletion of ARGs. We did not find evidence of taxonomic or ARG restructuring of soil microbiota with the application of manure to field soils, as soil communities remained resilient to manure-induced perturbation. IMPORTANCE The addition of dairy cow manure—stored in manure pits—to field soil has the potential to introduce not only organic nutrients but also mammalian microbial communities and antimicrobial resistance genes (ARGs) to soil communities. Using shotgun sequencing paired with functional metagenomics, we showed that microbial community composition changed between fresh manure and manure pit samples with a decrease in gut-associated pathobionts, while ARG abundance and diversity remained high. However, field soil communities were distinct from those in manure in both microbial taxonomic and ARG composition. These results broaden our understanding of the transfer of microbial communities in agricultural settings and suggest that field soil microbial communities are resilient against the deposition of ARGs or microbial communities from manure.

Antibiotics are deployed against bacterial pathogens, but their targeting of conserved microbial processes means they also collaterally perturb the commensal microbiome. To understand acute and persistent effects of antibiotics on the gut microbiota of healthy adult volunteers, we quantify microbiome dynamics before, during, and 6 months after exposure to 4 commonly used antibiotic regimens. We observe an acute decrease in species richness and culturable bacteria after antibiotics, with most healthy adult microbiomes returning to pre-treatment species richness after 2 months, but with an altered taxonomy, resistome, and metabolic output, as well as an increased antibiotic resistance burden. Azithromycin delays the recovery of species richness, resulting in greater compositional distance. A subset of volunteers experience a persistent reduction in microbiome diversity after antibiotics and share compositional similarities with patients hospitalized in intensive care units. These results improve our quantitative understanding of the impact of antibiotics on commensal microbiome dynamics, resilience, and recovery.

Commensal gut bacterial communities (microbiomes) are predicted to influence human health and disease1,2. Neonatal gut microbiomes are colonized with maternal and environmental flora and mature toward a stable composition over 2–3 years3,4. To study pre- and postnatal determinants of infant microbiome development, we analyzed 402 fecal metagenomes from 60 infants aged 0–8 months, using longitudinal generalized linear mixed models (GLMMs). Distinct microbiome signatures correlated with breastfeeding, formula ingredients, and maternal gestational weight gain (GWG). Amino acid synthesis pathway accretion in breastfed microbiomes complemented normative breastmilk composition. Prebiotic oligosaccharides, designed to promote breastfed-like microflora5, predicted functional pathways distinct from breastfed infant microbiomes. Soy formula in six infants was positively associated with Lachnospiraceae and pathways suggesting a short-chain fatty acid (SCFA)-rich environment, including glycerol to 1-butanol fermentation, which is potentially dysbiotic. GWG correlated with altered carbohydrate degradation and enriched vitamin synthesis pathways. Maternal and postnatal antibiotics predicted microbiome alterations, while delivery route had no persistent effects. Domestic water source correlates suggest water may be an underappreciated determinant of microbiome acquisition. Clinically important microbial pathways with statistically significant dietary correlates included dysbiotic markers6,7, core enterotype features8, and synthesis pathways for enteroprotective9 and immunomodulatory10,11 metabolites, epigenetic mediators1, and developmentally critical vitamins12, warranting further investigation.

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 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.

Background: Antimicrobial exposure is a significant risk factor for the development of antibiotic-resistant organisms (ARO); however, the depth and duration of this impact is not well described. The study goal is to define impact of antibiotics on the gut microbiome of healthy volunteers (HVs). Methods: HVs were randomized to receive either 5 days of levofloxacin (LVX), azithromycin (AZM), cefpodoxime (CPD), or AZM + CPD (Fig. 1). Stool samples were collected at 15 time points per patient before, during, and after antibiotics. Remnant stool samples from the microbiology laboratory were collected from patients admitted to the medical intensive care unit (MICU) as a comparison of the microbiome in a critically ill state. DNA was extracted from samples and was submitted for shotgun sequencing. Relative abundance, resistome, and metabolic pathway abundance of bacterial taxa were determined and statistical analysis conducted in R software. Results: In total, 289 stool specimens from 20 HVs, and 26 remnant stool specimens were obtained from patients admitted from the MICU (Fig. 1). Community diversity and richness decreased in the first week post-ABX for all HVs (P < .01). Linear discriminant analysis identified Bacteroides and Clostridium as taxonomic groups enriched after CPD, while AZM and LVX produced a relative abundance increase in diverse Firmicutes spp. Longitudinal tracking confirmed that after all antibiotics except LVX, HV microbiomes lost species diversity and shifted toward a state similar to that observed in MICU patients (Fig. 2). The gut microbiome of most HVs exhibited resiliency and returned to a higher diversity level similar to their starting point; however, 10% of HVs did not. Moreover, antibiotic-specific increases in resistance markers reveal innate resistance to β-lactams and macrolides within the gut microbiome of the HVs. Finally, HV microbiomes, which shifted toward a MICU-like taxonomic state, also clustered with microbial metabolic profiles from MICU patients.The HV microbial metabolic profiles were significantly enriched for important biosynthesis pathways producing chorismate and polysaccharides. MICU patient gut microbiomes were enriched for fatty acid regulation and quinolone biosynthesis, and for many degradation pathways important for different aspects of antibiotic resistance such as membrane integrity, alternative respiration, and antibiotic inactivation. Conclusions: Short courses of antibiotics can cause acute and chronic microbiome disruptions in HVs, as evidenced by decreased microbiome diversity and increases in specific innate resistance elements. These data support the need for antimicrobial stewardship to support rationale antibiotic use to prevent gut microbiome disruptions.Funding: CDC BAA 200-2016-91962Disclosures: None

Background Prophylactic cotrimoxazole treatment is recommended in human immunodeficiency virus (HIV)–exposed, uninfected (HEU) infants, but the effects of this treatment on developing HEU infant gut microbiotas and resistomes are largely undefined. Methods We analyzed whole-metagenome sequencing data from 163 longitudinally collected stool samples from 63 HEU infants randomized to receive (n = 34; CTX-T) or to not receive (n = 29; CTX-N) prophylactic cotrimoxazole treatment. We generated taxonomic, functional pathway, and resistance gene profiles for each sample and compared microbiome signatures between the CTX-T and CTX-N infants. Results Metagenomic analysis did not reveal significant differences in taxonomic or functional pathway α-diversity between CTX-T and CTX-N infants. In contrast, resistance gene prevalence (P = .00719) and α-diversity (P = .0045) increased in CTX-T infants. These differences increased over time for both resistance gene prevalence measured by log-normalized abundance (4-month mean, 0.71 [95% confidence interval {CI}, .2–1.2] and 6-month mean, 0.85 [95% CI, .1–1.7]) and α-diversity (P = .0045). Unlike α-diversity, interindividual gut microbiome taxonomic (mean, −0.11 [95% CI, −.15 to −.077]), functional taxonomic (mean, −0.050 [95% CI, −.084 to −.017]), and resistance gene (mean, −0.13 [95% CI, −.17 to −.099]) β-diversity decreased in CTX-T infants compared with CTX-N infants. These results are consistent with persistent antibiotic selection pressure. Conclusions Cotrimoxazole prophylaxis in HEU infants decreased gut microbiome β-diversity and increased antibiotic resistance gene α-diversity and prevalence. Antibiotic resistance is a growing threat, especially in low- and middle-income countries where the higher perinatal HIV exposure rates result in cotrimoxazole prophylaxis. Understanding effects from current HEU infant antibiotic prophylaxis guidelines will inform guideline revisions and efforts to reduce increasing antibiotic resistance.

Carbapenem-resistant Enterobacteriaceae (CRE) are an important public health and infection prevention threat. CRE are typically detected via phenotypic antimicrobial susceptibility testing (AST), for which interpretive standards were modified in recent years. Our objective was to measure the impact of breakpoint changes on AST interpretation for CRE. Zone sizes from disk diffusion AST for Enterobacteriaceae isolates recovered from clinical cultures over a 1-year period (n = 10,183) and CRE from clinical and environmental sources from the USA and Pakistan (n = 342) were evaluated. Results were interpreted according to historical (CLSI M100-S19) and current (CLSI M100-S29) breakpoints. Interpretive errors were calculated according to the FDA definitions. Using current breakpoints as the reference standard, 56 (17%) very major (false susceptibility) errors occurred for cefepime and 13 (45%) very major errors for meropenem interpretation using historical breakpoints in clinical isolates of Enterobacteriaceae, corresponding to 12 carbapenemase-producing CRE that would have been missed during the 1-year period. For confirmed blaKPC CP-CRE clinical and environmental isolates (n = 149), the very major error rate for historic breakpoints was 8%, 30%, 63%, and 0% for cefepime, meropenem, imipenem, and ertapenem, respectively. For blaKPC isolates, the use of historical breakpoints would have led to 42 (28%) reports of false susceptibility to meropenem. Failure to adopt updated AST breakpoints may lead to reports of false susceptibility for antimicrobials commonly used to treat Gram-negative infections and preclude recognition of CRE. Such errors could negatively impact patient care and hamper infection control and public health efforts.

Antimicrobial resistance extracts high morbidity, mortality and economic costs yearly by rendering bacteria immune to antibiotics. Identifying and understanding antimicrobial resistance are imperative for clinical practice to treat resistant infections and for public health efforts to limit the spread of resistance. Technologies such as next-generation sequencing are expanding our abilities to detect and study antimicrobial resistance. This Review provides a detailed overview of antimicrobial resistance identification and characterization methods, from traditional antimicrobial susceptibility testing to recent deep-learning methods. We focus on sequencing-based resistance discovery and discuss tools and databases used in antimicrobial resistance studies.

We sequenced a carbapenem-resistant Shewanella putrefaciens isolate cultured from the sink handle of a Pakistan hospital room. Assembly annotation indicates that the isolate has a chromosomal bla OXA-436 carbapenemase and a plasmid-borne bla NDM-1 gene. To our knowledge, this is the first report of a Shewanella species harboring bla NDM .

Carbapenems, our one-time silver bullet for multidrug resistant bacterial infections, are now threatened by widespread dissemination of carbapenem-resistant Enterobacteriaceae (CRE). Successful expansion of Enterobacteriaceae clonal groups and frequent horizontal gene transfer of carbapenemase expressing plasmids are causing increasing carbapenem resistance. Recent advances in genetic and phenotypic detection facilitate global surveillance of CRE diversity and prevalence. In particular, whole genome sequencing enabled efficient tracking, annotation, and study of genetic elements colocalized with carbapenemase genes on chromosomes and on plasmids. Improved characterization helps detail the co-occurrence of other antibiotic resistance genes in CRE isolates and helps identify pan-drug resistance mechanisms. The novel β-lactamase inhibitor, avibactam, combined with ceftazidime or aztreonam, is a promising CRE treatment compared to current colistin or tigecycline regimens. To halt increasing CRE-associated morbidity and mortality, we must continue quality, cooperative monitoring and urgently investigate novel treatments.

Cancer metastasis is an invasive process that involves the transplantation of cells into new environments. Since human placentation is also invasive, hypotheses about a relationship between invasive placentation in eutherian mammals and metastasis have been proposed. The relationship between metastatic cancer and invasive placentation is usually presented in terms of antagonistic pleiotropy. According to this hypothesis, evolution of invasive placentation also established the mechanisms for cancer metastasis. Here, in contrast, we argue that the secondary evolution of less invasive placentation in some mammalian lineages may have resulted in positive pleiotropic effects on cancer survival by lowering malignancy rates. These positive pleiotropic effects would manifest themselves as resistance to cancer cell invasion. To provide a preliminary test of this proposal, we re-analyze data from Priester and Mantel (Occurrence of tumors in domestic animals. Data from 12 United States and Canadian colleges of veterinary medicine. J Natl Cancer Inst 1971; 47: :1333-44) about malignancy rates in cows, horses, cats and dogs. From our analysis we found that equines and bovines, animals with less invasive placentation, have lower rates of metastatic cancer than felines and canines in skin and glandular epithelial cancers as well as connective tissue sarcomas. We conclude that a link between type of placentation and species-specific malignancy rates is more likely related to derived mechanisms that suppress invasion rather than different degrees of fetal placental aggressiveness.