Daily occupational exposure in swine farm alters human skin microbiota and antibiotic resistome.

Antimicrobial resistance (AMR) is a major threat to global public health, and antibiotic resistance genes (ARGs) are widely distributed across humans, animals, and environment. Farming environments are emerging as a key research area for ARGs and antibiotic resistant bacteria (ARB). While the skin is an important reservoir of ARGs and ARB, transmission mechanisms between farming environments and human skin remain unclear. Previous studies confirmed that swine farm environmental exposures alter skin microbiome, but the timeline of these changes is ill defined. To improve understanding of these changes and to determine the specific time, we designed a cohort study of swine farm workers and students through collected skin and environmental samples to explore the impact of daily occupational exposure in swine farm on human skin microbiome. Results indicated that exposure to livestock-associated environments where microorganisms are richer than school environment can reshape the human skin microbiome and antibiotic resistome. Exposure of 5 h was sufficient to modify the microbiome and ARG structure in workers' skin by enriching microorganisms and ARGs. These changes were preserved once formed. Further analysis indicated that ARGs carried by host microorganisms may transfer between the environment with workers' skin and have the potential to expand to the general population using farm workers as an ARG vector. These results raised concerns about potential transmission of ARGs to the broader community. Therefore, it is necessary to take corresponding intervention measures in the production process to reduce the possibility of ARGs and ARB transmission.

Acute and persistent effects of commonly used antibiotics on the gut microbiome and resistome in healthy adults.

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.

Structural and molecular rationale for the diversification of resistance mediated by the Antibiotic_NAT family.

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.

Destination shapes antibiotic resistance gene acquisitions, abundance increases, and diversity changes in Dutch travelers.

BACKGROUND: Antimicrobial-resistant bacteria and their antimicrobial resistance (AMR) genes can spread by hitchhiking in human guts. International travel can exacerbate this public health threat when travelers acquire AMR genes endemic to their destinations and bring them back to their home countries. Prior studies have demonstrated travel-related acquisition of specific opportunistic pathogens and AMR genes, but the extent and magnitude of travel's effects on the gut resistome remain largely unknown. METHODS: Using whole metagenomic shotgun sequencing, functional metagenomics, and Dirichlet multinomial mixture models, we investigated the abundance, diversity, function, resistome architecture, and context of AMR genes in the fecal microbiomes of 190 Dutch individuals, before and after travel to diverse international locations. RESULTS: Travel markedly increased the abundance and α-diversity of AMR genes in the travelers' gut resistome, and we determined that 56 unique AMR genes showed significant acquisition following international travel. These acquisition events were biased towards AMR genes with efflux, inactivation, and target replacement resistance mechanisms. Travel-induced shaping of the gut resistome had distinct correlations with geographical destination, so individuals returning to The Netherlands from the same destination country were more likely to have similar resistome features. Finally, we identified and detailed specific acquisition events of high-risk, mobile genetic element-associated AMR genes including qnr fluoroquinolone resistance genes, blaCTX-M family extended-spectrum ß-lactamases, and the plasmid-borne mcr-1 colistin resistance gene. CONCLUSIONS: Our results show that travel shapes the architecture of the human gut resistome and results in AMR gene acquisition against a variety of antimicrobial drug classes. These broad acquisitions highlight the putative risks that international travel poses to public health by gut resistome perturbation and the global spread of locally endemic AMR genes.

Manure Microbial Communities and Resistance Profiles Reconfigure after Transition to Manure Pits and Differ from Those in Fertilized Field Soil.

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.

Environmental remodeling of human gut microbiota and antibiotic resistome in livestock farms.

Anthropogenic environments have been implicated in enrichment and exchange of antibiotic resistance genes and bacteria. Here we study the impact of confined and controlled swine farm environments on temporal changes in the gut microbiome and resistome of veterinary students with occupational exposure for 3 months. By analyzing 16S rRNA and whole metagenome shotgun sequencing data in tandem with culture-based methods, we show that farm exposure shapes the gut microbiome of students, resulting in enrichment of potentially pathogenic taxa and antimicrobial resistance genes. Comparison of students' gut microbiomes and resistomes to farm workers' and environmental samples revealed extensive sharing of resistance genes and bacteria following exposure and after three months of their visit. Notably, antibiotic resistance genes were found in similar genetic contexts in student samples and farm environmental samples. Dynamic Bayesian network modeling predicted that the observed changes partially reverse over a 4-6 month period. Our results indicate that acute changes in a human's living environment can persistently shape their gut microbiota and antibiotic resistome.

Breakpoint beware: reliance on historical breakpoints for Enterobacteriaceae leads to discrepancies in interpretation of susceptibility testing for carbapenems and cephalosporins and gaps in detection of carbapenem-resistant organisms.

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.

Cotrimoxazole Prophylaxis Increases Resistance Gene Prevalence and α-Diversity but Decreases ß-Diversity in the Gut Microbiome of Human Immunodeficiency Virus-Exposed, Uninfected Infants.

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]) ß-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 ß-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.

Spatiotemporal dynamics of multidrug resistant bacteria on intensive care unit surfaces.

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.

Phenotypic and genotypic characterization of linezolid-resistant Enterococcus faecium from the USA and Pakistan.

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.

Sequencing-based methods and resources to study antimicrobial resistance.

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.

Genomic Characterization of Antibiotic Resistant Escherichia coli Isolated From Domestic Chickens in Pakistan.

Poultry husbandry is important for the economic health of Pakistan, but the Pakistani poultry industry is negatively impacted by infections from Escherichia coli. We performed Illumina whole genome sequencing on 92 E. coli isolates obtained from the livers of deceased chickens originating in five Pakistani geographical regions. Our analysis indicates that the isolates are predominantly from the B1 and A clade and harbor a diverse number of antibiotic resistance and virulence genes, with no linkage between phylogeny and antibiotic resistance gene presence but some association between phylogeny and virulence gene and SNP presence for the B1 and E phylogroups. The colistin resistance gene mcr-1 and the quinolone resistance gene qnrS1 were both found in 13/92 isolates. Alarmingly, 82/92 of the E. coli strains characterized in this study are multidrug resistant with 100% (92/92) resistance to lincomycin, 81.5% (75/92) to streptomycin, 79.3% (73/92) to ampicillin and 66.3% (61/92) to ciprofloxacin. These results provide a high-resolution analysis of poultry-associated E. coli isolates in an area with a high endemic burden of antibiotic resistance. Surveillance of antibiotic resistance in poultry associated E. coli isolates is an important pillar of the One Health concept to integrate analysis of potential pathogens in human, animal, and environmental niches.

Infant diet and maternal gestational weight gain predict early metabolic maturation of gut microbiomes.

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.

Superficieibacter electus gen. nov., sp. nov., an Extended-Spectrum ß-Lactamase Possessing Member of the Enterobacteriaceae Family, Isolated From Intensive Care Unit Surfaces.

Two Gram-negative bacilli strains, designated BP-1(T) and BP-2, were recovered from two different Intensive Care Unit surfaces during a longitudinal survey in Pakistan. Both strains were unidentified using the bioMerieux VITEK MS IVD v2.3.3 and Bruker BioTyper MALDI-TOF mass spectrometry platforms. To more precisely determine the taxonomic identity of BP-1(T) and BP-2, we employed a biochemical and phylogenomic approach. The 16S rRNA gene sequence of strain BP-1(T) had the highest identity to Citrobacter farmeri CDC 2991-81(T) (98.63%) Citrobacter amalonaticus CECT 863(T) (98.56%), Citrobacter sedlakii NBRC 105722(T) (97.74%) and Citrobacter rodentium NBRC 105723(T) (97.74%). The biochemical utilization scheme of BP-1(T) using the Analytic Profile Index for Enterobacteriaceae (API20E) indicated its enzymatic functions are unique within the Enterobacteriaceae but most closely resemble Kluyvera spp., Enterobacter cloacae and Citrobacter koseri/farmeri. Phylogenomic analysis of the shared genes between BP-1(T), BP-2 and type strains from Kluyvera, Citrobacter, Escherichia, Salmonella, Kosakonia, Siccibacter and Shigella indicate that BP-1(T) and BP-2 isolates form a distinct branch from these genera. Average Nucleotide Identity analysis indicates that BP-1(T) and BP-2 are the same species. The biochemical and phylogenomic analysis indicate strains BP-1(T) and BP-2 represent a novel species from a new genus within the Enterobacteriaceae family, for which the name Superficieibacter electus gen. nov., sp. nov., is proposed. The type strain is BP-1(T) (= ATCC BAA-2937, = NBRC 113412).

Draft Genome Sequence of the blaOXA-436- and blaNDM-1-Harboring Shewanella putrefaciens SA70 Isolate.

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 blaOXA-436 carbapenemase and a plasmid-borne blaNDM-1 gene. To our knowledge, this is the first report of a Shewanella species harboring blaNDM.

The rapid spread of carbapenem-resistant Enterobacteriaceae.

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 ß-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.

Malignant cancer and invasive placentation: A case for positive pleiotropy between endometrial and malignancy phenotypes.

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.