Antimicrobial Resistance in the Upper Respiratory Tract of Children Compared with Adults

ABSTRACTProphages are often involved in host survival strategies and contribute toward increasing the genetic diversity of the host genome. Prophages also drive horizontal propagation of various genes as vehicles. However, there are few retrospective studies contributing to the propagation of antimicrobial resistance (AMR) and virulence factor (VF) genes by prophage. We extracted the complete genome sequences of seven pathogens, including ESKAPE bacteria and Escherichia coli from a public database, and examined the distribution of both the AMR and VF genes in prophage-like regions. We found that the ratios of AMR and VF genes greatly varied among the seven species. More than 70% of Enterobacter cloacae strains had VF genes, but only 1.2% of Klebsiella pneumoniae strains had VF genes from prophages. AMR and VF genes are unlikely to exist together in the same prophage region except in E. coli and Staphylococcus aureus, and the distribution patterns of prophage types containing AMR genes are distinct from those of VF gene-carrying prophage types. AMR genes in the prophage were located near transposase and/or integrase. The prophage containing class 1 integrase possessed a significantly greater number of AMR genes than did prophages with no class 1 integrase. The results of this study present a comprehensive picture of AMR and VF genes present within, or close to, prophage-like elements and different prophage patterns between AMR- or VF-encoding prophage-like elements.IMPORTANCE Although we believe phages play an important role in horizontal gene transfer in exchanging genetic material, we do not know the distribution of the antimicrobial resistance (AMR) and/or virulence factor (VF) genes in prophages. We collected different prophage elements from the complete genome sequences of seven species—Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter cloacae, and Escherichia coli—and characterized the distribution of antimicrobial resistance and virulence genes located in the prophage region. While virulence genes in prophage were species specific, antimicrobial resistance genes in prophages were highly conserved in various species. An integron structure was detected within specific prophage regions such as P1-like prophage element. Maximum of 10 antimicrobial resistance genes were found in a single prophage region, suggesting that prophages act as a reservoir for antimicrobial resistance genes. The results of this study show the different characteristic structures between AMR- or VF-encoding prophages.

Antibiotic resistance is a global public health problem, and its association with reservoirs of antimicrobial resistance (AMR) genes in non-human sources, such as farm animals, including pigs, has raised concerns and highlighted the urgent need for monitoring. Mobile genetic elements, including plasmids, carrying different AMR genes have been reported in bacteria from pigs, in both sick and healthy animals. Therefore, studies focusing on clinically healthy animals are essential, as they can provide important information that improve actions to control antimicrobial resistance. In addition, it is crucial to understand the mechanisms involved in the spread of AMR genes in food-production animals. In this context, the present study aimed to characterize bacterial isolates of the Enterobacteriaceae family from clinically healthy pigs; evaluate the profile of antimicrobial susceptibility and AMR genes; investigate plasmids associated with AMR resistance; and investigate the participation of extracellular vesicles produced by multidrug-resistant (MDR) bacterial isolates as reservoirs of AMR genes. From the bacterial collection of 28 isolates, 96.4% (n = 27) were identified as belonging to the Escherichia coli species. All isolates were considered multidrug-resistant with multiple AMR genes. Horizontal transfer of the blaCTX-M gene to the recipient strain E. coli J53 was confirmed for nine MDR- E. coli isolates by conjugation assay, using ceftiofur as the selective agent. The ß-lactam resistance profile was confirmed in the transconjugants, in addition to the presence of the gene. Furthermore, three representative isolates were subsequently selected for genome sequencing and in silico characterization. As a result, consistent with their phenotypic profiles, these strains harbored multiple AMR genes and plasmid replicons belonging to different incompatibility groups (Inc). In silico analyses identified two hypothetical plasmids carrying the blaCTX-M gene and another carrying the blaTEM gene, all were conjugative and carrying additional AMR genes. To characterize the extracellular vesicles (EVs) produced by the selected bacteria, these were obtained by ultracentrifugation from the culture of isolates with and without ceftiofur and tetracycline as stress agents. As a result, treatment with tetracycline resulted in an increase in both the diameter and number of EVs compared to the control group. However, the EVs obtained from the control and ceftiofur groups carried a higher number of AMR genes, including the blaCTX-M 2, blaTEM, tetA, and tetB genes. These results demonstrate that EVs are capable of packaging and potentially spreading AMR genes even in the absence of antibiotics, suggesting that this mechanism may contribute to the spread of genes in the environment and in hosts. In conclusion, our results reveal that clinically healthy animals, regardless of the use of diets without antimicrobial additives, carry AMR genes of importance in animal and human clinical practice. They highlight the importance of investigating clinically healthy animals as reservoirs of AMR genes and reinforce the need for future studies focusing on EVs derived from MDR- E. coli of animal origin to better understand their contribution to the spread of antibiotic resistance in livestock. Keywords: horizontal multirresistance; one gene transfer; health vesiculation; food-producing animals

Co-occurrence of antimicrobial and metal resistance genes in pig feces and agricultural fields fertilized with slurry

Background/Objectives: Microbiomes surrounding mining sites have been found to harbor both antibiotic resistance genes and metal resistance genes. Within the "One Health" framework, which spans human, veterinary and environmental health, it is crucial to determine whether bacterial metal resistance (MR) genes can independently trigger antimicrobial resistance (AMR) or if they are linked to AMR genes and co-transferred horizontally. Methods and Results: Bacteria were isolated from an active and an inactive mining site in the alpine region of Austria. Most of the isolated bacteria harbored antimicrobial and metal resistance genes (88%). MALDI-TOF and whole genome sequencing (WGS) revealed that species from the Pseudomonadaceae family were the most identified, accounting for 32.5%. All Pseudomonas spp. carried AMR genes from the mex family, which encode multidrug efflux pumps. β-lactamase production encoded by bla genes were detected as the second most common (26%). The same AMR genes have often been detected within a particular bacterial genus. No tetracycline resistance gene has been identified. Among metal resistance genes, rufB (tellurium resistance) was the most prevalent (33%), followed by recGM (selenium resistance, 30%), copA (copper resistance, 26%), and mgtA (magnesium and cobalt resistance, 26%). Notably, the mer gene family (mercury resistance) was found exclusively in isolates from the inactive mining site (n = 6). In addition, genes associated with both antimicrobial and metal resistance, including arsBM, acrD, and the mer operon, were identified in 19 out of the 43 isolates. Conclusions: Bacteria isolated from mine water harbored both MR and AMR genes. Given the exceptional diversity of bacterial species in these settings, 16S rRNA gene sequence analysis is the recommended method for accurate species identification. Moreover, the presence of multi-drug transporters and transferable resistance genes against critically important antimicrobials such as fluoroquinolones and colistin identified in these environmental bacteria emphasizes the importance of retrieving environmental data within the "One Health" framework.

BackgroundThe presence of antimicrobial resistance and virulence genes in Escherichia coli allows them to survive and cause infections. The close contact between humans and pets can reinforce the risk of transmitting resistant and virulent bacteria between them.ObjectivesThis study aims to compare the patterns of the presence of tetracycline and streptomycin resistance genes, as well as important virulence genes in E. coli isolated from faeces of healthy dogs and their owners.MethodsPolymerase chain reactions were performed for detection of antimicrobial resistance (tetA, tetB, tetC, tetD, strA and strB) and virulence (fimH, iss, sitA and malX) genes in 144 faecal E. coli isolates from 28 dog–owner pairs and 16 humans who did not keep any pets as controls.ResultsAmong the investigated antimicrobial resistance and virulence genes, tetA (52.1%) and fimH (86.8%) genes had the highest prevalence. No statistically significant difference was found between the prevalence of antimicrobial resistance and virulence genes in isolates of dogs and their owners. In total, 46.4% of dog–owner pairs had the same patterns of presence or absence of six antimicrobial resistance genes, 50.0% had the same patterns of presence or absence of four virulence genes and 25.0% had the same patterns of presence or absence of all 10 tested genes.ConclusionThe presence of antimicrobial‐resistant virulent E. coli in humans and pets may predispose them to infections that are hard to cure with conventional antibiotics. Notable frequency of dogs’ and their owners’ E. coli isolates with similar patterns of antimicrobial resistance and virulence genes may indicate the possibility of sharing virulent antimicrobial resistant E. coli between them.

BackgroundMultidrug resistant (MDR) bacteria which resist at least three different antibiotic classes are serious threat to public health and patient treatment. Antimicrobial resistant (AMR) genes are often mediated through plasmids due to its horizontal transferability from bacteria to bacteria. Here, we assessed the prevalence of AMR genes in the USA by analyzing longitudinal K. pneumoniae plasmid genome data obtained from PATRIC (Pathosystems Resources Integration Center) database.MethodsThe PATRIC database have 176 K. pneumoniae plasmid genome data collected in 9 states of US from 2004 to 2019. The isolation source are various patient samples including urine, blood, etc. The sequence information was downloaded from GeneBank. The AMR genes on plasmids of K. pneumoniae were identified using open-source AMR database, Resfinder which search AMR genes for 16 types of antibiotic classes.ResultsMultidrug resistance was spread all over the US as most of isolates have AMR genes against more than one antibiotic classes, and some isolates contain against up to 8 antibiotic classes AMR genes. Most common AMR genes are against 9 classes including Aminoglycosides, beta-lactamase (carbapenem), Chloramphenicol, Macrolide, Quinolone, Rifampicin, Sulfonamide, Tetracycline, and Trimethoprim. Aminoglycosides and beta-lactamase including carbapenem resistance genes showed higher frequency than other classes of antibiotics. Carbapenem resistance genes, especially blaKPC showed higher frequency in east region of US including NY, PA, and VA (Fig 1A). While some resistances reduced over time, Aminoglycosides and beta-lactamase resistance genes are sharply increased (Fig 1B). Many isolates contain 3∼4 plasmids and plasmid amplicon types were various by isolates but no co-relation between plasmid types and AMR genes was observed.Figure 1A.The number of antibiotics resistance genes over the geographical region by the isolates. The states that have more than 3 isolates were included in the analysis.Figure IB.Changes in the number of antibiotics resistance genes over the years. Only time points that have more than 3 isolates been included in the analysis.ConclusionDatabase analysis of K. pneumoniae isolates from different regions and time provides insight of the prevalence of AMR genes. The prevalence of the multidrug resistance obtained from databases analysis showed the abundance of AMR genes regardless of the region or time, especially for Aminoglycoside and beta-lactamase. Bacterial sequence database such as PATRIC is a useful tool for tracking the existence and emergence of AMR genes.DisclosuresChetan Jinadatha, MD, MPH, AHRQ R01 Grant-5R01HS025598: Grant/Research Support|EOS Surfaces: Copper Coupons and materials for testing.

This study aimed to detect the cecal microbiome, antimicrobial resistance (AMR) and heavy metal resistance genes (MRGs) in fattening pigs raised under antibiotic-free (ABF) conditions compared with ordinary industrial pigs (control, C) using whole-genome shotgun sequencing. ABF pigs showed the enrichment of Prevotella (33%) and Lactobacillus (13%), whereas Escherichia coli (40%), Fusobacterium and Bacteroides (each at 4%) were notably observed in the C group. Distinct clusters of cecal microbiota of ABF and C pigs were revealed; however, microbiota of some C pigs (C1) appeared in the same cluster as ABF and were totally separated from the remaining C pigs (C2). For AMR genes, the highest abundance tet(Q) (35.7%) and mef(A) (12.7%) were markedly observed in the ABF group whereas tet(Q) (26.2%) and tet(W) (10.4%) were shown in the C group. tet(Q) was positively correlated to Prevotella in ABF and C1 samples. In the C2 group, the prominent tet(W) was positively correlated to Fusobacterium and Bacteroides Pigs have never received tetracycline but pregnant sows used chlortetracycline once 7 d before parturition. Chromosomal Cu and Zn resistance genes were also shown in both groups regardless the received Cu and Zn feed additives. A higher abundance of multi-metal resistance genes was observed in the C group (44%) compared with the ABF group (41%). In conclusion, the microbiome clusters in some C pigs were similar to that in ABF pigs. High abundant tetracycline resistance genes interrelated to major bacteria were observed in both ABF and C pigs. MRGs were also observed.IMPORTANCE: Owing to the increased problem of AMR in farm animals, raising farm animals without antibiotics is one method that could solve this problem. Our study showed that only some tetracycline and macrolide resistance genes, tet(Q), tet(W) and mef(A), were markedly abundant in ABF and C groups. The tet(Q) and tet(W) genes interrelated to different predominant bacteria in each group, showing the potential role of major bacteria as reservoirs of AMR genes. In addition, chromosomal Cu and Zn resistance genes were also observed in both pig groups, not depending on the use of Cu and Zn additives in both farms. The association of MRGs and AMR genotypes and phenotypes together with the method to re-sensitize bacteria to antibiotics should be studied further to unveil the cause of high resistance genes and solve the problems.

Simple SummaryThe alarming emergence of antimicrobial resistance (AMR) in human and veterinary medicine has activated awareness for monitoring the levels of AMR pollution in the environment and wildlife. European hedgehogs (Erinaceus europaeus) are common wild species habiting urban areas in Europe. In this study, the occurrence and distribution of extended-spectrum β-lactam (ESBL) resistant enterobacteria and AMR genes were assessed in wild European hedgehogs in Catalonia, NE Spain. The results showed that 36.8% of the animals were detected as carriers of β-lactamase/carbapenemase resistance genes, with a special occurrence of human nosocomial bacteria such as Klebsiella pneumoniae, Escherichia coli, and Citrobacter freundii. In addition, more than half of the enterobacteria presented a multidrug resistance (MDR) phenotype and 31% of the isolates had an extended XDR profile. No differences in the spatial distribution of animals with AMR genes were observed within the study region. The results of this study suggest that the close contact with human areas predispose the transmission of AMR genes to wild hedgehogs because they either inhabit and/or feed in an anthropogenic environment. In conclusion, hedgehogs could be good sentinels or bioindicators of AMR environmental pollution, especially in highly populated areas with high human activity.Wildlife has been suggested to be a good sentinel of environmental health because of its close interaction with human populations, domestic animals, and natural ecosystems. The alarming emergence of antimicrobial resistance (AMR) in human and veterinary medicine has activated/triggered the awareness of monitoring the levels of AMR pollution in wildlife. European hedgehogs (Erinaceus europaeus) are common wild species habiting urban areas in Europe. However, there are few studies conducted in hedgehogs as reservoirs of AMR bacteria or genes. The aim of this study was to assess the occurrence and distribution of ESBL, AmpC, and carbapenem-resistant enterobacteria and AMR genes in wild European hedgehogs in Catalonia, a densely populated region of NE Spain. A total of 115 hedgehogs admitted at the Wildlife Rehabilitation Center of Torreferrussa were studied. To our knowledge, this is the first description of β-lactam resistant enterobacteria in wild hedgehogs. Interestingly, 36.8% (42/114) of the animals were detected as carriers of β-lactamase/carbapenemase resistance genes. Klebsiella spp. (59.6%), and specifically K. pneumoniae (84.6%), were the bacteria with the highest proportion of resistance genes, followed by E. coli (34.6%) and C. freundii (5.8%). The most frequently detected genetic variants were blaCTX-M-15 (19.3%), blaSHV-28 (10.5%), blaCMY-1 (9.7%), blaCMY-2 (8.8%), and blaOXA-48 (1.7%). In addition, 52% (27/52) of the isolates presented a multidrug resistance (MDR) phenotype and 31% had an extended drug resistance (XDR) profile. No clustering of animals with AMR genes within the study region was shown in the spatial analysis, nor differences in the proportion of positive animals among regions, were detected. The results of this study suggest that wild European hedgehogs could be good sentinels of AMR environmental pollution, especially in areas with a high human population density, because they either inhabit and/or feed in an anthropogenic environment. In conclusion, it is crucial to raise awareness of the strong interconnection between habitats and compartments, and therefore this implies that AMR issues must be tackled under the One Health approach.

To support the role of insects as sustainable feed and food ingredients, evaluating their potential microbiological risk and safety is crucial. In this study, we investigated the presence of antimicrobial resistance (AMR) genes in selected live opportunistic pathogenic bacteria isolated during the rearing process from clinically healthy farm-reared crickets. Molecular analysis was performed by wholegenome sequencing of a total of 14 of these bacterial strains, 7 from house crickets (Acheta domesticus) and 7 from banded crickets (Gryllodes sigillatus), belonging to Enterobacteriaceae, Staphylococcaceae, Enterococcaceae, and Bacillaceae families. The β-lactam AMR genes (blaOXY2-6, blaACT-16, and blaSHV variants) were the most predominant genes identified, mainly in Enterobacteriaceae strains and in association with fosfomycin (fosA) and oqxAB efflux pump complexes. In addition, blaZ and mecA genes were detected in Bacillus cereus and Mammaliicoccus sciuri strains isolated from both insect species. Genetic mobile elements including IncFIA, IncFIB, IncHI1A, IncHI1B, rep13, and Col3M-like plasmids were detected in Klebsiella pneumoniae, Enterobacter hormaechei, Staphylococcus arlettae, and B. cereus, respectively. The results indicate that, not only in the final product but also during the insect-rearing process, microbial safety control, regarding the presence of pathogenic bacteria and AMR genes, is essential for effectively decreasing the microbiological risk between cricket batches within their environment and in terms of the related feed and food chain.

BackgroundAntibiotic use in livestock farming is thought to be a major contributor to the spread of antimicrobial resistance (AMR) genes in humans. However, quantitative data in this in this field are rare. To address this gap in the literature, we examined the prevalence of clinically important AMR genes before and after the introduction of chicken farming among women in rural Uganda.MethodsWe recruited a subset of women participating in a waitlist-randomized controlled trial of small-scale hybrid chicken farming in rural Uganda. Tetracycline is routinely administered to chicks during brooding. Stool samples before and one year after chicken introduction were obtained from six women randomized to the control arm, from five women randomized to the intervention arm, and from chickens. Microbial DNA was extracted from chicken and human stool and screened for 87 AMR genes using validated qPCR arrays (Qiagen).ResultsThe median age was 35 years. At baseline, 10 of the women reported animal contact, most commonly goats (n = 8), free ranging village chickens (n = 7), cats (n = 4), and dogs (n = 4). During baseline testing of the women’s stool, we detected 18 genes conferring AMR to aminoglycosides, fluoroquinolones, macrolides, lincosamides, streptogramin B, Class A-C β-lactamases and tetracycline efflux pumps. Chickens harbored 23 AMR genes from the same classes as found in humans, and were also found to have vancomycin resistance genes (Van B and C) and Group D β-lactamases (OXA-58 and OXA-10). At one year, six new AMR genes emerged in controls, including one present in chickens; CTX-M-1, a Class A β-lactamase. In contrast, seven new AMR genes emerged in the intervention group, including four present in chickens: SHV, SHV(238G240E), (Class A β lactamases) and QnrS, QnrB-5 (fluoroquinolone resistance genes). Two AMR genes gained by both control and intervention groups were not present in chickens.ConclusionWomen exposed to small-scale chicken farming acquired more AMR genes compared with unexposed participants. Chickens harbored many of the genes that emerged in humans. Introduction of antibiotic-treated animals may result in the transfer of AMR genes from animals to humans, even among humans exposed to a wide range of animals at baseline.DisclosuresAll authors: No reported disclosures.

Antibiotic use for livestock is presumed to be a contributor to the acquisition of antimicrobial resistance (AMR) genes in humans, yet studies do not capture AMR data before and after livestock introduction. We performed a feasibility study by recruiting a subset of women in a delayed-start randomized controlled trial of small-scale chicken farming to examine the prevalence of clinically-relevant AMR genes. Stool samples were obtained at baseline and one year post-randomization from five intervention women who received chickens at the start of the study, six control women who did not receive chickens until the end of the study, and from chickens provided to the control group at the end of the study. Stool was screened for 87 clinically significant AMR genes using a commercially available qPCR array (Qiagen). Chickens harbored 23 AMR genes from classes found in humans as well as additional vancomycin and β-lactamase resistance genes. AMR patterns between intervention and control women appeared more similar at baseline than one year post randomization (PERMANOVA R2 = 0.081, p = 0.61 at baseline, R2 = 0.186, p = 0.09 at 12 months) Women in the control group who had direct contact with the chickens sampled in the study had greater similarities in AMR gene patterns to chickens than those in the intervention group who did not have direct contact with chickens sampled (p = 0.01). However, at one year there was a trend towards increased similarity in AMR patterns between humans in both groups and the chickens sampled (p = 0.06). Studies designed to evaluate human AMR genes in the setting of animal exposure should account for high baseline AMR rates. Concomitant collection of animal, human, and environmental samples over time is recommended to determine the directionality and source of AMR genes. ClinicalTrials.gov Identifier NCT02619227.

Background Antibiotic resistance (AMR) is a crucial threat to human health and challenges the effectiveness of clinical interventions. Antibiotic resistance is often perpetuated by the indiscriminate use of antibiotics leading to selection pressure and the transfer of the resistance genes between humans, domestic animals, and the environment. Being the ultimate recipient of runoffs and effluents, the marine environment is a potential reservoir of Antimicrobial Resistance Genes (ARGs). Terrestrial input from anthropogenic activities such as the indiscriminate use of antibiotics drives the accumulation of ARGs in the marine environment. The dissemination of these genes in the marine environment is aided by Horizontal Gene Transfer (HGT) using Mobile Genetic elements (MGEs). Despite the reported evidence on the presence of ARGs in world oceans, antimicrobial resistance monitoring in the African marine environment remains limited. Methods This exploratory study conducted a bioinformatics-based screening for Antimicrobial Resistance Genes (ARGs) using secondary data from the European Nucleotide Archive (ENA). Antimicrobial Resistance Gene screening was done using the Resistance Gene Identifier and AMRFinderPlus tools. Results We found 38 different Antimicrobial Resistance Genes (ARGs) classified into 10 drug classes from the analyzed marine metagenomes. The most abundant genes identified include vanT and vanY belonging to the glycopeptide class, adeF in fluoroquinolone and tetracycline, blaOXA and blaSGM in the b-lactam class, and qacG in the small multidrug resistance group. Conclusion These findings underscore the crucial role of the marine environment in harbouring resistance genes, particularly in the African region, highlighting the urgent need to integrate environmental screening in the surveillance and monitoring programs of AMR.

The persistence of antimicrobial resistant (AMR) genes in the soil-environment is a concern, yet practices that mitigate AMR are poorly understood, especially in grasslands. Animal manures are widely deposited on grasslands, which are the largest agricultural land-use in the United States. These nutrient-rich manures may contain AMR genes. The aim of this study was to enumerate AMR genes in grassland soils following 14-years of poultry litter and cattle manure deposition and evaluate if best management practices (rotationally grazed with a riparian (RBR) area and a fenced riparian buffer strip (RBS), which excluded cattle grazing and poultry litter applications) relative to standard pasture management (continuously grazed (CG) and hayed (H)) minimize the presence and amount of AMR genes. Quantitative PCR (Q-PCR) was performed to enumerate four AMR genes (ermB, sulI, intlI, and blactx-m-32) in soil, cattle manure, and poultry litter environments. Six soil samples were additionally subjected to metagenomic sequencing and resistance genes were identified from assembled sequences. Following 14-years of continuous management, ermB, sulI, and intlI genes in soil were greatest (P < 0.05) in samples collected under long-term continuous grazing (relative to conservation best management practices), under suggesting overgrazing and continuous cattle manure deposition may increase AMR gene presence. In general, AMR gene prevalence increased downslope, suggesting potential lateral movement and accumulation based on landscape position. Poultry litter had lower abundance of AMR genes (ermB, sulI, and intlI) relative to cattle manure. Long-term applications of poultry litter increased the abundance of sulI and intlI genes in soil (P < 0.05). Similarly, metagenomic shotgun sequencing revealed a greater total number of AMR genes under long-term CG, while fewer AMR genes were found in H (no cattle manure) and RBS (no animal manure or poultry litter). Results indicate long-term conservation pasture management practices (e.g., RBS and RBR) and select animal manure (poultry litter inputs) may minimize the presence and abundance of AMR genes in grassland soils.

Antimicrobial resistance (AMR) is a growing global health concern, driven in part by antibiotic use in animal production systems. Despite its relevance, the microbiome and resistome of small ruminant farm environments remain largely underexplored. In this study, shotgun metagenomics was applied to environmental samples from 46 sheep, goat and mixed-species farms across 14 municipalities in central Portugal. Microbial profiling revealed a well-preserved microbiome with Pseudomonadota, Actinomycetota, Bacteroidota and Bacillota (syn. Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes respectively) as the most dominant phylum across different farm types. Regarding AMR, a total of 706 unique antimicrobial resistance genes (ARGs), covering 15 antibiotic classes, were detected. Tetracycline, aminoglycoside and macrolide resistance genes dominated across all samples, forming a conserved core resistome. While overall resistome profiles were broadly similar among farm types, significant differences were observed in specific ARG classes, such as pleuromutilin and fosfomycin. These findings highlight small ruminant farm environments as potential reservoirs of clinically relevant ARGs, including WHO highest priority critically important antimicrobial (HPCIA) resistance genes for macrolides (mph(c), erm(f), erm(b)) and fluoroquinolones (qnrD1), as well as critically important antimicrobial (CIA) resistance genes for glycopeptides (vanR-SC, vanR-O) and aminoglycosides (str, aadA), supporting the need to incorporate these environments into surveillance strategies.

Simple SummaryWorldwide, antimicrobial resistance (AMR) is of major concern for human and animal health since infections with multidrug-resistant bacteria are often more challenging and costly. In the family Staphyloccocaceae, the species Staphylococcusaureus in particular was reported to cause severe infections. Although most of the other Staphylococcaceae members were not shown to cause severe illnesses, the transmission of AMR genes to harmful species might take place. Therefore, the monitoring of AMR potential in different environments is of high relevance. Mammaliicocci on dairy farms might represent such an AMR gene reservoir. Thus, in this study, the AMR potential of mammaliicocci isolates from German dairy farms was investigated. Whole-genome sequencing (WGS) of the isolates was conducted to evaluate the phylogenetic relationship of the isolates and analyze AMR genes. In addition, antimicrobial susceptibility testing was performed to compare the AMR genotype with the phenotype. It turned out that mammaliicocci may harbor large numbers of different AMR genes and exhibit phenotypic resistance to various antibiotics. Since some AMR genes are likely located on mobile genetic elements, such as plasmids, AMR gene transmission between members of the Staphylococcaceae family might occur.Mammaliicocci might play a major role in antimicrobial resistance (AMR) gene transmission between organisms of the family Staphylococcaceae, such as the potentially pathogenic species Staphylococcus aureus. The interest of this study was to analyze AMR profiles of mammaliicocci from German dairy farms to evaluate the AMR transmission potential. In total, 65 mammaliicocci isolates from 17 dairy farms with a history of MRSA detection were analyzed for AMR genotypes and phenotypes using whole genome sequencing and antimicrobial susceptibility testing against 19 antibiotics. The various genotypic and phenotypic AMR profiles of mammaliicocci from German dairy farms indicated the simultaneous occurrence of several different strains on the farms. The isolates exhibited a non-wildtype phenotype to penicillin (58/64), cefoxitin (25/64), chloramphenicol (26/64), ciprofloxacin (25/64), clindamycin (49/64), erythromycin (17/64), fusidic acid (61/64), gentamicin (8/64), kanamycin (9/64), linezolid (1/64), mupirocin (4/64), rifampicin (1/64), sulfamethoxazol (1/64), streptomycin (20/64), quinupristin/dalfopristin (26/64), tetracycline (37/64), tiamulin (59/64), and trimethoprim (30/64). Corresponding AMR genes against several antimicrobial classes were detected. Linezolid resistance was associated with the cfr gene in the respective isolate. However, discrepancies between genotypic prediction and phenotypic resistance profiles, such as for fusidic acid and tiamulin, were also observed. In conclusion, mammaliicocci from dairy farms may carry a broad variety of antimicrobial resistance genes and exhibit non-wildtype phenotypes to several antimicrobial classes; therefore, they may represent an important source for horizontal gene transfer of AMR genes to pathogenic Staphylococcaceae.