Florfenicol and oxazolidone resistance status in livestock farms revealed by short- and long-read metagenomic sequencing. - Related Documents




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344201.0000Florfenicol and oxazolidone resistance status in livestock farms revealed by short- and long-read metagenomic sequencing. Antibiotic resistance genes (ARGs) as a novel type of environmental pollutant pose a health risk to humans. Oxazolidinones are one of the most important antibiotics for the treatment of Gram-positive bacterial infections in humans. Although oxazolidinones are not utilized in the livestock industry, florfenicol is commonly used on farms to treat bacterial infections, which may contribute to the spread of the cfr, optrA, and poxtA genes on farms. Using metagenomics sequencing, we looked into the antibiotic resistome context of florfenicol and oxazolidinone in 10 large-scale commercial farms in China. We identified 490 different resistance genes and 1,515 bacterial genera in the fecal samples obtained from 10 farms. Florfenicol-resistant Kurthia, Escherichia, and Proteus were widely present in these samples. The situation of florfenicol and oxazolidone resistance in pig farms is even more severe. The total number of genes and the abundance of drug resistance genes were higher in pigs than in chickens, including optrA and poxtA. All the samples we collected had a high abundance of fexA and floR. Through nanopore metagenomic analysis of the genetic environment, we found that plasmids, integrative and conjugative element (ICE), and transposons (Tn7-like and Tn558) may play an important role in the spread of floR, cfr, and optrA. Our findings suggest that florfenicol and oxazolidinone resistance genes have diverse genetic environments and are at risk of co-transmission with, for example, tetracycline and aminoglycoside resistance genes. The spread of florfenicol- and oxazolidinone-resistant bacteria on animal farms should be continuously monitored.202236338088
571810.9998A newly identified IncY plasmid from multi-drug-resistant Escherichia coli isolated from dairy cattle feces in Poland. Comprehensive whole-genome sequencing was performed on two multi-drug-resistant Escherichia coli strains isolated from cattle manure from a typical dairy farm in Poland in 2020. The identified strains are resistant to beta-lactams, aminoglycosides, tetracyclines, trimethoprim/sulfamethoxazole, and fluoroquinolones. The complete sequences of the harbored plasmids revealed antibiotic-resistance genes located within many mobile genetic elements (e.g., insertional sequences or transposons) and genes facilitating conjugal transfer or promoting horizontal gene transfer. These plasmids are hitherto undescribed. Similar plasmids have been identified, but not in Poland. The identified plasmids carried resistance genes, including the tetracycline resistance gene tet(A), aph family aminoglycoside resistance genes aph(3″)-lb and aph (6)-ld, beta-lactam resistance genes bla(TEM-1) and bla(CTX-M-15), sulfonamide resistance gene sul2, fluoroquinolone resistance gene qnrS1, and the trimethoprim resistance gene dfrA14. The characterized resistance plasmids were categorized into the IncY incompatibility group, indicating a high possibility for dissemination among the Enterobacteriaceae. While similar plasmids (99% identity) have been found in environmental and clinical samples, none have been identified in farm animals. These findings are significant within the One Health framework, as they underline the potential for antimicrobial-resistant E. coli from livestock and food sources to be transmitted to humans and vice versa. It highlights the need for careful monitoring and strategies to limit the spread of antibiotic resistance in the One Health approach. IMPORTANCE: This study reveals the identification of new strains of antibiotic-resistant Escherichia coli in cattle manure from a dairy farm in Poland, offering critical insights into the spread of drug resistance. Through whole-genome sequencing, researchers discovered novel plasmids within these bacteria, which carry genes resistant to multiple antibiotics. These findings are particularly alarming, as these plasmids can transfer between different bacterial species, potentially escalating the spread of antibiotic resistance. This research underscores the vital connection between the health of humans, animals, and the environment, emphasizing the concept of One Health. It points to the critical need for global vigilance and strategies to curb the proliferation of antibiotic resistance. By showcasing the presence of these strains and their advanced resistance mechanisms, the study calls for enhanced surveillance and preventive actions in both agricultural practices and healthcare settings to address the imminent challenge of antibiotic-resistant bacteria.202439012117
284520.9998Florfenicol administration in piglets co-selects for multiple antimicrobial resistance genes. Antimicrobial use in food-producing animals such as pigs is a significant issue due to its association with antimicrobial resistance. Florfenicol is a broad-spectrum phenicol antibiotic used in swine for various indications; however, its effect on the swine microbiome and resistome is largely unknown. This study investigated these effects in piglets treated intramuscularly with florfenicol at 1 and 7 days of age. Fecal samples were collected from treated (n = 30) and untreated (n = 30) pigs at nine different time points up until 140 days of age, and the fecal metagenomes were sequenced. The fecal microbiomes of the two groups of piglets were most dissimilar in the immediate period following florfenicol administration. These differences were driven in part by an increase in the relative abundance of Clostridium scindens, Enterococcus faecalis, and Escherichia spp. in the florfenicol-treated piglets and Fusobacterium spp., Pauljensenia hyovaginalis, and Ruminococcus gnavus in the control piglets. In addition to selecting for florfenicol resistance genes (floR, fexA, and fexB), florfenicol also selected for genes conferring resistance to the aminoglycosides, beta-lactams, or sulfonamides up until weaning at 21 days of age. Florfenicol-resistant Escherichia coli isolated from these piglets were found to carry a plasmid with floR, along with tet(A), aph(6)-Id, aph(3″)-Ib, sul2, and bla(TEM-1)/bla(CMY-2). A plasmid carrying fexB and poxtA (phenicols and oxazolidinones) was identified in florfenicol-resistant Enterococcus avium, Enterococcus faecium, and E. faecalis isolates from the treated piglets. This study highlights the potential for co-selection and perturbation of the fecal microbial community in pre-weaned piglets administered florfenicol.IMPORTANCEAntimicrobial use remains a serious challenge in food-animal production due to its linkage with antimicrobial resistance. Antimicrobial resistance can reduce the efficacy of veterinary treatment and can potentially be transferred to humans through the food chain or direct contact with animals and their environment. In this study, early-life florfenicol treatment in piglets altered the composition of the fecal microbiome and selected for many unrelated antimicrobial resistance genes up until weaning at 21 days of age. Part of this co-selection process appeared to involve an Escherichia coli plasmid carrying a florfenicol resistance gene along with genes conferring resistance to at least four other antimicrobial classes. In addition, florfenicol selected for certain genes that provide resistance to multiple antimicrobial classes, including the oxazolidinones. These results highlight that florfenicol can co-select for multiple antimicrobial resistance genes, and their presence on mobile genetic elements suggests the potential for transfer to other bacteria.202439584815
344830.9998Antimicrobial resistance genes and associated mobile genetic elements in Lactobacillales from various sources. Lactobacillales are commonly used in food products and as probiotics in animal and human medicine. Despite being generally recognized as safe, lactic acid bacteria may harbor a variety of antimicrobial resistance genes (ARGs), which may be transferable to human or veterinary pathogens, thus, may pose veterinary and public health concerns. This study investigates the resistome of Lactobacillales. A total of 4,286 whole-genome sequences were retrieved from NCBI RefSeq database. We screened ARGs in whole genome sequences and assessed if they are transmissible by plasmid transfer or by linkage to integrative mobile genetic elements. In the database, 335 strains were found to carry at least one ARG, and 194 strains carried at least one potentially transferable ARG. The most prevalent transferable ARG were tetM and tetW conferring antibiotic resistance to tetracycline. This study highlights the importance of the One Health concept by demonstrating the potential for Lactobacillales, commonly used in food products, to serve as reservoirs and vectors for ARGs.202338045025
344340.9998A hybrid DNA sequencing approach is needed to properly link genotype to phenotype in multi-drug resistant bacteria. Antibiotic resistance genes (ARGs) are now viewed as emerging contaminants posing a potential worldwide human health risk. The degree to which ARGs are transferred to other bacteria via mobile genetic elements (MGEs), including insertion sequences (ISs), plasmids, and phages, has a strong association with their likelihood to function as resistance transfer determinants. Consequently, understanding the structure and function of MGEs is paramount to assessing future health risks associated with ARGs in an environment subjected to strong antibiotic pressure. In this study we used whole genome sequencing, done using MinION and HiSeq platforms, to examine antibiotic resistance determinants among four multidrug resistant bacteria isolated from fish farm effluent in Jeju, South Korea. The combined data was used to ascertain the association between ARGs and MGEs. Hybrid assembly using HiSeq and MinION reads revealed the presence of IncFIB(K) and pVPH2 plasmids, whose sizes were verified using pulsed field gel electrophoresis. Twenty four ARGs and 95 MGEs were identified among the 955 coding sequences annotated on these plasmids. More importantly, 22 of 24 ARGs conferring resistance to various antibiotics were found to be located near MGEs, whereas about a half of the ARGs (11 out of 21) were so in chromosomes. Our results also suggest that the total phenotypic resistance exhibited by the isolates was mainly contributed by these putatively mobilizable ARGs. The study gives genomic insights into the origins of putatively mobilizable ARGs in bacteria subjected to selection pressure.202134330011
570650.9998Comparative Genomic Analysis of Enterococci across Sectors of the One Health Continuum. Enterococci are Gram-positive bacteria that can be isolated from a variety of environments including soil, water, plants, and the intestinal tract of humans and animals. Although they are considered commensals in humans, Enterococcus spp. are important opportunistic pathogens. Due to their presence and persistence in diverse environments, Enterococcus spp. are ideal for studying antimicrobial resistance (AMR) from the One Health perspective. We undertook a comparative genomic analysis of the virulome, resistome, mobilome, and the association between the resistome and mobilome of 246 E. faecium and 376 E. faecalis recovered from livestock (swine, beef cattle, poultry, dairy cattle), human clinical samples, municipal wastewater, and environmental sources. Comparative genomics of E. faecium and E. faecalis identified 31 and 34 different antimicrobial resistance genes (ARGs), with 62% and 68% of the isolates having plasmid-associated ARGs, respectively. Across the One Health continuum, tetracycline (tetL and tetM) and macrolide resistance (ermB) were commonly identified in E. faecium and E. faecalis. These ARGs were frequently associated with mobile genetic elements along with other ARGs conferring resistance against aminoglycosides [ant(6)-la, aph(3')-IIIa], lincosamides [lnuG, lsaE], and streptogramins (sat4). Study of the core E. faecium genome identified two main clades, clade 'A' and 'B', with clade A isolates primarily originating from humans and municipal wastewater and carrying more virulence genes and ARGs related to category I antimicrobials. Overall, despite differences in antimicrobial usage across the continuum, tetracycline and macrolide resistance genes persisted in all sectors.202336985300
284460.9998High throughput qPCR analyses suggest that Enterobacterales of French sheep and cow cheese rarely carry genes conferring resistances to critically important antibiotics for human medicine. Bacteria present in raw milk can carry acquired or intrinsic antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs). However, only a few studies have evaluated raw milk cheese as a potential reservoir of ARGs. This study thus aimed at providing new data regarding resistance markers present in raw milk cheese. Sheep (n = 360) and cow (n = 360) cheese samples produced in France were incubated in buffered peptone water supplemented with acriflavin or novobiocin; as corroborated by 16S metabarcoding, samples were enriched in Gram-negative bacteria since Escherichia coli and Hafnia alvei respectively accounted for 40 % and 20 % of the samples' microbiota. Screening of the samples for the presence of 30 ARGs and 16 MGEs by high throughput qPCR array showed that nine ARGs conferring resistances to 1st-generation beta-lactams, aminoglycosides, trimethoprim/sulfonamides and tetracyclines occurred in >75 % of both sheep and cow samples. This is neither surprising nor alarming since these resistance genes are widely spread across the One Health human, animal and environmental sectors. Conversely, genes conferring resistances to last-generations cephalosporins were rarely identified, while those conferring resistances to carbapenems or amikacin, which are restricted to human use, were never detected. Multiple MGEs were detected, the most frequent ones being IncF plasmids, confirming the potential transmission of ARGs. Our results are in line with the few studies of the resistome of milk or milk cheese showing that genes conferring resistances to 1st-generation beta-lactams, aminoglycosides and tetracyclines families are widespread, while those conferring resistances to critically important antibiotics are rare or absent.202337384974
573770.9998Survey of Colistin Resistance in Commensal Bacteria from Penaeus vannamei Farms in China. Aquatic environments are important reservoirs for drug resistance. Aquatic foods may act as carriers to lead antibiotic-resistant commensal bacteria into the human gastrointestinal system, then contacting gut microbiota and spreading antibiotic resistance. Here, several shrimp farms were investigated to identify colistin resistance among commensal bacteria of aquaculture. A total of 884 (41.6%) colistin-resistant isolates were identified among 2126 strains. Electroporation demonstrated that colistin-resistant fragments were present in some commensal bacteria that could be transferred to other bacteria. Most of the resistant bacteria were Bacillus spp., with 69.3% of the Bacillus species exhibiting multiple drug resistance. Bacillus licheniformis was prevalent, with 58 strains identified that comprised six sequence types (ST) based on multilocus sequence typing. Whole-genome sequencing and comparisons with previous B. licheniformis genomes revealed a high degree of genomic similarity among isolates from different regions. Thus, this species is widely distributed, and this study provides new insights into global antibiotic-resistant characteristics of B. licheniformis. Sequence analyses further revealed some of these strains are even pathogenic and virulent, suggesting the antibiotic resistance and hazards of commensal bacteria in aquaculture should be considered. Considering the "One Health" perspective, improved monitoring of aquatic food is needed to prevent the spread of drug-resistant commensal bacteria from food-associated bacteria to humans.202337297388
344680.9998Prevalence and transmission risk of colistin and multidrug resistance in long-distance coastal aquaculture. Due to the wide use of antibiotics, intensive aquaculture farms have been recognized as a significant reservoir of antibiotic resistomes. Although the prevalence of colistin resistance genes and multidrug-resistant bacteria (MDRB) has been documented, empirical evidence for the transmission of colistin and multidrug resistance between bacterial communities in aquaculture farms through horizontal gene transfer (HGT) is lacking. Here, we report the prevalence and transmission risk of colistin and multidrug resistance in 27 aquaculture water samples from 9 aquaculture zones from over 5000 km of subtropical coastlines in southern China. The colistin resistance gene mcr-1, mobile genetic element (MGE) intl1 and 13 typical antibiotic resistance genes (ARGs) were prevalent in all the aquaculture water samples. Most types of antibiotic (especially colistin) resistance are transmissible in bacterial communities based on evidence from laboratory conjugation and transformation experiments. Diverse MDRB were detected in most of the aquaculture water samples, and a strain with high-level colistin resistance, named Ralstonia pickettii MCR, was isolated. The risk of horizontal transfer of the colistin resistance of R. pickettii MCR through conjugation and transformation was low, but the colistin resistance could be steadily transmitted to offspring through vertical transfer. The findings have important implications for the future regulation of antibiotic use in aquaculture farms globally to address the growing threat posed by antibiotic resistance to human health.202337935916
344790.9998The Impact of Urban Pollution on Plasmid-Mediated Resistance Acquisition in Enterobacteria from a Tropical River. Background: The exposure of environmental bacteria to contaminants in aquatic ecosystems accelerates the dissemination of antibiotic-resistance genes (ARGs) through horizontal gene transfer (HGT). Methods: In this study, we sampled three locations along a contamination gradient of a polluted river, focusing on isolating Enterobacteria from the surface waters to investigate the relationship between urban pollution and antibiotic resistance. The genomes of 15 isolates (5 per site) were sequenced to identify plasmid-borne ARGs and their association with resistance phenotypes. Results: Isolates from the site with the highest contamination (Site 3) showeda larger number of ARGs, plasmids, and resistance phenotypes. Notably, one of the isolates analyzed, E. coli A231-12, exhibited phenotypic resistance to seven antibiotics, presumably conferred by a single plasmid carrying 12 ARGs. Comparative analysis of this plasmid revealed its close evolutionary relationship with another IncH plasmid hosted by Salmonella enterica, underscoring its high ARG burden in the aquatic environment. Other plasmids identified in our isolates carried sul and dfrA genes, conferring resistance to trimethoprim/sulfamethoxazole, a commonly prescribed antibiotic combination in clinical settings. Conclusions: These results highlight the critical need to expand research on the link between pollution and plasmid-mediated antimicrobial resistance in aquatic ecosystems, which can act as reservoirs of ARGs.202439596782
4966100.9998Whole Genome Analysis of 335 New Bacterial Species from Human Microbiota Reveals a Huge Reservoir of Transferable Antibiotic Resistance Determinants. BACKGROUND: The emergence and diffusion of strains of pathogenic bacteria resistant to antibiotics constitutes a real public health challenge. Antibiotic resistance genes (ARGs) can be carried by both pathogenic and non-pathogenic bacteria, including commensal bacteria from the human microbiota, which require special monitoring in the fight against antimicrobial resistance. METHODS: We analyzed the proteomes of 335 new bacterial species from human microbiota to estimate its whole range of ARGs using the BLAST program against ARGs reference databases. RESULTS: We found 278 bacteria that harbor a total of 883 potential ARGs with the following distribution: 264 macrolides-lincosamides-streptogramin, 195 aminoglycosides, 156 tetracyclines, 58 β-lactamases, 58 fosfomycin, 51 glycopeptides, 36 nitroimidazoles, 33 phenicols and 32 rifamycin. Furthermore, evolutionary analyses revealed the potential horizontal transfer with pathogenic bacteria involving mobile genetic elements such as transposase and plasmid. We identified many ARGs that may represent new variants in fosfomycin and β-lactams resistance. CONCLUSION: These findings show that new bacterial species from human microbiota should be considered as an important reservoir of ARGs that can be transferred to pathogenic bacteria. In vitro analyses of their phenotypic potential are required to improve our understanding of the functional role of this bacterial community in the development of antibiotic resistance.202235216256
4968110.9998Mobile genetic elements drive the multidrug resistance and spread of Salmonella serotypes along a poultry meat production line. The presence of mobile genetic elements in Salmonella isolated from a chicken farm constitutes a potential risk for the appearance of emerging bacteria present in the food industry. These elements contribute to increased pathogenicity and antimicrobial resistance through genes that are related to the formation of biofilms and resistance genes contained in plasmids, integrons, and transposons. One hundred and thirty-three Salmonella isolates from different stages of the production line, such as feed manufacturing, hatchery, broiler farm, poultry farm, and slaughterhouse, were identified, serotyped and sequenced. The most predominant serotype was Salmonella Infantis. Phylogenetic analyses demonstrated that the diversity and spread of strains in the pipeline are serotype-independent, and that isolates belonging to the same serotype are very closely related genetically. On the other hand, Salmonella Infantis isolates carried the pESI IncFIB plasmid harboring a wide variety of resistance genes, all linked to mobile genetic elements, and among carriers of these plasmids, the antibiograms showed differences in resistance profiles and this linked to a variety in plasmid structure, similarly observed in the diversity of Salmonella Heidelberg isolates carrying the IncI1-Iα plasmid. Mobile genetic elements encoding resistance and virulence genes also contributed to the differences in gene content. Antibiotic resistance genotypes were matched closely by the resistance phenotypes, with high frequency of tetracycline, aminoglycosides, and cephalosporins resistance. In conclusion, the contamination in the poultry industry is described throughout the entire production line, with mobile genetic elements leading to multi-drug resistant bacteria, thus promoting survival when challenged with various antimicrobial compounds.202337007466
4518120.9998Resistome, Mobilome and Virulome Analysis of Shewanella algae and Vibrio spp. Strains Isolated in Italian Aquaculture Centers. Antimicrobial resistance is a major public health concern restricted not only to healthcare settings but also to veterinary and environmental ones. In this study, we analyzed, by whole genome sequencing (WGS) the resistome, mobilome and virulome of 12 multidrug-resistant (MDR) marine strains belonging to Shewanellaceae and Vibrionaceae families collected at aquaculture centers in Italy. The results evidenced the presence of several resistance mechanisms including enzyme and efflux pump systems conferring resistance to beta-lactams, quinolones, tetracyclines, macrolides, polymyxins, chloramphenicol, fosfomycin, erythromycin, detergents and heavy metals. Mobilome analysis did not find circular elements but class I integrons, integrative and conjugative element (ICE) associated modules, prophages and different insertion sequence (IS) family transposases. These mobile genetic elements (MGEs) are usually present in other aquatic bacteria but also in Enterobacteriaceae suggesting their transferability among autochthonous and allochthonous bacteria of the resilient microbiota. Regarding the presence of virulence factors, hemolytic activity was detected both in the Shewanella algae and in Vibrio spp. strains. To conclude, these data indicate the role as a reservoir of resistance and virulence genes in the environment of the aquatic microbiota present in the examined Italian fish farms that potentially might be transferred to bacteria of medical interest.202032326629
3353130.9998Plasmid and integron-associated antibiotic resistance in Escherichia coli isolated from domestic wastewater treatment plants. The rapid dissemination of antibiotic resistance genes (ARGs) represents a significant global threat, with wastewater treatment plants (WWTPs) playing an important role as reservoirs and propagation hubs. In this study, we performed whole-genome sequencing and bioinformatic analyses on eight multidrug-resistant Escherichia coli isolates previously obtained from domestic WWTPs in Costa Rica. We identified 61 ARGs (23 unique), with 40 located on plasmids, and 21 on chromosomal sequences, seven of which were within integrons. Several ARGs were associated with resistance to clinically and veterinary important antibiotics, including sulfamethoxazole/trimethoprim, beta-lactams, and tetracyclines. One hundred twenty-one virulence-associated genes (29 unique) were detected, with 16 located on plasmids. Notably, the presence of virulence factors such as ompT and hlyF genes alongside ARGs on plasmids underscores the transmissible pathogenic potential of WWTP-associated E. coli strains. These findings highlight the role of small domestic WWTPs in disseminating pathogenic and multidrug-resistant bacteria and their mobile genetic elements, emphasizing the need for further research to understand how these discharges impact aquatic environments.202540246693
3356140.9998Conjugative multiple-antibiotic resistance plasmids in Escherichia coli isolated from environmental waters contaminated by human faecal wastes. AIMS: To better understand the involvement of faecal contamination in the dissemination of antibiotic resistance genes, we investigated the genetic supports of resistances in nine multi-resistant Escherichia coli strains originating from human faecal contamination, and isolated from three different aquatic environments used for producing drinking water. METHODS AND RESULTS: Seven strains harboured at least one large plasmid that we have characterized (size, antibiotic resistance patterns, incompatibility group, capacity of autotransfer, presence of integron). Most of these plasmids were conjugative and carried numerous resistances. One of the plasmids studied, belonging to the IncP incompatibility group, was able to transfer by conjugation to Pseudomonas fluorescens and Aeromonas sp. Only two of the plasmids we studied carried class 1 and/or 2 integron(s). CONCLUSIONS: Conjugative plasmids isolated from multi-resistant E. coli strains explained most of the resistances of their host strains and probably contribute to the spread of antibiotic resistance genes coming from human faecal contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: These results highlight the key role played by plasmids in the multi-resistance phenotype of faecal bacteria and the diversity of these genetic structures. Contaminated water, especially accidentally contaminated drinking water, could be a path back to humans for these plasmids.201525387599
3557150.9998Characterization of the variable region in the class 1 integron of antimicrobial-resistant Escherichia coli isolated from surface water. Fecal bacteria are considered to be a potential reservoir of antimicrobial resistance genes in the aquatic environment and could horizontally transfer these genes to autochthonous bacteria when carried on transferable and/or mobile genetic elements. Such circulation of resistance genes constitutes a latent public health hazard. The aim of this study was to characterize the variable region of the class 1 integron and relate its genetic content to resistance patterns observed in antimicrobial-resistant Escherichia coli isolated from the surface waters of Patos Lagoon, Southern Brazil. Genetic diversity of the isolates and presence of the qacEΔ1 gene, which confers resistance to quaternary ammonium compounds, were also investigated. A total of 27 isolates were analyzed. The variable region harbored dfrA17, dfrA1 and dfrA12 genes, which confer resistance to trimethoprim, and aadA1, aadA5 and aadA22 genes that encode resistance to streptomycin/spectinomycin. Most of the isolates were considered resistant to quaternary ammonium compounds and all of them carried the qacEΔ1 gene at the 3' conserved segment of the integron. ERIC-PCR analyses of E. coli isolates that presented the integrons showed great genetic diversity, indicating diverse sources of contamination in this environment. These results suggest that fecal bacteria with class 1 integrons in aquatic environments are potentially important reservoirs of antibiotic-resistance genes and may transfer these elements to other bacteria that are capable of infecting humans.201626991286
3444160.9998Multidrug resistance in bacteria associated with leafy greens and soil in urban agriculture systems. Urban farms and community gardens support local food production, though these agroecosystems can contain emerging environmental contaminants that may contribute to the dissemination of antimicrobial resistance (AMR). Our previous research enumerated AMR bacteria associated with leafy vegetable production environments in the greater Washington, D.C. area, identifying >100 isolates with multidrug-resistant (MDR) phenotypes. Here, we performed whole genome sequencing analysis of 87 of these strains recovered from leafy greens (n=29), root zone soil (n=42), and bulk soil (n=16) to comprehensively characterize their MDR genotypes, including taxonomy and any encoded ARGs, stress response genes, and mobile genetic elements (MGEs; e.g., plasmids, phages, conjugative elements). The MDR isolates spanned 4 phyla and 14 genera, with the majority identified as Pseudomonas (n = 29), Serratia (n = 22), Providencia (n = 11), and Bacillus (n = 11). Most of the ARGs were linked to multidrug efflux, while other abundant ARG classes reflected resistance to beta-lactams and tetracyclines. While the genotypes were often conserved within respective species and even genera, the observed phenotypes within taxonomic groups slightly varied, suggesting the potential roles of uncharacterized genetic elements in MDR function. Moreover, all of the MDR isolates encoded at least one gene annotated as a MGE, and there were 19 distinct ARGs located within 5,000 bp upstream or downstream of these sequences, suggesting potential implications for mobilization. Overall, our results indicate that the MDR bacteria in urban agriculture systems, including on fresh produce, are dominated by general soil-associated taxa that carry diverse ARGs and MGEs.202541059364
3360170.9997Gentamicin resistance genes in environmental bacteria: prevalence and transfer. A comprehensive multiphasic survey of the prevalence and transfer of gentamicin resistance (Gm(r)) genes in different non-clinical environments has been performed. We were interested to find out whether Gm(r) genes described from clinical isolates can be detected in different environmental habitats and whether hot spots can be identified. Furthermore, this study aimed to evaluate the impact of selective pressure on the abundance and mobility of resistance genes. The study included samples from soils, rhizospheres, piggery manure, faeces from cattle, laying and broiler chickens, municipal and hospital sewage water, and coastal water. Six clusters of genes coding for Gm-modifying enzymes (aac(3)-I, aac(3)-II/VI, aac(3)-III/IV, aac(6')-II/Ib, ant(2'')-I, aph(2'')-I) were identified based on a database comparison and primer systems for each gene cluster were developed. Gm-resistant bacteria isolated from the different environments had a different taxonomic composition. In only 34 of 207 isolates, mainly originating from sewage, faeces and coastal water polluted with wastewater, were known Gm(r) genes corresponding to five of the six clusters detected. The strains belonged to genera in which the genes had previously been detected (Enterobacteriaceae, Pseudomonas, Acinetobacter) but also to phylogenetically distant bacteria, such as members of the CFB group, alpha- and beta-Proteobacteria. Gm(r) genes located on mobile genetic elements (MGE) could be captured in exogenous isolations into recipients belonging to alpha-, beta- and gamma-Proteobacteria from all environments except for soil. A high proportion of the MGE, conferring Gm resistance isolated from sewage, were identified as IncPbeta plasmids. Molecular detection of Gm(r) genes, and broad host range plasmid-specific sequences (IncP-1, IncN, IncW and IncQ) in environmental DNA indicated a habitat-specific dissemination. A high abundance and diversity of Gm(r) genes could be shown for samples from faeces (broilers, layers, cattle), from sewage, from seawater, collected close to a wastewater outflow, and from piggery manure. In the latter samples all six clusters of Gm(r) genes could be detected. The different kinds of selective pressure studied here seemed to enhance the abundance of MGE, while an effect on Gm(r) genes was not obvious.200219709289
1933180.9997Antibiotic Resistance Genes Occurrence in Conventional and Antibiotic-Free Poultry Farming, Italy. Antimicrobial resistance is a complex and widespread problem threatening human and animal health. In poultry farms, a wide distribution of resistant bacteria and their relative genes is described worldwide, including in Italy. In this paper, a comparison of resistance gene distribution in litter samples, recovered from four conventional and four antibiotic-free broiler flocks, was performed to highlight any influence of farming systems on the spreading and maintenance of resistance determinants. Conventional PCR tests, targeting the resistance genes related to the most used antibiotics in poultry farming, along with some critically important antibiotics for human medicine, were applied. In conventional farms, n. 10 out of n. 30 investigated genes were present in at least one sample, the most abundant fragments being the tet genes specific for tetracyclines, followed by those for aminoglycosides and chloramphenicol. All conventional samples resulted negative for colistin, carbapenems, and vancomycin resistance genes. A similar trend was observed for antibiotic-free herds, with n. 13 out of n. 30 amplified genes, while a positivity for the mcr-1 gene, specific for colistin, was observed in one antibiotic-free flock. The statistical analysis revealed a significant difference for the tetM gene, which was found more frequently in the antibiotic-free category. The analysis carried out in this study allowed us to obtain new data about the distribution of resistance patterns in the poultry industry in relation to farming types. The PCR test is a quick and non-expensive laboratory tool for the environmental monitoring of resistance determinants identifying potential indicators of AMR dissemination.202236139170
2843190.9997High Throughput Screening of Antimicrobial Resistance Genes in Gram-Negative Seafood Bacteria. From a global view of antimicrobial resistance over different sectors, seafood and the marine environment are often considered as potential reservoirs of antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs); however, there are few studies and sparse results on this sector. This study aims to provide new data and insights regarding the content of resistance markers in various seafood samples and sources, and therefore the potential exposure to humans in a global One Health approach. An innovative high throughput qPCR screening was developed and validated in order to simultaneously investigate the presence of 41 ARGs and 33 MGEs including plasmid replicons, integrons, and insertion sequences in Gram-negative bacteria. Analysis of 268 seafood isolates from the bacterial microflora of cod (n = 24), shellfish (n = 66), flat fishes (n = 53), shrimp (n = 10), and horse mackerel (n = 115) show the occurrence of sul-1, ant(3″)-Ia, aph(3')-Ia, strA, strB, dfrA1, qnrA, and bla(CTX-M-9) genes in Pseudomonas spp., Providencia spp., Klebsiella spp., Proteus spp., and Shewanella spp. isolates and the presence of MGEs in all bacterial species investigated. We found that the occurrence of MGE may be associated with the seafood type and the environmental, farming, and harvest conditions. Moreover, even if MGE were detected in half of the seafood isolates investigated, association with ARG was only identified for twelve isolates. The results corroborate the hypothesis that the incidence of antimicrobial-resistant bacteria (ARB) and ARG decreases with increasing distance from potential sources of fecal contamination. This unique and original high throughput micro-array designed for the screening of ARG and MGE in Gram-negative bacteria could be easily implementable for monitoring antimicrobial resistance gene markers in diverse contexts.202235744743