Abundance Of Antimicrobial Resistance Genes Research Articles

Occurrence and distribution of antimicrobial resistance genes in the soil of an industrial park in China: A metagenomics survey.

As zoned areas of industries, industrial parks have great impacts on the environment. Several studies have demonstrated that chemical compounds and heavy metals released from industrial parks can contaminate soil, water, and air. However, as an emerging pollutant, antimicrobial resistance genes (ARGs) in industrial parks have not yet been investigated. Here, we collected soil samples from 35 sites in an industrial park in China and applied a metagenomics strategy to profile the ARGs and virulence factors (VFs). We further compared the relative abundance of ARGs between the sites (TZ_31-35) located in a beta-lactam antimicrobial-producing factory and other sites (TZ_1-30) in this industrial park. Metagenomic sequencing and assembly generated 14, 383, 065 contigs and 17, 631, 051 open reading frames (ORFs). Taxonomy annotation revealed Proteobacteria and Actinobacteria as the most abundant phylum and class, respectively. The 32 pathogenic bacterial genera listed in the virulence factor database (VFDB) were all identified from the soil metagenomes in this industrial park. In total, 685,354 ARGs (3.89% of the ORFs) and 272,694 virulence factors (VFs) (1.55% of the ORFs) were annotated. These ARGs exhibited resistance to several critically important antimicrobials, such as rifampins, fluroquinolones, and beta-lactams. In addition, no significant difference in the relative abundance of ARGs was observed between sites TZ_31-35 and TZ_1-30, indicating that ARGs have already disseminated widely in this industrial park. The present study gave us a better understanding of the whole picture of the resistome and virulome in the soil of the industrial park and suggested that we should treat the industrial park as a whole in the surveillance and maintenance of ARGs.

Study design, rationale and methods of the Revitalising Informal Settlements and their Environments (RISE) study: a cluster randomised controlled trial to evaluate environmental and human health impacts of a water-sensitive intervention in informal settlements in Indonesia and Fiji

IntroductionIncreasing urban populations have led to the growth of informal settlements, with contaminated environments linked to poor human health through a range of interlinked pathways. Here, we describe the design...

Temporal Resistome and Microbial Community Dynamics in an Intensive Aquaculture Facility with Prophylactic Antimicrobial Treatment.

Excessive use of antimicrobials in aquaculture is concerning, given possible environmental ramifications and the potential contribution to the spread of antimicrobial resistance (AR). In this study, we explored seasonal abundance of antimicrobial resistance genes and bacterial community composition in the water column of an intensive aquaculture pond stocked with Silver Carp (Hypophthalmichthys molitrix) prophylactically treated with sulfamethoprim (25% sulfadiazine; 5% trimethoprim), relative to an adjacent unstocked reservoir. Bacterial community composition was monitored using high-throughput sequencing of 16S rRNA gene amplicons in eight sampling profiles to determine seasonal dynamics, representing principal stages in the fish fattening cycle. In tandem, qPCR was applied to assess relative abundance of selected antimicrobial resistance genes (sul1, sul2, dfrA1, tetA and blaTEM) and class-1 integrons (int1). Concomitantly, resistomes were extrapolated from shotgun metagenomes in representative profiles. Analyses revealed increased relative abundance of sulfonamide and tetracycline resistance genes in fishpond-03, relative to pre-stocking and reservoir levels, whereas no significant differences were observed for genes encoding resistance to antimicrobials that were not used in the fishpond-03. Seasons strongly dictated bacterial community composition, with high abundance of cyanobacteria in summer and increased relative abundance of Flavobacterium in the winter. Our results indicate that prophylactic use of sulfonamides in intensive aquaculture ponds facilitates resistance suggesting that prophylactic use of these antimicrobials in aquaculture should be restricted.

The distribution of microbiomes and resistomes across farm environments in conventional and organic dairy herds in Pennsylvania

BackgroundAntimicrobial resistance is a serious concern. Although the widespread use of antimicrobials in livestock has exacerbated the emergence and dissemination of antimicrobial resistance genes (ARG) in farm environments, little is known about whether antimicrobial use affects distribution of ARG in livestock systems. This study compared the distribution of microbiomes and resistomes (collections of ARG) across different farm sectors in dairy herds that differed in their use of antimicrobials. Feces from heifers, non-lactating, and lactating cows, manure storage, and soil from three conventional (antimicrobials used to treat cows) and three organic (no antimicrobials used for at least four years) farms in Pennsylvania were sampled. Samples were extracted for genomic DNA, processed, sequenced on the Illumina NextSeq platform, and analyzed for microbial community and resistome profiles using established procedures.ResultsMicrobial communities and resistome profiles clustered by sample type across all farms. Overall, abundance and diversity of ARG in feces was significantly higher in conventional herds compared to organic herds. The ARG conferring resistance to betalactams, macrolide-lincosamide-streptogramin (MLS), and tetracyclines were significantly higher in fecal samples of dairy cows from conventional herds compared to organic herds. Regardless of farm type, all manure storage samples had greater diversity (albeit low abundance) of ARG conferring resistance to aminoglycosides, tetracyclines, MLS, multidrug resistance, and phenicol. All soil samples had lower abundance of ARG compared to feces, manure, and lagoon samples and were comprised of ARG conferring resistance to aminoglycosides, glycopeptides, and multi-drug resistance. The distribution of ARG is likely driven by the composition of microbiota in the respective sample types.ConclusionsAntimicrobial use on farms significantly influenced specific groups of ARG in feces but not in manure storage or soil samples.

Antimicrobial use and production system shape the fecal, environmental, and slurry resistomes of pig farms

BackgroundThe global threat of antimicrobial resistance (AMR) is a One Health problem impacted by antimicrobial use (AMU) for human and livestock applications. Extensive Iberian swine production is based on a more sustainable and eco-friendly management system, providing an excellent opportunity to evaluate how sustained differences in AMU impact the resistome, not only in the animals but also on the farm environment. Here, we evaluate the resistome footprint of an extensive pig farming system, maintained for decades, as compared to that of industrialized intensive pig farming by analyzing 105 fecal, environmental and slurry metagenomes from 38 farms.ResultsOur results evidence a significantly higher abundance of antimicrobial resistance genes (ARGs) on intensive farms and a link between AMU and AMR to certain antimicrobial classes. We observed differences in the resistome across sample types, with a higher richness and dispersion of ARGs within environmental samples than on those from feces or slurry. Indeed, a deeper analysis revealed that differences among the three sample types were defined by taxa-ARGs associations. Interestingly, mobilome analyses revealed that the observed AMR differences between intensive and extensive farms could be linked to differences in the abundance of mobile genetic elements (MGEs). Thus, while there were no differences in the abundance of chromosomal-associated ARGs between intensive and extensive herds, a significantly higher abundance of integrons in the environment and plasmids, regardless of the sample type, was detected on intensive farms.ConclusionsOverall, this study shows how AMU, production system, and sample type influence, mainly through MGEs, the profile and dispersion of ARGs in pig production.-mkotjguAeV5vmuT7Jtr8vVideo

A Cross-Sectional Study of Dairy Cattle Metagenomes Reveals Increased Antimicrobial Resistance in Animals Farmed in a Heavy Metal Contaminated Environment.

The use of heavy metals in economic and social development can create an accumulation of toxic waste in the environment. High concentrations of heavy metals can damage human and animal health, lead to the development of antibiotic resistance, and possibly change in bovine microbiota. It is important to investigate the influence of heavy metals in food systems to determine potential harmful effects environmental heavy metal contamination on human health. Because of a mining dam rupture, 43 million cubic meters of iron ore waste flowed into the Doce river basin surrounding Mariana City, Brazil, in 2015. Following this environmental disaster, we investigated the consequences of long-term exposure to contaminated drinking water on the microbiome and resistome of dairy cattle. We identified bacterial antimicrobial resistance (AMR) genes in the feces, rumen fluid, and nasopharynx of 16 dairy cattle 4 years after the environmental disaster. Cattle had been continuously exposed to heavy metal contaminated water until sample collection (A) and compared them to analogous samples from 16 dairy cattle in an unaffected farm, 356 km away (B). The microbiome and resistome of farm A and farm B differed in many aspects. The distribution of genes present in the cattle’s nasopharynx, rumen, and feces conferring AMR was highly heterogeneous, and most genes were present in only a few samples. The relative abundance and prevalence (presence/absence) of AMR genes were higher in farm A than in farm B. Samples from farm A had a higher prevalence (presence) of genes conferring resistance to multiple drugs, metals, biocides, and multi-compound resistance. Fecal samples had a higher relative abundance of AMR genes, followed by rumen fluid samples, and the nasopharynx had the lowest relative abundance of AMR genes detected. Metagenome functional annotation suggested that selective pressures of heavy metal exposure potentially skewed pathway diversity toward fewer, more specialized functions. This is the first study that evaluates the consequences of a Brazilian environmental accident with mining ore dam failure in the microbiome of dairy cows. Our findings suggest that the long-term persistence of heavy metals in the environment may result in differences in the microbiota and enrichment of antimicrobial-resistant bacteria. Our results also suggest that AMR genes are most readily detected in fecal samples compared to rumen and nasopharyngeal samples which had relatively lower bacterial read counts. Since heavy metal contamination has an effect on the animal microbiome, environmental management is warranted to protect the food system from hazardous consequences.

Metagenomic analysis of gut microbiome and resistome of diarrheal fecal samples from Kolkata, India, reveals the core and variable microbiota including signatures of microbial dark matter

BackgroundMetagenomic analysis of the gut microbiome and resistome is instrumental for understanding the dynamics of diarrheal pathogenesis and antimicrobial resistance transmission (AMR). Metagenomic sequencing of 20 diarrheal fecal samples from Kolkata was conducted to understand the core and variable gut microbiota. Five of these samples were used for resistome analysis. The pilot study was conducted to determine a microbiota signature and the source of antimicrobial resistance genes (ARGs) in the diarrheal gut.Results16S rRNA amplicon sequencing was performed using Illumina MiSeq platform and analysed using the MGnify pipeline. The Genome Taxonomy Database (GTDB-Tk) was used for bacterial taxonomic identification. Diarrheal etiology was determined by culture method. Phylum Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria were consistently present in 20 samples. Firmicutes was the most abundant phylum in 11 samples. The Bacteroidetes/Firmicutes ratio was less than 1 in 18 samples. 584 genera were observed. 18 of these were present in all the 20 samples. Proteobacteria was the dominant phylum in 6 samples associated with Vibrio cholerae infection. Conservation of operational taxonomic units (OTUs) among all the samples indicated the existence of a core microbiome. Asymptomatic carriage of pathogens like Vibrio cholerae and Helicobacter pylori was found. Signature of Candidate phyla or “microbial dark matter” occurred. Significant correlation of relative abundance of bacterial families of commensals and pathogens were found. Whole-genome sequencing (WGS) on Illumina MiSeq system and assembly of raw reads using metaSPAdes v3.9.1 was performed to study the resistome of 5 samples. ABRicate was used to assign ARG function. 491 resistance determinants were identified. In 80% of the samples tetracycline resistance was the most abundant resistance determinant. High abundance of ARGs against β-lactams, aminoglycosides, quinolones and macrolides was found. Eschericia sp. was the major contributor of ARGs.ConclusionsThis is the first comparative study of the gut microbiome associated with different diarrheal pathogens. It presents the first catalogue of different bacterial taxa representing the core and variable microbiome in acute diarrheal patients. The study helped to define a trend in the gut microbiota signature associated with diarrhea and revealed which ARGs are abundantly present and the metagenome-assembled genomes (MAGs) contributing to AMR.

Reservoirs of antimicrobial resistance genes in retail raw milk

BackgroundIt has been estimated that at least 3% of the USA population consumes unpasteurized (raw) milk from animal sources, and the demand to legalize raw milk sales continues to increase. However, consumption of raw milk can cause foodborne illness and be a source of bacteria containing transferrable antimicrobial resistance genes (ARGs). To obtain a comprehensive understanding of the microbiome and antibiotic resistome in both raw and processed milk, we systematically analyzed 2034 retail milk samples including unpasteurized milk and pasteurized milk via vat pasteurization, high-temperature-short-time pasteurization, and ultra-pasteurization from the United States using complementary culture-based, 16S rRNA gene, and metagenomic sequencing techniques.ResultsRaw milk samples had the highest prevalence of viable bacteria which were measured as all aerobic bacteria, coliform, and Escherichia coli counts, and their microbiota was distinct from other types of milk. 16S rRNA gene sequencing revealed that Pseudomonadaceae dominated raw milk with limited levels of lactic acid bacteria. Among all milk samples, the microbiota remained stable with constant bacterial populations when stored at 4 °C. In contrast, storage at room temperature dramatically enriched the bacterial populations present in raw milk samples and, in parallel, significantly increased the richness and abundance of ARGs. Metagenomic sequencing indicated raw milk possessed dramatically more ARGs than pasteurized milk, and a conjugation assay documented the active transfer of blaCMY-2, one ceftazidime resistance gene present in raw milk-borne E. coli, across bacterial species. The room temperature-enriched resistome differed in raw milk from distinct geographic locations, a difference likely associated with regionally distinct milk microbiota.ConclusionDespite advertised “probiotic” effects, our results indicate that raw milk microbiota has minimal lactic acid bacteria. In addition, retail raw milk serves as a reservoir of ARGs, populations of which are readily amplified by spontaneous fermentation. There is an increased need to understand potential food safety risks from improper transportation and storage of raw milk with regard to ARGs.E4kXQjxU9tgRorkuFR9RTiVideo

Antimicrobial Resistance in Swine Fecal Specimens Across Different Farm Management Systems.

Antimicrobial use in agricultural animals is known to be associated with increases in antimicrobial resistance. Most prior studies have utilized culture and susceptibility testing of select organisms to document these phenomena. In this study we aimed to detect 66 antimicrobial resistance (AMR) genes for 10 antimicrobial agent classes directly in swine fecal samples using our previously developed antimicrobial resistance TaqMan array card (AMR-TAC) across three different swine farm management systems. This included 38 extensive antimicrobial use (both in treatment and feed), 30 limited antimicrobial use (treatment only), and 30 no antimicrobial use farms. The number of resistance genes detected in extensive antimicrobial use farms was higher than in limited and no antimicrobial use farms (28.2 genes ± 4.2 vs. 24.0 genes ± 4.1 and 22.8 genes ± 3.6, respectively, p < 0.05). A principal component analysis and hierarchical clustering of the AMR gene data showed the extensive use farm samples were disparate from the limited and no antimicrobial use farms. The prevalence of resistance genes in extensive use farms was significantly higher than the other farm categories for 18 resistance genes including blaSHV, blaCTX–M1 group, blaCTX–M9 group, blaVEB, blaCMY2–LAT, aac(6′)-lb-cr, qnrB1, gyrA83L-E. coli, armA, rmtB, aac(3)-IIa, mphA, 23S rRNA 2075G-Campylobacter spp., mcr-1, catA1, floR, dfrA5-14, and dfrA17. These genotypic findings were supported by phenotypic susceptibility results on fecal E. coli isolates. To examine the timing of AMR gene abundance in swine farms, we also performed a longitudinal study in pigs. The results showed that AMR prevalence occurred both early, presumably from mothers, as well as after weaning, presumably from the environment. In summary, detection of AMR genes directly in fecal samples can be used to qualitatively and quantitatively monitor AMR in swine farms.

Toward Antibiotic Stewardship: Route of Antibiotic Administration Impacts the Microbiota and Resistance Gene Diversity in Swine Feces.

Oral antibiotics are a critical tool for fighting bacterial infections, yet their use can have negative consequences, such as the disturbance of healthy gut bacterial communities and the dissemination of antibiotic residues in feces. Altering antibiotic administration route may limit negative impacts on intestinal microbiota and reduce selective pressure for antimicrobial resistance genes (ARG) persistence and mobility. Thus, a study was performed in pigs to evaluate route of therapeutic oxytetracycline (oxytet) administration, an antibiotic commonly used in the U.S. swine industry, on intestinal microbial diversity and ARG abundance. Given that oral antibiotics would be in direct contact with intestinal bacteria, we hypothesized that oral administration would cause a major shift in intestinal bacterial community structure when compared to injected antibiotic. We further postulated that the impact would extend to the diversity and abundance of ARG in swine feces. At approximately 3 weeks-of-age, piglets were separated into three groups (n = 21–22 per group) with two groups receiving oxytet (one via injection and the second via feed) and a third non-medicated group. Oxytet levels in the plasma indicated injected antibiotic resulted in a spike 1 day after administration, which decreased over time, though oxytet was still detected in plasma 14 days after injection. Conversely, in-feed oxytet delivery resulted in lower but less variable oxytet levels in circulation and high concentrations in feces. Similar trends were observed in microbial community changes regardless of route of oxytet administration; however, the impact on the microbial community was more pronounced at all time points and in all samples with in-feed administration. Fecal ARG abundance was increased with in-feed administration over injected, with genes for tetracycline and aminoglycoside resistance enriched specifically in the feces of the in-feed group. Sequencing of plasmid-enriched samples revealed multiple genetic contexts for the resistance genes detected and highlighted the potential role of small plasmids in the movement of antibiotic resistance genes. The findings are informative for disease management in food animals, but also manure management and antibiotic therapy in human medicine for improved antibiotic stewardship.

Occurrence and abundance of clinically relevant antimicrobial resistance genes in environmental samples after the Brumadinho dam disaster, Brazil

On January 25th 2019, the structure damming a pond containing ore mining wastes and iron burst at Brumadinho City, Brazil. About 11.7 million m3 of a tailings-mud mixture was released from the dam, causing destruction along 300 km of the Paraopeba River toward the São Francisco River. The environments with a high content of metals may provide a suitable environment for horizontal gene transfer, including antimicrobial resistance genes (ARGs). Therefore, this study aimed to detect and quantify clinically relevant ARGs in environmental samples after the Brumadinho dam disaster. Soil, sediment, and water samples were collected within 300 km of the Brumadinho dam disaster at unaffected and affected sites. Physical-chemical parameters of water samples were measured. Total DNA was extracted and 65 clinically relevant ARGs were researched by PCR. The most prevalent ARGs were selected for real-time quantitative PCR analysis. The average of the physical-chemical parameters was higher in the affected sites when compared to the unaffected sites, especially turbidity, concentration of Fe and Al. A total of 387 amplicons from 29 ARGs were detected, which confer resistance to β-lactams, quinolones, aminoglycosides, tetracyclines, sulphonamides, phenicols, macrolides, glycopeptides, and polymyxins, including extended-spectrum β-lactamases-encoding genes, and mcr-7.1. The sul1 gene had higher total concentrations than blaTEM, tetB and qnrB in the environmental samples, and the diversity and abundance of ARGs increased at the sites affected by the Brumadinho dam disaster. Therefore, we point out that the contamination by the Brumadinho dam disaster tailings resulted in an increase in the amount and abundance of ARGs in the environment.

Molecular Analysis of Selected Resistance Determinants in Diarrheal Fecal Samples Collected From Kolkata, India Reveals an Abundance of Resistance Genes and the Potential Role of the Microbiota in Its Dissemination.

Twenty-five diarrheal fecal samples from Kolkata were examined to determine the relative abundance of antimicrobial resistance genes (ARGs) against eight common classes of antibiotics with polymerase chain reaction (PCR) and Sanger sequencing. Relative abundance of an ARG was calculated as the percentage of fecal samples showing the presence of that particular ARG. The frequency of occurrence of resistance marker against each class of antibiotic was calculated as the percentage of fecal samples carrying at least one resistance marker for that particular class of antimicrobials. Antibiogram of Vibrio cholerae (V. cholerae) O1 strains isolated from four of these samples was obtained by disc diffusion method and was compared with the ARG profile of corresponding fecal samples from which the strains were isolated. A 464 bp amplicon of the V3-V4 region of bacterial 16S rDNA was obtained by PCR from 9 of these 25 samples using the primer pair S-D-Bact-0341-b-S-17 and S-D-Bact-0785-a-A-21 and sequenced to determine the major operational taxonomic unit (OTU). These 9 samples represented diarrhea due to diverse etiology and also unresolved etiology as determined by culture method. We conclude that the diarrheal intestinal microbiome has a common gene pool of ARGs against the major classes of antibiotics and may be serving as a reservoir of ARG dissemination. ARG profile of cholera stool showed that ARGs present in the gut of cholera patients may be transferred to the V. cholerae genome and pose a serious threat to the treatment of cholera by triggering resistance against potential drugs to which contemporary strains of V. cholerae were found to be sensitive in the present study. Fecal samples which were culture negative for diarrheal pathogens we tested also carried ARGs and OTU. Abundance of resistance markers against macrolides, tetracyclines, and aminoglycosides was the highest. Phylum Proteobacteria was the most abundant OTU suggesting proteobacterial blooms characteristic of disturbed gut microflora. Our study is the first comparative study of ARG profile of diarrheal samples with varying etiologic agent revealing the presence of ARGs against the most important classes of antibiotics in the gut of diarrheal patients by common, robust molecular methods, which are easily accessible by molecular epidemiological laboratories worldwide.

Manure Compost Is a Potential Source of Tetracycline-Resistant Escherichia coli and Tetracycline Resistance Genes in Japanese Farms.

Manure compost has been thought of as a potential important route of transmission of antimicrobial-resistant bacteria (ARB) and antimicrobial resistance genes (ARGs) from livestock to humans. To clarify the abundance of ARB and ARGs, ARB and ARGs were quantitatively determined in tetracycline-resistant Escherichia coli (harboring the tetA gene)-spiked feces in simulated composts. In the simulated composts, the concentration of spiked E. coli decreased below the detection limit at day 7. The tetA gene remained in manure compost for 20 days, although the levels of the gene decreased. Next, to clarify the field conditions of manure compost in Japan, the quantities of tetracycline-resistant bacteria, tetracycline resistance genes, and residual tetracyclines were determined using field-manure-matured composts in livestock farms. Tetracycline-resistant bacteria were detected in 54.5% of tested matured compost (6/11 farms). The copy number of the tetA gene and the concentrations of residual tetracyclines in field manure compost were significantly correlated. These results suggest that the use of antimicrobials in livestock constitutes a selective pressure, not only in livestock feces but also in manure compost. The appropriate use of antimicrobials in livestock and treatment of manure compost are important for avoiding the spread of ARB and ARGs.

Abundance and diversity of resistomes differ between healthy human oral cavities and gut

The global threat of antimicrobial resistance has driven the use of high-throughput sequencing techniques to monitor the profile of resistance genes, known as the resistome, in microbial populations. The human oral cavity contains a poorly explored reservoir of these genes. Here we analyse and compare the resistome profiles of 788 oral cavities worldwide with paired stool metagenomes. We find country and body site-specific differences in the prevalence of antimicrobial resistance genes, classes and mechanisms in oral and stool samples. Within individuals, the highest abundances of antimicrobial resistance genes are found in the oral cavity, but the oral cavity contains a lower diversity of resistance genes compared to the gut. Additionally, co-occurrence analysis shows contrasting ARG-species associations between saliva and stool samples. Maintenance and persistence of antimicrobial resistance is likely to vary across different body sites. Thus, we highlight the importance of characterising the resistome across body sites to uncover the antimicrobial resistance potential in the human body.

Resistance to change: AMR gene dynamics on a commercial pig farm with high antimicrobial usage

Group antimicrobial administration is used to control disease in livestock, but we have little insight into how this impacts antimicrobial resistance (AMR) gene dynamics. Here, a longitudinal study was carried out during a single production cycle on a commercial pig unit with high historic and current antimicrobial usage. Quantitative PCR, 16S rRNA gene metabarcoding and shotgun metagenomic sequencing were used to track faecal AMR gene abundance and diversity and microbiome alpha diversity. Shotgun metagenomic sequencing identified 144 AMR genes in total, with higher AMR gene diversity present in young pigs compared to dry sows. Irrespective of in-feed antibiotic treatment or changes in microbiome diversity, mean AMR gene copy number was consistently high, with some AMR genes present at copy numbers comparable to the bacterial 16S rRNA gene. In conclusion, AMR gene prevalence and abundance were not influenced by antibiotic use, either during the production cycle or following whole-herd medication. The high levels of certain genes indicate they are widely disseminated throughout the microbial population, potentially aiding stability. Despite the high and relatively stable levels of resistance genes against the main antimicrobials used, these compounds continue to control production limiting diseases on this unit.

Antimicrobial Resistance in Retail Ground Beef with and Without a “Raised Without Antibiotics” Claim

ObjectivesThe occurrences of human bacterial infections complicated by antimicrobial resistance (AMR) have increased in recent decades. Concerns have been raised that food-animal production practices that incorporate antimicrobials contribute significantly to human AMR exposures since food-animal production accounts for approximately 81% of U.S. antimicrobial consumption by mass. Although empirical studies comparing AMR levels in meat products, including ground beef, are scant ground beef products with Raised without Antibiotics (RWA) label claims are perceived to harbor less AMR than “conventional” (CONV) products with no label claims regarding antimicrobial use. The objective of this research was to determine AMR levels in retail ground beef with and without an RWA label claims.Materials and MethodsRetail ground beef samples were obtained from 6 U.S. cities. Samples were obtained on the following dates: 9/18/2017, 10/30/2017, 11/27/2017. 1/29/2018. 3/5/2018, and 6/11/2018. A total of 599 samples were obtained. Samples with a “Raised without Antibiotics” or USDA Organic claim (N = 299) were assigned to the RWA production system. Samples lacking a “Raised without Antibiotics” claim (N = 300) were assigned to the CONV production system. Each sample was cultured for the detection of five antimicrobial resistant bacteria (ARB). Genomic DNA was isolated from each sample and qPCR was used to determine the abundance of ten antimicrobial resistance genes (ARGs). The impacts of production system and city on ARB detection were assessed by the Likelihood-ratio chi-squared test. The impacts of production system and city on ARG abundance was assessed by two-way ANOVA.ResultsTetracycline-resistant Escherichia coli (CONV = 46.3%; RWA = 34.4%) and erythromycin-resistant Enterococcus (CONV = 48.0%; RWA = 37.5%) were more frequently (P &lt; 0.01) detected in CONV. Detection of third generation cephalosporin-resistant E. coli (CONV = 5.7%; RWA = 1.0%), vancomycin-resistant Enterococcus (CONV = 0.0%; RWA = 0.0%) and methicillin-resistant Staphylococcus aureus (CONV = 1.3%; RWA = 0.7%) did not differ (P = 1.00). The blaCTX-M ARG was more abundant in CONV (2.4 vs. 2.1 log copies/gram, P = 0.01) but the tet(A) (2.4 vs. 2.5 log copies/gram, P = 0.02) and tet(M) (3.6 vs. 3.9 log copies/gram, P &lt; 0.01) ARGs were more abundant in RWA. aadA1, blaCMY-2, mecA, erm(B), and tet(B) abundances did not differ significantly (Fig. 5) (P &gt; 0.05). Abundances of aac (6’)-Ie-aph (2”)-Ia and blaKPC-2 were not analyzed since they were quantified in less than 5% of the samples.ConclusionU.S. retail CONV and RWA ground beef harbor generally similar levels of AMR since only 5 of 15 AMR measurements were statistically different between production systems. Three AMR measurements were higher in CONV, while 2 AMR measurements were higher in RWA. These results are in general agreement with a recently published study authored by our group that examined antimicrobial resistance in CONV and RWA ground beef obtained from U.S. foodservice suppliers (Vikram et al., J. Food Prot. 81:2007–2018. 2018.). Together these studies suggest that antimicrobial use during U.S. cattle production has minimal to no impact on human exposure to AMR via ground beef.Figure 5.

Predicting effects of changed antimicrobial usage on the abundance of antimicrobial resistance genes in finisher’ gut microbiomes

It is accepted that usage of antimicrobials (AMs) in food animals causes the emergence and spread of antimicrobial resistance (AMR) in this sector, while also contributing to the burden of AMR in humans. Curbing the increasing occurrence of AMR in food animals requires in-depth knowledge of the quantitative relationship between antimicrobial usage (AMU) and AMR to achieve desired resistance reductions from interventions targeting AMU. In the observational study, the relationships between lifetime AMU in 83 finisher batches from Danish farms and the AMR gene abundances of seven antimicrobial classes in their gut microbiomes were quantified using multi-variable linear regression models. These relationships and the national lifetime AMU in pigs were included in the predictive modelling that allowed for testing of scenarios with changed lifetime AMU for finishers produced in Denmark in 2014. A total of 50 farms from the observational study were included in validating the observational study and the predictive modelling. The results from the observational study showed that the relationship was linear, and that the parenteral usage of AMs had a high effect on specific AM-classes of resistance, whereas the peroral usage had a lower but broader effect on several classes. Three different scenarios of changed lifetime AMU were simulated in the predictive modelling. When all tetracycline usage ceased, the predicted interval reductions of aminoglycoside, lincosamide and tetracycline resistance were 4–42 %, 0–8 % and 9–18 %, respectively. When the peroral tetracycline usage of the 10 % highest users was replaced with peroral macrolide usage, the tetracycline resistance fell by 1–2 % and the macrolide and MLSb resistance increased by 5–8 %. When all extended-spectrum penicillin usage was replaced with parenteral lincosamide usage, the beta-lactam resistance fell by 2–7 %, but the lincosamide usage and resistance increased by 194 % and 10–45 %, respectively. The external validation provided results within the 95 % CI of the predictive modelling outcome at national level, while the external validation at farm level was less accurate. In conclusion, interventions targeting AMU will reduce AMR abundance, though differently depending on the targeted AM-class and provided the reduction of one AM-class usage is not replaced with usage of another AM-class. Predicting several classes of AMR gene abundance simultaneously will support stakeholders when deciding on interventions targeting AMU in the finisher production to avoid adverse and unforeseen effects on the AMR abundance. This study provides a sound predictive modelling framework for further development, including the dynamics of AMU on AMR in finishers at national level.

Role of wastewater treatment plants on environmental abundance of Antimicrobial Resistance Genes in Chilean rivers.

Point sources such as wastewater treatment plants (WWTPs) commonly discharge their effluent into rivers. Their waste may include antibiotic residues, disinfectants, antibiotic resistant bacteria (ARB), and Antimicrobial Resistance Genes (ARG). There is evidence that ARG can be found in the natural environment, but attribution to specific point sources is lacking. The goal of this study was to assess the release and dissemination of ARG from three WWTPs in southern Chile via two pathways: through the river systems, and through wild birds. A longitudinal study was conducted, collecting river sediment samples at different distances both upstream and downstream from each WWTP. Wild birds were sampled from around one of the WWTPs once a month for 13 months. A microfluidic q-PCR approach was used to quantify 48 genes covering different molecular mechanisms of resistance, and data was analyzed using ordination methods and linear mixed regression models. There was a statistically significant increase downstream from the WWTPs (p < 0.05) for 17 ARG, but the downstream dissemination through the rivers was not clear. Beta-lactamase genes blaKPC, blaTEM, and blaSHV were the most abundant in birds, with higher abundance of blaSHV in migratory species compared to resident species (p < 0.05). The gene profile was more similar between the migratory and resident bird groups compared to the WWTP gene profile. While results from this study indicate an influence of WWTPs on ARG abundance in the rivers, the biological significance of this increase and the extent of the WWTPs influence are unclear. In addition, wild birds were found to play a role in disseminating ARG, although association to the specific WWTP could not be ascertained.

Comparative genomic analysis reveals a high prevalence of inter-species in vivo transfer of carbapenem-resistance plasmids in patients with haematological malignancies

ObjectivesConjugative gene transfer has been considered as one of the driving factors in the transmission and dissemination of multidrug resistance in bacteria. The abundance of antimicrobial resistance genes and bacteria in the gut microbiome may provide the ideal platform for plasmid exchange. Systematic data on in vivo horizontal gene transfer (HGT) and its frequency are scarce. Materials and methodsOne hundred and ninety-six carbapenem-resistant gram-negative bacilli (CRGNBs) from 179 patients (158 inpatients and 21 outpatients) between January 2016 and April 2017 were analysed retrospectively. Alignment of plasmid content for 32 isolates from 16 patients with multiple CRGNB species was performed from whole-genome sequencing (WGS) data. ResultsSixteen of the 179 patients (8.9%) were colonized and/or infected with more than one CRGNB species; 11/179 (6.1%) were colonized by multiple carbapenem-resistant Enterobacteriaceae (CREs) and 5/179 (2.8%) by carbapenem-resistant non-fermenters (CRNFs) and CREs. WGS suggested interspecies transfer as the predominant mechanism rather than independent acquisition in 8/10 patients (80%, one non-recoverable isolate) with multiple CREs but not in CRNF–CRE combinations; 30/158 inpatients (20%) had underlying haematological malignancies, and they are more likely to exhibit multiple CRGNB strains (OR 3.0, 95%CI 0.98–8.89, p 0.05) and CRE strains (OR 3.9, 95%CI 1.02–14.58, p 0.04) during hospital stay compared to other patient groups. ConclusionOur data give insight into the occurrence of natural in vivo HGT in a clinical setting. Better understanding of HGT will help optimize containment measures and may guide antibiotic stewardship programmes.

Exploring the profile of antimicrobial resistance genes harboring by bacteriophage in chicken feces

Bacteriophage may play an important role in antimicrobial resistance genes (ARGs) transmission. However, the contribution of bacteriophage to the spread of ARGs in environment, especially in poultry farm environment, is rarely known. In this study, the prevalence of ARGs in bacteriophage DNA was investigated in chicken feces from 30 different poultry farms in China. Then the abundance of the aac(6′)-Ib-cr, blaCTX-M, ermB, floR, mcr-1, sul1, tetM and intI1 genes was determined by qPCR in bacteriophage and compared with certain representative plasmid DNA samples. The results showed that 12 ARGs (aac(6′)-Ib-cr, aph(3′)-IIIa, blaCTX-M, ermB, ermF, floR, mcr-1, qnrS, sul1, sul2, vanA, tetM genes) and class 1 integron gene intI1 were detected in bacteriophage DNA fraction. The sul1, tetM and aac(6′)-Ib-cr genes were most prevalent with high detection rates of 77%, 61% and 55%, respectively. To our best knowledge, this study firstly reported the presence of the mcr-1 gene in bacteriophage DNA derived from farms environments. We found that the gene copy (GC) numbers of the aac(6′)-Ib-cr, ermB and sul1 genes were as high as 5.47, 5.22 and 5.54 log10 GC/g, respectively. Both the prevalence and abundance of ARGs in broiler fecal wastes were also generally higher than in laying hens. In addition, although the GC numbers of the aac(6′)-Ib-cr, floR and tetM genes in plasmid DNA was higher than that in phage DNA fraction by 4.68, 3.59 and 3.9 orders of magnitude, respectively, the absolute abundances of the blaCTX-M and mcr-1 genes in phage DNA were close to or even higher than that in plasmid DNA at farm SIL2, SIL4 and SIB1. As potential vessels for ARGs, bacteriophage could not be ignored due to their unique extracellular persistence in environments. Overall, this is the first comprehensive survey about bacteriophage carried ARGs from farms in different regions in China.