Multiple Antimicrobial Resistance Genes Research Articles

Toxins and mobile antimicrobial resistance genes in Bacillus probiotics constitute a potential risk for One Health

Probiotic microbes conferring health benefits to the hosts have attracted great attention. However, the safety of probiotics is not guaranteed, although the increasing widespread use of probiotics with excellent overall safety records. Here, we performed a systematic evaluation of the safety of commercial Bacillus probiotics intended for usage in humans, animals, plants, aquaculture and environment in China. Nearly half of the 65 isolated Bacillus spp. strains from these commercial probiotic products were capable of producing hazardous toxins. Infections with the representative isolates could cause sepsis, intestinal inflammation and liver injury in different mouse models. Additionally, these isolates harbor multiple antimicrobial resistance genes coupled with mobile genetic elements. Collectively, the capability for producing various toxins and harboring mobile antimicrobial resistance genes in Bacillus probiotics indicates a potential risk for One Health.

Emerging challenges of whole-genome-sequencing–powered epidemiological surveillance of globally distributed clonal groups of bacterial infections, giving Acinetobacter baumannii ST195 as an example

Whole-genome sequencing (WGS) has revolutionized the genotyping of bacterial pathogens and is expected to become the new gold standard for tracing the transmissions of bacterial infectious diseases for public health purposes. However, it is still unexpectedly demanding to employ WGS for global epidemiological surveillance because of the high degree of similarity between the genomes of intercontinental isolates. The aim of this study was to utilize genomically derived bioinformatics analysis to identify globally distributed A. baumannii ST195 lineage and differentiation outbreaks to address this issue. The genomic sequences and their related epidemiological metadata of 2850 A. baumannii isolates were recruited from NCBI Genbank database. Assignment into sequence type (Oxford scheme) and lineage (global clone 2/CC92) were performed. A total of 91 ST195 A. baumannii isolates were subsequently classified to perform the bacterial source tracking analysis by implementing both core genome MLST (cgMLST) and core genome SNP (cgSNP) strategy that were integrated in our recently updated BacWGSTdb 2.0 server. Antibiotic resistance genes were identified using the ResFinder database. The ST195 A. baumannii isolates distributed widely in eight countries and harboured multiple antimicrobial resistance genes simultaneously. In most cases, the bacterial isolates recovered from geographically distant sources may present less genomic sequence similarity, i.e., the phylogenetic relationship between these ST195 isolates worldwide was roughly congruent with their country of isolation. However, a few isolates collected from distant geographic regions were revealed to possess smaller genetic distances (less than 8 loci or 20 SNPs) than the threshold without an observable epidemiological link. Our study highlights the emerging challenges entailed in the WGS-powered epidemiological surveillance of globally distributed clonal groups. Standardization is urgently required before WGS can be routinely applied to infectious diseases outbreak investigations.

Characterization of NDM-5- and CTX-M-55-coproducing Escherichia coli GSH8M-2 isolated from the effluent of a wastewater treatment plant in Tokyo Bay.

PurposeNew Delhi metallo-β-lactamase (NDM)-5-producing Enterobacteriaceae have been detected in rivers, sewage, and effluents from wastewater treatment plants (WWTPs). Environmental contamination due to discharged effluents is of particular concern as NDM variants may be released into waterways, thereby posing a risk to humans. In this study, we collected effluent samples from a WWTP discharged into a canal in Tokyo Bay, Japan.MethodsTesting included the complete genome sequencing of Escherichia coli GSH8M-2 isolated from the effluent as well as a gene network analysis.ResultsThe complete genome sequencing of GSH8M-2 revealed that it was an NDM-5-producing E. coli strain sequence type ST542, which carries multiple antimicrobial resistance genes for β-lactams, quinolone, tetracycline, trimethoprim-sulfamethoxazole, florfenicol/chloramphenicol, kanamycin, and fosfomycin. The blaNDM-5 gene was found in the IncX3 replicon plasmid pGSH8M-2-4. Gene network analysis using 142 IncX3 plasmid sequences suggested that pGSH8M-2-4 is related to both clinical isolates of E. coli and Klebsiella species in Eastern Asia. GSH8M-2 also carries the blaCTX-M-55 gene in IncX1 plasmid pGSH8M-2-3.ConclusionThis is the first report of environmental NDM-5-producing E. coli isolated from a WWTP in Japan. NDM-5 detection is markedly increasing in veterinary and clinical settings, suggesting that dual β-lactamases, such as NDM-5 and CTX-M-55, might be acquired through multiple steps in environment settings. Environmental contamination through WWTP effluents that contain producers of NDM variants could be an emerging potential health hazard. Thus, regular monitoring of WWTP effluents is important for the detection of antimicrobial-resistant bacteria that may be released into the waterways and nearby communities.

Microevolution of antimicrobial resistance and biofilm formation of Salmonella Typhimurium during persistence on pig farms

Salmonella Typhimurium and its monophasic variant S. 4,[5],12:i:- are the dominant serotypes associated with pigs in many countries. We investigated their population structure on nine farms using whole genome sequencing, and their genotypic and phenotypic variation. The population structure revealed the presence of phylogenetically distinct clades consisting of closely related clones of S. Typhimurium or S. 4,[5],12:i:- on each pig farm, that persisted between production cycles. All the S. 4,[5],12:i:- strains carried the Salmonella genomic island-4 (SGI-4), which confers resistance to heavy metals, and half of the strains contained the mTmV prophage, harbouring the sopE virulence gene. Most clonal groups were highly drug resistant due to the presence of multiple antimicrobial resistance (AMR) genes, and two clades exhibited evidence of recent on-farm plasmid-mediated acquisition of additional AMR genes, including an IncHI2 plasmid. Biofilm formation was highly variable but had a strong phylogenetic signature. Strains capable of forming biofilm with the greatest biomass were from the S. 4,[5],12:i:- and S. Typhimurium DT104 clades, the two dominant pandemic clones found over the last 25 years. On-farm microevolution resulted in enhanced biofilm formation in subsequent production cycle.

Comparative Genomic Analysis of Virulence, Antimicrobial Resistance, and Plasmid Profiles of Salmonella Dublin Isolated from Sick Cattle, Retail Beef, and Humans in the United States.

Salmonella enterica serovar Dublin is a host-adapted serotype associated with typhoidal disease in cattle. While rare in humans, it usually causes severe illness, including bacteremia. In the United States, Salmonella Dublin has become one of the most multidrug-resistant (MDR) serotypes. To understand the genetic elements that are associated with virulence and resistance, we sequenced 61 isolates of Salmonella Dublin (49 from sick cattle and 12 from retail beef) using the Illumina MiSeq and closed 5 genomes using the PacBio sequencing platform. Genomic data of eight human isolates were also downloaded from NCBI (National Center for Biotechnology Information) for comparative analysis. Fifteen Salmonella pathogenicity islands (SPIs) and a spv operon (spvRABCD), which encodes important virulence factors, were identified in all 69 (100%) isolates. The 15 SPIs were located on the chromosome of the 5 closed genomes, with each of these isolates also carrying 1 or 2 plasmids with sizes between 36 and 329 kb. Multiple antimicrobial resistance genes (ARGs), including blaCMY-2, blaTEM-1B, aadA12, aph(3')-Ia, aph(3')-Ic, strA, strB, floR, sul1, sul2, and tet(A), along with spv operons were identified on these plasmids. Comprehensive antimicrobial resistance genotypes were determined, including 17 genes encoding resistance to 5 different classes of antimicrobials, and mutations in the housekeeping gene (gyrA) associated with resistance or decreased susceptibility to fluoroquinolones. Together these data revealed that this panel of Salmonella Dublin commonly carried 15 SPIs, MDR/virulence plasmids, and ARGs against several classes of antimicrobials. Such genomic elements may make important contributions to the severity of disease and treatment failures in Salmonella Dublin infections in both humans and cattle.

Exploration of antibiotic resistance risks in a veterinary teaching hospital with Oxford Nanopore long read sequencing.

The Oxford Nanopore MinION DNA sequencing device can produce large amounts of long sequences, typically several kilobases, within a few hours. This long read capacity was exploited to detect antimicrobial resistance genes (ARGs) in a large veterinary teaching hospital environment, and to assess their taxonomic origin, genetic organisation and association with mobilisation markers concurrently. Samples were collected on eight occasions between November 2016 and May 2017 (inclusive) in a longitudinal study. Nanopore sequencing was performed on total DNA extracted from the samples after a minimal enrichment step in broth. Many ARGs present in the veterinary hospital environment could potentially confer resistance to antimicrobials widely used in treating infections of companion animals, including aminoglycosides, extended-spectrum beta-lactams, sulphonamides, macrolides, and tetracyclines. High-risk ARGs, defined here as single or multiple ARGs associated with pathogenic bacterial species or with mobile genetic elements, were shared between the intensive care unit (ICU) patient cages, a dedicated laundry trolley and a floor cleaning mop-bucket. By contrast, a floor surface from an office corridor without animal contact and located outside the veterinary hospital did not contain such high-risk ARGs. Relative abundances of high-risk ARGs and co-localisation of these genes on the same sequence read were higher in the laundry trolley and mop bucket samples, compared to the ICU cages, suggesting that amplification of ARGs is likely to occur in the collection points for hospital waste. These findings have prompted the implementation of targeted intervention measures in the veterinary hospital to mitigate the risks of transferring clinically important ARGs between sites and to improve biosecurity practices in the facility.

Data-Driven Analysis of Antimicrobial Resistance in Foodborne Pathogens from Six States within the US.

Foodborne pathogens cause thousands of illnesses across the US each year. However, these pathogens gain resistance to the antimicrobials that are commonly used to treat them. Typically, antimicrobial resistance is caused by mechanisms encoded by multiple antimicrobial-resistance genes. These are carried through pathogens found in foods such as meats. It is, thus, important to study the genes that are most related to antimicrobial resistance, the pathogens, and the meats carrying antimicrobial-resistance genes. This information can be further used to correlate the antimicrobial-resistance genes found in humans for improving human health. Therefore, we perform the first multivariate statistical analysis of the antimicrobial-resistance gene data provided in the NCBI Pathogen Detection Isolates Browser database, covering six states that are geographically either in close proximity to one another (i.e., Pennsylvania (PA), Maryland (MD), and New York (NY)) or far (i.e., New Mexico (NM), Minnesota (MN), and California (CA)). Hundreds of multidimensional data points were projected onto a two-dimensional space that was specified by the first and second principal components, which were then categorized with a hierarchical clustering approach. It turns out that aadA, aph(3’’), aph(3’’)-Ib, aph(6)-I, aph(6)-Id, bla, blaCMY, tet, tet(A), and sul2 constructed the assembly of ten genes that were most commonly involved in antimicrobial resistance in these six states. While geographically close states like PA, MD and NY share more similar antimicrobial-resistance genes, geographically far states like NM, MN, and CA also contain most of these common antimicrobial-resistance genes. One potential reason for this spread of antimicrobial-resistance genes beyond the geographic limitation is that animal meats like chicken and turkey act as the carriers for the nationwide spread of these genes.

Application of whole genome sequencing to query a potential outbreak of Elizabethkingia anophelis in Ontario, Canada.

Bioinformatic analysis of whole genome sequence (WGS) data is emerging as a tool to provide powerful insights for clinical microbiology. We used WGS data to investigate the genetic diversity of clinical isolates of the bacterial pathogen Elizabethkingia anophelis to query the existence of a single-strain outbreak in Ontario, Canada. The Public Health Ontario Laboratory (PHOL) provides reference identification of clinical isolates of bacteria for Ontario and prior to 2016 had not identified E. anophelis . In the wake of the Wisconsin outbreak of 2015–2016 for which a source was never elucidated, the identification of E. anophelis from clinical specimens from five Ontario patients gave reason to question the presence of an outbreak. Genomic comparisons based on core genome multi-locus sequence typing conclusively refuted the existence of an outbreak, since the 5 Ontario isolates were genetically dissimilar, representing at least 3 distinct sub-lineages scattered among a set of 39 previously characterized isolates. Further interrogation of the genomic data revealed multiple antimicrobial resistance genes. Retrospective reidentification via rpoB sequence analysis of 22 clinical isolates of Elizabethkingia spp. collected by PHOL from 2010 to 2018 demonstrated that E. anophelis was isolated from clinical specimens as early as 2010. The uptick in E. anophelis in Ontario was not due to an outbreak or increased incidence of the pathogen, but rather enhanced laboratory identification techniques and improved sequence databases. This study demonstrates the usefulness of WGS analysis as a public health tool to quickly rule out the existence of clonally related case clusters of bacterial pathogens indicative of single-strain outbreaks.

Whole-genome sequencing of Escherichia coli isolated from contaminated meat samples collected from the Northern Region of Ghana reveals the presence of multiple antimicrobial resistance genes.

This study reports the draft genomes of 14 Escherichia coli isolated from contaminated meat samples collected from the Northern Region of Ghana in order to determine the presence of antimicrobial resistance genes (ARGs) and genetic relatedness of the isolates. The 14 E. coli isolates were of beef (n=3), mutton (n=2), chevon (n=3), local chicken (n=3) and guinea fowl (n=3) origin. Whole-genome sequencing (WGS) was performed using an Illumina MiSeq sequencer. Double-disk synergy test (DDST) was also used to confirm the production of extended-spectrum β-lactamase (ESBL). WGS confirmed the identity of all of the E. coli isolates. All of the isolates contained at least one ARG and 57.1% (8/14) of them were multidrug-resistant (MDR). The mdf(A) gene was most common ARG, found in all 14 isolates. DDST confirmed the production of an ESBL in a MDR E. coli of guinea fowl origin. The sequence types (STs) varied among the E. coli isolates, with the exception of three isolates of ST155. Similarly, the serotypes of the E. coli isolates from meat sample were genetically diverse. Eleven different plasmid sequences were detected in ten of the isolates. E. coli from contaminated meat sources in Ghana possessed multiple ARGs and were genetically diverse. To the best of our knowledge, this is the first work on WGS of E. coli isolated from various meat samples in the study area. The sequence data add to data base for epidemiological studies.

Complete Sequences of Multiple-Drug Resistant IncHI2 ST3 Plasmids in Escherichia coli of Porcine Origin in Australia

IncHI2 ST3 plasmids are known carriers of multiple antimicrobial resistance genes. Complete plasmid sequences from multiple drug resistant Escherichia coli circulating in Australian swine is however limited. Here we sequenced two related IncHI2 ST3 plasmids, pSDE-SvHI2 and pSDC-F2_12BHI2, from phylogenetically unrelated multiple-drug resistant Escherichia coli strains SvETEC (CC23:O157:H19) and F2_12B (ST93:O7:H4) from geographically disparate pig production operations in New South Wales, Australia. Unicycler was used to co-assemble short read (Illumina) and long read (PacBio SMRT) nucleotide sequence data. The plasmids encoded three drug-resistance loci, two of which carried class 1 integrons. One integron, hosting drfA12-orfF-aadA2, was within a hybrid Tn1721/21, with the second residing within a copper/silver resistance transposon, comprising part of an atypical sul3-associated structure. The third resistance locus was flanked by IS15DI and encoded neomycin resistance (neoR). An oqx-encoding transposon (quinolone resistance), similar in structure to Tn6010, was identified only in pSDC-F2_12BHI2. Both plasmids showed high sequence identity to plasmid pSTM6-275, recently described in Salmonella enterica serotype 1,4,[5],12:i:- that has risen to prominence and become endemic in Australia. IncHI2 ST3 plasmids circulating in commensal and pathogenic E. coli from Australian swine belong to a lineage of plasmids often in association with sul3 and host multiple complex antibiotic and metal resistance structures, formed in part by IS26.

Draft genome sequences of three clinical isolates of teicoplanin-resistant Staphylococcus epidermidis from patients without prior exposure to glycopeptide antibiotics.

The aim of this study was to analyse the DNA sequences of three teicoplanin-resistant Staphylococcus epidermidis isolates collected from patients not previously treated with glycopeptide antibiotics. The minimum inhibitory concentrations (MICs) of 12 antibiotics, including teicoplanin and vancomycin, were determined by the broth microdilution method. Genomic DNA was isolated, was sequenced by HiSeqX paired-end sequencing and was assembled into draft genome sequences using MyPro pipeline. Analysis of the draft genome sequences demonstrated that the teicoplanin-resistant S. epidermidis isolates belonged to multilocus sequence typing (MLST) sequence types ST5 and ST87 and encoded multiple antimicrobial resistance genes, including the methicillin resistance gene mecA. This report highlights the risk of dissemination of S. epidermidis strains resistant to a wide range of clinically important antibiotics.

Inhibiting plasmid mobility: The effect of isothiocyanates on bacterial conjugation

Bacterial conjugation is the main mechanism for the transfer of multiple antimicrobial resistance genes among pathogenic micro-organisms. This process may be controlled by compounds that inhibit bacterial conjugation. In this study, the effects of allyl isothiocyanate, l-sulforaphane, benzyl isothiocyanate, phenylethyl isothiocyanate and 4-methoxyphenyl isothiocyanate on the conjugation of broad-host-range plasmids harbouring various antimicrobial resistance genes in Escherichia coli were investigated, namely plasmids pKM101 (IncN), TP114 (IncI2), pUB307 (IncP) and the low-copy-number plasmid R7K (IncW). Benzyl isothiocyanate (32 mg/L) significantly reduced conjugal transfer of pKM101, TP114 and pUB307 to 0.3 ± 0.6%, 10.7 ± 3.3% and 6.5 ± 1.0%, respectively. l-sulforaphane (16 mg/L; transfer frequency 21.5 ± 5.1%) and 4-methoxyphenyl isothiocyanate (100 mg/L; transfer frequency 5.2 ± 2.8%) were the only compounds showing anti-conjugal specificity by actively reducing the transfer of R7K and pUB307, respectively.

Protocols of Conjugative Plasmid Transfer in Salmonella: Plate, Broth, and Filter Mating Approaches.

Bacterial conjugation is a natural process that allows for horizontal transmission of DNA from one bacterium to another. Several plasmids carry transposons that encode multiple antimicrobial and metal resistance genes. Conjugative plasmid transfer requires intimate cell-to-cell contacts between the donor and the recipient. Self-conjugative plasmids harbor tra genes which facilitate plasmid transfer from donor to recipient bacterial strain. Here we describe different methods of conjugative plasmid transfers via conjugation.

Prospective investigation of carbapenem-resistant Klebsiella pneumonia transmission among the staff, environment and patients in five major intensive care units, Beijing

Following the alarming outbreak of carbapenem-resistant Klebsiella pneumonia (CRKP) in five intensive care units (ICUs) of a tertiary care hospital in China, a prospective investigation of CRKP colonized/infected patients was conducted. To describe the diffusion and transmission of CRKP among epidemiologically linked ICU patients, staff and environment. Enhanced CRKP infected/colonized case monitoring was performed by the real-time nosocomial infection surveillance system (RT-NISS). The immediate surroundings of each CRKP patient bed unit and the staff hands/gloves/gowns were sampled and then evaluated for the presence of CRKP. Antimicrobial susceptibility tests, pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS) were used to identify and to characterize these isolates. Among 2750 patients monitored, 67 CRKP patients were newly labeled and 11 patients' CRKP isolates were available. A total of 31.34% (21/67) bed units were positive at one or more surrounding surfaces, 7.99% (49/613) environmental samples and 3.57% (4/112) ICU staff samples were CRKP positive. The selected CRKP isolates (N=64) exhibited intermediate to high resistance levels to the antibiotics tested apart from colistin and tigecycline. RT-NISS data combined with MLST and PFGE revealed nine likely transmission clusters. WGS analysis of these CRKP isolates revealed extensive sharing of multiple antimicrobial resistance genes and plasmid replicons among these isolates. Two carbapenemase genes blaKPC-2 (62/64) and blaOXA-48 (2/64) were identified. These CRKP isolates carried one or more plasmid replicons. The contamination of ICU environment and staff's hands, gloves or gowns is frequent with CRKP patients. Our study also supports the hypothesis that an association between environmental contamination and transmission of CRKP bacteria in ICUs.

ARGDIT: a validation and integration toolkit for Antimicrobial Resistance Gene Databases.

Antimicrobial resistance is currently one of the main challenges in public health due to the excessive use of antimicrobials in medical treatments and agriculture. The advancements in high-throughput next-generation sequencing and development of bioinformatics tools allow simultaneous detection and identification of antimicrobial resistance genes (ARGs) from clinical, food and environment samples, to monitor the prevalence and track the dissemination of these ARGs. Such analyses are however reliant on a comprehensive database of ARGs with accurate sequence content and annotation. Most of the current ARG databases are therefore manually curated, but this is a time-consuming process and the resulting curation errors could be hard to detect. Several secondary ARG databases consolidate contents from different source ARG databases, and hence modifications in the primary databases might not be propagated and updated promptly in the secondary ARG databases. To address these problems, a validation and integration toolkit called ARGDIT was developed to validate ARG database fidelity, and merge multiple primary ARG databases into a single consolidated secondary ARG database with optional automated sequence re-annotation. Experimental results demonstrated the effectiveness of this toolkit in identifying errors such as sequence annotation typos in current ARG databases and generating an integrated non-redundant ARG database with structured annotation. A toolkit-oriented workflow is also proposed to minimize the efforts in validating, curating and merging multiple ARG protein or coding sequence databases. Database developers therefore benefit from faster update cycles and lower costs for database maintenance, while ARG pipeline users can easily evaluate the reference ARG database quality. ARGDIT is available at https://github.com/phglab/ARGDIT. Supplementary data are available at Bioinformatics online.

A novel integrative conjugative element mediates transfer of multi-drug resistance between Streptococcus suis strains of different serotypes

Streptococcus suis represents a key antibiotic resistance gene reservoir and an important pathogen for humans and animals. Resistance can be spread through horizontal gene transfer of chromosome-borne mobile genetic elements; however, the exact mechanism by which this occurs remains poorly understood. In the present study, we identified and characterized a novel 82-kb integrative conjugative element (ICE) named ICESsuCZ130302 from the virulent S. suis strain CZ130302. It carries genes that provide resistance to multiple antibiotics, such as tetracycline, doxycycline, erythromycin, lincomycin, neomycin, and kanamycin. It also contains a nisin biosynthesis gene cluster, a toxin-antitoxin system, a type IV secretion system, and an integrase and excisase system. The mobile element can be excised from the chromosome, circulized, and transferred via conjugation from serotype Chz strain CZ130302 to serotype 2 strain P1/7, where it confers resistance to the aforementioned antimicrobial agents. The full length ICE, where multiple antimicrobial resistance genes accumulated, was further identified to be naturally transferred between different serotypes strains of S. suis. This finding illustrates how such elements represent a potential means by which antimicrobial resistance is introduced to a wide range of bacteria of veterinary and medical significance.

Small Antimicrobial Resistance Plasmids in Livestock-Associated Methicillin-Resistant Staphylococcus aureus CC398.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) isolates of the clonal complex 398 are often resistant to a number of antimicrobial agents. Studies on the genetic basis of antimicrobial resistance in these bacteria identified SCCmec cassettes, various transposons and plasmids of different sizes that harbor antimicrobial resistance genes. While large plasmids that carry multiple antimicrobial resistance genes – occasionally together with heavy metal resistance genes and/or virulence genes – are frequently seen in LA-MRSA ST398, certain resistance genes are also associated with small plasmids of up to 15 kb in size. These small resistance plasmids usually carry only one, but in rare cases also two or three antimicrobial resistance genes. In the current review, we focus on small plasmids that carry the macrolide-lincosamide-streptogramin B resistance genes erm(C) or erm(T), the lincosamide resistance gene lnu(A), the pleuromutilin-lincosamide-streptogramin A resistance genes vga(A) or vga(C), the spectinomycin resistance gene spd, the apramycin resistance gene apmA, or the trimethoprim resistance gene dfrK. The detailed analysis of the structure of these plasmids allows comparisons with similar plasmids found in other staphylococci and underlines in many cases an exchange of such plasmids between LA-MRSA ST398 and other staphylococci including also coagulase-negative staphylococci.

Molecular Characterization of Nasal Methicillin-Resistant Staphylococcus aureus Isolates Showing Increasing Prevalence of Mupirocin Resistance and Associated Multidrug Resistance following Attempted Decolonization.

Sequential methicillin-resistant Staphylococcus aureus (MRSA) isolates from patients following attempted mupirocin nasal decolonization showed an increase in mupirocin resistance (MR) from 6.6% to 20%. MR isolates from patients who failed decolonization yielded indistinguishable spa types and carried multiple antimicrobial and antiseptic resistance genes, which may guide infection control and prevention.

Antimicrobial resistance in large clostridial toxin-negative, binary toxin-positive Clostridium difficile ribotypes

Antimicrobial resistance (AMR) is commonly found in Clostridium difficile strains and plays a major role in strain evolution. We have previously reported the isolation of large clostridial toxin-negative, binary toxin-producing (A−B-CDT+) C. difficile strains from colonised (and in some instances diarrhoeic) food animals, as well as from patients with diarrhoea. To further characterise these strains, we investigated the phenotypic and genotypic AMR profiles of a diverse collection of A−B-CDT+C. difficile strains. The in vitro activities of 10 antimicrobial agents were determined for 148 A−B-CDT+C. difficile strains using an agar dilution methodology. Whole-genome sequencing and in silico genotyping was performed on 53 isolates to identify AMR genes. All strains were susceptible to vancomycin, metronidazole and fidaxomicin, antimicrobials currently considered first-line treatments for C. difficile infection (CDI). Differences in antimicrobial phenotypes between PCR ribotypes (RTs) were observed but were minimal. Phenotypic resistance was observed in 13 isolates to tetracycline (TetR, MIC = 16 mg/L), moxifloxacin (MxfR, MIC = 16 mg/L), erythromycin (EryR, MIC ≥128 mg/L) and clindamycin (CliR, MIC = 8 mg/L). The MxfR strain (RT033) possessed mutations in gyrA/B, while the TetR (RT033) strain contained a tetM gene carried on the conjugative transposon Tn6190. All EryR and CliR strains (RT033, QX521) were negative for the erythromycin ribosomal methylase gene ermB, suggesting a possible alternative mechanism of resistance. This work describes the presence of multiple AMR genes in A−B-CDT+C. difficile strains and provides the first comprehensive analysis of the AMR repertoire in these lineages isolated from human, animal, food and environmental sources.

Environmental Dissemination of Multidrug Methicillin-Resistant Staphylococcus sciuri After Application of Manure from Commercial Swine Production Systems.

The deposition of manure originating from food animal farms in the environment can lead to the dissemination of antimicrobial-resistant (AMR) bacterial foodborne pathogens, thereby potentially impacting human health. The objective of our study was to determine the dissemination of multidrug methicillin-resistant Staphylococcus sciuri (MDR-MRSS) in the environment after land application of manure on commercial swine farms. A total of 400 environmental samples (40 manure and 360 soil) were collected after repeated sampling from four commercial swine farms located in North Carolina (n = 1) and Iowa (n = 3) in the United States. At each farm, we collected 10 manure and 40 soil samples (20 samples before and after 2 h of manure application) from four plots (five soil samples/plot) on day 0. Subsequently, 20 soil samples were collected on day 7, 14, and 21 from the same plots. A total of 67 (16.75%) MRSS were isolated from the 400 samples. The prevalence in soil and manure was 13.33% (48/360) and 47.5% (19/40), respectively. Prevalence was highest in the soil samples collected after 2 h of manure application on day 0 and decreased subsequently on 7, 14, and 21 days. Antimicrobial susceptibility testing was done against a panel of 12 antibiotics. A majority of S. sciuri isolates exhibited resistance against ampicillin (AMP; 95.5%), penicillin (PEN; 95.5%), clindamycin (CLI; 95.5%), cefoxitin (FOX; 92.5%), ceftiofur (XNL; 92.5%), tetracycline (TET; 86.56%), and erythromycin (ERY; 50.74%). The MDR pattern AMP FOX CLI PEN TET XNL (n = 24; 35.8%) was the most commonly observed. We detected multiple AMR genes, including mecA, aac(6'), Ie-aph(2″)Ia, tetM, tetK, mphC, ermA, ermB, and ermC. Pulsed-field gel electrophoresis clustered isolates from different sample collection days from the same farm into one group. Overall, our study identifies swine manure as an important reservoir of MDR-MRSS and highlights its dissemination in the environment upon spreading of manure.