Nanopore sequencing for identification and characterization of antimicrobial-resistant Escherichia coli and Salmonella spp. from tilapia and shrimp sold at wet markets in Dhaka, Bangladesh.

Wet markets in low-and middle-income countries are often reported to have inadequate sanitation resulting in fecal contamination of sold produce. Consumption of contaminated wet market-sourced foods has been linked to individual illness and disease outbreaks. This pilot study, conducted in two major wet markets in Dhaka city, Bangladesh during a 4-month period in 2021 aimed to assess the occurrence and characteristics of Escherichia coli and non-typhoidal Salmonella spp. (NTS) from tilapia (Oreochromis niloticus) and shrimp (Penaeus monodon). Fifty-four individuals of each species were collected. The identity of the bacterial isolates was confirmed by PCR and their susceptibility toward 15 antimicrobials was tested by disk diffusion. The whole genome of 15 E. coli and nine Salmonella spp. were sequenced using Oxford Nanopore Technology. E. coli was present in 60-74% of tilapia muscle tissue and 41-44% of shrimp muscle tissue. Salmonella spp. was found in skin (29%) and gills (26%) of tilapia, and occasionally in muscle and intestinal samples of shrimp. The E. coli had several Multilocus sequence typing and serotypes and limited antimicrobial resistance (AMR) determinants, such as point mutations on glpT and pmrB. One E. coli (BD17) from tilapia carried resistance genes for beta-lactams, quinolones, and tetracycline. All the E. coli belonged to commensal phylogroups B1 and A and showed no Shiga-toxin and other virulence genes, confirming their commensal non-pathogenic status. Among the Salmonella isolates, five belonged to Kentucky serovar and had similar AMR genes and phenotypic resistance patterns. Three strains of this serovar were ST198, often associated with human disease, carried the same resistance genes, and were genetically related to strains from the region. The two undetermined sequence types of S. Kentucky were distantly related and positioned in a separate phylogenetic clade. Two Brunei serovar isolates, one Augustenborg isolate, and one Hartford isolate showed different resistance profiles. This study revealed high fecal contamination levels in tilapia and shrimp sold at two main wet markets in Dhaka. Together with the occurrence of Salmonella spp., including S. Kentucky ST198, a well-known human pathogen, these results stress the need to improve hygienic practices and sanitation standards at markets to improve food safety and protect consumer health.

Molecular characterization of antimicrobial resistance in Brachyspira species isolated from UK chickens: Identification of novel variants of pleuromutilin and beta-lactam resistance genes.

Brachyspira species are Gram negative, anaerobic bacteria that colonise the gut of many animals, including poultry. In poultry, Brachyspira species can be commensal (B. innocens, B. murdochii, 'B. pulli') or pathogenic (B. pilosicoli, B. intermedia, B. alvinipulli or rarely B. hyodysenteriae), the latter causing avian intestinal spirochaetosis (AIS). Antimicrobial therapy options for treatment is limited, frequently involving administration of the pleuromutilin, tiamulin, in water. In this study 38 Brachyspira isolates from chickens in the UK, representing both commensal and pathogenic species, were whole genome sequenced to identify antimicrobial resistance (AMR) mechanisms and the minimum inhibitory concentration (MIC) to a number of antimicrobials was also determined. We identified several new variants of blaOXA in B. pilosicoli and B. pulli isolates, and variations in tva which led to two new tva variants in B.murdochii and B.pulli. A number of isolates also harboured mutations known to encode AMR in the 16S and 23S rRNA genes. The percentage of isolates that were genotypically multi-drug resistance (MDR) was 16%, with the most common resistance profile being: tetracycline, pleuromutilin and beta-lactam, which were found in three 'B. pulli' and one B. pilosicoli. There was good correlation with the genotype and the corresponding antibiotic MIC phenotypes: pleuromutilins (tiamulin and valnemulin), macrolides (tylosin and tylvalosin), lincomycin and doxycycline. The occurrence of resistance determinants identified in this study in pathogenic Brachyspira, especially those which were MDR, is likely to impact treatment of AIS and clearance of infections on farm.

Prevalence, antibiotic resistance, and genomic characterisation of Campylobacter spp. in retail chicken in Hanoi, Vietnam.

Campylobacter spp. are a leading cause of bacterial foodborne zoonosis worldwide, with poultry meat and products recognised as a significant source of human infection. In Vietnam there are few data regarding the occurrence, antimicrobial resistance, and genomic diversity of Campylobacter in poultry and poultry meat. The aim of this study was to estimate the prevalence of Campylobacter in chicken meat at retail in Hanoi, determine antimicrobial sensitivities of the Campylobacter isolated, and assess their genetic diversity. A total of 120 chicken meat samples were collected from eight traditional retail markets (n=80) and four supermarkets (n=40). Campylobacter was isolated following ISO 10272-1 : 2017 and identification verified by PCR. The prevalence of Campylobacter was 38.3 % (46/120) and C. coli was the most prevalent species in both retail markets (74 %) and supermarkets (88 %). The minimum inhibitory concentrations for ciprofloxacin, erythromycin, gentamicin, nalidixic acid, streptomycin, and tetracycline were determined by broth microdilution for 32 isolates. All characterised Campylobacter were resistant to ciprofloxacin, nalidixic acid, and tetracycline, with corresponding resistance determinants detected in the sequenced genomes. Most C. coli were multidrug resistant (24/28) and two harboured the erythromycin resistance gene ermB on a multiple drug-resistance genomic island, a potential mechanism for dissemination of resistance. The 32 isolates belonged to clonal complexes associated with both poultry and people, such as CC828 for C. coli. These results contribute to the One Health approach for addressing Campylobacter in Vietnam by providing detailed new insights into a main source of human infection and can inform the design of future surveillance approaches.

Prevalence and genomic characterization of Salmonella isolates from commercial chicken eggs retailed in traditional markets in Ghana.

Salmonella enterica are important foodborne bacterial pathogens globally associated with poultry. Exposure to Salmonella-contaminated eggs and egg-related products is a major risk for human salmonellosis. Presently, there is a huge data gap regarding the prevalence and circulating serovars of Salmonella in chicken eggs sold in Ghana. In this study, 2,304 eggs (pools of six per sample unit) collected from informal markets in Accra, Kumasi and Tamale, representing the three ecological belts across Ghana, were tested for Salmonella. Antimicrobial susceptibility testing and Whole Genome Sequencing (WGS) of the isolates were performed using standard microdilution protocols and the Illumina NextSeq platform, respectively. The total prevalence of Salmonella was 5.5% with a higher rate of contamination in eggshell (4.9%) over egg content (1.8%). The serovars identified were S. Ajiobo (n = 1), S. Chester (n = 6), S. Hader (n = 7), S. enteritidis (n = 2); and S. I 4:b:- (n = 8). WGS analysis revealed varied sequence types (STs) that were serovar specific. The S. I 4:b:- isolates had a novel ST (ST8938), suggesting a local origin. The two S. enteritidis isolates belonged to ST11 and were identified with an invasive lineage of a global epidemic clade. All isolates were susceptible to ampicillin, azithromycin, cefotaxime, ceftazidime, gentamicin, meropenem, and tigecycline. The phenotypic resistance profiles to seven antimicrobials: chloramphenicol (13%), ciprofloxacin (94%), and nalidixic acid (94%), colistin (13%), trimethoprim (50%) sulfamethoxazole (50%) and tetracycline (50%) corresponded with the presence of antimicrobial resistance (AMR) determinants including quinolones (gyrA (D87N), qnrB81), aminoglycosides (aadA1), (aph(3")-Ib aph(6)-Id), tetracyclines (tet(A)), phenicols (catA1), trimethoprim (dfrA14 and dfrA1). The S. enteritidis and S. Chester isolates were multidrug resistant (MDR). Several virulence factors were identified, notably cytolethal distending toxin (cdtB gene), rck, pef and spv that may promote host invasion and disease progression in humans. The findings from this study indicate the presence of multidrug resistant and virulent strains of Salmonella serovars in Ghanaian chicken eggs, with the potential to cause human infections. This is a critical baseline information that could be used for Salmonella risk assessment in the egg food chain to mitigate potential future outbreaks.

Multidrug-resistant non-typhoidal Salmonella of public health significance recovered from migratory birds in Bangladesh.

Non-typhoidal Salmonella provides an exemplar for the One Health approach as it encompasses public and animal health, food safety, and environmental considerations. The contribution of environmental aspects is currently less well-defined. The purpose of this study was to determine the carriage occurrence of non-typhoidal Salmonella in migratory birds in Bangladesh and assess the potential significance to public and animal health. Cloacal swabs (N = 453) were collected in the years 2018-2020 from Tanguar and Hakaluki Haors, important wetland ecosystems in Northeastern Bangladesh. The prevalence of Salmonella was 13.5% (61 positive swabs). Classical serotyping identified six serovars: Salmonella enterica subsp. enterica serovars Perth, Kentucky, Albany, Infantis, Weltevreden, and Brancaster. Resistance towards 14 antimicrobials was assessed by broth microdilution minimum inhibitory concentration determination and the antimicrobial resistance (AMR) genotype established by whole-genome sequencing. S. Perth and S. Weltevreden isolates were susceptible and harbored no acquired AMR genes. Isolates from the remaining serovars were multidrug resistant, commonly possessing resistance to tetracycline, ampicillin, chloramphenicol, sulfamethoxazole, trimethoprim, and ciprofloxacin. Salmonella resistant to ciprofloxacin meets WHO criteria for priority pathogens. There was excellent concordance between resistance phenotype and the presence of corresponding AMR genes, many of which reside on Salmonella Genomic Islands. High-level ciprofloxacin resistance correlated with the presence of mutations in the chromosomal gyrB and/or parC genes. The S. Kentucky isolates were ST198, a widely distributed multidrug-resistant lineage reported in humans and animals, and constituting an ongoing risk to public health worldwide. We have demonstrated that multidrug-resistant non-typhoidal Salmonella of public health significance can be recovered from migratory birds. A potential for risk can manifest through direct interaction, transmission to food-producing livestock on farms, and dissemination via the long range migratory movements of birds. Risks can be mitigated by measures including continued surveillance and implementation of good farm biosecurity practices.

Determination of antimicrobial use in commercial poultry farms in Plateau and Oyo States, Nigeria.

BACKGROUND: Indiscriminate use of antimicrobials for the prevention and treatment of bacterial infection in animals is a common practice in Nigeria as in other developing countries. These antimicrobials are purchased over the counter without restrictions and often administered in form of medicated feedstuffs. In Nigeria, like most developing countries, antimicrobial prescription data are not routinely collected or reported at the farm level, instead import data are used in reporting antimicrobial consumption. Farmers can be useful sources of data on the use of antimicrobial agents by class, animal species, production type and age. The objective of the study was to determine the knowledge, attitude and practices of poultry farmers on antimicrobial resistance and to generate data on antimicrobial use (AMU) in poultry farms in Plateau and Oyo states in accordance with the guidelines of the World Organization for Animal Health (WOAH). METHODS: A questionnaire used by the Food and Agriculture Organization (FAO) of the United Nations in Ghana was adopted and modified to collect data on the knowledge, attitude and practices of farmers on AMR and AMU and to collect AMU data from selected poultry farms. A focus group discussion (FGD) was conducted in Plateau state with poultry farmers and representatives from the state veterinary services, using a checklist. The aim of the FGD was to have an idea on antimicrobial use among poultry farmers and to generate additional questions that might be added to the questionnaire. Stratified random sampling technique was used to select 50 farms from Plateau and Oyo states, using the list of registered poultry farms in the two states as sampling frame. RESULTS: Ninety eight percent (98%) of farmers gave antibiotics as prophylactic treatment to day old chicks. There were 47 different products used in the two states within the study period. We observed that five classes of antibiotics (Tetracyclines, Penicillins, Aminoglycosides, Polypeptides and Fluoroquinolone) were used in the two states. A total of 351 kg of active ingredients from seven different classes, namely: tetracyclines, penicillins, aminoglycosides, polypeptide, fluoroquinolones, amphenicol and macrolides were recorded from the two states. Some products contained cocktail of antibiotics, having up to six different classes with very high concentration of active ingredients which are not in the list of registered antimicrobials reported to WOAH. CONCLUSION: The concept used for this survey proved that the approach can be applied for AMU surveillance in the animal health sector. It also provided insight on farmers' knowledge and practices with regards to the use of antimicrobials which is missing in the national import data. The need for "stronger" antibiotics was identified as one of the drivers of antibiotic resistance.

Establishing a marine monitoring programme to assess antibiotic resistance: A case study from the Gulf Cooperation Council (GCC) region.

The World Health Organization considers antimicrobial resistance as one of the most pressing global issues which poses a fundamental threat to human health, development, and security. Due to demographic and environmental factors, the marine environment of the Gulf Cooperation Council (GCC) region may be particularly susceptible to the threat of antimicrobial resistance. However, there is currently little information on the presence of AMR in the GCC marine environment to inform the design of appropriate targeted surveillance activities. The objective of this study was to develop, implement and conduct a rapid regional baseline monitoring survey of the presence of AMR in the GCC marine environment, through the analysis of seawater collected from high-risk areas across four GCC states: (Bahrain, Oman, Kuwait, and the United Arab Emirates). 560 Escherichia coli strains were analysed as part of this monitoring programme between December 2018 and May 2019. Multi-drug resistance (resistance to three or more structural classes of antimicrobials) was observed in 32.5% of tested isolates. High levels of reduced susceptibility to ampicillin (29.6%), nalidixic acid (27.9%), tetracycline (27.5%), sulfamethoxazole (22.5%) and trimethoprim (22.5%) were observed. Reduced susceptibility to the high priority critically important antimicrobials: azithromycin (9.3%), ceftazidime (12.7%), cefotaxime (12.7%), ciprofloxacin (44.6%), gentamicin (2.7%) and tigecycline (0.5%), was also noted. A subset of 173 isolates was whole genome sequenced, and high carriage rates of qnrS1 (60/173) and bla CTX-M-15 (45/173) were observed, correlating with reduced susceptibility to the fluoroquinolones and third generation cephalosporins, respectively. This study is important because of the resistance patterns observed, the demonstrated utility in applying genomic-based approaches to routine microbiological monitoring, and the overall establishment of a transnational AMR surveillance framework focussed on coastal and marine environments.

Molecular characterization of extended spectrum cephalosporin resistant Escherichia coli isolated from livestock and in-contact humans in Southeast Nigeria.

The rise in antimicrobial resistance (AMR) in bacteria is reducing therapeutic options for livestock and human health, with a paucity of information globally. To fill this gap, a One-Health approach was taken by sampling livestock on farms (n = 52), abattoir (n = 8), and animal markets (n = 10), and in-contact humans in Southeast Nigeria. Extended spectrum cephalosporin (ESC)-resistant (ESC-R) Escherichia coli was selectively cultured from 975 healthy livestock faecal swabs, and hand swabs from in-contact humans. Antimicrobial susceptibility testing (AST) was performed on all ESC-R E. coli. For isolates showing a multi-drug resistance (MDR) phenotype (n = 196), quantitative real-time PCR (qPCR) was performed for confirmation of extended-spectrum ß-lactamase (ESBL) and carbapenemase genes. Whole-genome sequencing (WGS) was performed on a subset (n = 157) for detailed molecular characterisation. The results showed ESC-R E. coli was present in 41.2% of samples, with AST results indicating 48.8% of isolates were phenotypically MDR. qPCR confirmed presence of ESBL genes, with bla CTX-M present in all but others in a subset [bla TEM (62.8%) and bla SHV (0.5%)] of isolates; none harboured transferable carbapenemase genes. Multi-locus sequence typing identified 34 Sequence Types (ST) distributed among different sampling levels; ST196 carrying bla CTX-M-55 was predominant in chickens. Large numbers of single nucleotide polymorphisms (SNPs) in the core genome of isolates, even within the same clade by phylogenetic analysis, indicated high genetic diversity. AMR genotyping indicated the predominant bla CTX-M variant was bla CTX-M-15 (87.9%), although bla CTX-M-55, bla CTX-M-64, and bla CTX-M-65 were present; it was notable that bla CTX-M-1, common in livestock, was absent. Other predominant AMR genes included: sul2, qnrS1, strB, bla TEM-1b, tetA-v2, and dfrA14, with prevalence varying according to host livestock species. A bla CTX-M-15 harbouring plasmid from livestock isolates in Ebonyi showed high sequence identity to one from river/sewage water in India, indicating this ESBL plasmid to be globally disseminated, being present beyond the river environment. In conclusion, ESC-R E. coli was widespread in livestock and in-contact humans from Southeast Nigeria. WGS data indicated the isolates were genetically highly diverse, probably representing true diversity of wild type E. coli; they were likely to be MDR with several harbouring bla CTX-M-15. Surprisingly, human isolates had highest numbers of AMR genes and pigs the least.

High prevalence of vancomycin non-susceptible and multi-drug resistant enterococci in farmed animals and fresh retail meats in Bangladesh.

The emergence of antimicrobial resistant Enterococcus spp., a main cause of untreatable nosocomial infection, in food animals and dissemination to humans is a public health risk. The study was performed to determine the prevalence and antimicrobial resistance, and virulence characteristics of Enterococcus faecalis and Enterococcus faecium in food animals and meats in Bangladesh. Enterococcus spp., were confirmed using sodA gene specific PCR, and antimicrobial resistance and virulence properties were characterized by PCR. Enterococcus spp. were recovered from 57% of the collected samples (n = 201/352). Farm samples yielded significantly higher (p ≤ 0.05) prevalence (62%) than that of retail meat samples (41%). E. faecalis (52%) is most frequently isolated species. Greater proportions of isolates exhibited resistance to tetracycline (74%), erythromycin (65%) and ciprofloxacin (34%). Fifty-one isolates are vancomycin non-susceptible enterococci (VNSE), of which forty-seven are MDR and twenty are linezolid resistant, a last line drug for VNSE. Virulence factors such as gelatinase (gelE), aggregation factor (asa1) and sex pheromone (cpd) are detected along with vancomycin resistance gene (vanA, vanB and vanC2/C3) in VNSE isolates. The high prevalence of MDR enterococci in food animals and retail meats may cause consumers infections with concomitant reduction of available therapeutic options.

Whole-Genome Sequencing of Brachyspira hyodysenteriae Isolates From England and Wales Reveals Similarities to European Isolates and Mutations Associated With Reduced Sensitivity to Antimicrobials.

Brachyspira hyodysenteriae is the principal cause of swine dysentery, a disease that threatens economic productivity of pigs in many countries as it can spread readily within and between farms, and only a small number of antimicrobials are authorized for treatment of pigs. In this study, we performed whole-genome sequencing (WGS) of 81 B. hyodysenteriae archived at the Animal and Plant Health Agency (APHA) from diagnostic submissions and herd monitoring in England and Wales between 2004 and 2015. The resulting genome sequences were analyzed alongside 34 genomes we previously published. Multi-locus sequence typing (MLST) showed a diverse population with 32 sequence types (STs) among the 115 APHA isolates, 25 of them identified only in England; while also confirming that the dominant European clonal complexes, CC8 and CC52, were common in the United Kingdom. A core-genome SNP tree typically clustered the isolates by ST, with isolates from some STs detected only within a specific region in England, although others were more widespread, suggesting transmission between different regions. Also, some STs were more conserved in their core genome than others, despite these isolates being from different holdings, regions and years. Minimum inhibitory concentrations to commonly used antimicrobials (Tiamulin, Valnemulin, Doxycycline, Lincomycin, Tylosin, Tylvalosin) were determined for 82 of the genome-sequenced isolates; genomic analysis revealed mutations generally correlated well with the corresponding resistance phenotype. There was a major swine dysentery intervention program in 2009-2010, and antimicrobial survival curves showed a significant reduction in sensitivity to tiamulin and valnemulin in isolates collected in and after 2010, compared to earlier isolates. This correlated with a significant increase in post-2009 isolates harboring the pleuromutilin resistance gene tva(A), which if present, may facilitate higher levels of resistance. The reduction in susceptibility of Brachyspira from diagnostic submissions to pleuromutilins, emphasizes the need for prudent treatment, control and eradication strategies.

Retrospective analysis of necrotizing typhlitis cases associated with Brachyspira spp. in British rheas.

This paper describes a retrospective analysis of necrotizing typhlitis in common rheas (Rhea americana) diagnosed in the United Kingdom by the Animal & Plant Health Agency (APHA). From January 2008 to January 2020, seven cases of spirochaetal typhlitis associated with Brachyspira spp. were identified using the Veterinary Investigation Diagnosis Analysis database. Gross examination was combined with selective anaerobic culture, polymerase chain reaction, and histopathology to diagnose typhlitis associated with spirochaetal infection. Whole-genome sequencing was subsequently utilized on archived isolates from six of the seven submissions, overcoming issues with traditional testing methods and yielded gains in the identification of Brachyspira to species level. Brachyspira hyodysenteriae, an organism traditionally associated with typhlitis in rheas, was isolated in three sequenced submissions. One of these also demonstrated co-infection with Brachyspira intermedia. Brachyspira suanatina, Brachyspira hampsonii, and Brachyspira alvinipulli were identified by sequencing as single infections in the remaining three animals. This report demonstrates the ability of Brachyspira species other than B. hyodysenteriae to colonize the caeca of rheas presenting with typhlitis. Additionally, the B. alvinipulli isolate harboured a tva(A) gene, indicating higher potential pleuromutilin resistance, which has not previously been described in this Brachyspira species. This study discusses the epidemiology of examined cases and examines the potential role other species may play in these outbreaks.

Use of whole genome sequencing of commensal Escherichia coli in pigs for antimicrobial resistance surveillance, United Kingdom, 2018.

BackgroundSurveillance of commensal Escherichia coli, a possible reservoir of antimicrobial resistance (AMR) genes, is important as they pose a risk to human and animal health. Most surveillance activities rely on phenotypic characterisation, but whole genome sequencing (WGS) presents an alternative.AimIn this retrospective study, we tested 515 E. coli isolated from pigs to evaluate the use of WGS to predict resistance phenotype.MethodsMinimum inhibitory concentration (MIC) was determined for nine antimicrobials of clinical and veterinary importance. Deviation from wild-type, fully-susceptible MIC was assessed using European Committee on Antimicrobial Susceptibility Testing (EUCAST) epidemiological cut-off (ECOFF) values. Presence of AMR genes and mutations were determined using APHA SeqFinder. Statistical two-by-two table analysis and Cohen's kappa (k) test were applied to assess genotype and phenotype concordance.ResultsOverall, correlation of WGS with susceptibility to the nine antimicrobials was 98.9% for test specificity, and 97.5% for the positive predictive value of a test. The overall kappa score (k = 0.914) indicated AMR gene presence was highly predictive of reduced susceptibility and showed excellent correlation with MIC. However, there was variation for each antimicrobial; five showed excellent correlation; four very good and one moderate. Suggested ECOFF adjustments increased concordance between genotypic data and kappa values for four antimicrobials.ConclusionWGS is a powerful tool for accurately predicting AMR that can be used for national surveillance purposes. Additionally, it can detect resistance genes from a wider panel of antimicrobials whose phenotypes are currently not monitored but may be of importance in the future.

Weakly haemolytic variants of Brachyspira hyodysenteriae newly emerged in Europe belong to a distinct subclade with unique genetic properties.

Brachyspira (B.) hyodysenteriae is widespread globally, and can cause mucohaemorrhagic colitis (swine dysentery, SD) with severe economic impact in infected herds. Typical strains of B. hyodysenteriae are strongly haemolytic on blood agar, and the haemolytic activity is believed to contribute to virulence in vivo. However, recently there have been reports of atypical weakly haemolytic isolates of B. hyodysenteriae (whBh). In this study, 34 European whBh and 82 strongly haemolytic isolates were subjected to comparative genomic analysis. A phylogenetic tree constructed using core single nucleotide polymorphisms showed that the whBh formed a distinct sub-clade. All eight genes previously associated with haemolysis in B. hyodysenteriae were present in the whBh. No consistent patterns of amino acid substitutions for all whBh were found in these genes. In contrast, a genome region containing six coding sequences (CDSs) had consistent nucleotide sequence differences between strongly and whBh isolates. Two CDSs were predicted to encode ABC transporter proteins, and a TolC family protein, which may have a role in the export of haemolysins from B. hyodysenteriae. Another difference in this region was the presence of three CDSs in whBh that are pseudogenes in strongly haemolytic isolates. One of the intact CDSs from whBh encoded a predicted PadR-like transcriptional repressor that may play a role in repression of haemolysis functions. In summary, a sub-clade of whBh isolates has emerged in Europe, and several genomic differences, that potentially explain the weakly haemolytic phenotype, were identified. These markers may provide targets for discriminatory molecular tests needed in SD surveillance.

Multidrug-Resistant Salmonella enterica Isolated from Food Animal and Foodstuff May Also Be Less Susceptible to Heavy Metals.

Salmonella enterica is a foodborne pathogen showing increasing multidrug resistance (MDR). We characterized the antimicrobial resistance (AMR) genotype using microarrays in a panel of 105 nontyphoidal S. enterica isolated from food animals and foodstuff. Nineteen isolates were chosen on the basis of their MDR and virulence for determination of heavy metal susceptibilities and screened by polymerase chain reaction for heavy metal resistance genes. Whole-genome sequencing (WGS) was performed on three isolates carrying clinically important AMR genes and the cdtB toxin gene to detect other heavy metal resistance mechanisms, and conjugation assays were performed to evaluate transfer of AMR/toxin genes with heavy metal resistance genes. AMR genotyping results showed isolates harbored between 1 and 12 mobile AMR genes, with 58% being classified as MDR. The tested subset of isolates showed reduced susceptibility to zinc (78%), copper (68%), silver (63%), arsenic (47%), and tellurite (26%); phenotypes that could be attributed to zitB (n = 32%), pcoA/pcoD (n = 32%), tcrB (n = 16%), arsB (n = 16%), silA/silE (n = 42%), and terF (n = 26%) genes. WGS confirmed the presence of other heavy metal resistance genes such as copA, cusA, and czcD. Isolates often harbored multiple heavy metal resistance genes. Two strains (Sal25 and Sal368) were able to conjugate with Escherichia coli J53 at a relatively high frequency (∼10-4 colony-forming units per recipient). Transformants selected in the presence of copper harbored either an IncHI2 (J53/Sal25 transconjugant) or IncF (J53/Sal368 transconjugant) plasmid with decreased susceptibilities to tellurite, zinc, copper, cobalt, arsenic, lead, mercury, and silver. blaCTX-M-1 and mcr-1 genes were also transferred to one transconjugant, and tet(M) and blaTEM-1 genes to the other. This work shows the presence of a diversity of AMR genes in this zoonotic pathogen, and suggests that heavy metals may contribute to selection of clinically important ones through the food chain, such as the plasmid-mediated colistin resistance gene mcr-1.

Identification of a New Antimicrobial Resistance Gene Provides Fresh Insights Into Pleuromutilin Resistance in Brachyspira hyodysenteriae, Aetiological Agent of Swine Dysentery.

Brachyspira hyodysenteriae is the aetiological agent of swine dysentery, a globally distributed disease that causes profound economic loss, impedes the free trade and movement of animals, and has significant impact on pig health. Infection is generally treated with antibiotics of which pleuromutilins, such as tiamulin, are widely used for this purpose, but reports of resistance worldwide threaten continued effective control. In Brachyspira hyodysenteriae pleuromutilin resistance has been associated with mutations in chromosomal genes encoding ribosome-associated functions, however the dynamics of resistance acquisition are poorly understood, compromising stewardship efforts to preserve pleuromutilin effectiveness. In this study we undertook whole genome sequencing (WGS) and phenotypic susceptibility testing of 34 UK field isolates and 3 control strains to investigate pleuromutilin resistance in Brachyspira hyodysenteriae. Genome-wide association studies identified a new pleuromutilin resistance gene, tva(A) (tiamulin valnemulin antibiotic resistance), encoding a predicted ABC-F transporter. In vitro culture of isolates in the presence of inhibitory or sub-inhibitory concentrations of tiamulin showed that tva(A) confers reduced pleuromutilin susceptibility that does not lead to clinical resistance but facilitates the development of higher-level resistance via mutations in genes encoding ribosome-associated functions. Genome sequencing of antibiotic-exposed isolates identified both new and previously described mutations in chromosomal genes associated with reduced pleuromutilin susceptibility, including the 23S rRNA gene and rplC, which encodes the L3 ribosomal protein. Interesting three antibiotic-exposed isolates harboured mutations in fusA, encoding Elongation Factor G, a gene not previously associated with pleuromutilin resistance. A longitudinal molecular epidemiological examination of two episodes of swine dysentery at the same farm indicated that tva(A) contributed to development of tiamulin resistance in vivo in a manner consistent with that seen experimentally in vitro. The in vitro studies further showed that tva(A) broadened the mutant selection window and raised the mutant prevention concentration above reported in vivo antibiotic concentrations obtained when administered at certain doses. We show how the identification and characterisation of tva(A), a new marker for pleuromutilin resistance, provides evidence to inform treatment regimes and reduce the development of resistance to this class of highly important antimicrobial agents.

An In Vitro Chicken Gut Model Demonstrates Transfer of a Multidrug Resistance Plasmid from Salmonella to Commensal Escherichia coli.

The chicken gastrointestinal tract is richly populated by commensal bacteria that fulfill various beneficial roles for the host, including helping to resist colonization by pathogens. It can also facilitate the conjugative transfer of multidrug resistance (MDR) plasmids between commensal and pathogenic bacteria which is a significant public and animal health concern as it may affect our ability to treat bacterial infections. We used an in vitro chemostat system to approximate the chicken cecal microbiota, simulate colonization by an MDR Salmonella pathogen, and examine the dynamics of transfer of its MDR plasmid harboring several genes, including the extended-spectrum beta-lactamase blaCTX-M1 We also evaluated the impact of cefotaxime administration on plasmid transfer and microbial diversity. Bacterial community profiles obtained by culture-independent methods showed that Salmonella inoculation resulted in no significant changes to bacterial community alpha diversity and beta diversity, whereas administration of cefotaxime caused significant alterations to both measures of diversity, which largely recovered. MDR plasmid transfer from Salmonella to commensal Escherichia coli was demonstrated by PCR and whole-genome sequencing of isolates purified from agar plates containing cefotaxime. Transfer occurred to seven E. coli sequence types at high rates, even in the absence of cefotaxime, with resistant strains isolated within 3 days. Our chemostat system provides a good representation of bacterial interactions, including antibiotic resistance transfer in vivo It can be used as an ethical and relatively inexpensive approach to model dissemination of antibiotic resistance within the gut of any animal or human and refine interventions that mitigate its spread before employing in vivo studies.IMPORTANCE The spread of antimicrobial resistance presents a grave threat to public health and animal health and is affecting our ability to respond to bacterial infections. Transfer of antimicrobial resistance via plasmid exchange is of particular concern as it enables unrelated bacteria to acquire resistance. The gastrointestinal tract is replete with bacteria and provides an environment for plasmid transfer between commensals and pathogens. Here we use the chicken gut microbiota as an exemplar to model the effects of bacterial infection, antibiotic administration, and plasmid transfer. We show that transfer of a multidrug-resistant plasmid from the zoonotic pathogen Salmonella to commensal Escherichia coli occurs at a high rate, even in the absence of antibiotic administration. Our work demonstrates that the in vitro gut model provides a powerful screening tool that can be used to assess and refine interventions that mitigate the spread of antibiotic resistance in the gut before undertaking animal studies.

Detection of the florfenicol resistance gene floR in Chryseobacterium isolates from rainbow trout. Exception to the general rule?

Bacteria from the family Flavobacteriaceae often show low susceptibility to antibiotics. With the exception of two Chryseobacterium spp. isolates that were positive for the florfenicol resistance gene floR, no clinical resistance genes were identified by microarray in 36 Flavobacteriaceae isolates from salmonid fish that could grow in ≥ 4 mg/L florfenicol. Whole genome sequence analysis of the floR positive isolates revealed the presence of a region that contained the antimicrobial resistance genes floR, a tet(X) tetracycline resistance gene, a streptothricin resistance gene and a chloramphenicol acetyltransferase gene. In silico analysis of 377 published genomes for Flavobacteriaceae isolates from a range of sources confirmed that well-characterised resistance gene cassettes were not widely distributed in bacteria from this group. Efflux pump-mediated decreased susceptibility to a range of antimicrobials was confirmed in both floR positive isolates using an efflux pump inhibitor (phenylalanine-arginine ß-naphthylamide) assay. The floR isolates possessed putative virulence factors, including production of siderophores and haemolysins, and were mildly pathogenic in rainbow trout. Results support the suggestion that, despite the detection of floR, susceptibility to antimicrobials in Flavobacteriaceae is mostly mediated via intrinsic mechanisms rather than the horizontally acquired resistance genes more normally associated with Gram-negative bacterial pathogens such as Enterobacteriaceae.

Microarray Evaluation of Antimicrobial Resistance and Virulence of Escherichia coli Isolates from Portuguese Poultry.

The presence of antimicrobial resistance and virulence factors of 174 Escherichia coli strains isolated from healthy Portuguese Gallus gallus was evaluated. Resistance profiles were determined against 33 antimicrobials by microbroth dilution. Resistance was prevalent for tetracycline (70%) and ampicillin (63%). Extended-spectrum beta-lactamase (ESBL) phenotype was observed in 18% of the isolates. Multidrug resistance was found in 56% of isolates. A subset of 74 isolates were screened by DNA microarrays for the carriage of 88 antibiotic resistance genes and 62 virulence genes. Overall, 37 different resistance genes were detected. The most common were tet(A) (72%), blaTEM (68%), and sul1 (47%), while 21% isolates harbored an ESBL gene (blaCTX-M group 1, group 2, or group 9). Of these, 96% carried the increased serum survival (iss) virulence gene, while 89% presented the enterobactin siderophore receptor protein (iroN), 70% the temperature-sensitive hemagglutinin (tsh), and 68% the long polar fimbriae (lpfA) virulence genes associated with extraintestinal pathogenic E. coli. In conclusion, prevalence of antibiotic resistant E. coli from the microbiota of Portuguese chickens was high, including to extended spectrum cephalosporins. The majority of isolates seems to have the potential to trigger extraintestinal human infection due to the presence of some virulence genes. However, the absence of genes specific for enteropathogenic E. coli reduces the risk for human intestinal infection.

Impact of Ciprofloxacin and Clindamycin Administration on Gram-Negative Bacteria Isolated from Healthy Volunteers and Characterization of the Resistance Genes They Harbor.

The aim of this study was to assess the impact of ciprofloxacin, clindamycin, and placebo administration on culturable Gram-negative isolates and the antibiotic resistance genes they harbor. Saliva and fecal samples were collected from healthy human volunteers before and at intervals, up to 1 year after antibiotic administration. Samples were plated on selective and nonselective media to monitor changes in different colony types or bacterial species. Following ciprofloxacin administration, there was a decrease of Escherichia coli in feces and after clindamycin administration a decrease of Bacteroides in feces and Leptotrichia in saliva, which all returned to pretreatment levels within 1 to 4 months. Ciprofloxacin administration also resulted in an increase in ciprofloxacin-resistant Veillonella in saliva, which persisted for 12 months. Additionally, 949 aerobic and anaerobic isolates purified from ciprofloxacin- and clindamycin-containing plates were screened for the presence of resistance genes. Resistance gene carriage was widespread in isolates from all three treatment groups, and no association was observed between genes and antibiotic administration. Although the anaerobic component of the microbiota was not a major reservoir of aerobe-associated antimicrobial resistance (AMR) genes, we detected the sulfonamide resistance gene sul2 in anaerobic isolates. The longitudinal nature of the study allowed identification of distinct Escherichia coli clones harboring multiple resistance genes, including one carrying an extended-spectrum ß-lactamase blaCTX-M group 9 gene, which persisted in the gut for up to 4 months. This study provided insight into the effects of antibiotic administration on healthy microbiota and the diversity of resistance genes harbored therein.