Antimicrobial resistance and geographical distribution of Staphylococcus sp. isolated from whiting (Merlangius merlangus) and seawater in the English Channel and the North sea.

Staphylococcus is a significant food safety hazard. The marine environment serves as a source of food for humans and is subject to various human-induced discharges, which may contain Staphylococcus strains associated with antimicrobial resistance (AMR). The aim of this study was to assess the occurrence and geographical distribution of AMR Staphylococcus isolates in seawater and whiting (Merlangius merlangus) samples collected from the English Channel and the North Sea. We isolated and identified 238 Staphylococcus strains, including 12 coagulase-positive (CoPs) and 226 coagulase-negative (CoNs) strains. All CoPs isolates exhibited resistance to at least one of the 16 antibiotics tested. Among the CoNs strains, 52% demonstrated resistance to at least one antibiotic, and 7 isolates were classified as multi-drug resistant (MDR). In these MDR strains, we identified AMR genes that confirmed the resistance phenotype, as well as other AMR genes, such as quaternary ammonium resistance. One CoNS strain carried 9 AMR genes, including both antibiotic and biocide resistance genes. By mapping the AMR phenotypes, we demonstrated that rivers had a local influence, particularly near the English coast, on the occurrence of AMR Staphylococcus. The analysis of marine environmental parameters revealed that turbidity and phosphate concentration were implicated in the occurrence of AMR Staphylococcus. Our findings underscore the crucial role of wild whiting and seawater in the dissemination of AMR Staphylococcus within the marine environment, thereby posing a risk to human health.

A Glimpse at the Anti-Phage Defenses Landscape in the Foodborne Pathogen Salmonella enterica subsp. enterica serovar Typhimurium.

Bacteriophages, which specifically infect and kill bacteria, are currently used as additives to control pathogens such as Salmonella in human food (PhageGuard S®) or animal feed (SalmoFREE®, Bafasal®). Indeed, salmonellosis is among the most important zoonotic foodborne illnesses. The presence of anti-phage defenses protecting bacteria against phage infection could impair phage applications aiming at reducing the burden of foodborne pathogens such as Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) to the food industry. In this study, the landscape of S. Typhimurium anti-phage defenses was bioinformatically investigated in publicly available genomes using the webserver PADLOC. The primary anti-phage systems identified in S. Typhimurium use nucleic acid degradation and abortive infection mechanisms. Reference systems were identified on an integrative and conjugative element, a transposon, a putative integrative and mobilizable element, and prophages. Additionally, the mobile genetic elements (MGEs) containing a subset of anti-phage systems were found in the Salmonella enterica species. Lastly, the MGEs alone were also identified in the Enterobacteriaceae family. The presented diversity assessment of the anti-phage defenses and investigation of their dissemination through MGEs in S. Typhimurium constitute a first step towards the design of preventive measures against the spread of phage resistance that may hinder phage applications.

High Throughput Screening of Antimicrobial Resistance Genes in Gram-Negative Seafood Bacteria.

From a global view of antimicrobial resistance over different sectors, seafood and the marine environment are often considered as potential reservoirs of antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs); however, there are few studies and sparse results on this sector. This study aims to provide new data and insights regarding the content of resistance markers in various seafood samples and sources, and therefore the potential exposure to humans in a global One Health approach. An innovative high throughput qPCR screening was developed and validated in order to simultaneously investigate the presence of 41 ARGs and 33 MGEs including plasmid replicons, integrons, and insertion sequences in Gram-negative bacteria. Analysis of 268 seafood isolates from the bacterial microflora of cod (n = 24), shellfish (n = 66), flat fishes (n = 53), shrimp (n = 10), and horse mackerel (n = 115) show the occurrence of sul-1, ant(3″)-Ia, aph(3')-Ia, strA, strB, dfrA1, qnrA, and blaCTX-M-9 genes in Pseudomonas spp., Providencia spp., Klebsiella spp., Proteus spp., and Shewanella spp. isolates and the presence of MGEs in all bacterial species investigated. We found that the occurrence of MGE may be associated with the seafood type and the environmental, farming, and harvest conditions. Moreover, even if MGE were detected in half of the seafood isolates investigated, association with ARG was only identified for twelve isolates. The results corroborate the hypothesis that the incidence of antimicrobial-resistant bacteria (ARB) and ARG decreases with increasing distance from potential sources of fecal contamination. This unique and original high throughput micro-array designed for the screening of ARG and MGE in Gram-negative bacteria could be easily implementable for monitoring antimicrobial resistance gene markers in diverse contexts.

Multicentre evaluation of a selective isolation protocol for detection of mcr-positive E. coli and Salmonella spp. in food-producing animals and meat.

This study was conducted to evaluate the performance of a screening protocol to detect and isolate mcr-positive Escherichia coli and Salmonella spp. from animal caecal content and meat samples. We used a multicentre approach involving 12 laboratories from nine European countries. All participants applied the same methodology combining a multiplex PCR performed on DNA extracted from a pre-enrichment step, followed by a selective culture step on three commercially available chromogenic agar plates. The test panel was composed of two negative samples and four samples artificially contaminated with E. coli and Salmonella spp. respectively harbouring mcr-1 or mcr-3 and mcr-4 or mcr-5 genes. PCR screening resulted in a specificity of 100% and a sensitivity of 83%. Sensitivity of each agar medium to detect mcr-positive colistin-resistant E. coli or Salmonella spp. strains was 86% for CHROMID® Colistin R, 75% for CHROMagarTM COL-APSE and 70% for COLISTIGRAM. This combined method was effective to detect and isolate most of the E. coli or Salmonella spp. strains harbouring different mcr genes from food-producing animals and food products and might thus be used as a harmonized protocol for the screening of mcr genes in food-producing animals and food products in Europe.

A European multicenter evaluation study to investigate the performance on commercially available selective agar plates for the detection of carbapenemase producing Enterobacteriaceae.

The European Food Safety Authority (EFSA) advised to prioritize monitoring carbapenemase producing Enterobacteriaceae (CPE) in food producing animals. Therefore, this study evaluated the performance of different commercially available selective agars for the detection of CPE using spiked pig caecal and turkey meat samples and the proposed EFSA cultivation protocol. Eleven laboratories from nine countries received eight samples (four caecal and four meat samples). For each matrix, three samples contained approximately 100 CFU/g CPE, and one sample lacked CPE. After overnight enrichment in buffered peptone water, broths were spread upon Brilliance™ CRE Agar (1), CHROMID® CARBA (2), CHROMagar™ mSuperCARBA™ (3), Chromatic™ CRE (4), CHROMID® OXA-48 (5) and Chromatic™ OXA-48 (6). From plates with suspected growth, one to three colonies were selected for species identification, confirmation of carbapenem resistance and detection of carbapenemase encoding genes, by methods available at participating laboratories. Of the eleven participating laboratories, seven reported species identification, susceptibility tests and genotyping on isolates from all selective agar plates. Agars 2, 4 and 5 performed best, with 100% sensitivity. For agar 3, a sensitivity of 96% was recorded, while agar 1 and 6 performed with 75% and 43% sensitivity, respectively. More background flora was noticed for turkey meat samples than pig caecal samples. Based on this limited set of samples, most commercially available agars performed adequately. The results indicate, however, that OXA-48-like and non-OXA-48-like producers perform very differently, and one should consider which CPE strains are of interest to culture when choosing agar type.

Comparison of Phenotypical Antimicrobial Resistance between Clinical and Non-Clinical E. coli Isolates from Broilers, Turkeys and Calves in Four European Countries.

Livestock data on antimicrobial resistance (AMR) are commonly collected from bacterial populations of clinical and non-clinical isolates. In contrast to data on non-clinical isolates from livestock, data on clinical isolates are not harmonized in Europe. The Normalized Resistance Interpretation (NRI) method was applied to overcome the lack of harmonization of laboratory methods and interpretation rules between monitoring systems. Statistical analyses were performed to identify associations between the isolate type (clinical vs. non-clinical) and resistance to four antimicrobials (ampicillin, tetracycline, gentamicin, and nalidixic acid) per animal category in Germany and France. Additional statistical analyses comparing clinical and non-clinical isolates were performed with the available data on the same antimicrobial panel and animal categories from the UK and Norway. Higher resistance prevalence was found in clinical isolates compared to non-clinical isolates from calves to all antimicrobials included in Germany and France. It was also found for gentamicin in broilers from France. In contrast, in broilers and turkeys from Germany and France and in broilers from the UK, a higher resistance level to ampicillin and tetracycline in non-clinical isolates was encountered. This was also found in resistance to gentamicin in isolates from turkeys in Germany. Resistance differed within countries and across years, which was partially in line with differences in antimicrobial use patterns. Differences in AMR between clinical and non-clinical isolates of Escherichia coli are associated with animal category (broiler, calf, and turkey) and specific antimicrobials. The NRI method allowed comparing results of non-harmonized AMR systems and might be useful until international harmonization is achieved.

Surveillance of Extended-Spectrum ß-Lactamase-, Cephalosporinase- and Carbapenemase-Producing Gram-Negative Bacteria in Raw Milk Filters and Healthy Dairy Cattle in Three Farms in Île-de-France, France.

The aim of this work was to test a surveillance protocol able to detect extended-spectrum ß-lactamase (ESBL)-, cephalosporinase (AmpC)- and carbapenemase (CP)-producing gram-negative bacteria in three conveniently chosen dairy farms with known prior occurrences of ESBL- and CP-producing strains. The protocol was applied monthly for a year. At each visit, 10 healthy lactating dairy cows were rectally swabbed, and raw milk filters (RMFs) were sampled in two of the three farms. Bacterial isolation was based on a first screening step with MacConkey agar supplemented with 1 mg/L cefotaxime and commercial carbapenem-supplemented media. We failed to detect CP-producing strains but showed that ESBL-Escherichia strains, found in one farm only (13 strains), were closely associated with multi-drug resistance (12 out of 13). The limited number of conveniently selected farms and the fact that RMFs could not be retrieved from one of them limit the validity of our findings. Still, our results illustrate that ESBL-status changes monthly based on fecal swabs and negative herds should be qualified as "unsuspected" as proposed by previous authors. Although surveillance of farm statuses based on RMF analysis could theoretically allow for a better sensitivity than individual swabs, we failed to illustrate it as both farms where RMFs could be retrieved were constantly negative. Determination of CP herd-level status based on RMFs and our surveillance protocol was hindered by the presence of intrinsically resistant bacteria or strains cumulating multiple non-CP resistance mechanisms which means our protocol is not specific enough for routine monitoring of CP in dairy farms.

Source Attribution Study of Sporadic Salmonella Derby Cases in France.

Salmonella enterica subsp. enterica serovar Derby is one of the most frequent causes of gastroenteritis in humans. In Europe, this pathogen is one of the top five most commonly reported serovars in human cases. In France, S. Derby has been among the ten most frequently isolated serovars in humans since the year 2000. The main animal hosts of this serovar are pigs and poultry, and white meat is the main source of human contamination. We have previously shown that this serovar is polyphyletic and that three distinct genetic lineages of S. Derby cohabit in France. Two of them are associated with pork and one with poultry. In this study, we conducted a source attribution study based on single nucleotide polymorphism analysis of a large collection of 440 S. Derby human and non-human isolates collected in 2014-2015, to determine the contribution of each lineage to human contamination. In France, the two lineages associated with pork strains, and corresponding to the multilocus sequence typing (MLST) profiles ST39-ST40 and ST682 were responsible for 94% of human contaminations. Interestingly, the ST40 profile is responsible for the majority of human cases (71%). An analysis of epidemiologic data and the structure of the pork sector in France allowed us to explain the spread and the sporadic pattern of human cases that occurred in the studied period.

Decrease in fluoroquinolone use in French poultry and pig production and changes in resistance among E. coli and Campylobacter.

This paper presents the impact on antimicrobial resistance (AMR) in poultry and pig bacteria of the French EcoAntibio plan, a public policy to reduce antimicrobial use in animals. The analysis was performed using sales data of veterinary antimicrobials and AMR data from bacteria obtained at slaughterhouse and from diseased animals. From 2011-2018, fluoroquinolones exposure decreased by 71.5 % for poultry and 89.7 % for pigs. For Campylobacter jejuni isolated from broilers at slaughterhouses, ciprofloxacin resistance increased from 51 % in 2010 to 63 % in 2018, whereas for turkeys the percentages varied from 56 % in 2014 to 63 % in 2018. For commensal E. coli isolated from the caecal content of broilers at slaughterhouses, the resistance to ciprofloxacin - assessed using an epidemiological cut-off value - increased in broiler isolates from 30.7 % in 2010 to 38.1 % in 2018. In turkeys, the percentage of resistant E. coli isolates decreased from 21.3 % in 2014 to 15.2 % in 2018, whereas in pigs, it increased from 1.9 % in 2009 to 5.5 % in 2017. However, for E. coli isolated from diseased animals, when the breakpoints of 2018 were applied, resistance to fluoroquinolones significantly decreased between 2010 and 2018 from 9.0%-5.4% for broilers/hens, from 7.4 % to 3.4 % for turkeys and from 9.4 % to 3.6 % for pigs. These data show that the major, rapid decrease in the exposition to fluoroquinolones had contrasting effects on resistance in the diverse bacterial collections. Co-selection or fitness of resistant strains may explain why changes in AMR do not always closely mirror changes in use.

Occurrence of Third-Generation Cephalosporins-Resistant Klebsiella pneumoniae in Fresh Fruits and Vegetables Purchased at Markets in Algeria.

This study aimed to characterize third-generation cephalosporin (3GC)-resistant Klebsiella pneumoniae isolated from fresh fruits and vegetables purchased at Bejaia city, Algeria. K. pneumoniae isolates were identified by MALDI-TOF. Susceptibility to antibiotics was tested by the disk diffusion method. Whole genome sequencing (WGS) was carried out to determine sequence type (ST), plasmid incompatibility group (Inc.), and acquired antimicrobial resistance gene presence. A total of 13 3GC-resistant K. pneumoniae strains were isolated. WGS identified blaCTX-M-15 in 11 extended-spectrum-beta-lactamases (ESBL)-K. pneumoniae and blaDHA-1 in 2 AmpC-K. pneumoniae. The aac(6')lb-cr gene was identified in 8 out of 13 isolates. Multilocus sequence typing (MLST) evidenced five different STs, namely ST14, ST45, ST219, ST236, and ST882. MDR K. pneumoniae contaminated fresh fruits and vegetables, often eaten raw and inappropriately washed, may represent an underestimated public health threat. This study highlights that hygiene measures during harvesting and retail process are of utmost importance to limit further ESBL/AmpC spread to the consumer households.

Phenotypic and Genotypic Antimicrobial Resistance Traits of Vibrio cholerae Non-O1/Non-O139 Isolated From a Large Austrian Lake Frequently Associated With Cases of Human Infection.

Vibrio cholerae belonging to serogroups other than O1 and O139 are opportunistic pathogens which cause infections with a variety of clinical symptoms. Due to the increasing number of V. cholerae non-O1/non-O139 infections in association with recreational waters in the past two decades, they have received increasing attention in recent literature and by public health authorities. Since the treatment of choice is the administration of antibiotics, we investigated the distribution of antimicrobial resistance properties in a V. cholerae non-O1/non-O139 population in a large Austrian lake intensively used for recreation and in epidemiologically linked clinical isolates. In total, 82 environmental isolates - selected on the basis of comprehensive phylogenetic information - and nine clinical isolates were analyzed for their phenotypic antimicrobial susceptibility. The genomes of 46 environmental and eight clinical strains were screened for known genetic antimicrobial resistance traits in CARD and ResFinder databases. In general, antimicrobial susceptibility of the investigated V. cholerae population was high. The environmental strains were susceptible against most of the 16 tested antibiotics, except sulfonamides (97.5% resistant strains), streptomycin (39% resistant) and ampicillin (20.7% resistant). Clinical isolates partly showed additional resistance to amoxicillin-clavulanic acid. Genome analysis showed that crp, a regulator of multidrug efflux genes, and the bicyclomycin/multidrug efflux system of V. cholerae were present in all isolates. Nine isolates additionally carried variants of bla CARB-7 and bla CARB-9, determinants of beta-lactam resistance and six isolates carried catB9, a determinant of phenicol resistance. Three isolates had both bla CARB-7 and catB9. In 27 isolates, five out of six subfamilies of the MATE-family were present. For all isolates no genes conferring resistance to aminoglycosides, macrolides and sulfonamides were detected. The apparent lack of either known antimicrobial resistance traits or mobile genetic elements indicates that in cholera non-epidemic regions of the world, V. cholerae non-O1/non-O139 play a minor role as a reservoir of resistance in the environment. The discrepancies between the phenotypic and genome-based antimicrobial resistance assessment show that for V. cholerae non-O1/non-O139, resistance databases are currently inappropriate for an assessment of antimicrobial resistance. Continuous collection of both data over time may solve such discrepancies between genotype and phenotype in the future.

Global phylogenomics of multidrug-resistant Salmonella enterica serotype Kentucky ST198.

Salmonella enterica serotype Kentucky can be a common causative agent of salmonellosis, usually associated with consumption of contaminated poultry. Antimicrobial resistance (AMR) to multiple drugs, including ciprofloxacin, is an emerging problem within this serotype. We used whole-genome sequencing (WGS) to investigate the phylogenetic structure and AMR content of 121 S.enterica serotype Kentucky sequence type 198 isolates from five continents. Population structure was inferred using phylogenomic analysis and whole genomes were compared to investigate changes in gene content, with a focus on acquired AMR genes. Our analysis showed that multidrug-resistant (MDR) S.enterica serotype Kentucky isolates belonged to a single lineage, which we estimate emerged circa 1989 following the acquisition of the AMR-associated Salmonella genomic island (SGI) 1 (variant SGI1-K) conferring resistance to ampicillin, streptomycin, gentamicin, sulfamethoxazole and tetracycline. Phylogeographical analysis indicates this clone emerged in Egypt before disseminating into Northern, Southern and Western Africa, then to the Middle East, Asia and the European Union. The MDR clone has since accumulated various substitution mutations in the quinolone-resistance-determining regions (QRDRs) of DNA gyrase (gyrA) and DNA topoisomerase IV (parC), such that most strains carry three QRDR mutations which together confer resistance to ciprofloxacin. The majority of AMR genes in the S. enterica serotype Kentucky genomes were carried either on plasmids or SGI structures. Remarkably, each genome of the MDR clone carried a different SGI1-K derivative structure; this variation could be attributed to IS26-mediated insertions and deletions, which appear to have hampered previous attempts to trace the clone's evolution using sub-WGS resolution approaches. Several different AMR plasmids were also identified, encoding resistance to chloramphenicol, third-generation cephalosporins, carbapenems and/or azithromycin. These results indicate that most MDR S. enterica serotype Kentucky circulating globally result from the clonal expansion of a single lineage that acquired chromosomal AMR genes 30 years ago, and has continued to diversify and accumulate additional resistances to last-line oral antimicrobials. This article contains data hosted by Microreact.

Genetic Diversity of Salmonella Derby from the Poultry Sector in Europe.

Salmonella Derby (S. Derby) is emerging in Europe as a predominant serovar in fattening turkey flocks. This serovar was recorded as being predominant in the turkey sector in 2014 in the United Kingdom (UK). Only two years later, in 2016, it was also recorded in the turkey and broiler sectors in Ireland and Spain. These S. Derby isolates were characterised as members of the multilocus sequence type (MLST) profile 71 (ST71). For the first time, we characterise by whole genome sequencing (WGS) analysis a panel of 90 S. Derby ST71 genomes to understand the routes of transmission of this emerging pathogen within the poultry/turkey food trade. Selected panel included strains isolated as early as 2010 in five leading European g countries for turkey meat production. Twenty-one of the 90 genomes were extracted from a public database-Enterobase. Five of these originated from the United States (n=3), China (n=1) and Taiwan (n=1) isolated between 1986 and 2016. A phylogenomic analysis at the core-genome level revealed the presence of three groups. The largest group contained 97.5% of the European strains and included both, turkey and human isolates that were genetically related by an average of 35 ± 15 single nucleotide polymorphism substitutions (SNPs). To illustrate the diversity, the presence of antimicrobial resistance genes and phages were characteised in 30, S. Derby ST71 genomes, including 11 belonging to this study This study revealed an emergent turkey-related S. Derby ST71 clone circulating in at least five European countries (the UK, Germany, Poland, Italy, and France) since 2010 that causes human gastroenteritis. A matter of concern is the identification of a gyrA mutation involved in resistance to quinolone, present in the Italian genomes. Interestingly, the diversity of phages seems to be related to the geographic origins. These results constitute a baseline for following the spread of this emerging pathogen and identifying appropriate monitoring and prevention measures.

Correction for Sévellec et al., "Complete Genome Sequence of Salmonella enterica subsp. enterica Serotype Derby, Associated with the Pork Sector in France".

[This corrects the article DOI: 10.1128/MRA.01027-18.].

Complete Genome Sequence of Salmonella enterica subsp. enterica Serotype Derby, Associated with the Pork Sector in France.

In the European Union, Salmonella enterica subsp. enterica serovar Derby is the most abundant serotype isolated from pork. Recent studies have shown that this serotype is polyphyletic. However, one main genomic lineage, characterized by sequence type 40 (ST40), the presence of the Salmonella pathogenicity island 23, and showing resistance to streptomycin, sulphonamides, and tetracycline (STR-SSS-TET), is pork associated. Here, we describe the complete genome sequence of a strain from this lineage isolated in France.

Polyphyletic Nature of Salmonella enterica Serotype Derby and Lineage-Specific Host-Association Revealed by Genome-Wide Analysis.

In France, Salmonella Derby is one of the most prevalent serotypes in pork and poultry meat. Since 2006, it has ranked among the 10 most frequent Salmonella serotypes isolated in humans. In previous publications, Salmonella Derby isolates have been characterized by pulsed field gel electrophoresis (PFGE) and antimicrobial resistance (AMR) profiles revealing the existence of different pulsotypes and AMR phenotypic groups. However, these results suffer from the low discriminatory power of these typing methods. In the present study, we built a collection of 140 strains of S. Derby collected in France from 2014 to 2015 representative of the pork and poultry food sectors. The whole collection was characterized using whole genome sequencing (WGS), providing a significant contribution to the knowledge of this underrepresented serotype, with few genomes available in public databases. The genetic diversity of the S. Derby strains was analyzed by single-nucleotide polymorphism (SNP). We also investigated AMR by both genome and phenotype, the main Salmonella pathogenicity island (SPI) and the fimH gene sequences. Our results show that this S. Derby collection is spread across four different lineages genetically distant by an average of 15k SNPs. These lineages correspond to four multilocus sequence typing (MLST) types (ST39, ST40, ST71, and ST682), which were found to be associated with specific animal hosts: pork and poultry. While the ST71 and ST682 strains are pansusceptible, ST40 isolates are characterized by the multidrug resistant profile STR-SSS-TET. Considering virulence determinants, only ST39 and ST40 present the SPI-23, which has previously been associated with pork enterocyte invasion. Furthermore, the pork ST682 isolates were found to carry mutations in the fimH sequence that could participate in the host tropism of this group. Our phylogenetic analysis demonstrates the polyphyletic nature of the Salmonella serotype Derby and provides an opportunity to identify genetic factors associated with host adaptation and markers for the monitoring of these different lineages within the corresponding animal sectors. The recognition of these four lineages is of primary importance for epidemiological surveillance throughout the food production chains and constitutes the first step toward refining monitoring and preventing dispersal of this pathogen.

Bacillus cereus, a serious cause of nosocomial infections: Epidemiologic and genetic survey.

Bacillus cereus is the 2nd most frequent bacterial agent responsible for food-borne outbreaks in France and the 3rd in Europe. In addition, local and systemic infections have been reported, mainly describing individual cases or single hospital setting. The real incidence of such infection is unknown and information on genetic and phenotypic characteristics of the incriminated strains is generally scarce. We performed an extensive study of B. cereus strains isolated from patients and hospital environments from nine hospitals during a 5-year study, giving an overview of the consequences, sources and pathogenic patterns of B. cereus clinical infections. We demonstrated the occurrence of several hospital-cross-contaminations. Identical B. cereus strains were recovered from different patients and hospital environments for up to 2 years. We also clearly revealed the occurrence of inter hospital contaminations by the same strain. These cases represent the first documented events of nosocomial epidemy by B. cereus responsible for intra and inter hospitals contaminations. Indeed, contamination of different patients with the same strain of B. cereus was so far never shown. In addition, we propose a scheme for the characterization of B. cereus based on biochemical properties and genetic identification and highlight that main genetic signatures may carry a high pathogenic potential. Moreover, the characterization of antibiotic resistance shows an acquired resistance phenotype for rifampicin. This may provide indication to adjust the antibiotic treatment and care of patients.

Multiplex PCR for detection of plasmid-mediated colistin resistance determinants, mcr-1, mcr-2, mcr-3, mcr-4 and mcr-5 for surveillance purposes.

Background and aimPlasmid-mediated colistin resistance mechanisms have been identified worldwide in the past years. A multiplex polymerase chain reaction (PCR) protocol for detection of all currently known transferable colistin resistance genes (mcr-1 to mcr-5, and variants) in Enterobacteriaceae was developed for surveillance or research purposes. Methods: We designed four new primer pairs to amplify mcr-1, mcr-2, mcr-3 and mcr-4 gene products and used the originally described primers for mcr-5 to obtain a stepwise separation of ca 200 bp between amplicons. The primer pairs and amplification conditions allow for single or multiple detection of all currently described mcr genes and their variants present in Enterobacteriaceae. The protocol was validated testing 49 European Escherichia coli and Salmonella isolates of animal origin. Results: Multiplex PCR results in bovine and porcine isolates from Spain, Germany, France and Italy showed full concordance with whole genome sequence data. The method was able to detect mcr-1, mcr-3 and mcr-4 as singletons or in different combinations as they were present in the test isolates. One new mcr-4 variant, mcr-4.3, was also identified. Conclusions: This method allows rapid identification of mcr-positive bacteria and overcomes the challenges of phenotypic detection of colistin resistance. The multiplex PCR should be particularly interesting in settings or laboratories with limited resources for performing genetic analysis as it provides information on the mechanism of colistin resistance without requiring genome sequencing.

Illustrative examples of probable transfer of resistance determinants from food animals to humans: Streptothricins, glycopeptides, and colistin.

Use, overuse, and misuse of antimicrobials contributes to selection and dissemination of bacterial resistance determinants that may be transferred to humans and constitute a global public health concern. Because of the continued emergence and expansion of antimicrobial resistance, combined with the lack of novel antimicrobial agents, efforts are underway to preserve the efficacy of current available life-saving antimicrobials in humans. As a result, uses of medically important antimicrobials in food animal production have generated debate and led to calls to reduce both antimicrobial use and the need for use. This manuscript, commissioned by the World Health Organization (WHO) to help inform the development of the WHO guidelines on the use of medically important antimicrobials in food animals, includes three illustrations of antimicrobial use in food animal production that has contributed to the selection-and subsequent transfer-of resistance determinants from food animals to humans. Herein, antimicrobial use and the epidemiology of bacterial resistance are described for streptothricins, glycopeptides, and colistin. Taken together, these historical and current narratives reinforce the need for actions that will preserve the efficacy of antimicrobials.