Antimicrobial resistance (AMR) is a serious threat to global public health, but obtaining representative data on AMR for healthy human populations is difficult. Here, we use metagenomic analysis of untreated sewage to characterize the bacterial resistome from 79 sites in 60 countries. We find systematic differences in abundance and diversity of AMR genes between Europe/North-America/Oceania and Africa/Asia/South-America. Antimicrobial use data and bacterial taxonomy only explains a minor part of the AMR variation that we observe. We find no evidence for cross-selection between antimicrobial classes, or for effect of air travel between sites. However, AMR gene abundance strongly correlates with socio-economic, health and environmental factors, which we use to predict AMR gene abundances in all countries in the world. Our findings suggest that global AMR gene diversity and abundance vary by region, and that improving sanitation and health could potentially limit the global burden of AMR. We propose metagenomic analysis of sewage as an ethically acceptable and economically feasible approach for continuous global surveillance and prediction of AMR.

Background and aim: Plasmid-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-4.6**, 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.

Salmonella enterica is commonly acquired from contaminated food and is an important cause of illness worldwide. Interventions are needed to control Salmonella; subtyping Salmonella by serotyping is useful for targeting such interventions. We, therefore, analyzed the global distribution of the 15 most frequently identified serovars of Salmonella isolated from humans from 2001 to 2007 in laboratories from 37 countries that participated in World Health Organization Global Foodborne Infections Network and demonstrated serotyping proficiency in the Global Foodborne Infections Network External Quality Assurance System. In all regions throughout the study period, with the exception of the Oceania and North American regions, Salmonella serovars Enteritidis and Typhimurium ranked as the most common and second most common serovar, respectively. In the North American and Oceania (Australia and New Zealand) regions, Salmonella serovar Typhimurium was the most common serovar reported, and Salmonella serovar Enteritidis was the second most common serovar. During the study period, the proportion of Salmonella isolates reported from humans that were Salmonella serovar Enteritidis was 43.5% (range: 40.6% [2007] to 44.9% [2003]), and Salmonella serovar Typhimurium was 17.1% (range: 15% [2007] to 18.9% [2001]). Salmonella serovars Newport (mainly observed in Latin and North American and European countries), Infantis (dominating in all regions), Virchow (mainly observed in Asian, European, and Oceanic countries), Hadar (profound in European countries), and Agona (intense in Latin and North American and European countries) were also frequently isolated with an overall proportion of 3.5%, 1.8%, 1.5%, 1.5%, and 0.8%, respectively. There were large differences in the most commonly isolated serovars between regions, but lesser differences between countries within the same region. The results also highlight the complexity of the global epidemiology of Salmonella and the need and importance for improving monitoring data of those serovars of highest epidemiologic importance.

Plasmid-mediated mobilized colistin resistance is currently known to be caused by phosphoethanolamine transferases termed MCR-1, MCR-2, MCR-3 and MCR-4. However, this study focuses on the dissection of a novel resistance mechanism in mcr-1-, mcr-2- and mcr-3-negative d-tartrate fermenting Salmonella enterica subsp. enterica serovar Paratyphi B (Salmonella Paratyphi B dTa+) isolates with colistin MIC values >2 mg/L. A selected isolate from the strain collection of the German National Reference Laboratory for Salmonella was investigated by WGS and bioinformatical analysis to identify novel phosphoethanolamine transferase genes involved in colistin resistance. Subsequently PCR screening, S1-PFGE and DNA-DNA hybridization were performed to analyse the prevalence and location of the identified mcr-5 gene. Cloning and transformation experiments in Escherichia coli DH5α and Salmonella Paratyphi B dTa+ control strains were carried out and the activity of MCR-5 was determined in vitro by MIC testing. In this study, we identified a novel phosphoethanolamine transferase in 14 mcr-1-, mcr-2- and mcr-3-negative Salmonella Paratyphi B dTa+ isolates with colistin MIC values >2 mg/L that were received during 2011–13. The respective gene, further termed as mcr-5 (1644 bp), is part of a 7337 bp transposon of the Tn3 family and usually located on related multi-copy ColE-type plasmids. Interestingly, in one isolate an additional subclone with a chromosomal location of the mcr-5 transposon was observed. Our findings suggest that the transfer of colistin-resistance-mediating phosphoethanolamine transferase genes from bacterial chromosomes to mobile genetic elements has occurred in multiple independent events raising concern regarding their variety, prevalence and impact on public health.

From 1995 to 2000, a total of 673 Enterococcus faecium and 1,088 Enterococcus faecalis isolates from pigs together with 856 E. faecium isolates from broilers were isolated and tested for susceptibility to four classes of antimicrobial agents used for growth promotion as part of the Danish program of monitoring for antimicrobial resistance. The four antimicrobials were avilamycin, erythromycin, vancomycin, and virginiamycin. Major changes in the use of antimicrobial agents for growth promotion have occurred during the last 6 years in Denmark. The government banned the use of avoparcin in 1995 and of virginiamycin in 1998. Furthermore, the producers have voluntarily stopped all use beginning in 1999. The avoparcin ban in 1995 was followed by a decrease in the occurrence of glycopeptide-resistant E. faecium (GRE) in broilers, from 72.7% in 1995 to 5.8% in 2000. The occurrence of glycopeptide resistance among isolates from pigs remained constant at around 20% from 1995 to 1997. It was shown that, in GRE from pigs, the genes encoding macrolide and glycopeptide resistance were genetically linked and that, following the decrease in the use of tylosin during 1998 and 1999, the occurrence of GRE in pigs decreased to 6.0% in 2000. From 1995 to 1997 the occurrence of erythromycin resistance among E. faecium and E. faecalis isolates from pigs was almost 90%. Use of tylosin decreased considerably during 1998 and 1999, and this decrease was followed by decreases in the occurrence of resistance to 46.7 and 28.1% among E. faecium and E. faecalis isolates from pigs, respectively. Erythromycin resistance among E. faecium isolates from broilers reached a maximum of 76.3% in 1997 but decreased to 12.7% in 2000 concomitantly with more limited use of virginiamycin. Use of virginiamycin increased from 1995 to 1997 and was followed by an increased occurrence of virginiamycin resistance among E. faecium isolates in broilers, from 27.3% in 1995 to 66.2% in 1997. In January 1998 the use of virginiamycin was banned in Denmark, and the occurrence of virginiamycin resistance decreased to 33.9% in 2000. Use of avilamycin increased from 1995 to 1996 and was followed by an increase in avilamycin resistance among E. faecium isolates from broilers, from 63.6% in 1995 to 77.4% in 1996. Since 1996 avilamycin usage has decreased, followed by a decrease in resistance to 4.8% in 2000. Our observations show that it is possible to reduce the occurrence of antimicrobial resistance in a national population of food animals when the selective pressure is removed. Cases in which resistance to vancomycin was linked to resistance to erythromycin were exceptions. In such cases resistance did not decrease until the use of both avoparcin and tylosin was limited.

The recent advancements in rapid and affordable DNA sequencing technologies have revolutionized diagnostic microbiology and microbial surveillance. The availability of bioinformatics tools and online accessible databases has been a prerequisite for this. We conducted a scientific literature review and here we present a description of examples of available tools and databases for antimicrobial resistance (AMR) detection and provide future perspectives and recommendations. At least 47 freely accessible bioinformatics resources for detection of AMR determinants in DNA or amino acid sequence data have been developed to date. These include, among others but not limited to, ARG-ANNOT, CARD, SRST2, MEGARes, Genefinder, ARIBA, KmerResistance, AMRFinder, and ResFinder. Bioinformatics resources differ for several parameters including type of accepted input data, presence/absence of software for search within a database of AMR determinants that can be specific to a tool or cloned from other resources, and for the search approach employed, which can be based on mapping or on alignment. As a consequence, each tool has strengths and limitations in sensitivity and specificity of detection of AMR determinants and in application, which for some of the tools have been highlighted in benchmarking exercises and scientific articles. The identified tools are either available at public genome data centers, from GitHub or can be run locally. NCBI and European Nucleotide Archive (ENA) provide possibilities for online submission of both sequencing and accompanying phenotypic antimicrobial susceptibility data, allowing for other researchers to further analyze data, and develop and test new tools. The advancement in whole genome sequencing and the application of online tools for real-time detection of AMR determinants are essential to identify control and prevention strategies to combat the increasing threat of AMR. Accessible tools and DNA sequence data are expanding, which will allow establishing global pathogen surveillance and AMR tracking based on genomics. There is however, a need for standardization of pipelines and databases as well as phenotypic predictions based on the data.