Exploring the epidemiology of mcr genes, genetic context and plasmids in Enterobacteriaceae originating from pigs and humans on farms in Thailand.

OBJECTIVES: In veterinary medicine, colistin has been widely used as therapeutic and prophylactic agent, and for growth promotion. However, colistin has been re-introduced into treatment of human MDR bacterial infections. We assessed the characteristics and spread of plasmid-borne colistin resistance among healthy pigs, workers with animal-contact and their household members in Thailand. METHODS: WGS and MIC data of 146 mcr-positive isolates from a cross-sectional One Health study were analysed. Long-read sequencing and conjugation were performed for selected isolates. RESULTS: mcr-carrying isolates were detected in 38% of pooled-pig samples and 16% of human faecal samples. Of 143 Escherichia coli and three Escherichia fergusonii, mcr-1, mcr-3, and mcr-9 variants were identified in 96 (65.8%), 61 (41.8%) and one (0.7%) isolate, respectively. Twelve E. coli co-harboured two mcr variants (mcr-1 and mcr-3). Clonal transmission was detected in five out of 164 farms. mcr-1 was mostly harboured by epidemic IncX4 and IncHI1 plasmids (89.9%). Conversely, mcr-3 was harboured by a range of different plasmids. Comparative plasmid studies suggested IncP and IncFII plasmids as possible endemic mcr-3 plasmids in Asian countries. Moreover, mcr-3 was associated with different mobile genetic elements including TnAs2, ISKpn40 and IS26/15DI. Detected genetic signatures (DRs) indicated recent mcr-3 transpositions, underlining the mobilizable nature of the mcr-3 cassette. CONCLUSIONS: The epidemiology of mcr and the possible evolution of successful plasmids and transposition modules should be carefully monitored. Of special concern is the growing number of different horizontal gene transferring pathways encompassing various transposable modules the mcr genes can be shared between bacteria.

Highly conserved composite transposon harbouring aerobactin iuc3 in Klebsiella pneumoniae from pigs.

Klebsiella pneumoniae is an important opportunistic pathogen associated with severe invasive disease in humans. Hypervirulent K. pneumoniae, which are K. pneumoniae with several acquired virulence determinants such as the siderophore aerobactin and others, are more prominent in countries in South and South-East Asia compared to European countries. This Klebsiella pathotype is capable of causing liver abscesses in immunocompetent persons in the community. K. pneumoniae has not been extensively studied in non-human niches. In the present study, K. pneumoniae isolated from caecal samples (n=299) from healthy fattening pigs in Norway were characterized with regard to population structure and virulence determinants. These data were compared to data from a previous study on K. pneumoniae from healthy pigs in Thailand. Lastly, an in-depth plasmid study on K. pneumoniae with aerobactin was performed. Culturing and whole-genome sequencing was applied to detect, confirm and characterize K. pneumoniae isolates. Phylogenetic analysis described the evolutionary relationship and diversity of the isolates, while virulence determinants and sequence types were detected with Kleborate. Long-read sequencing was applied to obtain the complete sequence of virulence plasmids harbouring aerobactin. A total of 48.8 % of the investigated Norwegian pig caecal samples (n=299) were positive for K. pneumoniae. Acquired virulence determinants were detected in 72.6 % of the isolates, the most prominent being aerobactin (69.2 %), all of which were iuc3. In contrast, only 4.6 % of the isolates from Thailand harboured aerobactin. The aerobactin operon was located on potentially conjugative IncFIBK/FIIK plasmids of varying sizes in isolates from both countries. A putative, highly conserved composite transposon with a mean length of 16.2 kb flanked by truncated IS3-family IS407-group insertion sequences was detected on these plasmids, harbouring the aerobactin operon as well as several genes that may confer increased fitness in mammalian hosts. This putative composite transposon was also detected in plasmids harboured by K. pneumoniae from several countries and sources, such as human clinical samples. The high occurrence of K. pneumoniae harbouring aerobactin in Norwegian pigs, taken together with international data, suggest that pigs are a reservoir for K. pneumoniae with iuc3. Truncation of the flanking ISKpn78-element suggest that the putative composite transposon has been permanently integrated into the plasmid, and that it is no longer mobilizable.

CReSIL: accurate identification of extrachromosomal circular DNA from long-read sequences.

Extrachromosomal circular DNA (eccDNA) of chromosomal origin is found in many eukaryotic species and cell types, including cancer, where eccDNAs with oncogenes drive tumorigenesis. Most studies of eccDNA employ short-read sequencing for their identification. However, short-read sequencing cannot resolve the complexity of genomic repeats, which can lead to missing eccDNA products. Long-read sequencing technologies provide an alternative to constructing complete eccDNA maps. We present a software suite, Construction-based Rolling-circle-amplification for eccDNA Sequence Identification and Location (CReSIL), to identify and characterize eccDNA from long-read sequences. CReSIL's performance in identifying eccDNA, with a minimum F1 score of 0.98, is superior to the other bioinformatic tools based on simulated data. CReSIL provides many useful features for genomic annotation, which can be used to infer eccDNA function and Circos visualization for eccDNA architecture investigation. We demonstrated CReSIL's capability in several long-read sequencing datasets, including datasets enriched for eccDNA and whole genome datasets from cells containing large eccDNA products. In conclusion, the CReSIL suite software is a versatile tool for investigating complex and simple eccDNA in eukaryotic cells.

Genome-associations of extended-spectrum ß-lactamase producing (ESBL) or AmpC producing E. coli in small and medium pig farms from Khon Kaen province, Thailand.

Thailand is undergoing rapid intensification of livestock production where small subsistence farms and medium sized commercial farms coexist. In medium farms, antimicrobials are prescribed by a veterinarian, whereas in small farms antimicrobial use remains largely unsupervised. The impact of these differences as well as other farming practices on the emergence and composition of antimicrobial resistance genes (ARGs) remains largely unknown. We analyzed 363 genomes of extended-spectrum ß-lactamase producing (ESBL) and/or AmpC producing Escherichia coli recovered from humans and pigs at small and medium farms from the Khon Kaen province, Thailand. We tested for genome-wide associations to identify links between ARGs, host, and farm size. Pig isolates from small farms were associated with mcr and qnr genes conferring resistance to colistin and fluoroquinolones, respectively. In contrast, pig isolates from medium farms were associated with ARGs conferring resistance to drugs commonly used on medium farms (i.e., streptomycin). ESBL plasmids from small farms co-carried ARGs conferring resistance to critically important antimicrobials more frequently compared to plasmid from medium farms. Frequent ARG combinations included blaCTX-M-55 + qnrS1 (29.8% vs 17.5% in small and medium farms, respectively), blaCTX-M-55 + qnrS1 + mcr-3.19 (5% vs 0%), blaCTX-M-14 + qnrS1 (9.3% vs 6.2%), and blaCTX-M-14 + qnrS1 + mcr-1.1 (3.1% vs 0%). The co-location on plasmids of ARGs conferring resistance to critically important antimicrobials as defined by the World Health Organization is concerning, and actions to curb their spread are urgently needed. Legislation on limiting antimicrobial sales and initiatives to better inform farmers and veterinarians on appropriate antimicrobial usage and farm biosecurity could help reduce antimicrobial use on farms.

Exploring Klebsiella pneumoniae in Healthy Poultry Reveals High Genetic Diversity, Good Biofilm-Forming Abilities and Higher Prevalence in Turkeys Than Broilers.

Klebsiella pneumoniae is a well-studied human pathogen for which antimicrobial resistant and hypervirulent clones have emerged globally. K. pneumoniae is also present in a variety of environmental niches, but currently there is a lack of knowledge on the occurrence and characteristics of K. pneumoniae from non-human sources. Certain environmental niches, e.g., animals, may be associated with high K. pneumoniae abundance, and these can constitute a reservoir for further transmission of strains and genetic elements. The aim of this study was to explore and characterize K. pneumoniae from healthy broilers and turkeys. A total of 511 cecal samples (broiler n = 356, turkey n = 155), included in the Norwegian monitoring program for antimicrobial resistance (AMR) in the veterinary sector (NORM-VET) in 2018, were screened for K. pneumoniae by culturing on SCAI agar. K. pneumoniae was detected in 207 (40.5%) samples. Among the broiler samples, 25.8% were positive for K. pneumoniae, in contrast to turkey with 74.2% positive samples (p < 0.01). Antibiotic susceptibility testing was performed, in addition to investigating biofilm production. Whole genome sequencing was performed on 203 K. pneumoniae isolates, and analysis was performed utilizing comparative genomics tools. The genomes grouped into 66 sequence types (STs), with ST35, ST4710 and ST37 being the most prevalent at 13.8%, 7.4%, and 5.4%, respectively. The overall AMR occurrence was low, with only 11.3% of the isolates showing both pheno- and genotypic resistance. Genes encoding aerobactin, salmochelin or yersiniabactin were detected in 47 (23.2%) genomes. Fifteen hypervirulent genomes belonging to ST4710 and isolated from turkey were identified. These all encoded the siderophore virulence loci iuc5 and iro5 on an IncF plasmid. Isolates from both poultry species displayed good biofilm-forming abilities with an average of OD595 0.69 and 0.64. To conclude, the occurrence of K. pneumoniae in turkey was significantly higher than in broiler, indicating that turkey might be an important zoonotic reservoir for K. pneumoniae compared to broilers. Furthermore, our results show a highly diverse K. pneumoniae population in poultry, low levels of antimicrobial resistance, good biofilm-forming abilities and a novel hypervirulent ST4710 clone circulating in the turkey population.

Corrigendum: bla CTX-M-1/IncI1-Iγ Plasmids Circulating in Escherichia coli From Norwegian Broiler Production Are Related, but Distinguishable.

[This corrects the article DOI: 10.3389/fmicb.2020.00333.].

Geographic Drivers of Antimicrobial Use and Resistance in Pigs in Khon Kaen Province, Thailand.

In Thailand, pig production has increased considerably in the last decades to meet a growing demand for pork. Antimicrobials are used routinely in intensive pig production to treat infections and increase productivity. However, the use of antimicrobials also contributes to the rise of antimicrobial resistance with potential consequences for animal and human health. Here, we quantify the association between antimicrobial use and resistance rates in extensive and intensive farms with a focus on geographic proximity between farm and drugstores. Of the 164 enrolled farms, 79% reported using antimicrobials for disease prevention, treatment, or as a feed additive. Antimicrobial-resistant E. coli were present in 63% of farms. These drugs included critically important antimicrobials, such as quinolones and penicillins. Medium-scale farms with intensive animal production practices showed higher resistance rates than small-scale farms with extensive practices. Farms with drug-resistant Escherichia coli were located closer to drugstores and a had a higher proportion of disease than farms without drug-resistant E. coli. We found no association between the presence of resistance in humans and antimicrobial use in pigs. Our findings call for actions to improve herd health to reduce the need for antimicrobials and systematic training of veterinarians and drugstore owners on judicious use of antimicrobials in animals to mitigate resistance.

Exploring the Antibiotic Resistance Burden in Livestock, Livestock Handlers and Their Non-Livestock Handling Contacts: A One Health Perspective.

Antibiotics are freqeuently used in the livestock sector in low- and middle-income countries for treatment, prophylaxis, and growth promotion. However, there is limited information into the zoonotic prevalence and dissemination patterns of antimicrobial resistance (AMR) within these environments. In this study we used pig farming in Thailand as a model to explore AMR; 156 pig farms were included, comprising of small-sized (<50 sows) and medium-sized (≥100 sows) farms, where bacterial isolates were selectively cultured from animal rectal and human fecal samples. Bacterial isolates were subjected to antimicrobial susceptibility testing (AST), and whole-genome sequencing. Our results indicate extensive zoonotic sharing of antibiotic resistance genes (ARGs) by horizontal gene transfer. Resistance to multiple antibiotics was observed with higher prevalence in medium-scale farms. Zoonotic transmission of colistin resistance in small-scale farms had a dissemination gradient from pigs to handlers to non-livestock contacts. We highly recommend reducing the antimicrobial use in animals' feeds and medications, especially the last resort drug colistin.

Characterization of Klebsiella pneumoniae complex isolates from pigs and humans in farms in Thailand: population genomic structure, antibiotic resistance and virulence genes.

OBJECTIVES: To define characteristics of Klebsiella pneumoniae complex (hereafter KP) isolates from healthy pigs, farm workers and their household members in Thailand. METHODS: A total of 839 individual rectal swabs from pigs on 164 farms and 271 faecal samples of humans working on pig farms and persons living in the same household in Khon Kaen, Thailand were screened for gut colonization by KP. Genomic sequences were investigated for antibiotic resistance and virulence genes. Phylogenetic analyses were performed in addition to comparison with isolates from previous studies from Thailand. RESULTS: KP was detected in approximately 50% of pig and human samples. In total, 253 KP isolates were obtained: 39% from pigs, 34% from farmers and 26% from individuals living on the same farm but without animal contact. MLST revealed high genetic diversity with 196 different STs distributed over four phylogroups (Kp1 to Kp4). Low prevalence of ESBL-KP (7.5%) and colistin-resistant KP (3.2%) was observed among pigs and humans. Remarkably, four convergent MDR and hypervirulent strains were observed: one from pigs (ST290) and three from humans [ST35, ST3415 (strain 90CP1), ST17 (strain 90CM2)]. Sharing of KP clones among pigs and humans was identified for some STs including ST4788, ST661, ST3541 and ST29. CONCLUSIONS: The study indicated a low prevalence of ESBL and mcr genes among KP isolated from pigs and healthy humans in Thailand and suggested the possibility of zoonotic transmission for a subset of circulating KP clones.

Corrigendum: bla CTX-M-1 /IncI1-Iγ Plasmids Circulating in Escherichia coli From Norwegian Broiler Production Are Related, but Distinguishable.

[This corrects the article DOI: 10.3389/fmicb.2020.00333.].

Comparative genomics of two Shewanella xiamenensis strains isolated from a pilgrim before and during travels to the Hajj.

BACKGROUND: Shewanella xiamenensis has been reported in water environment and in patients and can act as the originator of oxacillinase in gram-negative bacteria. In order to assess genome plasticity and its functional properties related diarrhea symptoms in pilgrim, comparisons of draft genome sequences of the two isolates were conducted with other closely related genomes. RESULTS: We isolated S. xiamenensis 111B and 111D strains from a pilgrim before travels to the Hajj and during travels with diarrhea symptom, respectively. Whole-genome sequencing showed that draft genome size of 111B strain was 5,008,191 bp, containing 49 kb of a putative plasmid. The genome size of 111D was 4,964,295 bp containing 225 kb of a putative plasmid that shared the backbone sequences with the hospital wastewater strain T17. Comparatively, two Hajj strains are identical at 97.3% identity and 98.7% coverage. They are closely related to river water strain, AS58 by SNPs analysis. Notably, a novel blaOXA-48 allele blaOXA-547 was identified in 111D, sharing 99.5% identity with blaOXA-546 and blaOXA-894. Multiple copies of virulence specific genes, such as capsular polysaccharide biosynthesis, O-antigen and lasB (vibriolysin related gene) have been identified specifically in 111D, but absent in 111B strain. CONCLUSIONS: The whole genome sequences of S. xiamenensis strain 111B and 111D, including comparative genomic analysis, highlight here the potential for virulence factors that might be related to the cause of diarrhea in humans and also indicate the possible acquisition of pathogenic bacteria, including antibiotic resistance genes or plasmids during the Hajj.

Antimicrobial Resistance in Fecal Escherichia coli from Humans and Pigs at Farms at Different Levels of Intensification.

The overall aim of the current study was to test the hypotheses that (i) antibiotic resistance in bacteria were more frequent in clinically health pigs in intensified company owned, medium-scale farms (MSFs) (100-500 sows) than in pigs in family-owned, small-scale farms (SSFs) (1-50 sows) and (ii) that farmers working at the MSFs were more prone to attain antibiotic resistant bacteria than farmers working at SSFs. The study was conducted in North-Eastern Thailand, comprising fecal Escherichia coli isolates from pigs, farmers working with the pigs (contact humans) and persons living in the same household as the farmer (non-contact humans) at 51 MSFs and 113 SSFs. Samples from all farms were also screened for methicillin-resistant staphylococcus aureus (MRSA), which was not detected in pig samples, but was found in one human sample. Susceptibility was tested by disc-diffusion for seven antibiotics commonly used in the study area. Resistance in pig isolates from MSFs were more frequent for chloramphenicol which (P < 0.001), trimethoprim/sulfamethoxazole (P < 0.001) and gentamicin (P < 0.05) compared with isolates from SSFs, whereas the opposite was true for tetracycline (P < 0.01). Resistance in the human isolates was lower than those in the isolates from pigs for tetracycline, trimethoprim/sulfamethoxazole and chloramphenicol (P < 0.001). The frequency of resistance in the contact human samples from SSFs and MSFs did not differ. There was no difference between isolates from contact and non-contact humans for any of the tested antibiotics. Multidrug resistance in isolates from pigs was 26%, significantly higher (P < 0.01) than the 13% from humans. The data indicate that (i) resistance to antibiotics, including those critical and highly important for human medicine, were more common in fecal E. coli from pigs at the MSFs than at the SSFs, whereas (ii) the resistance in fecal E. coli from pig farmers seemed not to be influenced by the level of intensification of the farm they were working at.

blaCTX-M-1/IncI1-Iγ Plasmids Circulating in Escherichia coli From Norwegian Broiler Production Are Related, but Distinguishable.

Escherichia coli carrying blaCTX-M-1 mediating resistance to extended-spectrum cephalosporins was recently described as a new genotype in Norwegian broiler production. The aim of this study was to characterize these isolates (n = 31) in order to determine whether the emergence of the genotype was caused by clonal expansion or horizontal dissemination of blaCTX-M-1-carrying plasmids. All included isolates were subjected to whole genome sequencing. Plasmid transferability was determined by conjugation, and plasmid replicons in the transconjugants were described using PCR-based replicon typing. Plasmid sizes were determined using S1 nuclease digestion. Plasmids in a subset of strains were reconstructed and compared to plasmids from broiler production in other European countries. The isolates belonged to nine different sequence types (STs), with the largest group being ST57 (n = 12). The vast majority of blaCTX-M-1-carrying plasmids were conjugative. All transconjugants were positive for the IncI1-Iγ replicon, and several also harbored the IncFIB replicon. Highly similar plasmids were present in different E. coli STs. Additionally, high similarity to previously published plasmids was detected. A reconstructed plasmid from an ST57 isolate harbored both IncI1-Iγ and IncFIB replicons and was considered to be co-integrated. The presence of one large plasmid was confirmed by S1 nuclease digestion. Our results show that dissemination of blaCTX-M-1 in Norwegian broiler production is due to both clonal expansion and horizontal transfer of plasmids carrying blaCTX-M-1. The blaCTX-M-1/IncI1-Iγ plasmids grouped into two main lineages, namely clonal complex (CC)-3 and CC-7. The genetic diversity at both strain and plasmid level indicates multiple introductions to Norway. We also show that the blaCTX-M-1 plasmids circulating in Norwegian broiler production are highly similar to plasmids previously described in other countries.

Investigation of urban birds as source of ß-lactamase-producing Gram-negative bacteria in Marseille city, France.

BACKGROUND: We investigate here the presence of multidrug-resistant bacteria isolated from stool samples of yellow-legged gulls and chickens (n = 136) in urban parks and beaches of Marseille, France. Bacterial isolation was performed on selective media, including MacConkey agar with ceftriaxone and LBJMR medium. Antibiotic resistance genes, including extended-spectrum ß-lactamases (ESBL) (i.e. blaCTX-M, blaTEM and blaSHV), carbapenemases (blaKPC, blaVIM, blaNDM, blaOXA-23, blaOXA-24, blaOXA-48 and blaOXA-58) and colistin resistance genes (mcr-1 to mcr-5) were screened by real-time PCR and standard PCR and sequenced when found. RESULTS: Of the 136 stools samples collected, seven ESBL-producing Gram-negative bacteria (BGN) and 12 colistin-resistant Enterobacteriaceae were isolated. Among them, five ESBL-producing Escherichia coli and eight colistin-resistant Hafnia alvei strains were identified. Four blaTEM-1 genes were detected in yellow-legged gulls and chickens. Three CTX-M-15 genes were detected in yellow-legged gulls and pigeons, and one CTX-M-1 in a yellow-legged gull. No mcr-1 to mcr-5 gene were detected in colistin-resistant isolates. Genotyping of E. coli strains revealed four different sequence types already described in humans and animals and one new sequence type. CONCLUSIONS: Urban birds, which are believed to have no contact with antibiotics appear as potential source of ESBL genes. Our findings highlight the important role of urban birds in the proliferation of multidrug-resistant bacteria and also the possible zoonotic transmission of such bacteria from wild birds to humans.

Characterisation of blaOXA-538, a new variant of blaOXA-48, in Shewanella xiamenensis isolated from river water in Algeria.

OBJECTIVES: In this study, the presence of carbapenemase genes in Shewanella xiamenensis strains isolated from river water in Béjaïa, Algeria, was investigated. METHODS: Four isolates of S. xiamenensis were isolated from water from Soummam River. The isolates were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) and sequencing of the 16S rRNA and gyrB genes. Isolates were subjected to antimicrobial susceptibility testing. Carbapenemase production was screened using phenotypic tests. PCR and sequencing were used to identify carbapenemase genes in the isolates. The genetic context of the blaOXA-48-like gene was investigated by sequencing the whole genome of strain AS58. RESULTS: All four S. xiamenensis strains harboured blaOXA-48-like genes. They exhibited different resistance patterns and had imipenem minimum inhibitory concentrations (MICs) of ≥0.5mg/L. Sequencing of blaOXA-48-like genes from the S. xiamenensis isolates showed that two strains harboured blaOXA-181, one strain harboured blaOXA-199 and one strain exhibited a new variant of the blaOXA-48-like gene, named blaOXA-538. This new variant shared 98% nucleotide identity with blaOXA-162, with three amino acid changes (G201A, A213G and I219F). Conjugation assays with Escherichia coli J53 recipient were performed but no transconjugants were obtained. Analysis of the genome of AS58 Touati strain confirmed the chromosomal location of the blaOXA-538 gene. CONCLUSION: This study showed that environmental water holds a diversity of S. xiamenensis strains harbouring blaOXA-48-like genes and may play an important role in the dissemination and spread of these genes from the environment to humans.

LBJMR medium: a new polyvalent culture medium for isolating and selecting vancomycin and colistin-resistant bacteria.

BACKGROUND: Multi-drug resistant bacteria are a phenomenon which is on the increase around the world, particularly with the emergence of colistin-resistant Enterobacteriaceae and vancomycin-resistant enterococci strains. The recent discovery of a plasmid-mediated colistin resistance with the description of the transferable mcr-1 gene raised concerns about the need for an efficient detection method for these pathogens, to isolate infected patients as early as possible. The LBJMR medium was developed to screen for all polymyxin-resistant Gram-negative bacteria, including mcr-1 positive isolates, and vancomycin-resistant Gram-positive bacteria. RESULTS: The LBJMR medium was developed by adding colistin sulfate salt at a low concentration (4 µg/mL) and vancomycin (50 µg/mL), with glucose (7.5 g/L) as a fermentative substrate, to a Purple Agar Base (31 g/L). A total of 143 bacterial strains were used to evaluate this universal culture medium, and the sensitivity and specificity of detection were 100% for the growth of resistant strains. 68 stool samples were cultured on LBJMR, and both colistin-resistant Gram-negative and vancomycin-resistant Gram-positive strains were specifically detected. CONCLUSIONS: The LBJMR medium is a multipurpose selective medium which makes it possible to identify bacteria of interest from clinical samples and to isolate contaminated patients in hospital settings. This is a simple medium that could be easily used for screening in clinical microbiology laboratories.

Emergence of drug resistant bacteria at the Hajj: A systematic review.

BACKGROUND: Hajj is the annual mass gathering of Muslims, and is a reservoir and potential source of bacterial transmission. The emergence of bacterial transmission, including multi-drug resistance (MDR) bacteria, during Hajj has not been systematically assessed. METHODS: Articles in Pubmed, Scopus, and Google scholar were identified using controlled words relating to antibiotic resistance (AR) at the Hajj from January 2002 to January 2017. Eligible studies were identified by two researchers. AR patterns of bacteria were obtained for each study. RESULTS: We included 31 publications involving pilgrims, Hajj workers or local patients attending hospitals in Mecca, Mina, and the Medina area. Most of these publications provided antibiotic susceptibility results. Ten of them used the PCR approach to identify AR genes. MRSA carriage was reported in pilgrims and food handlers at a rate of 20%. Low rates of vancomycin-resistant gram-positive bacteria were reported in pilgrims and patients. The prevalence of third-generation cephalosporin-resistant bacteria was common in the Hajj region. Across all studies, carbapenem-resistant bacteria were detected in fewer than 10% of E.coli isolates tested but up to 100% in K. pneumoniae and A. baumannii. Colistin-resistant Salmonella enterica, including mcr-1 colistin-resistant E.coli and K.pneumoniae were only detected in the pilgrim cohorts. CONCLUSION: This study provides an overview of the prevalence of MDR bacteria at the Hajj. Pilgrims are at high risk of AR bacterial transmission and may carry and transfer these bacteria when returning to their home countries. Thus, pilgrims should be instructed by health care practitioners about hygiene practices aiming at reducing traveler's diarrhea and limited use of antibiotics during travel in order to reduce the risk of MDR bacterial transmission.