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Abstract Background Antimicrobial resistance has been identified as a major threat to global health. The pig food chain is considered an important source of antimicrobial resistance genes (ARGs). However, there is still a lack of knowledge on the dispersion of ARGs in pig production system, including the external environment. Results In the present study, we longitudinally followed one swine farm located in Italy from the weaning phase to the slaughterhouse to comprehensively assess the diversity of ARGs, their diffusion, and the bacteria associated with them. We obtained shotgun metagenomic sequences from 294 samples, including pig feces, farm environment, soil around the farm, wastewater, and slaughterhouse environment. We identified a total of 530 species-level genome bins (SGBs), which allowed us to assess the dispersion of microorganisms and their associated ARGs in the farm system. We identified 309 SGBs being shared between the animals gut microbiome, the internal and external farm environments. Specifically, these SGBs were characterized by a diverse and complex resistome, with ARGs active against 18 different classes of antibiotic compounds, well matching antibiotic use in the pig food chain in Europe. Conclusions Collectively, our results highlight the urgency to implement more effective countermeasures to limit the dispersion of ARGs in the pig food systems and the relevance of metagenomics-based approaches to monitor the spread of ARGs for the safety of the farm working environment and the surrounding ecosystems.

ABSTRACTAntimicrobial resistance (AMR) poses an escalating global public health threat. Canals are essential in Thailand, including the capital city, Bangkok, as agricultural and daily water sources. However, the characteristic and antimicrobial-resistance properties of the bacteria in the urban canals have never been elucidated. This study employed whole genome sequencing to characterize 30 genomes of a causal pathogenic bacteria, Salmonella enterica, isolated from Bangkok canal water between 2016 and 2020. The dominant serotype was Salmonella Agona. In total, 35 AMR genes and 30 chromosomal-mediated gene mutations were identified, in which 21 strains carried both acquired genes and mutations associated with fluoroquinolone resistance. Virulence factors associated with invasion, adhesion, and survival during infection were detected in all study strains. 75.9% of the study stains were multidrug-resistant and all the strains harbored the necessary virulence factors associated with salmonellosis. One strain carried 20 resistance genes, including mcr-3.1, mutations in GyrA, ParC, and ParE, and typhoid toxin-associated genes. Fifteen plasmid replicon types were detected, with Col(pHAD28) being the most common type. Comparative analysis of nine S. Agona from Bangkok and 167 from public databases revealed that specific clonal lineages of S. Agona might have been circulating between canal water and food sources in Thailand and globally. These findings provide insight into potential pathogens in the aquatic ecosystem and support the inclusion of environmental samples into comprehensive AMR surveillance initiatives as part of a One Health approach. This approach aids in comprehending the rise and dissemination of AMR and devising sustainable intervention strategies.IMPORTANCEBangkok is the capital city of Thailand and home to a large canal network that serves the city in various ways. The presence of pathogenic and antimicrobial-resistant Salmonella is alarming and poses a significant public health risk. The present study is the first characterization of the genomic of Salmonella strains from Bangkok canal water. Twenty-two of 29 strains (75.9%) were multidrug-resistant Salmonella and all the strains carried essential virulence factors for pathogenesis. Various plasmid types were identified in these strains, potentially facilitating the horizontal transfer of AMR genes. Additional investigations indicated a potential circulation of S. Agona between canal water and food sources in Thailand. The current study underscores the role of environmental water in an urban city as a reservoir of pathogens and these data obtained can serve as a basis for public health risk assessment and help shape intervention strategies to combat AMR challenges in Thailand.

Abstract Background The advances and decreasing economical cost of whole genome sequencing (WGS), will soon make this technology available for routine infectious disease epidemiology. In epidemiological studies, outbreak isolates have very little diversity and require extensive genomic analysis to differentiate and classify isolates. One of the successfully and broadly used methods is analysis of single nucletide polymorphisms (SNPs). Currently, there are different tools and methods to identify SNPs including various options and cut-off values. Furthermore, all current methods require bioinformatic skills. Thus, we lack a standard and simple automatic tool to determine SNPs and construct phylogenetic tree from WGS data. Results Here we introduce snpTree, a server for online-automatic SNPs analysis. This tool is composed of different SNPs analysis suites, perl and python scripts. snpTree can identify SNPs and construct phylogenetic trees from WGS as well as from assembled genomes or contigs. WGS data in fastq format are aligned to reference genomes by BWA while contigs in fasta format are processed by Nucmer. SNPs are concatenated based on position on reference genome and a tree is constructed from concatenated SNPs using FastTree and a perl script. The online server was implemented by HTML, Java and python script. The server was evaluated using four published bacterial WGS data sets (V. cholerae, S. aureus CC398, S. Typhimurium and M. tuberculosis). The evalution results for the first three cases was consistent and concordant for both raw reads and assembled genomes. In the latter case the original publication involved extensive filtering of SNPs, which could not be repeated using snpTree. Conclusions The snpTree server is an easy to use option for rapid standardised and automatic SNP analysis in epidemiological studies also for users with limited bioinformatic experience. The web server is freely accessible at http://www.cbs.dtu.dk/services/snpTree-1.0/.

Abstract Background Certain clonal complexes (CCs) of Klebsiella pneumoniae such as CC147 (ST147 and ST392) are major drivers of bla NDM dissemination across the world. ST147 has repeatedly reported from our geographical region, but its population dynamics and evolutionary trajectories need to be further studied. Methods Comparative genomic analysis of 51 carbapenem-nonsusceptible strains as well as three hypervirulent K. pneumoniae (hvKp) recovered during 16-months of surveillance was performed using various bioinformatics tools. We investigated the genetic proximity of our ST147 strains with publicly available corresponding genomes deposited globally and from neighbor countries in our geographic region. Results While IncL/M plasmid harboring bla OXA-48 was distributed among divergent clones, bla NDM-1 was circulated by twenty of the 25 CC147 dominant clone and were mostly recovered from the ICU. The NDM-1 core structure was bracketed by a single isoform of mobile genetic elements (MGEs) [ΔISKpn26-NDM-TnAs3-ΔIS3000-Tn5403] and was located on Col440I plasmid in 68.7% of ST392. However, various arrangements of MGEs including MITESen1/MITESen1 composite transposon or combination of MITESen1/ISSen4/IS903B/IS5/ISEhe3 on IncFIb (pB171) were identified in ST147. It seems that ST392 circulated bla NDM-1 in 2018 before being gradually replaced by ST147 from the middle to the end of sample collection in 2019. ST147 strains possessed the highest number of resistance markers and showed high genetic similarity with four public genomes that harbored bla NDM-1 on the same replicon type. Mainly, there was a convergence between clusters and isolated neighboring countries in the minimum-spanning tree. A conserved arrangement of resistance markers/MGEs was linked to methyltransferase armA which was embedded in class 1 integron in 8 isolates of ST147/ST48 high-risk clones. Conclusion Our findings highlight the dynamic nature of bla NDM-1 transmission among K. pneumoniae in Iran that occurs both clonally and horizontally via various combinations of MGEs. This is the first analysis of Iranian ST147/NDM + clone in the global context.

Abstract Background Technological advances in high throughput genome sequencing are making whole genome sequencing (WGS) available as a routine tool for bacterial typing. Standardized procedures for identification of relevant genes and of variation are needed to enable comparison between studies and over time. The core genes--the genes that are conserved in all (or most) members of a genus or species--are potentially good candidates for investigating genomic variation in phylogeny and epidemiology. Results We identify a set of 2,882 core genes clusters based on 73 publicly available Salmonella enterica genomes and evaluate their value as typing targets, comparing whole genome typing and traditional methods such as 16S and MLST. A consensus tree based on variation of core genes gives much better resolution than 16S and MLST; the pan-genome family tree is similar to the consensus tree, but with higher confidence. The core genes can be divided into two categories: a few highly variable genes and a larger set of conserved core genes, with low variance. For the most variable core genes, the variance in amino acid sequences is higher than for the corresponding nucleotide sequences, suggesting that there is a positive selection towards mutations leading to amino acid changes. Conclusions Genomic variation within the core genome is useful for investigating molecular evolution and providing candidate genes for bacterial genome typing. Identification of genes with different degrees of variation is important especially in trend analysis.

Abstract Background Many species of marine shrimp in the Family Penaeidae, viz. Penaeus (Litopenaeus) vannamei, Penaeus monodon, Penaeus (Fenneropenaeus) chinensis, and Penaeus (Marsupenaeus) japonicus, are animals of economic importance in the aquaculture industry. Yet information about their DNA and protein sequences is lacking. In order to predict their collective proteome, we combined over 270,000 available EST and cDNA sequences from the 4 shrimp species with all protein sequences of Drosophila melanogaster and Caenorhabditis elegans. EST data from 4 other crustaceans, the crab Carcinus maenas, the lobster Homarus americanus (Decapoda), the water flea Daphnia pulex, and the brine shrimp Artemia franciscana were also used. Findings Similarity searches from EST collections of the 4 shrimp species matched 64% of the protein sequences of the fruit fly, but only 45% of nematode proteins, indicating that the shrimp proteome content is more similar to that of an insect than a nematode. Combined results with 4 additional non-shrimp crustaceans increased matching to 78% of fruit fly and 56% of nematode proteins, suggesting that present shrimp EST collections still lack sequences for many conserved crustacean proteins. Analysis of matching data revealed the presence of 4 EST groups from shrimp, namely sequences for proteins that are both fruit fly-like and nematode-like, fruit fly-like only, nematode-like only, and non-matching. Gene ontology profiles of proteins for the 3 matching EST groups were analyzed. For non-matching ESTs, a small fraction matched protein sequences from other species in the UniProt database, including other crustacean-specific proteins. Conclusions Shrimp ESTs indicated that the shrimp proteome is comprised of sub-populations of proteins similar to those common to both insect and nematode models, those present specifically in either model, or neither. Combining small EST collections from related species to compensate for their small size allowed prediction of conserved expressed protein components encoded by their uncharacterized genomes. The organized data should be useful for transferring annotation data from model species into shrimp data and for further studies on shrimp proteins with particular functions or groups.

Abstract The market for the application of probiotics as a livestock health improvement supplement has increased in recent years. However, most of the available products are quality-controlled using low-resolution techniques and un-curated databases, resulting in misidentification and incorrect product labels. In this work, we deployed two workflows and compared results obtained by full-length 16S rRNA genes (16S) and metagenomic (Meta) data to investigate their reliability for the microbial composition of both liquid and solid forms of animal probiotic products using Oxford Nanopore long-read-only (without short-read). Our result revealed that 16S amplicon data permits to detect the bacterial microbiota even with the low abundance in the samples. Moreover, the 16S approach has the potential to provide species-level resolution for prokaryotes but not for assessing yeast communities. Whereas, Meta data has more power to recover of high-quality metagenome-assembled genomes that enables detailed exploration of both bacterial and yeast populations, as well as antimicrobial resistance genes, and functional genes in the population. Our findings clearly demonstrate that implementing these workflows with long-read-only monitoring could be applied to assessing the quality and safety of probiotic products for animals and evaluating the quality of probiotic products on the market. This would benefit the sustained growth of the livestock probiotic industry.

ABSTRACT Listeria monocytogenes is a Gram-positive, facultative intracellular foodborne pathogen capable of causing severe, invasive illness (listeriosis). Three serotypes, 1/2a, 1/2b, and 4b, are leading contributors to human listeriosis, with 4b including the major hypervirulent clones. The multiplex PCR scheme developed by Doumith and collaborators employs primers targeting specific lineages (e.g., lineage II-specific lmo0737, lineage I-specific LMOf2365_2059) or serotypes (e.g., serotype 4b-specific LMOf2365_1900). The Doumith scheme (DS) is extensively employed for molecular serotyping of L. monocytogenes due to its high accuracy, relative ease, and affordability. However, for certain strains, the DS serotype designations are in conflict with those relying on antibody-based schemes or whole-genome sequence (WGS) analysis. In the current study, all 27 tested serotype 4b strains with sequence type 782 (ST782) within the hypervirulent clonal complex 2 (CC2) were designated 1/2b/3b using the DS. These strains lacked the serotype 4b-specific gene LMOf2365_1900, while retaining LMOf2365_2059, which, together with prs, yields the DS 1/2b/3b profile. Furthermore, 15 serotype 1/2a strains of four STs, mostly from water, were designated 1/2b/3b using the DS. These strains lacked the lmo0737 cassette but harbored genomic islands with LMOf2365_2059, thus yielding the DS 1/2b/3b profile. Lastly, we investigated a novel, dual 1/2a-1/2b profile obtained using the DS with 21 serotype 1/2a strains of four STs harboring both the lmo0737 cassette and genomic islands with LMOf2365_2059. The findings suggest that for certain strains and clones of L. monocytogenes the DS designations should be viewed with caution and complemented with alternative tools, e.g., traditional serotyping or WGS analysis. IMPORTANCE Listeria monocytogenes is a foodborne pathogen responsible for severe illness (listeriosis), especially in pregnant women and their fetuses, immunocompromised individuals, and the elderly. Three serotypes, 1/2a, 1/2b, and 4b, account for most human listeriosis, with certain serotype 4b clonal complexes (CCs) overrepresented in human disease. Serotyping remains extensively employed in Listeria epidemiologic investigations, and a multiplex PCR-based serotyping scheme is widely used. However, the PCR gene targets can be lost or gained via horizontal gene transfer, leading to novel PCR profiles without known serotype designations or to incorrect serotype assignments. Thus, an entire serotype 4b clone of the hypervirulent CC2 would be misidentified as serotype 1/2b, and several strains of serotype 1/2a would be identified as serotype 1/2b. Such challenges are especially common in novel clones from underexplored habitats, e.g., wildlife and surface water. The findings suggest caution in application of molecular serotyping, while highlighting Listeria’s diversity and potential for horizontal gene transfer.

Abstract The emergence of antimicrobial resistance (AMR) is one of the biggest health threats globally. In addition, the use of antimicrobial drugs in humans and livestock is considered an important driver of antimicrobial resistance. The commensal microbiota, and especially the intestinal microbiota, has been shown to have an important role in the emergence of AMR. Mobile genetic elements (MGEs) also play a central role in facilitating the acquisition and spread of AMR genes. We isolated Escherichia coli (n = 627) from fecal samples in respectively 25 poultry, 28 swine, and 15 veal calf herds from 6 European countries to investigate the phylogeny of E. coli at country, animal host and farm levels. Furthermore, we examine the evolution of AMR in E. coli genomes including an association with virulence genes, plasmids and MGEs. We compared the abundance metrics retrieved from metagenomic sequencing and whole genome sequenced of E. coli isolates from the same fecal samples and farms. The E. coli isolates in this study indicated no clonality or clustering based on country of origin and genetic markers; AMR, and MGEs. Nonetheless, mobile genetic elements play a role in the acquisition of AMR and virulence genes. Additionally, an abundance of AMR was agreeable between metagenomic and whole genome sequencing analysis for several AMR classes in poultry fecal samples suggesting that metagenomics could be used as an indicator for surveillance of AMR in E. coli isolates and vice versa.

Staphylococcus aureus is a common cause of infection in humans and animals, including bovine mastitis, globally. The objective of this study was to genetically characterize a collection of S. aureus isolates recovered from milk and nasal swabs from humans with and without animal contact (bovine = 43, human = 12). Using whole genome sequencing (NextSeq550), isolates were sequence typed, screened for antimicrobial resistance and virulence genes and examined for possible inter-species host transmission. Multi locus sequence typing (MLST) and single nucleotide polymorphism (SNP)-based phylogeny revealed 14 different sequence types, including the following six novel sequence types: ST7840, 7841, 7845, 7846, 7847, and 7848. The SNP tree confirmed that MLST clustering occurred most commonly within CC97, CC5477, and CC152. ResFinder analysis revealed five common antibiotic resistance genes, namely tet(K), blaZ, dfrG, erm©, and str, encoding for different antibiotics. mecA was discovered in one human isolate only. Multidrug resistance was observed in 25% of the isolates, predominantly in CC152 (7/8) and CC121 (3/4). Known bovine S. aureus (CC97) were collected in humans and known human S. aureus lineages (CC152) were collected in cattle; additionally, when these were compared to bovine-isolated CC97 and human-isolated CC152, respectively, no genetic distinction could be observed. This is suggestive of inter-host transmission and supports the need for surveillance of the human–animal interface.

Abstract Background A novel Brucella strain closely related to Brucella (B.) melitensis biovar (bv) 3 was found in Croatian cattle during testing within a brucellosis eradication programme. Case presentation Standardised serological, brucellin skin test, bacteriological and molecular diagnostic screening for Brucella infection led to positive detection in one dairy cattle herd. Three isolates from that herd were identified to species level using the Bruce ladder method. Initially, two strains were typed as B. melitensis and one as B. abortus, but multiplex PCR based on IS711 and the Suis ladder showed that all of them to belong to B. melitensis, and the combination of whole-genome and multi-locus sequencing as well as Multi-Locus Variable numbers of tandem repeats Analysis (MLVA) highlighted a strong proximity within the phylogenetic branch of B. melitensis strains previously isolated from Croatia, Albania, Kosovo and Bosnia and Herzegovina. Two isolates were determined to be B. melitensis bv. 3, while the third showed a unique phylogenetic profile, growth profile on dyes and bacteriophage typing results. This isolate contained the 609-bp omp31 sequence, but not the 723-bp omp31 sequence present in the two isolates of B. melitensis bv. 3. Conclusions Identification of a novel Brucella variant in this geographic region is predictable given the historic endemicity of brucellosis. The emergence of a new variant may reflect a combination of high prevalence among domestic ruminants and humans as well as weak eradication strategies. The zoonotic potential, reservoirs and transmission pathways of this and other Brucella variants should be explored.

Abstract Bacteriophages, or phages, are ubiquitous bacterial and archaeal viruses with an estimated total global population of 1031. It is well-known that wherever there are bacteria, their phage counterparts will be found, aiding in shaping the bacterial population. The present study used metagenomic data from global influent sewage in 79 cities in 60 countries to identify phages associated with bacteria and to explore their potential role in antimicrobial resistance gene (ARG) dissemination. The reads were mapped to known databases for bacteriophages and their abundances determined and correlated to geographic origin and the countries socio-economic status, as well as the abundances of bacterial species and ARG. We found that some phages were not equally distributed on a global scale, but their distribution was rather dictated by region and the socioeconomic status of the specific countries. This study provides a preliminary insight into the global and regional distribution of phages and their potential impact on the transmission of ARGs between bacteria. Moreover, the findings may indicate that phages in sewage could have adopted a lytic lifestyle, meaning that most may not be associated with bacteria and instead may be widely distributed as free-living phages, which are known to persist longer in the environment than their hosts. In addition, a significant correlation between phages and ARGs was obtained, indicating that phages may play a role in ARG dissemination. However, further analyses are needed to establish the true relationship between phages and ARGs due to a low abundance of the phages identified.

Listeria monocytogenes is a Gram-positive pathogen responsible for the severe foodborne disease listeriosis. A chromosomal hotspot between lmo0301 and lmo0305 has been noted to harbor diverse restriction modification (RM) systems. Here, we analyzed 872 L. monocytogenes genomes to better understand the prevalence and types of RM systems in this region, designated the immigration control region (ICR). Type I, II, III and IV RM systems were found in 86.1% of strains inside the ICR and in 22.5% of strains flanking the ICR. ICR content was completely conserved within the same multilocus sequence typing-based sequence type (ST), but the same RM system could be identified in diverse STs. The intra-ST conservation of ICR content suggests that this region may drive the emergence of new STs and promote clone stability. Sau3AI-like, LmoJ2 and LmoJ3 type II RM systems as well as type I EcoKI-like, and type IV AspBHI-like and mcrB-like systems accounted for all RM systems in the ICR. A Sau3AI-like type II RM system with specificity for GATC was harbored in the ICR of many STs, including all strains of the ancient, ubiquitous ST1. The extreme paucity of GATC recognition sites in lytic phages may reflect ancient adaptation of these phages to preempt resistance associated with the widely distributed Sau3AI-like systems. These findings indicate that the ICR has a high propensity for RM systems which are intraclonaly conserved and may impact bacteriophage susceptibility as well as ST emergence and stability.

ObjectivesImplementing whole-genome sequencing (WGS) technologies in clinical microbiology laboratories can increase the amount and quality of information available for healthcare practitioners. In this study, we analysed the applicability of this method and determined the distribution of bacterial species processed in clinical settings in Denmark.MethodsWe performed a point-prevalence study of all bacterial isolates (n = 2,009) processed and reported in the Clinical Microbiology Laboratories in Denmark in one day in January 2018. We compared species identification as performed by classical methods (MALDI-TOF) and by bioinformatics analysis (KmerFinder and rMLST) of WGS (Illumina NextSeq) data. We compared the national point-prevalence of bacterial isolates observed in clinical settings with the research attention given to those same genera in scientific literature.ResultsThe most prevalent bacterium was Escherichia coli isolated from urine (n = 646), followed by Staphylococcus spp. from skin or soft tissues (n = 197). The distribution of bacterial species throughout the country was not homogeneous. We observed concordance of species identification for all methods in 95.7% (n = 1,919) of isolates, furthermore obtaining concordance for 99.7% (n = 1,999) at genus level. The number of scientific publications in the country did not correlate with the number of bacterial isolates of each genera analysed in this study.ConclusionsWGS technologies have the potential to be applied in clinical settings for routine diagnostics purposes. This study also showed that bioinformatics databases should be continuously improved and results from local point-prevalence surveys should not be applied at national levels without previously determining possible regional variations.

Food authentication is a rapidly growing field driven by increasing public awareness of food quality and safety. Foods containing herbs are particularly prone to industrial fraud and adulteration. Several methodologies are currently used to evaluate food authenticity. DNA-based technologies have increased focus, with DNA barcoding the most widely used. DNA barcoding is based on the sequencing and comparison of orthologous DNA regions from all species in a sample, but the approach is limited by its low resolution to distinguish closely-related species. Here we developed a customised database and bioinformatics pipeline (Herbs Authenticity - GitHub) to identify herbal ingredients implemented as a metagenomics approach for plant-derived product authenticity testing. We evaluated the accuracy of the method by using publicly available plant genomes and databases to allow the construction of our customised database barcodes, which were also complemented with entries from publicly available resources (iBOL and ENA). The pipeline performance was then tested with new 47 de novo partly sequenced whole plant genomes or barcodes as query sequences. Our results show that using our mapping algorithm with the customised barcode database correctly identifies the main components of a wide range of plant-derived samples, albeit with variable additional noise across samples depending on the tested samples and barcodes. Our result also show that at the current stage the usefulness of metagenomics is limited by the availability of reference sequences and the needed sequencing depth. However, this method shows promise for evaluating the authenticity of different herbal products provided that the method is further refined to increase the qualitative and quantitative accuracy.

Escherichia coli

Colistin is a last-resort drug for the treatment of infections by carbapenem-resistant Enterobacteriaceae, and the emergence of colistin resistance poses a serious clinical challenge. The aim of this study was to investigate the occurrence of colistin-resistant Escherichia coli in retail meat in Southern Italy in 2018–2020. Of 570 samples, 147 contained E. coli. Two out of 147 (1.4%) E. coli showed a non-wild-type phenotype to colistin and harboured mcr-1. mcr-1 was also detected in a wild-type isolate, resulting in a 2% mcr prevalence. mcr-1-positive isolates originated from turkey meat collected in Apulia (n = 2) and Basilicata (n = 1). A whole-genome sequencing analysis confirmed mcr-1.2 and mcr-1.1 in two and one isolate, respectively. The strains were diverse, belonging to three multi-locus sequence types (ST354, ST410, SLV of ST10) and harbouring genes mediating resistance to antimicrobials in two, six and seven classes. mcr-1 was carried by IncX4 plasmids with high nucleotide similarity to IncX4 plasmids harbouring mcr-1.2 and mcr-1.1 in Enterobacterales from different sources and geographical regions. This is the first study reporting updates on E. coli non-wild-type to colistin from retail meat in Southern Italy, highlighting the importance of phenotypic and genotypic antimicrobial resistance surveillance to contain the dissemination of mcr among E. coli.

Obtaining data on antimicrobial resistance (AMR) from healthy human populations is difficult. Here, Hendriksen et al. use metagenomic analysis to obtain AMR data from untreated sewage from 79 sites in 60 countries, finding correlations with socio-economic, health and environmental factors.

ABSTRACT Antimicrobial resistance (AMR) is a major threat to global health, and it is crucial to understand the epidemiological aspects in order to predict the emergence and propagation of AMR genes. The aim of this study was to assess the variability and medium-term AMR trends within the mostly healthy human population of a single city. We monitored over 36 months (November 2015 to November 2018) the AMR level in the city of Copenhagen, Denmark, by taking bi-weekly sewage samples from the inlets of the three main water treatment plants, extracting the DNA, performing metagenomic sequencing, and read-mapping against a database of known AMR genes. We found that the AMR level was surprisingly stable with no periodic variability and no signs of drift over the measured period. We found, however, that the seemingly random variations at each site correlate in time with each other, suggesting that the variations we see are due to real environmental changes in the occurrence of AMR. IMPORTANCE The Copenhagen sewage resistome is surprisingly stable in time. The implication is that, at least for cities that are comparable to Copenhagen in terms of sewer infrastructure, few or even single samples provide a robust picture of the resistome within a city.

Antimicrobial resistance (AMR) in bacteria is a complex subject, why one need to look at this phenomenon from a wider and holistic perspective. The extensive use of the same antimicrobial classes in human and veterinary medicine as well as horticulture is one of the main drivers for the AMR selection. Here, we applied shotgun metagenomics to investigate the AMR epidemiology in several animal species including farm animals, which are often exposed to antimicrobial treatment opposed to an unique set of wild animals that seems not to be subjected to antimicrobial pressure. The comparison of the domestic and wild animals allowed to investigate the possible anthropogenic impact on AMR spread. Inclusion of animals with different feeding behaviors (carnivores, omnivores) enabled to further assess which AMR genes that thrives within the food chain. We tested fecal samples not only of intensively produced chickens, turkeys, and pigs, but also of wild animals such as wild boars, red foxes, and rodents. A multi-directional approach mapping obtained sequences to several databases provided insight into the occurrence of the different AMR genes. The method applied enabled also analysis of other factors that may influence AMR of intestinal microbiome such as diet. Our findings confirmed higher levels of AMR in farm animals than in wildlife. The results also revealed the potential of wildlife in the AMR dissemination. Particularly in red foxes, we found evidence of several AMR genes conferring resistance to critically important antimicrobials like quinolones and cephalosporins. In contrast, the lowest abundance of AMR was observed in rodents originating from natural environment with presumed limited exposure to antimicrobials. Shotgun metagenomics enabled us to demonstrate that discrepancies between AMR profiles found in the intestinal microbiome of various animals probably resulted from the different antimicrobial exposure, habitats, and behavior of the tested animal species.

BackgroundTanzania is one of seven countries with the highest disease burden caused by cholera in Africa. We studied the evolution of Vibrio cholerae O1 isolated in Tanzania during the past three decades.Methodology/principal findingsGenome-wide analysis was performed to characterize V. cholerae O1 responsible for the Tanzanian 2015-2017 outbreak along with strains causing outbreaks in the country for the past three decades. The genomes were further analyzed in a global context of 590 strains of the seventh cholera pandemic (7PET), as well as environmental isolates from Lake Victoria. All Tanzanian cholera outbreaks were caused by the 7PET lineage. The T5 sub-lineage (ctxB3) dominated outbreaks until 1997, followed by the T10 atypical El Tor (ctxB1) up to 2015, which were replaced by the T13 atypical El Tor of the current third wave (ctxB7) causing most cholera outbreaks until 2017 with T13 being phylogenetically related to strains from East African countries, Yemen and Lake Victoria. The strains were less drug resistant with approximate 10-kb deletions found in the SXT element, which encodes resistance to sulfamethoxazole and trimethoprim. Nucleotide deletions were observed in the CTX prophage of some strains, which warrants further virulence studies. Outbreak strains share 90% of core genes with V. cholerae O1 from Lake Victoria with as low as three SNPs difference and a significantly similar accessory genome, composed of genomic islands namely the CTX prophage, Vibrio Pathogenicity Islands; toxin co-regulated pilus biosynthesis proteins and the SXT-ICE element.Conclusion/significanceCharacterization of V. cholerae O1 from Tanzania reveals genetic diversity of the 7PET lineage composed of T5, T10 and T13 sub-lineages with introductions of new sequence types from neighboring countries. The presence of these sub-lineages in environmental isolates suggests that the African Great Lakes may serve as aquatic reservoirs for survival of V. cholerae O1 favoring continuous human exposure.

Candida albicans and Candida glabrata are opportunistic fungal pathogens with increasing incidence worldwide and higher-than-expected prevalence in Denmark. We whole-genome sequenced yeast isolates collected from Danish Clinical Microbiology Laboratories to obtain an overview of the Candida population in the country. The majority of the 30 C. albicans isolates were found to belong to three globally prevalent clades, and, with one exception, the remaining isolates were also predicted to cluster with samples from other geographical locations. Similarly, most of the eight C. glabrata isolates were predicted to be prevalent subtypes. Antifungal susceptibility testing proved all C. albicans isolates to be susceptible to both azoles and echinocandins. Two C. glabrata isolates presented azole-resistant phenotypes, yet all were susceptible to echinocandins. There is no indication of causality between population structure and resistance phenotypes for either species.

The emergence of plasmid-mediated colistin resistance (mcr genes) threatens the effectiveness of polymyxins, which are last-resort drugs to treat infections by multidrug- and carbapenem-resistant Gram-negative bacteria. Based on the occurrence of colistin resistance the aims of the study were to determine possible resistance mechanisms and then characterize the mcr-positive Escherichia coli. The research used material from the Polish national and EU harmonized antimicrobial resistance (AMR) monitoring programs. A total of 5,878 commensal E. coli from fecal samples of turkeys, chickens, pigs, and cattle collected in 2011–2016 were screened by minimum inhibitory concentration (MIC) determination for the presence of resistance to colistin (R) defined as R > 2 mg/L. Strains with MIC = 2 mg/L isolated in 2014–2016 were also included. A total of 128 isolates were obtained, and most (66.3%) had colistin MIC of 2 mg/L. PCR revealed mcr-1 in 80 (62.5%) isolates recovered from 61 turkeys, 11 broilers, 2 laying hens, 1 pig, and 1 bovine. No other mcr-type genes (including mcr-2 to -5) were detected. Whole-genome sequencing (WGS) of the mcr-1–positive isolates showed high diversity in the multi-locus sequence types (MLST) of E. coli, plasmid replicons, and AMR and virulence genes. Generally mcr-1.1 was detected on the same contig as the IncX4 (76.3%) and IncHI2 (6.3%) replicons. One isolate harbored mcr-1.1 on the chromosome. Various extended-spectrum beta-lactamase (blaSHV–12, blaCTX–M–1, blaCTX–M–15, blaTEM–30, blaTEM–52, and blaTEM–135) and quinolone resistance genes (qnrS1, qnrB19, and chromosomal gyrA, parC, and parE mutations) were present in the mcr-1.1–positive E. coli. A total of 49 sequence types (ST) were identified, ST354, ST359, ST48, and ST617 predominating. One isolate, identified as ST189, belonged to atypical enteropathogenic E. coli. Our findings show that mcr-1.1 has spread widely among production animals in Poland, particularly in turkeys and appears to be transferable mainly by IncX4 and IncHI2 plasmids spread across diverse E. coli lineages. Interestingly, most of these mcr-1–positive E. coli would remain undetected using phenotypic methods with the current epidemiological cut-off value (ECOFF). The appearance and spread of mcr-1 among various animals, but notably in turkeys, might be considered a food chain, and public health hazard.

Colistin resistance by mobilisable mcr genes has been described in bacteria of food-animal origin worldwide, which has raised public health concerns about its potential foodborne transmission to human pathogenic bacteria. Here we provide baseline information on the molecular epidemiology of colistin-resistant, mcr-positive Escherichia coli and Salmonella isolates in food-producing animals in Italy in 2014-2015. A total 678, 861 and 236 indicator E. coli, Extended Spectrum Beta-Lactamase (ESBL)/AmpC-producing E. coli, and Salmonella isolates, respectively, were tested for colistin susceptibility. These isolates were collected according to the EU harmonized antimicrobial resistance monitoring program and are representative of at least 90 and 80% of the Italian poultry (broiler chickens and turkeys) and livestock (pigs and bovines < 12 months) production, respectively. Whole genome sequencing by Illumina technology and bioinformatics (Center for Genomic Epidemiology pipeline) were used to type 42 mcr-positive isolates by PCR. Colistin resistance was mainly observed in the ESBL/AmpC E. coli population, and was present in 25.9, 5.3, 6.5, and 3.9% of such isolates in turkeys, broilers, pigs, and bovines, respectively. Most colistin-resistant isolates (141/161, 87.5%) harbored genes of the mcr-1 group. mcr-1 was also detected in a small proportion of Salmonella isolates (3/146, 2.0%) in turkeys. Additional mcr types were mcr-3 in four ESBL-producing E. coli from bovines, and two mcr-4 in ESBL (n = 1) and indicator E. coli (n = 1) from pigs and bovines. We describe notable diversity of mcr variants with predominance of mcr-1.1 and mcr-1.2 on conjugative IncX4 plasmids in E. coli and in Salmonella serovars Typhimurium, Newport, Blockley from turkey. A new variant, mcr-1.13 was detected in the chromosome in E. coli in turkey and pig isolates. Additionally, we describe mcr-3.2 and mcr-4.3 in E. coli from bovines, and mcr-4.2 in E. coli from pigs. These findings elucidate the epidemiology of colistin resistance in food-producing animals in Italy along with its genetic background, and highlight the likelihood of mcr horizontal transfer between commensal bacteria and major food-borne pathogens (Salmonella) within the same type of productions. Thorough action and strategies are needed in order to mitigate the risk of mcr transfer to humans, in a “One Health” perspective.

Cholera is still an important public health problem in several countries, including Thailand. In this study, a collection of clinical and environmental V. cholerae serogroup O1, O139, and non-O1/non-O139 strains originating from Thailand (1983 to 2013) was characterized to determine phenotypic and genotypic traits and to investigate the genetic relatedness. Using a combination of conventional methods and whole genome sequencing (WGS), 78 V. cholerae strains were identified. WGS was used to determine the serogroup, biotype, virulence, mobile genetic elements, and antimicrobial resistance genes using online bioinformatics tools. In addition, phenotypic antimicrobial resistance was determined by the minimal inhibitory concentration (MIC) test. The 78 V. cholerae strains belonged to the following serogroups O1: (n = 44), O139 (n = 16) and non-O1/non-O139 (n = 18). Interestingly, we found that the typical El Tor O1 strains were the major cause of clinical cholera during 1983-2000 with two Classical O1 strains detected in 2000. In 2004-2010, the El Tor variant strains revealed genotypes of the Classical biotype possessing either only ctxB or both ctxB and rstR while they harbored tcpA of the El Tor biotype. Thirty O1 and eleven O139 clinical strains carried CTXϕ (Cholera toxin) and tcpA as well four different pathogenic islands (PAIs). Beside non-O1/non-O139, the O1 environmental strains also presented chxA and Type Three Secretion System (TTSS). The in silico MultiLocus Sequence Typing (MLST) discriminated the O1 and O139 clinical strains from other serogroups and environmental strains. ST69 was dominant in the clinical strains belonging to the 7th pandemic clone. Non-O1/non-O139 and environmental strains showed various novel STs indicating genetic variation. Multidrug-resistant (MDR) strains were observed and conferred resistance to ampicillin, azithromycin, nalidixic acid, sulfamethoxazole, tetracycline, and trimethoprim and harboured variants of the SXT elements. For the first time since 1986, the presence of V. cholerae O1 Classical was reported causing cholera outbreaks in Thailand. In addition, we found that V. cholerae O1 El Tor variant and O139 were pre-dominating the pathogenic strains in Thailand. Using WGS and bioinformatic tools to analyze both historical and contemporary V. cholerae circulating in Thailand provided a more detailed understanding of the V. cholerae epidemiology, which ultimately could be applied for control measures and management of cholera in Thailand.

Bacteriophages represent an alternative solution to control bacterial infections. When interacting, bacteria and phage can evolve, and this relationship is described as antagonistic coevolution, a pattern that does not fit all models. In this work, the model consisted of a microcosm of Salmonella enterica serovar Enteritidis and φSan23 phage. Samples were taken for 12 days every 48 h. Bacteria and phage samples were collected; and isolated bacteria from each time point were challenged against phages from previous, contemporary, and subsequent time points. The phage plaque tests, with the genomics analyses, showed a mutational asymmetry dynamic in favor of the bacteria instead of antagonistic coevolution. This is important for future phage-therapy applications, so we decided to explore the population dynamics of Salmonella under different conditions: pressure of one phage, a combination of phages, and phages plus an antibiotic. The data from cultures with single and multiple phages, and antibiotics, were used to create a mathematical model exploring population and resistance dynamics of Salmonella under these treatments, suggesting a nonlethal, growth-inhibiting antibiotic may decrease resistance to phage-therapy cocktails. These data provide a deep insight into bacterial dynamics under different conditions and serve as additional criteria to select phages and antibiotics for phage-therapy.

ABSTRACT Salmonella enterica subsp. enterica bacteria are highly diverse foodborne pathogens that are subdivided into more than 1,500 serovars. The diversity is believed to result from mutational evolution, as well as intra- and interspecies recombination that potentially could be influenced by restriction-modification (RM) systems. The aim of this study was to investigate whether RM systems were linked to the evolution of Salmonella enterica subsp. enterica. The study included 221 Salmonella enterica genomes, of which 68 were de novo sequenced and 153 were public available genomes from ENA. The data set covered 97 different serovars of Salmonella enterica subsp. enterica and an additional five genomes from four other Salmonella subspecies as an outgroup for constructing the phylogenetic trees. The phylogenetic trees were constructed based on multiple alignment of core genes, as well as the presence or absence of pangenes. The topology of the trees was compared to the presence of RM systems, antimicrobial resistance (AMR) genes, Salmonella pathogenicity islands (SPIs), and plasmid replicons. We did not observe any correlation between evolution and the RM systems in S. enterica subsp. enterica. However, sublineage correlations and serovar-specific patterns were observed. Additionally, we conclude that plasmid replicons, SPIs, and AMR were all better correlated to serovars than to RM systems. This study suggests a limited influence of RM systems on the evolution of Salmonella enterica subsp. enterica, which could be due to the conjugational mode of horizontal gene transfer in Salmonella. Thus, we conclude that other factors must be involved in shaping the evolution of bacteria. IMPORTANCE The evolution of bacterial pathogens, their plasticity and ability to rapidly change and adapt to new surroundings are crucial for understanding the epidemiology and public health. With the application of genomics, it became clear that horizontal gene transfer played a key role in evolution. To understand the evolution and diversification of pathogens, we need to understand the processes that drive the horizontal gene transfer. Restriction-modification systems are thought to cause rearrangements within the chromosome, as well as act as a barrier to horizontal gene transfer. However, here we show that the correlation between restriction-modification systems and evolution in other bacterial species does not apply to Salmonella enterica subsp. enterica. In summary, from this work, we conclude that other mechanisms might be involved in controlling and shaping the evolution of Salmonella enterica subsp. enterica.

Salmonella enterica is a common cause of minor and large food borne outbreaks. To achieve successful and nearly 'real-time' monitoring and identification of outbreaks, reliable sub-typing is essential. Whole genome sequencing (WGS) shows great promises for using as a routine epidemiological typing tool. Here we evaluate WGS for typing of S. Typhimurium including different approaches for analyzing and comparing the data. A collection of 34 S. Typhimurium isolates was sequenced. This consisted of 18 isolates from six outbreaks and 16 epidemiologically unrelated background strains. In addition, 8 S. Enteritidis and 5 S. Derby were also sequenced and used for comparison. A number of different bioinformatics approaches were applied on the data; including pan-genome tree, k-mer tree, nucleotide difference tree and SNP tree. The outcome of each approach was evaluated in relation to the association of the isolates to specific outbreaks. The pan-genome tree clustered 65% of the S. Typhimurium isolates according to the pre-defined epidemiology, the k-mer tree 88%, the nucleotide difference tree 100% and the SNP tree 100% of the strains within S. Typhimurium. The resulting outcome of the four phylogenetic analyses were also compared to PFGE revealing that WGS typing achieved the greater performance than the traditional method. In conclusion, for S. Typhimurium, SNP analysis and nucleotide difference approach of WGS data seem to be the superior methods for epidemiological typing compared to other phylogenetic analytic approaches that may be used on WGS. These approaches were also superior to the more classical typing method, PFGE. Our study also indicates that WGS alone is insufficient to determine whether strains are related or un-related to outbreaks. This still requires the combination of epidemiological data and whole genome sequencing results.

Multidrug-resistant Salmonella enterica serovar Typhimurium ST313 has emerged in sub-Saharan Africa causing severe infections in humans. Therefore, it has been speculated that this specific sequence type, ST313, carries factors associated with increased pathogenicity. We assessed the role in virulence of a gene with a yet unknown function, st313-td, detected in ST313 through comparative genomics. Additionally, the structure of the genomic island ST313-GI, harbouring the gene was determined. The gene st313-td was cloned into wild type S. Typhimurium 4/74 (4/74-C) as well as knocked out in S. Typhimurium ST313 02-03/002 (Δst313-td) followed by complementation (02-03/002-C). Δst313-td was less virulent in mice following i.p. challenge than the wild type and this phenotype could be partly complemented in trans, indicating that st313-td plays a role during systemic infection. The gene st313-td was shown not to affect invasion of cultured epithelial cells, while the absence of the gene significantly affects uptake and intracellular survival within macrophages. The gene st313-td was proven to be strongly associated to invasiveness, harboured by 92.5% of S. Typhimurium blood isolates (n = 82) and 100% of S. Dublin strains (n = 50) analysed. On the contrary, S. Typhimurium isolates of animal and food origin (n = 82) did not carry st313-td. Six human, non-blood isolates of S. Typhimurium from Belarus, China and Nepal harboured the gene and belonged to sequence types ST398 and ST19. Our data showed a global presence of the st313-td gene and in other sequence types than ST313. The gene st313-td was shown to be expressed during logarithmic phase of growth in 14 selected Salmonella strains carrying the gene. This study reveals that st313-td plays a role in S. Typhimurium ST313 pathogenesis and adds another chapter to understanding of the virulence of S. Typhimurium and in particular of the emerging sequence type ST313.

Whole genome sequencing (WGS) shows great potential for real-time monitoring and identification of infectious disease outbreaks. However, rapid and reliable comparison of data generated in multiple laboratories and using multiple technologies is essential. So far studies have focused on using one technology because each technology has a systematic bias making integration of data generated from different platforms difficult. We developed two different procedures for identifying variable sites and inferring phylogenies in WGS data across multiple platforms. The methods were evaluated on three bacterial data sets and sequenced on three different platforms (Illumina, 454, Ion Torrent). We show that the methods are able to overcome the systematic biases caused by the sequencers and infer the expected phylogenies. It is concluded that the cause of the success of these new procedures is due to a validation of all informative sites that are included in the analysis. The procedures are available as web tools.

Abstract: Whole genome sequencing was utilized to investigate the genomic profile of Vibrio cholerae O1 strains, isolated from symptomatic patients in a low‐income urban area of Dhaka, Bangladesh. Comparative genomics using bioinformatics tools were applied to identify major virulence factors, biotype and antimicrobial resistance genes in three V. cholerae O1 strains (VC‐1, 2 and 3) isolated from two case patients. A phylogenetic SNP (single nucleotide polymorphism)‐based analysis was conducted to infer the relatedness to V. cholerae O1 strains isolated elsewhere. The V. cholerae strains were the El Tor variant carrying ctxB1 (standard classical genotype). SNP‐based global phylogeny revealed that the three isolates were strictly clonal and the closest neighbouring genomes were epidemic clones of V. cholerae O1 isolated in 2010 from cholera patients in Pakistan. All strains harboured the integrase gene of the SXT element (intSXT), antimicrobial resistance genes for aminoglycosides, phenicol, sulphonamide and trimethoprim except VC‐1 that lacked sulphonamide resistance genes. The multilocus sequence typing (MLST) revealed that the strains belonged to sequence type, ST69. The study provides knowledge on current genetic traits of clinical V. cholerae O1 circulating in urban household clusters of Bangladesh which may help in predicting emergence of new pandemic strains in Bangladesh. Significance and Impact of the Study: Vibrio cholerae has frequently experienced genetic changes with rapid evolution of pandemic clones in the Ganges Delta region. Whole genome sequencing can reveal genetic information of current pathogenic V. cholerae in Bangladesh which includes cefotaxime genotypes, virulence factors, altered antimicrobial resistance pattern as well as mobile genetic element compared to global pandemic strains. This study data could be used in planning future surveillance strategies in Ganges Delta region by informing new epidemiology of current outbreak strains. [ABSTRACT FROM AUTHOR]

Summary: The sources and modes of transmission of non‐typhoidal Salmonella particularly zoonotic transmission are poorly understood in Africa. This study compared phenotypic and genotypic characteristics of Salmonellae isolated from cattle and humans. Faecal samples of diarrhoeic patients (n = 234), and a healthy population (n = 160), beef cattle at slaughter (n = 250), farms (n = 72) and market (n = 100) were cultured for salmonellae and serotyping and antimicrobial susceptibility were determined. Whole‐genome sequence typing (WGST) of selected isolates and bioinformatic analysis were used to identify the multilocus sequence type (MLST), plasmid replicons, antimicrobial resistance genes and genetic relatedness by single nucleotide polymorphism (SNP) analysis. The Salmonella isolates, diarrhoeic patients (n = 17), healthy population (n = 13), cattle (abattoir, n = 67; farms, n = 10; market n = 5), revealed 49 serovars; some serovars were common to humans and cattle. Rare serovars were prevalent: Colindale (cattle and humans); Rubislaw and Bredeney (humans); and Dublin, Give, Eastbourne, Hadar, Marseille, Sundsvall, Bergen, Ekotedo, Carno and Ealing (cattle). The sequence types (ST) include ST 584, ST 198, ST 562 and ST 512 for S. Colindale, S. Kentucky S. Rubislaw and S. Urbana, respectively. Clonal cluster shared by cattle and human WGST isolates was not found. Antimicrobial resistance rates were generally low and towards only chloramphenicol, ampicillin, gentamicin, ciprofloxacin, tetracycline and streptomycin, range 2.7% (chloramphenicol) to 8.9% (streptomycin). Multiply resistant isolates included serovars Kentucky, 4,5,12:i:‐ and Typhimurium. The study presents a baseline description of the prevalence, serotypes, antimicrobial resistance phenotypes and genetic relatedness of Salmonella isolated from healthy and diarrhoeic humans, and cattle at harvest, on farm and at market. Cattle are a reservoir of diverse salmonellae with shared serovars with humans, but WGST does not support zoonotic transmission. Further study with larger samples is recommended to determine whether epidemiological link exists between cattle and humans. [ABSTRACT FROM AUTHOR]