Towards facilitated interpretation of shotgun metagenomics long-read sequencing data analyzed with KMA for the detection of bacterial pathogens and their antimicrobial resistance genes.

Metagenomic sequencing is a promising method that has the potential to revolutionize the world of pathogen detection and antimicrobial resistance (AMR) surveillance in food-producing environments. However, the analysis of the huge amount of data obtained requires performant bioinformatics tools and databases, with intuitive and straightforward interpretation. In this study, based on long-read metagenomics data of chicken fecal samples with a spike-in mock community, we proposed confidence levels for taxonomic identification and AMR gene detection, with interpretation guidelines, to help with the analysis of the output data generated by KMA, a popular k-mer read alignment tool. Additionally, we demonstrated that the completeness and diversity of the genomes present in the reference databases are key parameters for accurate and easy interpretation of the sequencing data. Finally, we explored whether KMA, in a two-step procedure, can be used to link the detected AMR genes to their bacterial host chromosome, both detected within the same long-reads. The confidence levels were successfully tested on 28 metagenomics datasets which were obtained with sequencing of real and spiked samples from fecal (chicken, pig, and buffalo) or food (minced beef and food enzyme products) origin. The methodology proposed in this study will facilitate the analysis of metagenomics sequencing datasets for KMA users. Ultimately, this will contribute to improvements in the rapid diagnosis and surveillance of pathogens and AMR genes in food-producing environments, as prioritized by the EU.

Comparing the transmission of carbapenemase-producing and extended-spectrum beta-lactamase-producing Escherichia coli between broiler chickens.

The emergence of carbapenemase-producing Enterobacteriaceae (CPE) is a threat to public health, because of their resistance to clinically important carbapenem antibiotics. The emergence of CPE in meat-producing animals is particularly worrying because consumption of meat contaminated with resistant bacteria comparable to CPE, such as extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, contributed to colonization in humans worldwide. Currently, no data on the transmission of CPE in livestock is available. We performed a transmission experiment to quantify the transmission of CPE between broilers to fill this knowledge gap and to compare the transmission rates of CPE and other antibiotic-resistant E. coli. A total of 180 Ross 308 broiler chickens were distributed over 12 pens on the day of hatch (day 0). On day 5, half of the 10 remaining chickens in each pen were orally inoculated with 5·102 colony-forming units of CPE, ESBL, or chloramphenicol-resistant E. coli (catA1). To evaluate the effect of antibiotic treatment, amoxicillin was given twice daily in drinking water in 6 of the 12 pens from days 2-6. Cloacal swabs of all animals were taken to determine the number of infectious broilers. We used a Bayesian hierarchical model to quantify the transmission of the E. coli strains. E. coli can survive in the environment and serve as a reservoir. Therefore, the susceptible-infectious transmission model was adapted to account for the transmission of resistant bacteria from the environment. In addition, the caecal microbiome was analyzed on day 5 and at the end of the experiment on day 14 to assess the relationship between the caecal microbiome and the transmission rates. The transmission rates of CPE were 52 - 68 per cent lower compared to ESBL and catA1, but it is not clear if these differences were caused by differences between the resistance genes or by other differences between the E. coli strains. Differences between the groups in transmission rates and microbiome diversity did not correspond to each other, indicating that differences in transmission rates were probably not caused by major differences in the community structure in the caecal microbiome. Amoxicillin treatment from day 2-6 increased the transmission rate more than three-fold in all inoculums. It also increased alpha-diversity compared to untreated animals on day 5, but not on day 14, suggesting only a temporary effect. Future research could incorporate more complex transmission models with different species of resistant bacteria into the Bayesian hierarchical model.

Implementation of WGS analysis of ESBL-producing Escherichia coli within EU AMR monitoring in livestock and meat.

BACKGROUND: As WGS comes of age, changes in EU legislation implemented in 2021 allow its usage for systematic monitoring of ESBL-producing Escherichia coli from livestock and meat, replacing phenotypic testing. Presently, phenotypic testing correlates well with antimicrobial resistance predicted from WGS data. WGS has added value in the wealth of additional information that is present in the data. OBJECTIVES: In this study we have detected the resistance phenotypes for a panel of antimicrobials while also analysing the molecular epidemiology of ESBL-producing E. coli. METHODS: Susceptibility testing was performed with broth microdilution of selectively isolated E. coli. Short-read WGS was performed in parallel and phenotypes predicted based on the sequence data, which was also used to determine the phylogeny of the isolates. RESULTS: The phenotypically determined resistance and the predicted resistance correlated 90%-100% for the different antimicrobial classes. Furthermore, clonal relationships were detected amongst ESBL-producing E. coli within livestock sectors and the meat produced by this sector. CONCLUSIONS: Further implementation of WGS analysis of ESBL/AmpC-producing E. coli within the AMR monitoring programme of EU member states and global surveillance programmes will contribute to determining the attribution of livestock in the prevalence of ESBL/AmpC-encoding E. coli in humans.

Building an International One Health Strain Level Database to Characterise the Epidemiology of AMR Threats: ESBL-AmpC Producing E. coli as An Example-Challenges and Perspectives.

Antimicrobial resistance (AMR) is one of the top public health threats nowadays. Among the most important AMR pathogens, Escherichia coli resistant to extended spectrum cephalosporins (ESC-EC) is a perfect example of the One Health problem due to its global distribution in animal, human, and environmental sources and its resistant phenotype, derived from the carriage of plasmid-borne extended-spectrum and AmpC ß-lactamases, which limits the choice of effective antimicrobial therapies. The epidemiology of ESC-EC infection is complex as a result of the multiple possible sources involved in its transmission, and its study would require databases ideally comprising information from animal (livestock, companion, wildlife), human, and environmental sources. Here, we present the steps taken to assemble a database with phenotypic and genetic information on 10,763 ESC-EC isolates retrieved from multiple sources provided by 13 partners located in eight European countries, in the frame of the DiSCoVeR Joint Research project funded by the One Health European Joint Programme (OH-EJP), along with its strengths and limitations. This database represents a first step to help in the assessment of different geographical and temporal trends and transmission dynamics in animals and humans. The work performed highlights aspects that should be considered in future international efforts, such as the one presented here.

Antibiotic resistance genes, mobile elements, virulence genes, and phages in cultivated ESBL-producing Escherichia coli of poultry origin in Kwara State, North Central Nigeria.

The paucity of information on the genomic diversity of drug-resistant bacteria in most food-producing animals, including poultry in Nigeria, has led to poor hazard characterization and the lack of critical control points to safeguard public health. Hence, this study used whole genome sequencing (WGS) to assess the presence and the diversity of antibiotic resistance genes, mobile genetic elements, virulence genes, and phages in Extended Spectrum Beta Lactamase producing Escherichia coli (ESBL - E. coli) isolates obtained from poultry via the EURL guideline of 2017 in Ilorin, Nigeria. The prevalence of ESBL - E. coli in poultry was 10.5 % (n = 37/354). The phenotypic antibiotic susceptibility testing showed that all the ESBL- E. coli isolates were multi-drug resistant (MDR). The in-silico analysis of the WGS raw-read data from 11 purposively selected isolates showed that the isolates had a wide array of ARGs that conferred resistance to beta-lactam antibiotics, and 8 other classes of antibiotics (fluoroquinolones, foliate pathway antagonists, aminoglycoside, phenicol, tetracycline, epoxide, macrolides, and rifamycin). All the ARGs were in the bacterial chromosome except in two isolates where plasmid-mediated quinolone resistance (PMQR) was detected. Two isolates carried the gyrAp.S83L mutation which confers resistance to certain fluoroquinolones. The mobilome consisted of several Col-plasmids and the predominant IncF plasmids belonged to the IncF64:A-:B27 sequence type. The virulome consisted of genes that function as adhesins, iron acquisition genes, toxins, and protectins. Intact phages were found in 8 of the 11 isolates and the phageome consisted of representatives of four families of viruses: Myoviridae (62.5 %, n = 5/8), Siphoviridae (37.5 %, n = 3/8), Inoviridae (12.5 %, n = 1), and Podoviridae (12.5 %, n = 1/8). ESBL - E. coli isolates harboured 1-5 intact phages and no ARGs were identified on any of the phages. Although five of the isolates belonged to phylogroup A, the isolates were diverse as they belonged to different serotype and sequence types. Our findings demonstrate the high genomic diversity of ESBL - E. coli of poultry origin in Ilorin, Nigeria. These diverse isolates harbor clinically relevant ARGs, mobile elements, virulence genes, and phages that may have detrimental zoonotic potentials on human health.

Horsing around: Escherichia coli ST1250 of equine origin harbouring epidemic IncHI1/ST9 plasmid with bla CTX-M-1 and an operon for short-chain fructooligosaccharides metabolism.

The relatedness of the equine-associated Escherichia coli ST1250 and its single- and double-locus variants (ST1250-SLV/DLV), obtained from horses in Europe, was studied by comparative genome analysis. A total of 54 isolates of E. coli ST1250 and ST1250-SLV/DLV from healthy and hospitalized horses across Europe [Czech Republic (n=23), the Netherlands (n=18), Germany (n=9), Denmark (n=3) and France (n=1)] from 2008-2017 were subjected to whole-genome sequencing. An additional 25 draft genome assemblies of E. coli ST1250 and ST1250-SLV/DLV were obtained from the public databases. The isolates were compared for genomic features, virulence genes, clade structure and plasmid content. The complete nucleotide sequences of eight IncHI1/ST9 and one IncHI1/ST2 plasmids were obtained using long-read sequencing by PacBio or MinION. In the collection of 79 isolates, only 10 were phylogenetically close (<8 SNP). The majority of isolates belonged to phylogroup B1 (73/79, 92.4%) and carried bla CTX-M-1 (58/79, 73.4%). The plasmid content of the isolates was dominated by IncHI1 of ST9 (56/62, 90.3%) and ST2 (6/62, 9.7%), while 84.5% (49/58) bla CTX-M-1 genes were associated with presence of IncHI1 replicon of ST9 and 6.9% (4/58) with IncHI1 replicon of ST2 within the corresponding isolates. The operon for the utilization of short chain fructooligosaccharides (fos operon) was present in 55 (55/79, 69.6%) isolates, and all of these carried IncHI1/ST9 plasmids. The eight complete IncHI1/ST9 plasmid sequences showed the presence of bla CTX-M-1 and the fos operon within the same molecule. Sequences of IncHI1/ST9 plasmids were highly conserved (>98% similarity) regardless of country of origin and varied only in the structure and integration site of MDR region. E. coli ST1250 and ST1250-SLV/DLV are phylogenetically-diverse strains associated with horses. A strong linkage of E. coli ST1250 with epidemic multi-drug resistance plasmid lineage IncHI1/ST9 carrying bla CTX-M-1 and the fos operon was identified.

Intracellular Transposition of Mobile Genetic Elements Associated with the Colistin Resistance Gene mcr-1.

Mobile colistin resistance (mcr) genes are often located on conjugative plasmids, where their association with insertion sequences enables intercellular and intracellular dissemination throughout bacterial replicons and populations. Multiple mcr genes have been discovered in every habitable continent, in many bacterial species, on both plasmids and integrated into the chromosome. Previously, we showed the intercellular transfer of mcr-1 on an IncI1 plasmid, pMCR-E2899, between strains of Escherichia coli. Characterizing the intracellular dynamics of mcr-1 transposition and recombination would further our understanding of how these important genes move through bacterial populations and whether interventions can be put in place to stop their spread. In this study, we aimed to characterize transfer events from the mcr-1-containing transposon Tn7511 (ISApl1-mcr-1-pap2-ISApl1), located on plasmid pMCR-E2899, using the pBACpAK entrapment vector. Following the transformation of pBACpAK into our DH5α-Azir/pMCR-E2899 transconjugant, we captured ISApl1 in pBACpAK multiple times and, for the first time, observed the ISApl1-mediated transfer of the mcr-1 transposon (Tn7511) into the chromosome of E. coli DH5α. Whole-genome sequencing allowed us to determine consensus insertion sites of ISApl1 and Tn7511 in this strain, and comparison of these sites allowed us to explain the transposition events observed. These observations reveal the consequences of ISApl1 transposition within and between multiple replicons of the same cell and show mcr-1 transposition within the cell as part of the novel transposon Tn7511. IMPORTANCE By analyzing the intracellular transfer of clinically relevant transposons, we can understand the dissemination and evolution of drug resistance conferring mobile genetic elements (MGEs) once a plasmid enters a cell following conjugation. This knowledge will help further our understanding of how these important genes move through bacterial populations. Utilizing the pBACpAK entrapment vector has allowed us to determine the mobility of the novel mcr-1-containing transposon Tn7511.

Detection of an IMI-2 carbapenemase-producing Enterobacter asburiae at a Swedish feed mill.

Occurrence of multidrug resistant Enterobacteriaceae in livestock is of concern as they can spread to humans. A potential introduction route for these bacteria to livestock could be animal feed. We therefore wanted to identify if Escherichia spp., Enterobacter spp., Klebsiella spp., or Raoutella spp. with transferable resistance to extended spectrum cephalosporins, carbapenems or colistin could be detected in the environment at feed mills in Sweden. A second aim was to compare detected isolates to previous described isolates from humans and animals in Sweden to establish relatedness which could indicate a potential transmission between sectors and feed mills as a source for antibiotic resistant bacteria. However, no isolates with transferable resistance to extended-cephalosporins or colistin could be identified, but one isolate belonging to the Enterobacter cloacae complex was shown to be carbapenem-resistant and showing carbapenemase-activity. Based on sequencing by both short-read Illumina and long-read Oxford Nanopore MinIon technologies it was shown that this isolate was an E. asburiae carrying a bla IMI-2 gene on a 216 Kbp plasmid, designated pSB89A/IMI-2, and contained the plasmid replicons IncFII, IncFIB, and a third replicon showing highest similarity to the IncFII(Yp). In addition, the plasmid contained genes for various functions such as plasmid segregation and stability, plasmid transfer and arsenical transport, but no additional antibiotic resistance genes. This isolate and the pSB89A/IMI-2 was compared to three human clinical isolates positive for bla IMI-2 available from the Swedish antibiotic monitoring program Swedres. It was shown that one of the human isolates carried a plasmid similar with regards to gene content to the pSB89A/IMI-2 except for the plasmid transfer system, but that the order of genes was different. The pSB89A/IMI-2 did however share the same transfer system as the bla IMI-2 carrying plasmids from the other two human isolates. The pSB89A/IMI-2 was also compared to previously published plasmids carrying bla IMI-2, but no identical plasmids could be identified. However, most shared part of the plasmid transfer system and DNA replication genes, and the bla IMI-2 gene was located next the transcription regulator imiR. The IS3-family insertion element downstream of imiR in the pSB89A was also related to the IS elements in other bla IMI-carrying plasmids.

cfr and fexA genes in methicillin-resistant Staphylococcus aureus from humans and livestock in the Netherlands.

Background: Although the Netherlands is a country with a low endemic level of methicillin-resistant Staphylococcus aureus (MRSA), a national MRSA surveillance has been in place since 1989. In 2003 livestock emerged as a major reservoir of MRSA and currently livestock-associated MRSA (clonal complex CC398) make up 25% of all surveillance isolates. To assess possible transfer of resistant strains or resistance genes, MRSA obtained from humans and animals were characterized in detail. Methods: The sequenced genomes of 6327 MRSA surveillance isolates from humans and from 332 CC398 isolates from livestock-related samples were analyzed and resistance genes were identified. Several isolates were subjected to long-read sequencing to reconstruct chromosomes and plasmids. Results: Here we show the presence of the multi-resistance gene cfr in seven CC398 isolates obtained from humans and in one CC398 isolate from a pig-farm dust sample. Cfr induces resistance against five antibiotic classes, which is true for all but two isolates. The isolates are genetically unrelated, and in seven of the isolates cfr are located on distinct plasmids. The fexA gene is found in 3.9% surveillance isolates and in 7.5% of the samples from livestock. There is considerable sequence variation of fexA and geographic origin of the fexA alleles. Conclusions: The rare cfr and fexA resistance genes are found in MRSA from humans and animals in the Netherlands, but there is no evidence for spread of resistant strains or resistance plasmids. The proportion of cfr-positive MRSA is low, but its presence is worrying and should be closely monitored.

Succession in the caecal microbiota of developing broilers colonised by extended-spectrum ß-lactamase-producing Escherichia coli.

BACKGROUND: Broilers are among the most common and dense poultry production systems, where antimicrobials have been used extensively to promote animal health and performance. The continuous usage of antimicrobials has contributed to the appearance of resistant bacteria, such as extended-spectrum ß-lactamase-producing Escherichia coli (ESBL-Ec). Here, we studied the ESBL-Ec prevalence and successional dynamics of the caecal microbiota of developing broilers in a commercial flock during their production life cycle (0-35 days). Broilers were categorised as ESBL-Ec colonised (ESBL-Ec+) or ESBL-Ec non-colonised (ESBL-Ec-) by selective culturing. Using 16S rRNA gene sequencing, we i. compared the richness, evenness and composition of the caecal microbiota of both broilers' groups and ii. assessed the combined role of age and ESBL-Ec status on the broilers' caecal microbiota. RESULTS: From day two, we observed an increasing linear trend in the proportions of ESBL-Ec throughout the broilers' production life cycle, X2 (1, N = 12) = 28.4, p < 0.001. Over time, the caecal microbiota richness was consistently higher in ESBL-Ec- broilers, but significant differences between both broilers' groups were found exclusively on day three (Wilcoxon rank-sum test, p = 0.016). Bray-Curtis distance-based RDA (BC-dbRDA) showed no explanatory power of ESBL-Ec status, while age explained 14% of the compositional variation of the caecal microbiota, F (2, 66) = 6.47, p = 0.001. CONCLUSIONS: This study assessed the role of ESBL-Ec in the successional dynamics of the caecal microbiota in developing broilers and showed that the presence of ESBL-Ec is associated with mild but consistent reductions in alpha diversity and with transient bacterial compositional differences. We also reported the clonal spread of ESBL-Ec and pointed to the farm environment as a likely source for ESBLs.

Longitudinal study on the prevalence of extended spectrum cephalosporins-resistant Escherichia coli colonization in Dutch veal farms.

A longitudinal study was performed to investigate the prevalence of Extended-Spectrum Cephalosporin-Resistant (ESC-R) Escherichia coli colonization in Dutch veal farms. Rectal swabs from 683 calves born in 13 Dutch dairy farms were collected one day prior to transportation to the veal farm at 14 or 28 days of age, and at 5 different time points 8 Dutch veal farms. In addition, characteristics of the calf, cows, and farm management were collected. Rectal swabs were selectively cultured for ESC-R E. coli. In total, 1202 ESC-R E. coli isolates were recovered. Overall, the prevalence of ESC-R E. coli increased from 24.4 % at one day prior to transportation to 57.3 % in week two after arrival of calves at the veal farm. No associations were found between the presence of ESC-R E. coli at the dairy or veal farm and age of transportation, sex and breed. The presence of ESC-R E. coli in week 6, 10, and 18 at the veal farm was positively associated with the presence of ESC-R E. coli in week 10, 18, and 24, respectively (p < 0.05). Individual antibiotic treatments applied before week 2 and 6 upon arrival to the veal farms tended to increase the ESC-R E. coli colonization frequency. Our results indicate that ESC-R E. coli colonization frequency substantially increases after arrival of calves on the veal farm. In addition to individual antibiotic treatments, it is considered likely that frequently applied batch antibiotic treatments are also implicated in the ESC-R E. coli colonization frequency.

Changes in Fecal Carriage of Extended-Spectrum ß-Lactamase Producing Enterobacterales in Dutch Veal Calves by Clonal Spread of Klebsiella pneumoniae.

This study aimed to characterize the changes in fecal carriage of Extended-Spectrum ß-Lactamase (ESBL) producing Enterobacterales (ESBL-PE) in a single Dutch veal calves. During the rearing period at the Dutch veal farm, a decrease in fecal carriage of cefotaxime-resistant Escherichia coli isolates was observed after 2 weeks at the veal farm, while an increase of cefotaxime-resistant Klebsiella pneumoniae isolates was demonstrated. E. coli and K. pneumoniae were isolated from rectal swabs collected from 110 veal calves in week 2, 6, 10, 18, and 24 after their arrival at the farm. ESBL-PE isolates were selectively cultured and identified by MALDI-TOF. ESBL genes were characterized by RT-PCR, PCRs, and amplicon sequencing. A total of 80 E. coli and 174 K. pneumoniae strains were isolated from 104 out of 110 veal calves. The prevalence of ESBL-E. coli decreased from week 2 (61%) to week 6 (7%), while an unexpected increase in ESBL-K. pneumoniae colonization was detected in week 6 (80%). The predominant ESBL genes detected in E. coli isolates were bla CTX-M-15 and the non-ESBL gene bla TEM-1a, while in K. pneumoniae bla CTX-M-14 gene was detected in all isolates. Four cefotaxime-resistant K. pneumoniae isolates were randomly selected and characterized in deep by transformation, PCR-based replicon typing, and whole-genome sequencing (WGS). The clonal relatedness of a subgroup of nine animals carrying K. pneumoniae ESBL genes was investigated by Multi Locus sequence typing (MLST). In four ESBL-K. pneumoniae isolates, bla CTX-M-14 was located on IncFIIK and IncFIINK plasmid replicons and the isolates were multi-drug resistant (MDR). MLST demonstrated a clonal spread of ESBL-K. pneumoniae ST107. To the best of our knowledge, this is the first study to report a change in fecal carriage of ESBL-PE over time in the same veal calf during the rearing period.

A role for ColV plasmids in the evolution of pathogenic Escherichia coli ST58.

Escherichia coli ST58 has recently emerged as a globally disseminated uropathogen that often progresses to sepsis. Unlike most pandemic extra-intestinal pathogenic E. coli (ExPEC), which belong to pathogenic phylogroup B2, ST58 belongs to the environmental/commensal phylogroup B1. Here, we present a pan-genomic analysis of a global collection of 752 ST58 isolates from diverse sources. We identify a large ST58 sub-lineage characterized by near ubiquitous carriage of ColV plasmids, which carry genes encoding virulence factors, and by a distinct accessory genome including genes typical of the Yersiniabactin High Pathogenicity Island. This sub-lineage includes three-quarters of all ExPEC sequences in our study and has a broad host range, although poultry and porcine sources predominate. By contrast, strains isolated from cattle often lack ColV plasmids. Our data indicate that ColV plasmid acquisition contributed to the divergence of the major ST58 sub-lineage, and different sub-lineages inhabit poultry, swine and cattle.

Chlamydia caviae in Swiss and Dutch Guinea Pigs-Occurrence and Genetic Diversity.

Chlamydia (C.) caviae is a known pathogen in guinea pigs, causing conjunctivitis, respiratory infections and abortions. Recently, a C. caviae-induced zoonotic link was identified as the etiology of severe community-acquired pneumonia in humans. Here, 784 conjunctival and rectal swabs originating from 260 guinea pigs and 110 rabbits from 64 husbandries in Switzerland, as well as 200 composite conjunctival swabs originating from 878 guinea pigs from 37 husbandries in The Netherlands were examined by real-time PCR followed by conventional PCR and sequencing. Chlamydiaceae were detected in 2.3% (18/784) and 12.5% (25/200) of all Swiss and Dutch samples, respectively. An overall C. caviae occurrence was detected in 2.7% (7/260) and 8.9% (78/878) of all Swiss and Dutch guinea pigs, respectively. OmpA genotyping of 64 C. caviae-positive samples resulted in 33 sequences sharing 100% nucleotide identity with the strains isolated from the zoonotic transmission cases in The Netherlands. However, all ompA sequences of this study were distinct from the C. caviae GPIC reference strain. C. caviae was not detected in rabbits but C. psittaci genotype A was identified in guinea pigs and rabbits, raising concerns about the importance of these animal species as novel zoonotic sources for C. psittaci.

Genetic and phenotypic analysis of the pathogenic potential of two novel Chlamydia gallinacea strains compared to Chlamydia psittaci.

Chlamydia gallinacea is an obligate intracellular bacterium that has recently been added to the family of Chlamydiaceae. C. gallinacea is genetically diverse, widespread in poultry and a suspected cause of pneumonia in slaughterhouse workers. In poultry, C. gallinacea infections appear asymptomatic, but studies about the pathogenic potential are limited. In this study two novel sequence types of C. gallinacea were isolated from apparently healthy chickens. Both isolates (NL_G47 and NL_F725) were closely related to each other and have at least 99.5% DNA sequence identity to C. gallinacea Type strain 08-1274/3. To gain further insight into the pathogenic potential, infection experiments in embryonated chicken eggs and comparative genomics with Chlamydia psittaci were performed. C. psittaci is a ubiquitous zoonotic pathogen of birds and mammals, and infection in poultry can result in severe systemic illness. In experiments with embryonated chicken eggs, C. gallinacea induced mortality was observed, potentially strain dependent, but lower compared to C. psittaci induced mortality. Comparative analyses confirmed all currently available C. gallinacea genomes possess the hallmark genes coding for known and potential virulence factors as found in C. psittaci albeit to a reduced number of orthologues or paralogs. The presence of potential virulence factors and the observed mortality in embryonated eggs indicates C. gallinacea should rather be considered as an opportunistic pathogen than an innocuous commensal.

Temperature and Nutrient Limitations Decrease Transfer of Conjugative IncP-1 Plasmid pKJK5 to Wild Escherichia coli Strains.

Plasmid-mediated dissemination of antibiotic resistance among fecal Enterobacteriaceae in natural ecosystems may contribute to the persistence of antibiotic resistance genes in anthropogenically impacted environments. Plasmid transfer frequencies measured under laboratory conditions might lead to overestimation of plasmid transfer potential in natural ecosystems. This study assessed differences in the conjugative transfer of an IncP-1 (pKJK5) plasmid to three natural Escherichia coli strains carrying extended-spectrum beta-lactamases, by filter mating. Matings were performed under optimal laboratory conditions (rich LB medium and 37°C) and environmentally relevant temperatures (25, 15 and 9°C) or nutrient regimes mimicking environmental conditions and limitations (synthetic wastewater and soil extract). Under optimal nutrient conditions and temperature, two recipients yielded high transfer frequencies (5 × 10-1) while the conjugation frequency of the third strain was 1000-fold lower. Decreasing mating temperatures to psychrophilic ranges led to lower transfer frequencies, albeit all three strains conjugated under all the tested temperatures. Low nutritive media caused significant decreases in transconjugants (-3 logs for synthetic wastewater; -6 logs for soil extract), where only one of the strains was able to produce detectable transconjugants. Collectively, this study highlights that despite less-than-optimal conditions, fecal organisms may transfer plasmids in the environment, but the transfer of pKJK5 between microorganisms is limited mainly by low nutrient conditions.

Genomic evolution of antimicrobial resistance in Escherichia coli.

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.

Latent class analysis to assess whole-genome sequencing versus broth microdilution for monitoring antimicrobial resistance in livestock.

Antimicrobial resistance (AMR) monitoring in animals is performed in commensal Escherichia coli, and other microorganisms relevant for human or veterinary health. Due to advances in the field and major reductions in cost, it is expected that whole-genome sequencing (WGS)-based antimicrobial susceptibility testing (AST) will (partly) replace culture-based AST. So far, no studies have been performed without using culture-based AST as the gold standard. Our aim was to use Bayesian latent class analysis to evaluate the accuracy of susceptibility testing of commensal E. coli by WGS-based AST versus culture-based AST as this test does not assume a gold standard. OpenBUGS was used to model two independent tests in three animal populations (N = 150, 50 bacterial isolates per population): veal calves, pigs, and broilers. This resulted in the first estimation of sensitivity and specificity of WGS-based AST versus culture-based AST to detect AMR without a gold standard. Both methods had high sensitivity (>0.92, lowest limit probability interval: 0.76) and specificity was generally high for both methods for all antimicrobial classes except for aminoglycosides and macrolides. We compared WGS results for different length and identity settings (%) of gene alignment and found few differences between the 60/90, 90/90 and 95/95 settings. We recommend to further investigate sensitivity and specificity of WGS-based AST by means of latent class analysis, especially for low-prevalent resistance.

Plasmids conferring resistance to extended-spectrum beta-lactamases including a rare IncN+IncR multireplicon carrying blaCTX-M-1 in Escherichia coli recovered from migrating barnacle geese ( Branta leucopsis).

Background: Increasing antimicrobial resistance (AMR) is a global threat and wild migratory birds may act as mediators of resistant bacteria across country borders. Our objective was to study extended-spectrum beta-lactamase (ESBL) and plasmid-encoded AmpC (pAmpC) producing Escherichia coli in barnacle geese using whole genome sequencing (WGS) and to identify plasmids harboring bla genes. Methods: Barnacle geese feces (n=200) were collected during fall 2017 and spring 2018 from an urban area in Helsinki, Finland. ESBL/AmpC-producing E. coli were recovered from nine samples (4.5%) and isolates were subjected to WGS on both short- and long-read sequencers, enabling hybrid assembly and determination of the genomic location of bla genes. Results: A rare multireplicon IncN+IncR was recovered from one isolate carrying bla CTX-M-1 in addition to aadA2b, lnu(F), and qnrS1. Moreover, rarely detected IncY plasmids in two isolates were found to harbor multiple resistance genes in addition to the human-associated bla CTX-M-15. Poultry-associated bla CMY-2 was identified from the widely distributed IncI1 and IncK plasmids from four different isolates. One isolate harbored an IncI1 plasmid with bla CTX-M-1 and flor. A chromosomal point mutation in the AmpC promoter was identified in one of the isolates. WGS analysis showed isolates carried multiple resistance and virulence genes and harbored multiple different plasmid replicons in addition to bla-carrying plasmids. Conclusions: Our findings suggest that wild migratory birds serve as a limited source of ESBL/AmpC-producing E. coli and may act as disseminators of the epidemic plasmid types IncI1 and IncK but also rarely detected plasmid types carrying multidrug resistance. Human and livestock-associated ESBL enzyme types were recovered from samples, suggesting a potential for interspecies transmission. WGS offers a thorough method for studying AMR from different sources and should be implemented more widely in the future for AMR surveillance and detection. Understanding plasmid epidemiology is vital for efforts to mitigate global AMR spread.