The genetic relationship between human and pet isolates: a core genome multilocus sequence analysis of multidrug-resistant bacteria.

INTRODUCTION: The global increase of multidrug-resistant organisms (MDROs) is one of the most urgent public health threats affecting both humans and animals. The One Health concept emphasizes the interconnectedness of human, animal and environmental health and highlights the need for integrated approaches to combat antimicrobial resistance (AMR). Although the sharing of environments and antimicrobial agents between companion animals and humans poses a risk for MDRO transmission, companion animals have been studied to a lesser extent than livestock animals. This study therefore used core genome multilocus sequence typing (cgMLST) to investigate the genetic relationships and putative transmission of MDROs between humans and pets. METHODS: This descriptive integrated typing study included 252 human isolates, 53 dog isolates and 10 cat isolates collected from 2019 to 2022 at the Charité University Hospital in Berlin, Germany. CgMLST was performed to characterize methicillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci and multidrug-resistant gram-negative bacteria. The genetic diversity of the MDROs of the different host populations was determined and compared based on sequence type and core genome complex type. RESULTS: Within this study the majority of samples from pets and humans was genetically distinct. However, for some isolates, the number of allelic differences identified by cgMLST was low. Two cases of putative household transmission or shared source of VR E. faecium and MDR E. coli between humans and pets were documented. CONCLUSIONS: The interaction between humans and their pets appears to play a minor role in the spread of the MDROs studied. However, further research is needed. This study emphasizes the importance of comprehensive molecular surveillance and a multidisciplinary One Health approach to understand and contain the spread of MDROs in human and animal populations. TRIAL REGISTRATION: The study is registered with the German Clinical Trials Register (DRKS00030009).

Population genomics uncovers global distribution, antimicrobial resistance, and virulence genes of the opportunistic pathogen Klebsiella aerogenes.

Klebsiella aerogenes is an understudied and clinically important pathogen. We therefore investigate its population structure by genome analysis aligned with metadata. We sequence 130 non-duplicated K. aerogenes clinical isolates and identify two inter-patient transmission events. We then retrieve all publicly available K. aerogenes genomes (n = 1,026, accessed by January 1, 2023) and analyze them with our 130 genomes. We develop a core-genome multi-locus sequence-typing scheme. We find that K. aerogenes is a species complex comprising four phylogroups undergoing evolutionary divergence, likely forming three species. We delineate remarkable clonal diversity and identify three worldwide-distributed carbapenemase-encoding clonal clusters, representing high-risk lineages. We uncover that K. aerogenes has an open genome equipped by a large arsenal of antimicrobial resistance genes. We identify two genetic regions specific for K. aerogenes, encoding a type VI secretion system and flagella/chemotaxis for motility, respectively, both contributing to the virulence. These results provide much-needed insights into the population structure and pan-genomes of K. aerogenes.

A multicenter study on accuracy and reproducibility of nanopore sequencing-based genotyping of bacterial pathogens.

Nanopore sequencing has shown the potential to democratize genomic pathogen surveillance due to its ease of use and low entry cost. However, recent genotyping studies showed discrepant results compared to gold-standard short-read sequencing. Furthermore, although essential for widespread application, the reproducibility of nanopore-only genotyping remains largely unresolved. In our multicenter performance study involving five laboratories, four public health-relevant bacterial species were sequenced with the latest R10.4.1 flow cells and V14 chemistry. Core genome MLST analysis of over 500 data sets revealed highly strain-specific typing errors in all species in each laboratory. Investigation of the methylation-related errors revealed consistent DNA motifs at error-prone sites across participants at read level. Depending on the frequency of incorrect target reads, this either leads to correct or incorrect typing, whereby only minimal frequency deviations can randomly determine the final result. PCR preamplification, recent basecalling model updates and an optimized polishing strategy notably diminished the non-reproducible typing. Our study highlights the potential for new errors to appear with each newly sequenced strain and lays the foundation for computational approaches to reduce such typing errors. In conclusion, our multicenter study shows the necessity for a new validation concept for nanopore sequencing-based, standardized bacterial typing, where single nucleotide accuracy is critical.

Genomic Analysis of Cronobacter condimenti s37: Identification of Resistance and Virulence Genes and Comparison with Other Cronobacter and Closely Related Species.

Cronobacter condimenti are environmental commensals that have not been associated with any clinical infections. To date, they are the least understood and described Cronobacter species within the genus. The objective of this study was to use a draft genome sequence (DGS) of the Cronobacter condimenti strain s37 to screen for genes encoding for antibiotic resistance, virulence, response to environmental stress, and biofilm formation. The strain was isolated in Poland from commercial small radish sprouts. This is the second genome of this species available in the GenBank database. The comparative genome analysis (cgMLST) of C. condimenti s37 with other Cronobacter spp. including the pathogenic species C. sakazakii and the plant-associated closely related genera Franconibacter and Siccibacter was also performed. The assembled and annotated genome of the C. condimenti s37 genome was 4,590,991 bp in length, with a total gene number of 4384, and a GC content of 55.7%. The s 37 genome encoded for genes associated with resistance to stressful environmental conditions (metal resistance genes: zinc, copper, osmotic regulation, and desiccation stress), 17 antimicrobial resistance genes encoding resistance to various classes of antibiotics and 50 genes encoding for the virulence factors. The latter were mainly genes associated with adhesion, chemotaxis, hemolysis, and biofilm formation. Cg-MLST analysis (3991 genes) revealed a greater similarity of C. condimenti s37 to S. turicensis, F. pulveris, and C. dublinensis than to other species of the genus Cronobacter. Studies on the diversity, pathogenicity, and virulence of Cronobacter species isolated from different sources are still insufficient and should certainly be continued. Especially the analysis of rare strains such as s37 is very important because it provides new information on the evolution of these bacteria. Comparative cgMLST analysis of s37 with other Cronobacter species, as well as closely related genera Franconibacter and Siccibacter, complements the knowledge on their adaptability to specific environments such as desiccation.

Development and implementation of a core genome multilocus sequence typing scheme for Haemophilus influenzae.

Haemophilus influenzae is part of the human nasopharyngeal microbiota and a pathogen causing invasive disease. The extensive genetic diversity observed in H. influenzae necessitates discriminatory analytical approaches to evaluate its population structure. This study developed a core genome multilocus sequence typing (cgMLST) scheme for H. influenzae using pangenome analysis tools and validated the cgMLST scheme using datasets consisting of complete reference genomes (N = 14) and high-quality draft H. influenzae genomes (N = 2297). The draft genome dataset was divided into a development dataset (N = 921) and a validation dataset (N = 1376). The development dataset was used to identify potential core genes, and the validation dataset was used to refine the final core gene list to ensure the reliability of the proposed cgMLST scheme. Functional classifications were made for all the resulting core genes. Phylogenetic analyses were performed using both allelic profiles and nucleotide sequence alignments of the core genome to test congruence, as assessed by Spearman's correlation and ordinary least square linear regression tests. Preliminary analyses using the development dataset identified 1067 core genes, which were refined to 1037 with the validation dataset. More than 70% of core genes were predicted to encode proteins essential for metabolism or genetic information processing. Phylogenetic and statistical analyses indicated that the core genome allelic profile accurately represented phylogenetic relatedness among the isolates (R 2 = 0.945). We used this cgMLST scheme to define a high-resolution population structure for H. influenzae, which enhances the genomic analysis of this clinically relevant human pathogen.

Development of the Pneumococcal Genome Library, a core genome multilocus sequence typing scheme, and a taxonomic life identification number barcoding system to investigate and define pneumococcal population structure.

Investigating the genomic epidemiology of major bacterial pathogens is integral to understanding transmission, evolution, colonization, disease, antimicrobial resistance and vaccine impact. Furthermore, the recent accumulation of large numbers of whole genome sequences for many bacterial species enhances the development of robust genome-wide typing schemes to define the overall bacterial population structure and lineages within it. Using the previously published data, we developed the Pneumococcal Genome Library (PGL), a curated dataset of 30 976 genomes and contextual data for carriage and disease pneumococci recovered between 1916 and 2018 in 82 countries. We leveraged the size and diversity of the PGL to develop a core genome multilocus sequence typing (cgMLST) scheme comprised of 1222 loci. Finally, using multilevel single-linkage clustering, we stratified pneumococci into hierarchical clusters based on allelic similarity thresholds and defined these with a taxonomic life identification number (LIN) barcoding system. The PGL, cgMLST scheme and LIN barcodes represent a high-quality genomic resource and fine-scale clustering approaches for the analysis of pneumococcal populations, which support the genomic epidemiology and surveillance of this leading global pathogen.

Three prolonged outbreaks of metallo-ß-lactamase-producing Pseudomonas aeruginosa in an Upper Austrian hospital, 2017-2023.

In spring 2022, an increase in metallo-ß-lactamase-producing Pseudomonas aeruginosa (MBL-Pa) infections was detected in a hospital in Upper Austria. To identify the source of infection and to stop further transmissions, an epidemiological outbreak investigation including whole-genome sequencing (WGS)-based typing was conducted. The final case definition included cases admitted to the hospital between 2020 and 2023 with an MBL-Pa in one of the three genomic clusters identified. In addition, the investigation was extended to include historical cases from 2017. Core genome multilocus sequence typing was performed to assess the genetic relatedness between the isolates. Fifty-four clinical P. aeruginosa isolates and eight P. aeruginosa isolates from the hospital environment were obtained. All but nine isolates grouped into one of three genomic clusters (ST235/blaVIM-1, ST111/blaVIM-2, or ST621/blaIMP-13), which were considered to be distinct, prolonged outbreaks involving 47 out of 52 cases. The most likely source of infection for cluster 1 (ST111/blaVIM-2) and cluster 2 (ST235/blaVIM-1) was sinks in the intensive care unit (ICU) washroom. Cluster 3 clone (ST621/blaIMP-13) could have originated in the urology ward in 2020 and then spread to the ICU years later. However, the nosocomial origin of this clone could not be proven. In March 2023, following the implementation of control measures (gowning, patient isolation, screening, and daily disinfection), no further MLB-Pa was detected, and the outbreaks were considered to be over. As ICUs play an important role in the transmission of P. aeruginosa, emphasis should be placed on genomic surveillance, infection prevention, and control in such wards. IMPORTANCE: The significance of our work lies in the successful resolution of three prolonged outbreaks of MBL-Pa infections in a hospital in Upper Austria. Through a comprehensive epidemiological investigation coupled with WGS-based typing of P. aeruginosa isolates, the study identified three distinct genomic clusters responsible for prolonged outbreaks involving 47 cases. The investigation pinpointed sinks in the ICU washroom as the likely source of infection for two of the clusters. The study demonstrates the effectiveness of control measures such as hand hygiene, gowning, patient isolation, screening, and disinfection in stopping further transmission and bringing the outbreaks to a close. This underscores the critical role of genomic surveillance and control measures, particularly in high-risk settings like ICUs, in reducing nosocomial transmission of MBL-Pa infections.

Comparison of the effectiveness of core genome multilocus sequence typing and polymerase chain reaction-based open reading frame typing in tracing nosocomial methicillin-resistant Staphylococcus aureus transmission.

INTRODUCTION: A clonal shift from staphylococcal cassette chromosome mec (SCCmec) type II/ST5 methicillin-resistant Staphylococcus aureus (MRSA) to SCCmec type IV/clonal complex (CC)1 MRSA has occurred rapidly in Japan. Our previous research in a geriatric hospital found SCCmec type IV/CC1 MRSA prevalence in long-term care wards. Due to intensive personal care requirements, frequent contact with healthcare providers can potentially cause unintentional nosocomial MRSA transmission. We performed polymerase chain reaction-based open reading frame typing (POT) and core genome multilocus sequence typing (cgMLST) to investigate the occurrence of nosocomial transmission and to compare the results of these methods. METHODS: POT and whole genome sequencing were performed in 83 MRSA isolates. Commercial automated software (Ridom SeqSphere+) was used to perform cgMLST. MRSA isolates with 0-8 allelic differences were considered related, and medical records were consulted in these cases. RESULTS: SCCmec type IV/CC1 MRSA was the most frequently detected clone (n = 56, 67.5 %), which was divided into 14 POT types, followed by SCCmec type I/ST8 (n = 9) and SCCmec type IV/ST8 (n = 8). Identical POT types were found across 7 of 11 wards. However, cgMLST analysis identified only three cases (six strains) of high genetic similarity, indicating nosocomial transmission; only one involved SCCmec type IV/CC1 (two strains). The mean allelic difference in the core genomes between strains with identical POT types in the same ward was 55.3 ± 22.0. CONCLUSIONS: The cgMLST method proved more effective for identifying nosocomial transmissions compared to POT, highlighting its utility in tracking MRSA spread in healthcare settings.

Antimicrobial Resistance Profile, Whole-Genome Sequencing and Core Genome Multilocus Sequence Typing of B. anthracis Isolates in Croatia from 2001 to 2022.

Bacillus anthracis, the causative agent of anthrax disease, is a worldwide threat to livestock, wildlife and public health. It is also considered one of the most important pathogens of bioterrorism. Rapid and reliable diagnosis and administration of antimicrobials are essential for effective anthrax treatment. In this study, we determined the in vitro susceptibilities of 40 isolates of B. anthracis isolated in Croatia over the recent two decades to 18 antimicrobials. Whole-genome sequencing was performed, and bioinformatics tools were used to determine virulence factors and antimicrobial resistance genes. Core genome-based multilocus sequence typing was used for isolate comparison and phylogenetic analysis. All isolates were susceptible to all antimicrobials recommended for post-exposure prophylaxis or anthrax therapy. Susceptibility was found to all other tested antimicrobials that are an alternative for primary therapy. We found two beta-lactamase genes, but their expression is not sufficient to confer resistance. In all isolates used in this study, we found 21 virulence genes, 8 of which are responsible for toxin and capsule production. As far as phylogenetic analysis is concerned, the B. anthracis isolates from Croatia are categorised into two clades. The first is clade A, subclade Trans Eurasia, and the other is clade B, subclade B2.

Development and implementation of a core genome multilocus sequence typing scheme for Yersinia enterocolitica: a tool for surveillance and outbreak detection.

Yersinia enterocolitica (Y. enterocolitica) is the most frequent etiological agent of yersiniosis and has been responsible for several national outbreaks in Norway and elsewhere. A standardized high-resolution method, such as core genome Multilocus Sequence Typing (cgMLST), is needed for pathogen traceability at the national and international levels. In this study, we developed and implemented a cgMLST scheme for Y. enterocolitica. We designed a cgMLST scheme in SeqSphere + using high-quality genomes from different Y. enterocolitica biotype sublineages. The scheme was validated if more than 95% of targets were found across all tested Y. enterocolitica: 563 Norwegian genomes collected between 2012 and 2022 and 327 genomes from public data sets. We applied the scheme to known outbreaks to establish a threshold for identifying major complex types (CTs) based on the number of allelic differences. The final cgMLST scheme included 2,582 genes with a median of 97.9% (interquartile range 97.6%-98.8%) targets found across all tested genomes. Analysis of outbreaks identified all outbreak strains using single linkage clustering at four allelic differences. This threshold identified 311 unique CTs in Norway, of which CT18, CT12, and CT5 were identified as the most frequently associated with outbreaks. The cgMLST scheme showed a very good performance in typing Y. enterocolitica using diverse data sources and was able to identify outbreak clusters. We recommend the implementation of this scheme nationally and internationally to facilitate Y. enterocolitica surveillance and improve outbreak response in national and cross-border outbreaks.

Molecular epidemiology of Brucella abortus isolated from the environment in Ningxia Hui autonomous region, China.

Brucellosis is among the key zoonotic infectious diseases in China, and The Ningxia Hui Autonomous Region represents a major endemic area, and it is one of the main causes of poverty in the region due to illness. In Ningxia, there is substantial research on Brucella melitensis, studies on the molecular epidemiology of Brucella abortus are notably scarce. Consequently, this study aims to undertake pathogenic isolation and molecular epidemiological research on Brucella abortus isolated from the environment in Ningxia, providing insights and evidence to advance the prevention and control measures for brucellosis in the region. Building on traditional pathogenic detection methods, this research employs whole-genome sequencing(WGS) techniques and bioinformatics software to conduct a phylogenetic comparison of Ningxia strains and strains of Brucella abortus from various geographical origins. The results indicate that four Brucella abortus strains are classified as biovar 3 and MLST type ST2. It is shown that the local strains were closer phylogenetic relationships with strains from Asian and European countries. The presence of Brucella abortus in certain environmental sectors of Ningxia indicates a risk of transmission from the environment to animals and subsequently to humans. In conclusion, the Brucella abortus exists in some farming environments in Ningxia, and exists for a long time. Therefore, it is necessary to strengthen the monitoring of the disinfection effect of the farming environment to provide a basis for the forward movement of the gate of brucellosis prevention and control.

Protracted transmission and persistence of ST80 vancomycin-resistant Enterococcus faecium clonal complex types CT2933, CT2932 and CT1916 in a large Irish hospital: a 39-month whole-genome sequencing study.

BACKGROUND: Vancomycin-resistant Enterococcus faecium (VREfm) are significant nosocomial pathogens. Sequence type (ST) 80 vanA-encoding VREfm predominate in Irish hospitals, but their transmission is poorly understood. AIMS: To investigate transmission and persistence of predominant complex type (CT) VREfm in two wards of an Irish hospital (H1) using whole-genome sequencing, and their intra- and inter-hospital dissemination. METHODS: Rectal screening (N = 330, September 2019 to December 2022) and environmental (N = 48, November 2022 to December 2022) E. faecium were investigated. Isolate relatedness was assessed by core-genome multi-locus sequence typing (cgMLST) and core-genome single nucleotide polymorphism (cgSNP) analysis. Likely transmission chains were identified using SeqTrack (https://graphsnp.fordelab.com/graphsnp) using cgSNP data and recovery location. Well-characterized E. faecium (N = 908) from seven Irish hospitals including H1 (June 2017 to July 2022) were also investigated. FINDINGS: Conventional MLST assigned isolates to nine STs (ST80, 82%). cgMLST identified three predominant ST80 CTs (CT2933, CT2932 and CT1916) (55% of isolates) of related isolates (≤20 allelic differences). cgSNP analysis differentiated these CTs into multiple distinct closely related genomic clusters (≤10 cgSNPs). Parisimonious network construction identified 55 likely inter- and intra-ward transmissions with epidemiological support between patients ≤30 days involving 73 isolates (≤10 cgSNPs) from seven genomic clusters. Numerous other likely transmissions over longer time periods without evident epidemiological links were identified, suggesting persistence and unidentified reservoirs contribute to dissemination. The three CTs predominated among E. faecium (N = 1286) in seven hospitals, highlighting inter-hospital spread without known epidemiological links. CONCLUSION: This study revealed the long-term intra- and inter-hospital dominance of three major CT ST80 VREfm lineages, widespread transmission and persistence, implicating unidentified reservoirs.

Association between Exposure to Leptospira spp. and Abortion in Mares in Croatia.

Leptospirosis is one of the most common zoonotic infections and a major problem in terms of both veterinary medicine and public health. However, the disease is under-recognised and under-diagnosed worldwide, particularly in horses. Clinical leptospirosis in horses is mainly associated with recurrent uveitis (ERU), which has recently been studied more intensively, and reproductive disorders, the epidemiology of which is still relatively poorly understood. To enhance our comprehension of abortions caused by leptospirosis in horses and to identify the causative strains, a serological study was carried out with subsequent molecular characterisation of the isolate obtained. Using the microscopic agglutination test (MAT), serum samples from mares that aborted and foetal fluids (when available) were tested for antibodies against Leptospira spp. Furthermore, bacteria isolation from kidney cultures was conducted. Of 97 mare serum samples, 21 (21.64%) tested positive, with Grippotyphosa and Pomona being the most frequently detected serogroups. A significantly higher seroprevalence was found in aborting mares compared to the healthy horse population from the same geographical area, as well as a pronounced seasonal variation. Leptospiral antibodies were not detected in any of the foetal fluids, but isolation was successful in 1 case out of 39 (2.56%). Genotyping by multilocus sequence typing (MLST) and core genome multilocus sequence typing (cgMLST) identified the obtained isolate as Leptospira kirschneri, serogroup Pomona, serovar Mozdok. Further surveillance and molecular typing of Leptospira strains causing abortion in horses would be invaluable in understanding the prevalence and impact of leptospirosis on equine reproductive health in Europe.

The mobilome of Staphylococcus aureus from wild ungulates reveals epidemiological links at the animal-human interface.

Staphylococcus aureus thrives at animal-human-environment interfaces. A large-scale work from our group indicated that antimicrobial resistance (AMR) in commensal S. aureus strains from wild ungulates is associated with agricultural land cover and livestock farming, raising the hypothesis that AMR genes in wildlife strains may originate from different hosts, namely via exchange of mobile genetic elements (MGE). In this work, we generate the largest available dataset of S. aureus draft genomes from wild ungulates in Portugal and explore their mobilome, which can determine important traits such as AMR, virulence, and host specificity, to understand MGE exchange. Core genome multi-locus sequence typing based on 98 newly generated draft genomes and 101 publicly available genomes from Portugal demonstrated that the genomic relatedness of S. aureus from wild ungulates assigned to livestock-associated sequence types (ST) is greater compared to wild ungulate isolates assigned to human-associated STs. Screening of host specificity determinants disclosed the unexpected presence in wildlife of the immune evasion cluster encoded in φSa3 prophage, described as a human-specific virulence determinant. Additionally, two plasmids, pAVX and pETB, previously associated with avian species and humans, respectively, and the Tn553 transposon were detected. Both pETB and Tn553 encode penicillin resistance through blaZ. Pangenome analysis of wild ungulate isolates shows a core genome fraction of 2133 genes, with isolates assigned to ST72 and ST3224 being distinguished from the remaining by MGEs, although there is no reported role of these in adaptation to wildlife. AMR related gene clusters found in the shell genome are directly linked to resistance against penicillin, macrolides, fosfomycin, and aminoglycosides, and they represent mobile ARGs. Altogether, our findings support epidemiological interactions of human and non-human hosts at interfaces, with MGE exchange, including AMR determinants, associated with putative indirect movements of S. aureus among human and wildlife hosts that might be bridged by livestock.

Surveillance of vancomycin-resistant enterococci reveals shift in dominating clusters from vanA to vanB Enterococcus faecium clusters, Denmark, 2015 to 2022.

BackgroundVancomycin-resistant enterococci (VRE) are increasing in Denmark and Europe. Linezolid and vancomycin-resistant enterococci (LVRE) are of concern, as treatment options are limited. Vancomycin-variable enterococci (VVE) harbour the vanA gene complex but are phenotypically vancomycin-susceptible.AimThe aim was to describe clonal shifts for VRE and VVE in Denmark between 2015 and 2022 and to investigate genotypic linezolid resistance among the VRE and VVE.MethodsFrom 2015 to 2022, 4,090 Danish clinical VRE and VVE isolates were whole genome sequenced. We extracted vancomycin resistance genes and sequence types (STs) from the sequencing data and performed core genome multilocus sequence typing (cgMLST) analysis for Enterococcus faecium. All isolates were tested for the presence of mutations or genes encoding linezolid resistance.ResultsIn total 99% of the VRE and VVE isolates were E. faecium. From 2015 through 2019, 91.1% of the VRE and VVE were vanA E. faecium. During 2020, to the number of vanB E. faecium increased to 254 of 509 VRE and VVE isolates. Between 2015 and 2022, seven E. faecium clusters dominated: ST80-CT14 vanA, ST117-CT24 vanA, ST203-CT859 vanA, ST1421-CT1134 vanA (VVE cluster), ST80-CT1064 vanA/vanB, ST117-CT36 vanB and ST80-CT2406 vanB. We detected 35 linezolid vancomycin-resistant E. faecium and eight linezolid-resistant VVEfm.ConclusionFrom 2015 to 2022, the numbers of VRE and VVE increased. The spread of the VVE cluster ST1421-CT1134 vanA E. faecium in Denmark is a concern, especially since VVE diagnostics are challenging. The finding of LVRE, although in small numbers, ia also a concern, as treatment options are limited.

Antimicrobial resistance and genetic relatedness among Campylobacter coli and Campylobacter jejuni from humans and retail chicken meat in Taiwan.

OBJECTIVES: Campylobacter is a significant zoonotic pathogen primarily transmitted through poultry. Our study aimed to assess antimicrobial resistance and genetic relationships among Campylobacter isolates from retail chicken meat and humans in Taiwan. METHODS: Campylobacter isolates were analysed using whole-genome sequencing to investigate their antimicrobial resistance, genetic determinants of resistance, and genotypes. RESULTS: Campylobacter coli and Campylobacter jejuni accounted for 44.9% and 55.1% of chicken meat isolates, and 11.4% and 88.6% of human isolates, respectively. C. coli displayed significantly higher resistance levels. Furthermore, isolates from chicken meat exhibited higher levels of resistance to most tested antimicrobials compared to isolates from humans. Multidrug resistance was observed in 96.3% of C. coli and 43.3% of C. jejuni isolates from chicken meat and 80.6% of C. coli and 15.8% of C. jejuni isolates from humans. Macrolide resistance was observed in 85.5% of C. coli isolates, primarily attributed to the erm(B) rather than the A2075G mutation in 23S rRNA. Among the 511 genomes, we identified 133 conventional MLST sequence types, indicating significant diversity among Campylobacter strains. Notably, hierarchical Core-genome multilocus sequence typing clustering, including HC0, HC5, and HC10, revealed a significant proportion of closely related isolates from chicken meat and humans. CONCLUSIONS: Our research highlights significant associations in antimicrobial resistance and genetic relatedness between Campylobacter isolates from chicken meat and humans in Taiwan. The genetic analysis data suggest that campylobacteriosis outbreaks may occur more frequently in Taiwan than previously assumed. Our study emphasizes the need for strategies to control multidrug-resistant strains and enhance outbreak prevention.

Antimicrobial resistance, virulence potential and genomic epidemiology of global genomes of the rare Salmonella enterica serovar Orion.

AIMS: Our aim is to characterize through whole-genome sequencing (WGS) the antimicrobial resistance (AMR) and heavy metal tolerance (HMT) genes content, plasmid presence, virulence potential and genomic diversity of the rare non-typhoid Salmonella enterica serovar Orion (S. Orion) from 19 countries of the African, American, Eastern Mediterranean, European, Southeastern Asia and Western Pacific regions. METHODS AND RESULTS: Totally 324 S. Orion genomes were screened for AMR, HMT and virulence genes, plasmids and Salmonella Pathogenicity Islands (SPIs). Genomic diversity was investigated using Multi-Locus Sequence Typing (MLST) and core-genome MLST (cgMLST). Efflux pump encoding genes mdsA and mdsB were present in all genomes analysed, while quinolone chromosomal point mutations and aminoglycoside, beta-lactam, colistin, lincosamide, macrolide, phenicol, sulphonamide, trimethoprim, tetracycline and disinfectant resistance genes were found in 0.3%-5.9%. A total of 17 genomes (5.2%) from Canada, the United Kingdom, the USA and Tanzania showed a potential multi-drug resistance profile. Gold tolerance genes golS and golT were detected in all genomes analysed, while arsenic, copper, mercury, silver and tellurium tolerance genes were found in 0.3%-35.5%. Col(MGD2) was the most frequently detected plasmid, in 15.4% of the genomes. Virulence genes related to adherence, macrophage induction, magnesium uptake, regulation, serum resistance, stress adaptation, type III secretion systems and six SPIs (1, 2, 3, 4, 5, 9, 12, 13, 14 and C63PI) were detected. ST639 was assigned to 89.2% of the S. Orion genomes, while cgMLST showed core-genome STs and clusters of strains specific by countries. CONCLUSION: The high virulence factor frequencies, the genomic similarity among some non-clinical and clinical strains circulating worldwide and the presence of a strain carrying a resistance gene against a last resource antimicrobial like colistin, highlight the potential risk of S. Orion strains for public health and food safety and reinforce the importance to not underestimate the potential hazard of rare non-typhoid Salmonella serovars.

Whole genome-based antimicrobial resistance, virulence, and phylogenetic characteristics of Trueperella pyogenes clinical isolates from humans and animals.

Trueperella pyogenes is an opportunistic zoonotic bacterial pathogen, whose antimicrobial resistance, virulence, and genetic relatedness between strains from animals and humans are barely studied. These characteristics were therefore analyzed for clinical T. pyogenes strains from 31 animals of 11 different species and 8 humans determining their complete circular genome sequence and antimicrobial susceptibility. The MICs of 19 antimicrobials including 3 antiseptics correlated to the resistance genes identified in silico within the genomes revealing a predominance of resistance to streptomycin (aadA9), sulfamethoxazole (sul1), and tetracycline (tet(33), tet(W/N/W)) among strains from humans and cattle. Additional resistance genes (erm(X), erm(56), cmx, drfA1, aadA1, aph(3'')-Ib (strA), aph(6)-Id (strB), aac(3)-IVa, aph(4)-Ia) were found only sporadically. The resistance genes were localized on genetic elements integrated into the chromosome. A cgMLST-based phylogenetic analysis revealed two major clusters each containing genetically diverse strains. The human strains showed the closest relatedness to strains from cattle. Virulence genes coding for fimbriae (fimA, fimC), neuroamidase (nanP, nanH), pyolysin (plo), and collagen binding protein (cbpA) were identified in strains from different hosts, but no correlation was observed between virulence factors and strain origin. The existence of resistance genes typically found in Gram-negative bacteria within the Gram-positive T. pyogenes indicates a wider capacity to adapt to antimicrobial selective pressure. Moreover, the presence of similar antimicrobial resistance profiles found in cattle and human strains as well as their closest relatedness suggests common zoonotic features and cattle as the potential source for human infections.

Core genome multilocus sequence typing (cgMLST) applicable to the monophyletic Klebsiella oxytoca species complex.

The environmental bacterium Klebsiella oxytoca displays an alarming increase of antibiotic-resistant strains that frequently cause outbreaks in intensive care units. Due to its prevalence in the environment and opportunistic presence in humans, molecular surveillance (including resistance marker screening) and high-resolution cluster analysis are of high relevance. Furthermore, K. oxytoca previously described in studies is rather a species complex (KoSC) than a single species comprising at least six closely related species that are not easily differentiated by standard typing methods. To reach a discriminatory power high enough to identify and resolve clusters within these species, whole genome sequencing is necessary. The resolution is achievable with core genome multilocus sequence typing (cgMLST) extending typing of a few housekeeping genes to thousands of core genome genes. CgMLST is highly standardized and provides a nomenclature enabling cross laboratory reproducibility and data exchange for routine diagnostics. Here, we established a cgMLST scheme not only capable of resolving the KoSC species but also producing reliable and consistent results for published outbreaks. Our cgMLST scheme consists of 2,536 core genome and 2,693 accessory genome targets, with a percentage of good cgMLST targets of 98.31% in 880 KoSC genomes downloaded from the National Center for Biotechnology Information (NCBI). We also validated resistance markers against known resistance gene patterns and successfully linked genetic results to phenotypically confirmed toxic strains carrying the til gene cluster. In conclusion, our novel cgMLST enables highly reproducible typing of four different clinically relevant species of the KoSC and thus facilitates molecular surveillance and cluster investigations.

Implications of ad-hoc molecular typing for infection control measures in a multi-cluster, multi-phenotypic Serratia marcescens outbreak in a neonatal intensive care unit.

BACKGROUND: Serratia marcescens is known to cause outbreaks in neonatal intensive care units (NICUs). Traditionally epidemiological data, antimicrobial resistance patterns and epidemiological typing have been used to guide infection prevention methods. Whole-genome sequencing (WGS) applications such as core-genome multi-locus sequence typing (cgMLST) applied during an outbreak would potentially yield more information. AIM: To use cgMLST to acquire detailed information on the source and spread of bacteria, enabling more efficient control measures during an S. marcescens outbreak at a NICU. METHODS: Neonates admitted to the NICU of the Leiden University Medical Center (LUMC) during an outbreak between September 2023 and January 2024, with S. marcescens being cultured, were included. Environmental samples were taken to search for a common source, antibiotic susceptibility testing was performed, and antimicrobial resistance genes were analysed. FINDINGS: S. marcescens strains from 17 of the 20 positive patients were available for molecular typing. The cgMLST scheme revealed five different complex types consisting of four separate clusters. Multiple clusters made an unidentified persistent environmental source as cause of the outbreak less likely, leading to a quick downscaling of infection prevention measures. Differences were shown in aminoglycoside resistance patterns of isolates within the same complex types and patients. CONCLUSION: The use of ad-hoc cgMLST provided timely data for rational decision-making during an S. marcescens outbreak at the NICU. Antibiotic phenotyping alone was found not to be suitable for studying clonal spread during this outbreak with S. marcescens.