Core Genome Single Nucleotide Polymorphism Research Articles

Whole-genome sequencing analyses and antibiotic resistance situation of 48 Helicobacter pylori strains isolated in Zhejiang, China

PurposeIn the Zhejiang region, research on Helicobacter pylori is lacking. The purpose of this study was to assess the extent of antibiotic resistance in H. pylori in this region, explore alternative methods for predicting the resistance patterns of H. pylori, and investigate the colonization of native gastric mucosa by other clades of H. pylori in the structure population of this bacterium.MethodsStrains were cultured under microaerobic conditions, and antimicrobial susceptibility testing (AST) was performed via agar dilution. Whole-genome sequencing (WGS) was performed via next-generation sequencing (NGS) technology. Epidemiological data including data from this study and reported articles from Zhejiang, China, were included. Further analyses based on AST, WGS, and epidemiological date include virulence genes, antibiotic resistance-related mutations, and phylogenetic trees based on 7 housekeeping genes and core-genome single nucleotide polymorphisms (SNPs).ResultsThe bacterial isolates in this study presented higher antibiotic resistance rates than previously reported, especially against levofloxacin and clarithromycin. The point mutation A2147G in 23 S rRNA is specific to clarithromycin resistance. Mutations at position/s 87 and/or 91 of the gyrA gene amino acid sequence are highly consistent with levofloxacin resistance highly. The point mutations C1707T in 23 S rRNA and E463K in the gyrB gene have not been previously documented in China. All the bacterial isolates belong to Asian branches in the structure population. The resistance rate to clarithromycin of isolates from hosts born after January 1, 1977 is statistically higher than that of hosts born before 1977.ConclusionEradication therapy based on AST results is urgently needed in Zhejiang. The point mutation A2147G in 23 S rRNA and point mutations in the gyrA gene at amino acid/s 87 and/or 91 are sufficient for predicting resistance to clarithromycin and levofloxacin, respectively. The isolate with the mutation E463K in the gyrB gene represents a significant contribution to the field. Mutations in 23 S rRNA may offer valuable insights into the dynamics of H. pylori transmission among hosts.

Potential Use of Fourier-Transform Infrared Spectroscopy as a Rapid Screening Tool for Investigating Nosocomial Outbreaks of ST-80 Vancomycin-Resistant Enterococcus faecium

Core genome single-nucleotide polymorphism phylogeny was used to characterise a nosocomial outbreak caused by ST-80 Vancomycin-resistant Enterococcus faecium (VREf). It identified 22 of 25 epidemiologically related isolates as belonging to an outbreak cluster. The use of Fourier-transform infrared (FT-IR) spectroscopy with a cluster-defining cut-off of 0.071 resulted in the correct classification of 21 of 22 phylogenetically related isolates in a single cluster. It successfully distinguished three phylogenetically unrelated isolates from the outbreak cluster, along with five ST-80 unrelated control isolates, and five isolates from a previous outbreak in May 2023, yielding only one mischaracterised environmental isolate. These findings support the potential use of FT-IR spectroscopy as a rapid screening tool to assist outbreak investigations. Notably, this study is the first to focus on the performance of FT-IR spectroscopy in the epidemiological analysis of VREf isolates with the same sequence type.

Genomic epidemiology of Clostridioides difficile Sequence Type 35 reveals intraspecies and interspecies clonal transmission

ABSTRACT Clostridioides difficile sequence type (ST) 35 has been found in humans and animals worldwide. However, its genomic epidemiology and clonal transmission have not been explored in detail. In this study, 176 C. difficile ST35 isolates from six countries were sequenced. Genomic diversity, clonal transmission and epidemiological data were analyzed. Sporulation and virulence capacities were measured. Four ribotypes (RT) were identified including RT046 (97.2%), RT656 (1.1%), RT427 (0.6%), and RT AI-78 (1.1%). Phylogenetic analysis of 176 ST35 genomes, along with 50 publicly available genomes, revealed two distinctive lineages without time-, region-, or source-dependent distribution. However, the distribution of antimicrobial resistance genes differed significantly between the two lineages. Nosocomial and communal transmission occurred in humans with the isolates differed by ≤ two core-genome single-nucleotide polymorphism (cgSNPs) and clonal circulation was found in pigs with the isolates differed by ≤ four cgSNPs. Notably, interspecies clonal transmission was identified among three patients with community acquired C. difficile infection and pigs with epidemiological links, differed by ≤ nine cgSNPs. Toxin B (TcdB) concentrations were significantly higher in human isolates compared to pig isolates, and ST35 isolates exhibited stronger sporulation capacities than other STs. Our study provided new genomic insights and epidemiological evidence of C. difficile ST35 intraspecies and interspecies clonal transmission, which can also be facilitated by its strong sporulation capacity.

Genomic analysis of Brucella isolates from animals and humans, Türkiye, 2010 to 2020.

BackgroundBrucellosis is a bacterial zoonosis causing severe illness in humans and animals and leading to economic losses in the livestock production in Türkiye and other endemic countries.AimWe aimed at investigating genomic differences of Brucella isolates from animals and humans in Türkiye.MethodsWe used whole genome sequencing (WGS) to assess the genetic diversity of Brucella isolates from 41 provinces in Türkiye and compared with isolates from other countries. We applied allele-based typing and core genome single nucleotide polymorphism (cgSNP) determination.ResultsOf the 106 Turkish Brucella isolates included, 57 were B. abortus and 49 were B. melitensis. One B. melitensis and two B. abortus isolates were identified as vaccine strains. Most (n = 55) B. abortus isolates clustered in three major branches, with no spatial discernible pattern. Of the B. melitensis isolates, 48 were assigned to the Eastern Mediterranean lineage with no discernible patterns between host species, location and sampling date. The Turkish isolates clustered with isolates from neighbouring countries such as Greece and Syria, but some also with isolates from human patients in European countries, like Germany, Norway and Sweden, suggesting that the source may be travel-related.ConclusionSeveral B. melitensis and B. abortus lineages are circulating in Türkiye. To decrease the prevalence and prevent brucellosis in animals and humans, stricter control measures are needed, particularly in areas where humans and animals have close contact. Furthermore, illegal transportation of animals across borders should be more closely controlled and regulated.

Genomic analysis of multi-drug resistant coagulase-negative staphylococci from healthy humans and animals revealed unusual mechanisms of resistance and CRISPR-Cas system.

Coagulase-negative staphylococci (CoNS) are evolving as major reservoirs and vectors of unusual and critical antimicrobialresistance (AMR) mechanisms. In this study, the genomic characterization of 26 multidrug-resistant (MDR)-CoNS (S. borealis, S. saprophyticus, S. sciuri, S. hominis, S. epidermidis, S. pasteuri, S. hyicus, S. simulans, S. haemolyticus, and S. arlettae) previously obtained from the nasal cavity of healthy nestling storks, humans who had no contact with animals, pigs, and pig farmers, as well as dogs and dog owners from Spain was performed. High-quality draft genomes obtained by Illumina sequencing technology were used to determine their resistome, virulome, mobile genetic elements, and CRISPR-Cas types. The relatedness of three CoNS species with publicly available genomes was assessed by core-genome single nucleotidepolymorphisms (SNPs). AMR genes to all classes of antibiotics in staphylococci were detected including unusual ones (mecC, ermT, and cfr), of which their corresponding genetic organizations were analyzed. About 96.1% of the MDR-CoNS strains harbored diverse adherence or immune evasion genes. Remarkably, one enterotoxin-C and -L-carrying S. epidermidis-ST595 strain from a nestling stork was detected. Moreover, various plasmid bound-biocide resistance genes (qacACGJ) were identified in 34.6% of the MDR-CoNS. Two genes that encode for cadmium and zinc resistance (cadD, czrC) were found, of which czrC predominated (42.3%). Complete CRISPR-Cas system was detected in 19.2% of the CoNS strains, of which cas-1, -2, and -9 predominated, especially in 75% of the S. borealis strains. The phylogenetic analysis identified clusters of related S. epidermidis lineages with those of other countries (SNP < 100). Also, highly related S. borealis isolates (SNP < 10) from pigs was confirmed for the first time in Spain. These findings showed that various ecological niches harbor CoNS that presented MDR phenotypes mediated by multiple AMR genes carried by mobile genetic elements with relatively low frequency of intact CRISPR-Cas systems. Furthermore, the transmission of some CoNS species in humans and animals is strongly suggested.

Clinical, phenotypic characterization and genomic analysis of the mucoid Acinetobacter baumannii from a teaching hospital

BackgroundAcinetobacter baumannii (A. baumannii) has become a significant nosocomial pathogen globally over the past decade due to the increasing prevalence of antibiotic-resistant isolates. The formation of the mucoid phenotype is a crucial adaptive defense response to external pressure, but the clinical, phenotypic and genotypic characteristics and their relationship with sequence types (ST) and K locus (KL) types remain unclear. MethodsIn this study, we screened a total of 736 A. baumannii isolates, from which we identified and characterized 13 mucoid isolates. The study explored the clinical characteristics of patients with mucoid isolates, investigated the mucoid phenotype, performed capsule observation, quantified capsule production, and assessed antimicrobial susceptibility. Subsequently, whole-genome sequencing (WGS) was used to analyze the sequence types (ST), loci for capsular polysaccharide (KL), antibiotic resistance genes, virulence genes, and core-genome single-nucleotide polymorphisms (SNPs). Additionally, the virulence of all mucoid strains was evaluated through serum resistance assay, biofilm-forming assay, and Galleria mellonella survival assay. ResultsAll mucoid A. baumannii isolates were found to be encapsulated and extremely drug-resistant. Among patients infected with these isolates, 92.3 % had pulmonary infections, and the 30-day mortality rate was 61.5 %. The analysis revealed that not all strains are highly virulent. Whole-genome sequencing (WGS) identified the sequence types as ST136, ST208, ST381, ST195, and ST281, and the capsular types as KL77, KL7, KL33, KL2, and KL3. The ST208 and KL7 isolates exhibited higher virulence and greater biofilm formation, with KL7 isolates also showing higher capsule production. Despite these differences, no significant variations in virulence genes were observed among the mucoid isolates, except for biofilm-associated and quorum-sensing genes. The highly virulent ST208/KL7 strains (AB276, AB313, and AB552) lacked biofilm-associated genes (csuA/BABCDE), indicating these genes do not directly cause differences in biofilm formation. ConclusionThe mucoid A. baumannii isolates were extensively drug-resistant, and infections caused by these isolates could lead to higher mortality. However, not all strains had high virulence, with variations likely related to specific sequence types (ST) and K locus (KL) types.

Prevalence and Characteristics of Salmonella from Tibetan Pigs in Tibet, China.

This study aimed to understand the epidemiological characteristics of Salmonella in Tibetan pigs. We isolated, identified, and examined via antimicrobial susceptibility testing on Salmonella from Tibetan pigs breeder farms and slaughterhouses in Tibet, China. A genetic evolutionary tree was constructed on the basis of whole genome sequencing (WGS). A total of 81 Salmonella isolates were isolated from 987 samples. The main serovars were Salmonella Typhimurium and Salmonella London in Tibetan pigs. The isolated Salmonella Typhimurium isolates subjected to antimicrobial susceptibility testing showed varying degrees of resistance to β-lactams, aminoglycosides, fluoroquinolones, sulfonamides, tetracyclines, and amphenicols. WGS analysis was performed on 20 Salmonella Typhimurium isolates in Tibet (n = 10), Jiangsu (n = 10), and 205 genome sequences downloaded from the Enterobase database to reveal their epidemiological and genetic characteristics. They were divided into two clusters based on core genome single-nucleotide polymorphisms: Cluster A with 112 isolates from Tibet and other regions in China and Cluster B with 113 isolates from Jiangsu and other regions. The isolates in Cluster A were further divided into two subclusters: A-1 with 40 isolates including Tibet and A-2 with 72 isolates from other regions. Virulence factors analysis revealed that all isolates from Tibet carried adeG, but this observation was not as common in Salmonella isolates from Jiangsu and other regions of China. Antibiotic resistance genes (ARGs) analysis showed that all isolates from Tibet carried blaTEM-55 and rmtB, which were absent in Salmonella isolates from Jiangsu and other regions of China. Genetic characteristic analysis and biofilm determination indicated that the biofilm formation capabilities of the isolates from Tibet were stronger than those of the isolates from Jiangsu and other regions of China. Our research revealed the epidemic patterns and genomic characteristics of Salmonella in Tibetan pigs and provided theoretical guidance for the prevention and control of local salmonellosis.

Rapid Whole Genome Characterization of High-Risk Pathogens Using Long-Read Sequencing to Identify Potential Healthcare Transmission.

Routine use of whole genome sequencing (WGS) has been shown to help identify transmission of pathogens causing healthcare-associated infections (HAIs). However, the current gold standard of short-read, Illumina-based WGS is labor and time-intensive. In light of recent improvements in long-read Oxford Nanopore Technologies (ONT) sequencing, we sought to establish a low resource utilization approach capable of providing accurate WGS-based comparisons of HAI pathogens within a time frame allowing for infection prevention and control (IPC) interventions. WGS was prospectively performed on antimicrobial-resistant pathogens at increased risk of potential healthcare transmission using the ONT MinION sequencer with R10.4.1 flow cells and Dorado basecalling algorithm. Potential transmission was assessed via Ridom SeqSphere+ for core genome multilocus sequence typing and MINTyper for reference-based core genome single nucleotide polymorphisms using previously published cut-off values. The accuracy of our ONT pipeline was determined relative to Illumina-based WGS data generated from the same genomic DNA sample. Over a six-month period, 242 bacterial isolates from 216 patients were sequenced by a single operator. Compared to the Illumina gold-standard data, our ONT pipeline achieved a Q score of 60 for assembled genomes, even with a coverage rate of as low as 40X. The mean time from initiating DNA extraction to complete genetic analysis was 2 days (IQR 2-3.25 days). We identified five potential transmission clusters comprising 21 isolates (8.7% of all sequenced strains). Combining ONT WGS data with epidemiological data, >70% (15/21) of the isolates originated from patients with potential healthcare transmission links. Via a stand-alone ONT pipeline, we detected potentially transmitted HAI pathogens rapidly and accurately, aligning closely with epidemiological data. Our low-resource method has the potential to assist in the efficient detection and deployment of preventative measures against HAI transmission.

An Evaluation of the Lineage of Brucella Isolates in Turkey by a Whole-Genome Single-Nucleotide Polymorphism Analysis.

Brucellosis is a disease caused by the Brucella (B.) species. It is a zoonotic disease that affects farm animals and causes economic losses in many countries worldwide. Brucella has the ability to persist in the environment and infect the host at low doses. Thus, it is more important to trace brucellosis outbreaks, identify their sources of infection, and interrupt their transmission. Some countries already have initial data, but most of these data are based on a Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA), which is completely unsuitable for studying the Brucella genome. Since brucellosis is an endemic disease in Turkey, this study aimed to examine the genome of Turkish Brucella isolates collected between 2018 and 2020, except for one isolate, which was from 2012. A total of 28 strains of B. melitensis (n = 15) and B. abortus (n = 13) were analyzed using a core-genome single-nucleotide polymorphism (cgSNP) analysis. A potential connection between the Turkish isolates and entries from Sweden, Israel, Syria, Austria, and India for B. melitensis was detected. For B. abortus, there may be potential associations with entries from China. This explains the tight ties found between Brucella strains from neighboring countries and isolates from Turkey. Therefore, it is recommended that strict measures be taken and the possible effects of uncontrolled animal introduction are emphasized.

Simultaneous clonal spread of NDM-1–producing Pseudomonas aeruginosa ST773 from Ukrainian patients in the Netherlands and Spain

ObjectivesWe describe the clonal spread of New Delhi metallo-β-lactamase (NDM) 1–producing Pseudomonas aeruginosa isolates belonging to the ST773 clone in Spain and the Netherlands, associated with the transfer of Ukrainian patients during the war. MethodsBetween March and December 2022, nine NDM-1–producing P. aeruginosa ST773 isolates were recovered from nine Ukrainian patients evacuated to two Spanish (n = 3) and five Dutch (n = 6) hospitals. Antimicrobial susceptibility testing was studied (Sensititre, Microscan, EUCAST-2023). Whole genome sequencing (Illumina, Oxford-Nanopore) was used to analyze the genetic relatedness, the resistome, and the prophage content. ResultsAll NDM-1–producing P. aeruginosa ST773 isolates exhibited resistance to all tested antimicrobials except colistin, aztreonam, and cefiderocol. Genomic analysis revealed that all isolates had an identical resistome and a chromosomally encoded integrative conjugative element carrying the blaNDM-1 gene. The core genome multilocus sequence typing and core genome single nucleotide polymorphisms analysis showed highly related isolates, irrespective of country of isolation, distant from other NDM-1-ST773 P. aeruginosa not collected in Ukraine. Both analysis revealed two closely related clusters, spanning the Spanish and Dutch isolates. In addition, a high content of prophages was identified in all strains, most of them in more than one isolate simultaneously, regardless of their origin country. Moreover, an identical phage tail-like bacteriocin cluster was identified in all NDM-1-ST773 P. aeruginosa. ConclusionsWe report a clonal dissemination of NDM-producing P. aeruginosa ST773 to the Netherlands and Spain associated with patients from Ukraine. Our work highlights the importance of genomic surveillance and to understand the dynamics of resistance in multidrug-resistant bacteria after the transfer of patients from conflict zones. International collaboration is crucial to address global antimicrobial resistance.

Use of Whole Genome Sequencing for Investigation of Potential Hospital-Acquired Vancomycin Resistant Enterococcus

Background: Whole genome sequencing (WGS) is a relatively new method for analyzing outbreaks and modes of transmission, particularly for multidrug resistant bacteria. This study sought to investigate clusters of patients with genetically related Vancomycin-Resistant Enterococcus spp. (VRE) bacteremia for shared hospital environmental exposures. Methods: All VRE blood culture isolates from patients from July 1, 2021 to June 30, 2022 underwent Illumina WGS. Core single nucleotide polymorphisms (SNPs) were identified, and multi-locus sequence typing (MLST) was performed across the VRE isolates. Clusters were defined as isolates with 15 or fewer core genome SNPs and were investigated for potential transmission routes. For each cluster, patients were evaluated in the 12 weeks before and after the first VRE isolate for shared hospital environmental exposures (hospital unit, patient rooms, procedural rooms, and radiology suites). Hospital units were comprised of patient rooms located geographically together on the same floor of the hospital. Results: A total of 82 VRE isolates underwent WGS. Thirty-eight (46%) clustered genetically with at least one other isolate. Clusters included 2 to 15 patients per group and represented 10 distinct MLST subgroups (Figure 1). Nine hundred and thirty-nine hospital environmental exposures were identified across the 38 patients. For each cluster, there was a total of 341 (36.3%) shared exposures. Shared environmental exposures occurred in radiology suites (35, 38.5%), patient rooms (32, 35.6%) and procedural rooms (23, 25.6%). Of the patients who shared the same hospital unit, 10 (31.3%) had the same patient room with 7 (70%) of them being in the emergency department (ED). Overall, the ED represented 7 (21.9%) of the shared hospital units. Each cluster had at least one shared hospital environmental exposure found. Conclusions: Use of WGS can help investigate outbreak clusters of resistant organisms such as VRE. In this study, nearly half of all VRE blood isolates were able to be segregated into clusters with at least one other isolate. Although VRE colonization of hospital rooms is well described, patient rooms represented the smallest proportion of shared hospital environmental exposures. This study thus suggests other environmental transmission routes such as radiology suites and procedural rooms warrant closer investigation.

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

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. 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. 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. 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. 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.

Genomic Characterization and Phylogenetic Analysis of Linezolid-Resistant Enterococcus from the Nostrils of Healthy Hosts Identifies Zoonotic Transmission

Linezolid resistance in Enterococcus spp. is increasingly considered critically important and a public health threat which mandates the need to understand their genomic contents and dissemination patterns. Here, we used whole-genome sequencing to characterize the resistome, virulome and mobile genetic elements of nine linezolid-resistant (LZDR) enterococci (seven optrA-E. faecalis, one poxtA-E. faecium and one optrA-E. casseliflavus) previously obtained from the nares of healthy dogs, pigs, pig farmers and tracheal samples of nestling storks in Spain. Also, the relatedness of the isolates with publicly available genomes was accessed by core-genome single nucleotide polymorphism (SNP) analysis. The optrA gene of the E. faecalis and E. casseliflavus isolates was located downstream of the fexA gene. The optrA gene in the E. casseliflavus isolate was carried in a plasmid (pURX4962), while those in the seven E. faecalis isolates were chromosomally located. The OptrA proteins were mostly variants of wild type (DP-2: Y176D/T481P; RDK: I104R/Y176D/E256K; DD-3: Y176D/G393D; and EDD: K3E/Y176D/G393D), except two that were wild type (one E. faecalis and one E. casseliflavus). The poxtA gene in the E. faecium isolate was found alone within its contig. The cfrD was upstream of ermB gene in the E. casseliflavus isolate and flanked by ISNCY and IS1216. All the LZDR enterococci carried plasmid rep genes (2–3) containing tetracycline, chloramphenicol and aminoglycoside resistance genes. All isolates except E. casseliflavus carried at least one intact prophage, of which E. faecalis-ST330 (X4957) from a pig carried the highest (n = 5). Tn6260 was associated with lnuG in E. faecalis-ST330 while Tn554 was with fexA in E. feaecalis-ST59 isolates. All except E. casseliflavus (n = 0) carried at least two metal resistance genes (MRGs), of which poxtA-carrying E. faecium-ST1739 isolate contained the most (arsA, copA, fief, ziaA, znuA, zosA, zupT, and zur). SNP-based analyses identified closely related optrA-E. faecalis isolates from a pig and a pig farmer on the same farm (SNP = 4). Moreover, optrA- carrying E. faecalis-ST32, -ST59, and -ST474 isolates from pigs were related to those previously described from humans (sick and healthy) and cattle in Spain, Belgium, and Switzerland (SNP range 43–86). These findings strongly suggest the transmission of LZDR-E. faecalis between a pig and a pig farmer and potential inter-country dissemination. These highlight the need to strengthen molecular surveillance of LZDR enterococci in all ecological niches and body parts to direct appropriate control strategies.

Antimicrobial resistance and genome characteristics of Salmonella enteritidis from Huzhou, China.

Salmonella enteritidis is a main pathogen responsible for sporadic outbreaks of gastroenteritis, and therefore is an important public health problem. This study investigated the drug resistance and genomic characteristics of S. enteritidis isolated from clinical and food sources in Huzhou, Zhejiang Province, China, from February 1, 2021, to December 30, 2023. In total, 43 S. enteritidis strains isolated during the study period were subjected to virulence gene, drug resistance gene, genetic correlation, antibiotic resistance, and multilocus sequence typing analyses. All 43 isolates were identified as ST11, and contained 108 virulence-related genes. Drug sensitivity analysis of the 43 isolates showed resistance rates of 100% to nalidixic acid and 90.70% to ampicillin and ampicillin/sulbactam. Multidrug resistance is a serious issue, with 81.40% of strains resistant to three or more antibacterial drugs. Genome sequencing indicated that S. enteritidis possessed 23 drug resistance genes, of which 14 were common to all 43 isolates. Phylogenetic analysis based on core genome single-nucleotide polymorphisms divided the 43 S. enteritidis strains into three clusters, with the 10 samples from an outbreak forming an independent branch located in cluster 3.

Genomic analysis of Clostridium perfringens type D isolates from goat farms

C. perfringens type D strains are the leading cause of enterotoxaemia in ruminants such as goats, sheep, and cattle. However, there has been no prior research on the genomic characteristics of C. perfringens type D strains from various regions in China. Here, we investigated the antibiotic resistance, genomic characteristics, and phylogenetic relationship of C. perfringens type D isolates recovered from goat farms in Shaanxi, Gansu, and Ningxia provinces. The antibiotic resistance test indicated that the isolates displayed high minimum inhibitory concentration (MIC) values to sulfafurazole, whereas the other antibiotics tested, such as penicillin, enrofloxacin, and florfenicol, worked well on them. Additionally, only tetracycline resistance genes [tetA(P) and tetB(P)] were identified from the isolates. A collective of 13 toxin genes, including etx and cpe were detected among the isolates. Sequence comparison revealed that the etx and cpe genes shared high sequence identities, and they could coexist on a pCW3-like plasmid, representing a potential risk to both animal breeding and public health. Phylogenetic analysis using core genome multi-locus sequence typing (cgMLST) and core genome single nucleotide polymorphisms (SNPs) revealed the close genetic relationship and potential regional/transregional transmission of the C. perfringens type D isolates in Shaanxi and Gansu provinces. Furthermore, pan-genomic analysis suggested the functional differences at the protein-coding gene level, although isolates from the same source shared a close genetic relationship. In conclusion, this study indicated the antibiotic resistance, virulence markers, potential transregional transmission, and genomic diversity of C. perfringens type D strains from various regions in China, which could provide references for the prevention of C. perfringens foodborne diseases and further research.

Cereulide production capacities and genetic properties of 31 emetic Bacillus cereus group strains

The highly potent toxin cereulide is a frequent cause of foodborne intoxications. This extremely resistant toxin is produced by Bacillus cereus group strains carrying the plasmid encoded cesHPTABCD gene cluster. It is known that the capacities to produce cereulide vary greatly between different strains but the genetic background of these variations is not clear.In this study, cereulide production capacities were associated with genetic characteristics. For this, cereulide levels in cultures of 31 strains were determined after incubation in tryptic soy broth for 24 h at 24 °C, 30 °C and 37 °C. Whole genome sequencing based data were used for an in-depth characterization of gene sequences related to cereulide production. The taxonomy, population structure and phylogenetic relationships of the strains were evaluated based on average nucleotide identity, multi-locus sequence typing (MLST), core genome MLST and single nucleotide polymorphism analyses. Despite a limited strain number, the approach of a genome wide association study (GWAS) was tested to link genetic variation with cereulide quantities.Our study confirms strain-dependent differences in cereulide production. For most strains, these differences were not explainable by sequence variations in the cesHPTABCD gene cluster or the regulatory genes abrB, spo0A, codY and pagRBc. Likewise, the population structure and phylogeny of the tested strains did not comprehensively reflect the cereulide production capacities. GWAS yielded first hints for associated proteins, while their possible effect on cereulide synthesis remains to be further investigated.

Epidemiological characteristics of human- and chicken-derived CTX-M-type extended-spectrum β-lactamase-producing Escherichia coli from China

Bacterial resistance to β-lactams is mainly attributed to CTX-M-type extended-spectrum β-lactamases (ESBLs). However, the predominant sequence type (ST) of blaCTX-M-carrying Escherichia coli (blaCTX-M-Ec) in chickens, an important food animal, in China and its contribution to human β-lactam resistance are not investigated. In this study, approximately 1808 chicken-derived strains collected from 10 provinces from 2012 to 2020 were screened for blaCTX-M-Ec, and 222 blaCTX-M-Ec were identified. Antimicrobial susceptibility tests, whole genome sequencing and conjugation experiment were performed. All quality-controlled 136 chicken-derived blaCTX-M-Ec and 1193 human-derived blaCTX-M-Ec genomes were downloaded from NCBI and EnteroBase to comprehensively analyze the prevalence of blaCTX-M-Ec in China. blaCTX-M-55 (153/358, 42.7% in chicken isolates; 312/1193, 26.2% in human isolates) and blaCTX-M-14 (92/358, 25.7% in chicken isolates; 450/1193, 37.7% in human isolates) were dominant in blaCTX-M-Ec. The STs of blaCTX-M-Ec were diverse and scattered, with ST155 (n = 21) and ST152 (n = 120) being the most abundant in chicken- and human-derived isolates, respectively. Few examples indicated that chicken- and human-derived blaCTX-M-Ec have 10 or less core genome single nucleotide polymorphisms (cgSNPs). Genetic environment analysis indicated that ISEcp1, IS26 and IS903B were closely associated with blaCTX-M transfer. The almost identical pc61–55 and pM-64–1161 indicated the possibility of plasmid-mediated transmission of blaCTX-M between humans and chickens. Although the genomes of most blaCTX-M-Ec isolated from chickens and humans were quite different, the prevalence and genetic environment of blaCTX-M variants in both hosts were convergent. CTX-M-mediated resistance is more likely to spread through horizontal gene transmission than bacterial clones.

Traces of pandemic fluoroquinolone-resistant Escherichia coli clone ST131 transmitted from human society to aquatic environments and wildlife in Japan

Transmission of antimicrobial-resistant bacteria among humans, animals, and the environment is a growing concern worldwide. The distribution of an international high-risk fluoroquinolone-resistant Escherichia coli clone, ST131, has been documented in clinical settings. However, the transmission of ST131 from humans to surrounding environments remains poorly elucidated. To comprehend the current situation and identify the source of ST131 in nature, we analyzed the genetic features of ST131 isolates from the aquatic environment (lake/river water) and wildlife (fox, raccoon, raccoon dog, and deer) and compared them with the features of isolates from humans in Japan using accessory and core genome single nucleotide polymorphism (SNP) analyses. We identified ST131 isolates belonging to the same phylotype and genome clusters (four of eight clusters were concomitant) with low SNP distance between the human isolates and those from the aquatic environment and wildlife. These findings warn of ST131 transmission between humans and the surrounding environment in Japan.

Transmission of ceftazidime-avibactam-resistant Escherichia coli among pets, veterinarians and animal hospital environment

Ceftazidime-avibactam (CZA) is a recently approved combination synthetic β-lactamase inhibitor used in human clinical medicine. Cases of CZA resistance in humans have already been reported, but limited research has investigated CZA resistance in pets. This study explored the prevalence and transmission of CZA-resistant Escherichia coli (CZAREC) among pets, their owners, veterinarians, and the environment in animal hospitals. A total of 5,419 clinical samples were collected from dogs and cats, along with samples from the environment (n = 5,843), veterinarians (n = 557), and pet owners (n = 368) in animal hospitals. From these samples, 760 Escherichia coli (E. coli) isolates were obtained, out of which 60 were identified as CZAREC. These included 34 isolates from the environment (9.14 %, n = 372), three from veterinarians (8.11 %, n = 37), and 23 from animals (6.82 %, n = 337). No CZAREC isolates were found in pet owners. The predominant sequence types of CZARECs were ST156 (n = 20), ST410 (n = 19) and ST101 (n = 7). Bayesian analysis revealed six clusters comprising 47 isolates from the hospital environment, pets, and veterinaries, displaying genetic relatedness of less than 100 core genome single nucleotide polymorphisms (cgSNPs) between any two isolates in each cluster. Some CZAREC isolates with high genetic similarity persisted in the same animal hospital for four to six months. Moreover, discriminant analysis of principal components indicated that most isolates from different hosts shared a genetic source in the human/dog/cat merged cluster. Overall, evidence of CZARECs transmission was found among pets, the environment, and veterinarians in animal hospitals. The findings emphasize the importance of monitoring CZARECs in the veterinary clinical setting to ensure the health of both pets and humans.

Evaluating Fourier-transform infrared spectroscopy with IR Biotyper as a faster and simpler method for investigating the sources of an outbreak of legionellosis.

Fourier-transform infrared (FTIR) spectroscopy using the IR Biotyper and core genome singlenucleotide polymorphism (cgSNP) analysis were performed on 12 Legionella isolates associated with an outbreak at a spa house in Kanagawa Prefecture, Japan, and 3 non-outbreak isolates. The discriminative power of FTIR spectroscopy for 48-h incubation conditions of L. pneumophila in this outbreak was lower than cgSNP-based typing but higher than serogroup typing. FTIR spectroscopy could screen outbreak isolates from a group of genetically related isolates and may be useful as an initial typing method in Legionella outbreak investigations.