Core Genome Multilocus Sequence Typing Research Articles

Characteristics of Clinical Isolates of Listeria monocytogenes in Sichuan, China, in 2022 Based on Whole Genome Sequencing Analysis.

Listeria monocytogenes is a foodborne pathogen. In 2022, we collected 15 strains of L. monocytogenes isolated from patients in some foodborne disease sentinel monitoring hospitals in Sichuan Province. Through whole genome sequencing (WGS), we obtained the virulence genes carried by the strains, multi-locus sequence typing (MLST), core genome MLST (cgMLST), clonal complex (CC), and serum groups and constructed a phylogenetic tree and minimum spanning tree with nonhuman strains. An analysis shows that all 15 strains of L. monocytogenes carry virulence genes LIPI-1 and LIPI-2, whereas the carrying rates of LIPI-3 and LIPI-4 virulence genes are relatively low. The MLST typing results showed a total of 10 sequence types (ST), including 10 CCs, with ST7 being the dominant type. The cgMLST clearly distinguishes strains of different lineages and CC types. The serum group is divided into three types: IIa, IIb, and IVb, with IIa being the dominant serum group. An analysis of antibiotic genes showed that all 15 strains carried FosX, lin, mprF, and norB with high carrying rates. The minimum inhibitory concentration results indicated that all were susceptible to eight antibiotics (ampicillin, penicillin, tetracycline, meropenem, erythromycin, vancomycin, ciprofloxacin, and trimethoprim-sulfamethoxazole). The analysis of strains isolated from different sources of Listeria revealed varying degrees of diversity, and the contamination of meat and environment within the province is closely related to clinical cases. L. monocytogenes isolated from clinical cases in Sichuan Province carry multiple virulence and antibiotic genes, with high potential pathogenicity. It is necessary to further strengthen the monitoring and control of food and environment by L. monocytogenes within Sichuan Province.

Automating the Illumina DNA library preparation kit for whole genome sequencing applications on the flowbot ONE liquid handler robot.

Whole-genome sequencing (WGS) is currently making its transition from research tool into routine (clinical) diagnostic practice. The workflow for WGS includes the highly labor-intensive library preparations (LP), one of the most critical steps in the WGS procedure. Here, we describe the automation of the LP on the flowbot ONE robot to minimize the risk of human error and reduce hands-on time (HOT). For this, the robot was equipped, programmed, and optimized to perform the Illumina DNA Prep automatically. Results obtained from 16 LP that were performed both manually and automatically showed comparable library DNA yields (median of 1.5-fold difference), similar assembly quality values, and 100% concordance on the final core genome multilocus sequence typing results. In addition, reproducibility of results was confirmed by re-processing eight of the 16 LPs using the automated workflow. With the automated workflow, the HOT was reduced to 25min compared to the 125min needed when performing eight LPs using the manual workflow. The turn-around time was 170 and 200min for the automated and manual workflow, respectively. In summary, the automated workflow on the flowbot ONE generates consistent results in terms of reliability and reproducibility, while significantly reducing HOT as compared to manual LP.

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.

Surveillance of stewed meat products in Shanghai, China: Importance of controlling processing environment contamination for Listeria monocytogenes

Human listeriosis is a foodborne disease caused by Listeria monocytogenes contamination. Most cases occur due to consumption of ready-to-eat (RTE) foods. Stewed meat in seasoning, a type of pre-packaged ready-to-eat (RTE) food, is a popular traditional product in China. We used whole genome sequencing to analyze core genome multilocus sequence typing (cgMLST), serogroups and multilocus typing sequence typing (ST) of 67 L. monocytogenes isolates. The L. monocytogenes serogroup IIb-ST5 was predominantly isolated from different sources and linked to stewed meat in seasoning of Brand A, B, and C with <7 allelic differences by cgMLST, suggesting that this kind of food may be a high-risk food causing sporadic cases of listeriosis. Furthermore, L. monocytogenes isolates from different batches of stewed meat in seasoning products processed in the same processing plant had <5 five allelic differences, confirming that contamination occurred during manufacture. Additionally, 16 L. monocytogenes isolates from Plant A were the same clone (0 allelic difference), which confirmed the strong ability of L. monocytogenes to survive in the processing environment. Thus, increased and frequent monitoring, as well as constant technical support for the control of L. monocytogenes in food processing plants is necessary.

A core genome multi-locus sequence typing scheme for Streptococcus uberis: an evolution in typing a genetically diverse pathogen.

Streptococcus uberis is a globally endemic and poorly controlled cause of bovine mastitis impacting the sustainability of the modern dairy industry. A core genome was derived from 579 newly sequenced S. uberis isolates, along with 305 publicly available genome sequences of S. uberis isolated from 11 countries around the world and used to develop a core genome multi-locus sequence typing (cgMLST) scheme. The S. uberis core genome comprised 1475 genes, and these were used to identify 1447 curated loci that were indexed into the cgMLST scheme. This was able to type 1012 of 1037 (>97  %) isolates used and differentiated the associated sequences into 932 discrete core genome sequence types (cgSTs). Analysis of the phylogenetic relationships of cgSTs revealed no clear clustering of isolates based on metadata such as disease status or year of isolation. Geographical clustering of cgSTs was limited to identification of a UK-centric clade, but cgSTs from UK isolates were also dispersed with those originating from other geographical regions across the entire phylogenetic topology. The cgMLST scheme offers a new tool for the detailed analysis of this globally important pathogen of dairy cattle. Initial analysis has re-emphasized and exemplified the genetically diverse nature of the global population of this opportunistic pathogen.

Genomic analysis of inter-hospital transmission of vancomycin-resistant Enterococcus faecium sequence type 80 isolated during an outbreak in Hiroshima, Japan.

Outbreaks caused by vancomycin-resistant enterococci that transcend jurisdictional boundaries are occurring worldwide. This study focused on a vancomycin-resistant enterococcus outbreak that occurred between 2018 and 2021 across two cities in Hiroshima, Japan. The study involved genetic and phylogenetic analyses using whole-genome sequencing of 103 isolates of vancomycin-resistant enterococci to identify the source and transmission routes of the outbreak. Phylogenetic analysis was performed using core genome multilocus sequence typing and core single-nucleotide polymorphisms; infection routes between hospitals were inferred using BadTrIP. The outbreak was caused by Enterococcus faecium sequence type (ST) 80 carrying the vanA plasmid, which was derived from strain A10290 isolated in India. Of the 103 isolates, 93 were E. faecium ST80 transmitted across hospitals. The circular vanA plasmid of the Hiroshima isolates was similar to the vanA plasmid of strain A10290 and transferred from E. faecium ST80 to other STs of E. faecium and other Enterococcus species by conjugation. The inferred transmission routes across hospitals suggest the existence of a central hospital serving as a hub, propagating vancomycin-resistant enterococci to multiple hospitals. Our study highlights the importance of early intervention at the key central hospital to prevent the spread of the infection to small medical facilities, such as nursing homes, with limited medical resources and a high number of vulnerable individuals.

Development and validation of a random forest algorithm for source attribution of animal and human Salmonella Typhimurium and monophasic variants of S. Typhimurium isolates in England and Wales utilising whole genome sequencing data.

Source attribution has traditionally involved combining epidemiological data with different pathogen characterisation methods, including 7-gene multi locus sequence typing (MLST) or serotyping, however, these approaches have limited resolution. In contrast, whole genome sequencing data provide an overview of the whole genome that can be used by attribution algorithms. Here, we applied a random forest (RF) algorithm to predict the primary sources of human clinical Salmonella Typhimurium (S. Typhimurium) and monophasic variants (monophasic S. Typhimurium) isolates. To this end, we utilised single nucleotide polymorphism diversity in the core genome MLST alleles obtained from 1,061 laboratory-confirmed human and animal S. Typhimurium and monophasic S. Typhimurium isolates as inputs into a RF model. The algorithm was used for supervised learning to classify 399 animal S. Typhimurium and monophasic S. Typhimurium isolates into one of eight distinct primary source classes comprising common livestock and pet animal species: cattle, pigs, sheep, other mammals (pets: mostly dogs and horses), broilers, layers, turkeys, and game birds (pheasants, quail, and pigeons). When applied to the training set animal isolates, model accuracy was 0.929 and kappa 0.905, whereas for the test set animal isolates, for which the primary source class information was withheld from the model, the accuracy was 0.779 and kappa 0.700. Subsequently, the model was applied to assign 662 human clinical cases to the eight primary source classes. In the dataset, 60/399 (15.0%) of the animal and 141/662 (21.3%) of the human isolates were associated with a known outbreak of S. Typhimurium definitive type (DT) 104. All but two of the 141 DT104 outbreak linked human isolates were correctly attributed by the model to the primary source classes identified as the origin of the DT104 outbreak. A model that was run without the clonal DT104 animal isolates produced largely congruent outputs (training set accuracy 0.989 and kappa 0.985; test set accuracy 0.781 and kappa 0.663). Overall, our results show that RF offers considerable promise as a suitable methodology for epidemiological tracking and source attribution for foodborne pathogens.

A Prolonged Outbreak of Enteric Fever Associated With Illegal Miners in the City of Matlosana, South Africa, November 2020-September 2022.

In South Africa, the annual incidence of enteric fever averaged 0.1 per 100 000 persons between 2003 and 2018. During 2021 an increase in the number of enteric fever cases was observed. An outbreak investigation was conducted to determine the magnitude and source of the outbreak. We performed a cross-sectional descriptive study. Data were collected through telephonic or face-to-face interviews with cases or proxies via a standardized case investigation form. Whole genome sequencing was performed on all Salmonella Typhi isolates. Drinking water samples were collected, tested, and analyzed. Descriptive analysis was performed with Microsoft Excel. Between January 2020 and September 2022, a cluster of 53 genetically highly related Salmonella Typhi isolates was identified from 5 provinces in South Africa. Isolates associated with the cluster showed ≤5 allelic differences, as determined following core genome multilocus sequence typing analysis. Most cases (60%, 32/53) were in the North West province. Males represented 68% (36/53). Of these, 72% (26/36) were aged 15 to 49 years, with a median age of 31 years. Where occupation was known within this age group, 78% (14/18) were illegal gold miners. Illegal miners reported illness onset while working underground. Five municipal tap water samples were tested and showed no evidence of fecal contamination. This outbreak predominantly affected illegal gold miners, likely due to the consumption of contaminated groundwater while working in a gold mine shaft. In addition, this investigation highlights the value of whole genome sequencing to detect clusters and support epidemiologic investigation of enteric fever outbreaks.

First isolation and genotyping of pathogenic Leptospira spp. from Austria

Leptospirosis is a globally distributed zoonotic disease. The standard serological test, known as Microscopic Agglutination Test (MAT), requires the use of live Leptospira strains. To enhance its sensitivity and specificity, the usage of locally circulating strains is recommended. However, to date, no local strain is available from Austria. This study aimed to isolate circulating Leptospira strains from cattle in Austria to enhance the performances of the routine serological test for both humans and animals. We used a statistical approach combined with a comprehensive literature search to profile cattle with greater risk of leptospirosis infection and implemented a targeted sampling between November 2021 and October 2022. Urine and/or kidney tissue were sampled from 410 cattle considered at higher risk of infection. Samples were inoculated into EMJH-STAFF culture media within 2–6 h and a real-time PCR targeting the lipL32 gene was used to confirm the presence/absence of pathogenic Leptospira in each sample. Isolates were further characterised by core genome multilocus sequence typing (cgMLST). Nine out of 429 samples tested positive by PCR, from which three isolates were successfully cultured and identified as Leptospira borgpetersenii serogroup Sejroe serovar Hardjobovis, cgMLST cluster 40. This is the first report on the isolation and genotyping of local zoonotic Leptospira in Austria, which holds the potential for a significant improvement in diagnostic performance in the country. Although the local strain was identified as a cattle-adapted serovar, it possesses significant zoonotic implications. Furthermore, this study contributes to a better understanding of the epidemiology of leptospirosis in Europe.

One Health surveillance of colistin-resistant Enterobacterales in Belgium and the Netherlands between 2017 and 2019.

Colistin serves as the last line of defense against multidrug resistant Gram-negative bacterial infections in both human and veterinary medicine. This study aimed to investigate the occurrence and spread of colistin-resistant Enterobacterales (ColR-E) using a One Health approach in Belgium and in the Netherlands. In a transnational research project, a total of 998 hospitalized patients, 1430 long-term care facility (LTCF) residents, 947 children attending day care centres, 1597 pigs and 1691 broilers were sampled for the presence of ColR-E in 2017 and 2018, followed by a second round twelve months later for hospitalized patients and animals. Colistin treatment incidence in livestock farms was used to determine the association between colistin use and resistance. Selective cultures and colistin minimum inhibitory concentrations (MIC) were employed to identify ColR-E. A combination of short-read and long-read sequencing was utilized to investigate the molecular characteristics of 562 colistin-resistant isolates. Core genome multi-locus sequence typing (cgMLST) was applied to examine potential transmission events. The presence of ColR-E was observed in all One Health sectors. In Dutch hospitalized patients, ColR-E proportions (11.3 and 11.8% in both measurements) were higher than in Belgian patients (4.4 and 7.9% in both measurements), while the occurrence of ColR-E in Belgian LTCF residents (10.2%) and children in day care centres (17.6%) was higher than in their Dutch counterparts (5.6% and 12.8%, respectively). Colistin use in pig farms was associated with the occurrence of colistin resistance. The percentage of pigs carrying ColR-E was 21.8 and 23.3% in Belgium and 14.6% and 8.9% in the Netherlands during both measurements. The proportion of broilers carrying ColR-E in the Netherlands (5.3 and 1.5%) was higher compared to Belgium (1.5 and 0.7%) in both measurements. mcr-harboring E. coli were detected in 17.4% (31/178) of the screened pigs from 7 Belgian pig farms. Concurrently, four human-related Enterobacter spp. isolates harbored mcr-9.1 and mcr-10 genes. The majority of colistin-resistant isolates (419/473, 88.6% E. coli; 126/166, 75.9% Klebsiella spp.; 50/75, 66.7% Enterobacter spp.) were susceptible to the critically important antibiotics (extended-spectrum cephalosporins, fluoroquinolones, carbapenems and aminoglycosides). Chromosomal colistin resistance mutations have been identified in globally prevalent high-risk clonal lineages, including E. coli ST131 (n = 17) and ST1193 (n = 4). Clonally related isolates were detected in different patients, healthy individuals and livestock animals of the same site suggesting local transmission. Clonal clustering of E. coli ST10 and K. pneumoniae ST45 was identified in different sites from both countries suggesting that these clones have the potential to spread colistin resistance through the human population or were acquired by exposure to a common (food) source. In pig farms, the continuous circulation of related isolates was observed over time. Inter-host transmission between humans and livestock animals was not detected. The findings of this study contribute to a broader understanding of ColR-E prevalence and the possible pathways of transmission, offering insights valuable to both academic research and public health policy development.

On the onset and dispersal of a major MDR TB clone among HIV-negative patients, Tunisia

BackgroundTo carry out a whole genome sequencing (WGS)-based investigation on the emergence and spread of the largest multidrug-resistant tuberculosis (MDR TB) outbreak that has been thriving among HIV-negative patients, Tunisia, since the early 2000s.MethodsWe performed phylogeographic analyses and molecular dating based on a WGS dataset representing 68 unique Mycobacterium tuberculosis isolates, covering almost the entire MDR TB outbreak for the time period 2001–2016.ResultsThe data indicate that the ancestor of the MDR TB outbreak emerged in the region of Bizerte, as early as 1974 (95% CI 1951–1985), from where it spread to other regions by 1992 (95% CI 1980–1996). Analysis of a minimum spanning tree based on core genome Multilocus Sequence Typing (cgMLST) uncovered the early spill-over of the fitness-compensated MDR TB strain from the prison into the general population. Indeed, cases with history of incarceration were found to be directly or indirectly linked to up to 22 new outbreak cases (32.35%) among the non-imprisoned population. By around 2008, the MDR TB outbreak strain had acquired additional resistance, leading to an XDR phenotype.ConclusionsWGS allowed refining our understanding of the emergence and evolution of the largest MDR TB outbreak in Tunisia, whose causative strain has been circulating silently for almost 26 years before. Our study lends further support to the critical role of prisons-related cases in the early spread of the outbreak among the general population. The shift to an XDR phenotype of such an epidemic clone prompts an urgent need to undertake drastic control measures.

Genetic epidemiology and plasmid-mediated transmission of mcr-1 by Escherichia coli ST155 from wastewater of long-term care facilities.

Long-term care facilities (LTCFs) for older people play an important and unique role in multidrug-resistant organism transmission. Herein, we investigated the genetic characteristics of mobile colistin resistance gene (mcr-1)-carrying Escherichia coli strains isolated from wastewater of LTCFs in Shanghai. Antimicrobial susceptibility test was carried out by agar dilution methods. Whole-genome sequencing and plasmid sequencing were conducted, and resistance genes and sequence types of colistin in E. coli isolates were analyzed. Core genome multilocus sequence typing (cgMLST) analysis was performed by the Ridom SeqSphere+ software. Phylogenetic tree through the maximum likelihood method was constructed by MEGA X. Out of 306 isolates, only 1 E. coli named ECSJ33 was found, and the plasmid pECSJ33 from ECSJ33 harbored the mcr-1 gene that was located with 59,080 bp belonging to IncI2 type. The plasmid pECSJ33 was capable of conjugation with an efficiency of 2.9 × 10-2. Bioinformatic analysis indicated pECSJ33 shared backbone with the previously reported mcr-1-harboring pHNGDF93 isolated from fish source. Moreover, the cgMLST analysis revealed that ECSJ33 belongs to different lineages from those reported from previous E. coli strains but shared high similarity to NCTC11129 in cluster 11. The phylogenetic tree revealed MCR-1 of ECSJ33 in this study was mostly of animal food origin and that they were closely related. Our study firstly reports detection of genome sequence of a multidrug-resistant mcr-1-harboring E. coli ST155 from wastewater of LTCF source in China. The data may prove that the plasmid pECSJ33 belongs to food origin and help to understand the antimicrobial resistance mechanisms and genomic features of colistin resistance under One Health approach.IMPORTANCEOne Escherichia coli named ECSJ33 was found from wastewater of a long-term care facility (LTCF) and the plasmid pECSJ33 from ECSJ33 harbored the mobile colistin resistance gene (mcr-1) that was located with 59,080 bp belonging to IncI2 type, which was capable of conjugation with an efficiency of 2.9 × 10-2. This paper firstly reports an mcr-1-carrying E. coli strain ST155 isolated from LTCF in China. Comparative genomics analysis indicated pECSJ33 shared backbone with the previously reported mcr-1-harboring pHNGDF93 isolated from fish source. The phylogenetic tree revealed MCR-1 protein of ECSJ33 in this study was mostly of animal food origin and that they were closely related. Therefore, the pECSJ33 could be considered as food-origin transmission mcr-1-harboring plasmid.

A 6-year retrospective study of clinical features, microbiology, and genomics of Shiga toxin-producing Escherichia coli in children who presented to a tertiary referral hospital in Costa Rica.

Shiga-toxin-producing Escherichia coli (STEC) is associated with diarrhea and hemolytic uremic syndrome (HUS). STEC infections in Costa Rica are rarely reported in children. We gathered all the records of STEC infections in children documented at the National Children's Hospital, a tertiary referral hospital, from 2015 to 2020. Clinical, microbiological, and genomic information were analyzed and summarized. A total of 3,768 diarrheal episodes were reviewed. Among them, 31 STEC were characterized (29 fecal, 1 urine, and 1 bloodstream infection). The prevalence of diarrheal disease due to STEC was estimated at 0.8% (n = 29/3,768), and HUS development was 6.4% (n = 2/31). The stx1 gene was found in 77% (n = 24/31) of STEC strains. In silico genomic predictions revealed a predominant prevalence of serotype O118/O152:H2, accompanied by a cluster exhibiting allele differences ranging from 33 to 8, using a core-genome multilocus sequence typing (cgMLST) approach. This is the first study using a genomic approach for STEC infections in Costa Rica.IMPORTANCEThis study provides a comprehensive description of clinical, microbiological, genomic, and demographic data from patients who attended the only pediatric hospital in Costa Rica with Shiga-toxin-producing Escherichia coli (STEC) infections. Despite the low prevalence of STEC infections, we found a predominant serotype O118/O152:H2, highlighting the pivotal role of genomics in understanding the epidemiology of public health threats such as STEC. Employing a genomic approach for this pathogen for the first time in Costa Rica, we identified a higher prevalence of STEC in children under 2 years old, especially those with gastrointestinal comorbidities, residing in densely populated regions. Limitations such as potential geographic bias and lack of strains due to direct molecular diagnostics are acknowledged, emphasizing the need for continued surveillance to uncover the true extent of circulating serotypes and potential outbreaks in Costa Rica.

Multiple interspecies recombination events documented by whole-genome sequencing in multidrug-resistant Haemophilus influenzae clinical isolates.

Haemophilus influenzae (Hi) was long known as an easy-to-treat bacterium, but increasing resistance against beta-lactams and other critically important antibiotics is now a growing concern. We describe here the whole-genome sequencing (WGS) analysis of three non-typeable Hi isolates received in 2018-2019 by the Belgian National Reference Centre (NRC) for Haemophilus influenzae, as they presented an unusual multi-resistant profile. All three isolates were sequenced by WGS and mapped to the reference isolate Hi Rd KW20. Shorten uptake signal sequences (USSs) known to be associated with homologous recombination were sought in ftsI, murE and murF genes, and inner partial sequences were compared against the blast nucleotide database to look for similarity with other Haemophilus species. Their antimicrobial resistance (AMR) genotype was studied. Core-genome multilocus sequence typing (MLST) was performed on the NTHi database pubMLST to place our isolates in the actual worldwide epidemiology. The isolates also harboured interspecies recombination patterns in the murF-murE-ftsI region involved in cell wall synthesis. The three isolates were multidrug resistant and two of them were also resistant to amoxicillin-clavulanic acid and showed a reduced susceptibility to meropenem. All three isolates belonged to the MLST clonal complex (CC) 422, and WGS revealed that the three were very similar. They harboured mobile genetic elements (carrying blaTEM-1B, mefA and msrD genes associated with resistance), mutations in gyrA and parC linked to fluoroquinolone resistance as well as remodelling events in ompP2 that might be related to lower carbapenem susceptibility. The Hi evolution towards antimicrobial multiresistance (AMR) is a complex and poorly understood phenomenon, although probably linked to a large degree to the presence of USSs and exchange within the family Pasteurellaceae. To better understand the respective roles of clonal expansion, horizontal gene transfers, spontaneous mutations and interspecies genetic rearrangements in shaping Hi AMR, both analysis of Hi communities over time within individuals and worldwide monitoring of non-typeable Hi causing infections should be conducted.

Salmonella Vitkin Outbreak Associated with Bearded Dragons, Canada and United States, 2020-2022.

We identified 2 cases of Salmonella enterica serovar Vitkin infection linked by whole-genome sequencing in infants in Ontario, Canada, during 2022. Both households of the infants reported having bearded dragons as pets. The outbreak strain was also isolated from an environmental sample collected from a patient's bearded dragon enclosure. Twelve cases were detected in the United States, and onset dates occurred during March 2021-September 2022 (isolates related to isolates from Canada within 0-9 allele differences by core-genome multilocus sequence typing). Most US patients (66.7%) were <1 year of age, and most (72.7%) had reported bearded dragon exposure. Hospitalization was reported for 5 (38.5%) of 13 patients. Traceback of bearded dragons identified at least 1 potential common supplier in Southeast Asia. Sharing rare serovar information and whole-genome sequencing data between Canada and the United States can assist in timely identification of outbreaks, including those that might not be detected through routine surveillance.

High performance Legionella pneumophila source attribution using genomics-based machine learning classification.

Fundamental to effective Legionnaires' disease outbreak control is the ability to rapidly identify the environmental source(s) of the causative agent, Legionella pneumophila. Genomics has revolutionized pathogen surveillance, but L. pneumophila has a complex ecology and population structure that can limit source inference based on standard core genome phylogenetics. Here, we present a powerful machine learning approach that assigns the geographical source of Legionnaires' disease outbreaks more accurately than current core genome comparisons. Models were developed upon 534 L. pneumophila genome sequences, including 149 genomes linked to 20 previously reported Legionnaires' disease outbreaks through detailed case investigations. Our classification models were developed in a cross-validation framework using only environmental L. pneumophila genomes. Assignments of clinical isolate geographic origins demonstrated high predictive sensitivity and specificity of the models, with no false positives or false negatives for 13 out of 20 outbreak groups, despite the presence of within-outbreak polyclonal population structure. Analysis of the same 534-genome panel with a conventional phylogenomic tree and a core genome multi-locus sequence type allelic distance-based classification approach revealed that our machine learning method had the highest overall classification performance-agreement with epidemiological information. Our multivariate statistical learning approach maximizes the use of genomic variation data and is thus well-suited for supporting Legionnaires' disease outbreak investigations.IMPORTANCEIdentifying the sources of Legionnaires' disease outbreaks is crucial for effective control. Current genomic methods, while useful, often fall short due to the complex ecology and population structure of Legionella pneumophila, the causative agent. Our study introduces a high-performing machine learning approach for more accurate geographical source attribution of Legionnaires' disease outbreaks. Developed using cross-validation on environmental L. pneumophila genomes, our models demonstrate excellent predictive sensitivity and specificity. Importantly, this new approach outperforms traditional methods like phylogenomic trees and core genome multi-locus sequence typing, proving more efficient at leveraging genomic variation data to infer outbreak sources. Our machine learning algorithms, harnessing both core and accessory genomic variation, offer significant promise in public health settings. By enabling rapid and precise source identification in Legionnaires' disease outbreaks, such approaches have the potential to expedite intervention efforts and curtail disease transmission.

Genomic surveillance of carbapenem-resistant Klebsiella pneumoniae reveals a prolonged outbreak of extensively drug-resistant ST147 NDM-1 during the COVID-19 pandemic in the Apulia region (Southern Italy)

The recent worldwide spread of New Delhi metallo-beta-lactamase-producing Klebsiella pneumoniae (NDM-KP) in health-care settings remains a concern. The aim of the study was to describe an outbreak of extensively drug-resistant ST147 NDM-1-KP in the Apulia region of Southern Italy that occurred between 2020 and 2022 through genomic surveillance of carbapenem-resistant Enterobacterales. A total of 459 carbapenem-resistant KP isolates collected from patients hospitalised with bloodstream infections were tested using a commercial multiplex real-time polymerase chain reaction to identify carbapenemase genes. A subset of 27 isolates was subjected to whole-genome sequencing. Core-genome multilocus sequence typing was performed by analysing a panel of 4884 genes. Molecular testing revealed that 104 (22.6%) isolates carried the carbapenemase NDM gene. Phylogenetic analysis of the 27 isolates subjected to whole-genome sequencing revealed high genetic relatedness among strains. All isolates were resistant to all first-line antibiotics. Virulome analysis identified the ybt locus, the two well-recognised virulence factors iucABCDiutA and rmpA, and the genes encoding the type 3 pilus virulence factor. Plasmids IncFIB(pkPHS1), IncFIB(pNDM-Mar), IncFIB(pQil), IncHI1B(pNDM-MAR), IncR, and Col(pHAD28) were identified in all isolates. Moreover, further analysis identified the IncFIB-type plasmid carrying the NDM-1 genes. The increasing circulation of extensively drug-resistant NDM-1 ST147 KP strains in Southern Italy in recent years is worrisome, because these clones pose a real risk, particularly in hospital settings. Genomic surveillance is a crucial tool for early identification of emerging threats such as the spread of high-risk pathogens. Rapid infection control measures and antimicrobial stewardship are key to preventing further spread of hypervirulent KP strains.

Antimicrobial Resistance in Enterococcus spp. Isolates from Red Foxes (Vulpes vulpes) in Latvia.

Antimicrobial resistance (AMR) is an emerging public health threat and is one of the One Health priorities for humans, animals, and environmental health. Red foxes (Vulpes vulpes) are a widespread predator species with great ecological significance, and they may serve as a sentinel of antimicrobial resistance in the general environment. The present study was carried out to detect antimicrobial resistance, antimicrobial resistance genes, and genetic diversity in faecal isolates of red foxes (Vulpes vulpes). In total, 34 Enterococcus isolates, including E. faecium (n = 17), E. faecalis (n = 12), E. durans (n = 3), and E. hirae (n = 2), were isolated. Antimicrobial resistance to 12 antimicrobial agents was detected with EUVENC panels using the minimum inhibitory concentration (MIC). The presence of antimicrobial resistance genes (ARGs) was determined using whole-genome sequencing (WGS). Resistance to tetracycline (6/34), erythromycin (3/34), ciprofloxacin (2/34), tigecycline (2/34), and daptomycin (2/34) was identified in 44% (15/34) of Enterococcus isolates, while all the isolates were found to be susceptible to ampicillin, chloramphenicol, gentamicin, linezolid, teicoplanin, and vancomycin. No multi-resistant Enterococcus spp. were detected. A total of 12 ARGs were identified in Enterococcus spp., with the presence of at least 1 ARG in every isolate. The identified ARGs encoded resistance to aminoglycosides (aac(6')-I, ant(6)-Ia, aac(6')-Iih and spw), tetracyclines (tet(M), tet(L) and tet(S)), and macrolide-lincosamide-streptogramin AB (lnu(B,G), lsa(A,E), and msr(C)), and their presence was associated with phenotypical resistance. Core genome multilocus sequence typing (cgMLST) revealed the high diversity of E. faecalis and E. faecium isolates, even within the same geographical area. The distribution of resistant Enterococcus spp. in wild foxes in Latvia highlights the importance of a One Health approach in tackling AMR.

Genomic insights into a blaNDM-5-carrying Escherichia coli ST167 isolate recovered from faecal sample of a healthy individual in China

ObjectivesSince its discovery, blaNDM-5 has spread widely amongst Escherichia coli strains in clinical patients, causing carbapenem resistance. Here we report the complete genome sequence of an NDM-5-producing E. coli strain isolated from the faecal sample of a healthy individual in Hangzhou, China. MethodsThe whole-genome sequence of E. coli CREC8 was obtained utilising both the Nanopore sequencer and the Illumina NovaSeq 6000 platform. Antimicrobial resistance genes, multilocus sequence typing, and plasmid replicons were identified using the BacWGSTdb server. The phylogenetic relationship between CREC8 and other E. coli strains was investigated using the core genome multilocus sequence typing (cgMLST) strategy. ResultsThe complete genome sequence of E. coli CREC8 consists of one chromosome and 7 plasmids. CREC8 belongs to ST167 according to the MLST scheme. Seven ARGs were identified, including carbapenem resistance gene blaNDM-5 which was located in an IncFIA/IncFII type plasmid. A total of 164 E. coli ST167 strains related to 25 countries across four continents can be retrieved from the NCBI database, 95 of them carrying the blaNDM gene with blaNDM-5 the most (N = 79). Phylogenetic analysis revealed a worldwide distribution of E. coli ST167 strains, with China having the highest prevalence (37%, 61/165). ConclusionIn summary, we reported a blaNDM-5-carrying E. coli ST167 strain isolated from a healthy individual in China. Such strains are more commonly isolated from hospitalised patients but are rarely isolated from healthy individuals. This indicates a further epidemic of carbapenem-resistant E. coli strains in the healthy population which needs our attention.

High Genetic Diversity in Third-Generation Cephalosporin-Resistant Escherichia coli in Wastewater Systems of Schleswig-Holstein.

The spread of multidrug-resistant bacteria from humans or livestock is a critical issue. However, the epidemiology of resistant pathogens across wastewater pathways is poorly understood. Therefore, we performed a detailed comparison of third-generation cephalosporin-resistant Escherichia coli (3GCREC) from wastewater treatment plants (WWTPs) to analyze dissemination pathways. A total of 172 3GCREC isolated from four WWTPs were characterized via whole genome sequencing. Clonal relatedness was determined using multi-locus sequence typing (MLST) and core genome MLST. Resistance genotypes and plasmid replicons were determined. A total of 68 MLST sequence types were observed with 28 closely related clusters. Resistance genes to eight antibiotic classes were detected. In fluoroquinolone-resistant isolates, resistance was associated with three-or-more point mutations in target genes. Typing revealed high genetic diversity with only a few clonal lineages present in all WWTPs. The distribution paths of individual lines could only be traced in exceptional cases with a lack of enrichment of certain lineages. Varying resistance genes and plasmids, as well as fluoroquinolone resistance-associated point mutations in individual isolates, further corroborated the high diversity of 3GCREC in WWTPs. In total, we observed high diversity of 3GCREC inside the tested WWTPs with proof of resistant strains being released into the environment even after treatment processes.