Genome Multilocus Sequence Typing Research Articles

Unraveling the impact of genome assembly on bacterial typing: a one health perspective

BackgroundIn the context of pathogen surveillance, it is crucial to ensure interoperability and harmonized data. Several surveillance systems are designed to compare bacteria and identify outbreak clusters based on core genome MultiLocus Sequence Typing (cgMLST). Among the different approaches available to generate bacterial cgMLST, our research used an assembly-based approach (chewBBACA tool).MethodsSimulations of short-read sequencing were conducted for 5 genomes of 27 pathogens of interest in animal, plant, and human health to evaluate the repeatability and reproducibility of cgMLST. Various quality parameters, such as read quality and depth of sequencing were applied, and several read simulations and genome assemblies were repeated using three tools: SPAdes, Unicycler and Shovill. In vitro sequencing were also used to evaluate assembly impact on cgMLST results, for six bacterial species: Bacillus thuringiensis, Listeria monocytogenes, Salmonella enterica, Staphylococcus aureus, Vibrio parahaemolyticus and Xylella fastidiosa.ResultsThe results highlighted variability in cgMLST, which not only related to the assembly tools, but also induced by the intrinsic composition of the genomes themselves. This variability observed in simulated sequencing was further validated with real data for six of the bacterial pathogens studied.ConclusionThis highlights that the intrinsic genome composition affects assembly and resulting cgMLST profiles, and that variability in bioinformatics tools can induce a bias in cgMLST profiles. In conclusion, we propose that the completeness of cgMLST schemes should be considered when clustering strains.

Molecular Characterization of Multidrug-Resistant and Hypervirulent New Delhi Metallo-Beta-Lactamase Klebsiella pneumoniae in Lazio, Italy: A Five-Year Retrospective Study

Background/Objectives: Antimicrobial resistance represents a challenge to public health systems because of the array of resistance and virulence mechanisms that lead to treatment failure and increased mortality rates. Although for years the main driver of carbapenem resistance in Italy has been the Klebsiella pneumoniae KPC carbapenemase, recent years have seen an increase in VIM and NDM metallo-beta-lactamases (MBLs). We conducted a five-year survey of New Delhi Metallo-beta-Lactamase (NDM)-producing Klebsiella pneumoniae (NDM-Kpn) clinical isolates from the Lazio region, Italy; the study aimed to elucidate the molecular mechanisms underpinning their resistant and virulent phenotype. Methods: Antimicrobial susceptibility was evaluated by automated systems and broth microdilution. In silico analysis of acquired resistance and virulence genes was performed using whole-genome sequencing (WGS), molecular typing through MLST, and core genome multi-locus sequence typing (cgMLST). Conclusions: A total of 126 clinical NDM-Kpn isolates were collected from 19 distinct hospitals in the Lazio region. Molecular analysis highlighted the existence of NDM-1 (108/126) and NDM-5 (18/126) variants, 18 Sequence Types (STs), and 15 Cluster Types (CTs). Notably, 31/126 isolates displayed a virulence score of 4, carrying ybt, ICEKp, iuc, and rmp genes. This study identified a variety of NDM-Kpn STs, mainly carrying the blaNDM-1 gene, with a significant number linked to high-risk clones. Of these isolates, 24.6% showed high-level resistance and virulence, emphasizing the risk of the spread of strains that combine multi-drug-resistance (MDR) and virulence. Proactive surveillance and international collaborations are needed to prevent the spread of high-risk clones, as well as further research into new antimicrobial agents to fight antibiotic resistance.

Antimicrobial resistance and clonal relationships of Salmonella enterica Serovar Gallinarum biovar pullorum strains isolated in China based on whole genome sequencing

BackgroundPullorum disease is a serious problem in many countries. Caused by Salmonella enterica serovar Gallinarum biovar Pullorum (S. Pullorum), it creates huge economic losses in the poultry industry. Although pullorum disease has been well-controlled in many developed countries, it is still a critical problem in developing countries. However, there is still a lack of information on S. Pullorum strains isolated from different regions and sources in China. The objective of this study was to supply the antimicrobial resistance patterns and clonal relationships of S. Pullorum from breeder chicken farms.MethodsIn this study, a total of 114 S. Pullorum strains recovered from 11 provinces and municipalities in China between 2020 and 2021 were selected. These 114 S. Pullorum strains were analyzed using whole genome sequencing (WGS). Antimicrobial resistance (AMR) was tested both by genotypic prediction using the WGS method and using disc diffusion to assess phenotypic AMR.ResultsThese 114 sequenced S. Pullorum strains were divided into three sequence types (STs), the dominant STs was ST92 (104/114). Further core genome multi-locus sequence typing analysis indicated that 114 S. Pullorum strains may have a close relationship, which could be clonally transmitted among different provinces and municipalities. Our results showed a close relationship between the S. Pullorum strains found in different regions, indicating these strains may have been transmitted in China a long time ago. Nearly all S. Pullorum strains 94.74% (n = 108) were resistant to at least one antimicrobial class, and 35.96% of the examined Salmonella strains were considered multiple drug resistant.ConclusionOverall, this study showed that S. Pullorum strains in China have a close genetic relationship in terms of antimicrobial resistance, suggesting widespread clonal transmission.

Construction of core genome multi-locus sequence typing schemes for population structure analyses of Nocardia species

Nocardia, a member of the Actinobacteria phylum, populates diverse habitats globally, with certain species being the cause of various clinical infections in humans. There is paucity of data regarding the population structure of this genus and of established genomic-based phylogenetic methods. We examined the whole genome sequences of 193 isolates spanning five major pathogenic Nocardia species sourced from public databases, encompassing diverse geographic regions. Using the chewBBACA pipeline, a species-specific core genome multilocus sequence typing (cgMLST) schema was created for N. cyriacigeorgica, N. farcinica, N. brasiliensis, N. wallacei, and N. abscessus. Additional genomic features that were examined included virulence factor (VF) profile, total length and open-reading frame count, the core genome length and core gene count, and GC content. Our findings indicated that: (i) N. brasiliensis diverges significantly from the other four species, underscoring its distinct evolutionary trajectory; (ii) the population structures of all species were polyclonal, with phylogenetic clustering occurring in the minority of isolates; (iii) clonal complexes were largely restricted to specific geographical locations, rather than demonstrating a global distribution, and (iv) initial evidence suggests no direct common-source transmission amongst the studied strains. Our study establishes a comprehensive genome-based phylogenetic methodology for population structure of Nocardia species.

Ralstonia solanacearum (phylotype II) isolated from Rosa spp. in the Netherlands is closely related to phylotype II isolates from other sources in the Netherlands and is virulent on potato

AbstractIn 2018, during an annual survey in the Netherlands, Ralstonia solanacearum phylotype II (phy II) was found in asymptomatic greenhouse rose plants at three geographic locations. These findings were remarkable, since previous findings of Ralstonia sp. in rose always concerned Ralstonia pseudosolanacearum phylotype I (phy I). Therefore, no information was available on the virulence of R. solanacearum phy II on rose. In this study, R. solanacearum phy II isolates PD 7421 and PD 7394, isolated in 2018 from asymptomatic ornamental rose (Rosa spp.), were assessed for their virulence in two rose cultivars (“Armando” and “Red Naomi”) at two temperatures. No typical symptoms were observed for PD 7421 and PD 7394 on the two rose cultivars, irrespective of the temperature. However, latent infections upon inoculation of these isolates on rose did occur. R. solanacearum phy II is known as a major potato pathogen, where it causes brown rot. Whole genome multilocus sequence typing analysis demonstrated that the isolates from rose were closely related to R. solanacearum phy II isolates previously found in seed potato and surface water in the Netherlands. Because of this close genetic relatedness, the virulence of PD 7421 and PD 7394 was also assessed in potato plants, where both isolates caused severe disease symptoms on the shoots as well as the daughter tubers. This implies that rose can act as a reservoir for R. solanacearum phy II and, in this way, can potentially be involved in spreading this bacterium.

Isolation of Virulent Leptospira Serogroup Australis Field Strains from Symptomatic Dogs for Canine Leptospiral Vaccine Development.

Leptospirosis is a widespread zoonosis caused by spirochaetes belonging to the pathogenic species of Leptospira, which are classified into more than 25 serogroups and 250 serovars. Vaccination can prevent the disease in dogs but offers incomplete efficacy because of a lack of cross-protection between serogroups. The aim of this study was to validate a robust recruitment and sampling process, with the objectives of isolating and typing circulating Leptospira pathogenic strains and then selecting those of proven virulence and pathogenicity for vaccine development. Blood and urine samples from dogs with clinical syndromes compatible with acute leptospirosis were sterilely collected and transported to a reference laboratory for a micro-agglutination test (MAT), PCR, and bacterial isolation. Isolated strains underwent molecular typing using RNA16S, variable-number tandem repeat (VNTR), and pulsed-field gel electrophoresis (PFGE). Subtyping was performed using core genome multilocus sequence typing (CgMLST). Among 64 included dogs, 41 had MAT and/or PCR results compatible with Leptospira infection, and 14 Leptospira strains were isolated. Based on molecular typing, 11 isolates were classified as L. interrogans serogroup Australis, serovar Bratislava, and 3 as serogroup Icterohaemorrhagiae, serovar Icterohaemorrhagiae. CgMLST subtyping revealed a diversity of clonal groups (CGs) distributed in several regional clusters. Besides validating a robust recruitment and sampling process, this study outlines the value of combining PCR and serological testing when suspecting leptospirosis and the usefulness of implementing molecular typing methods to identify circulating field strains. It also confirms the epidemiological importance of the Australis serogroup and allows for the collection of different highly pathogenic strains for vaccine development.

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

IntroductionThe 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.MethodsThis 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.ResultsWithin 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.ConclusionsThe 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 RegistrationThe study is registered with the German Clinical Trials Register (DRKS00030009).

Outbreak-associated Salmonella Baildon found in wastewater demonstrates how sewage monitoring can supplement traditional disease surveillance.

Non-typhoidal Salmonella is a common cause of gastroenteritis worldwide, but current non-typhoidal Salmonella surveillance is suboptimal. Here, we evaluated the utility of wastewater monitoring to enhance traditional surveillance for this foodborne pathogen. In June 2022, we tested raw sewage collected twice a week from two treatment plants in central Pennsylvania for non-typhoidal Salmonella and characterized isolates using whole-genome sequencing. We recovered 43 Salmonella isolates from wastewater samples, differentiated by genomic analysis into seven serovars: 16 Panama (37.2%), 9 Senftenberg (20.9%), 8 Baildon (18.6%), and 3 or fewer of four other serovars. We assessed genetic relatedness and epidemiologic links between these wastewater isolates with those from patients with salmonellosis. All S. Baildon isolates from wastewater were genetically similar to those associated with a known contemporaneous salmonellosis outbreak. S. Baildon from wastewater and 42 outbreak-related isolates in the national outbreak detection database had the same core genome multilocus sequence typing, and outbreak code differed by zero or one single polynucleotide polymorphism. One of the 42 outbreak-related isolates was obtained from a patient residing in the wastewater sample collection catchment area, which serves approximately 17000 people. S. Baildon is a rare serovar (reported in <1% cases nationally, over five years). Our study underscores the value of monitoring sewage from a defined population to supplement traditional surveillance methods for the evidence of Salmonella infections and to determine the extent of outbreaks.IMPORTANCEDuring the COVID-19 pandemic, monitoring for SARS-CoV-2 in wastewater was highly effective in identifying the variants of concern earlier than clinical surveillance methods. Here, we show that monitoring domestic sewage can also augment traditional reporting of foodborne illnesses to public health authorities. Our study detected multiple Salmonella enterica serovars in samples from two wastewater treatment plants in central Pennsylvania. Using whole-genome sequencing, we demonstrated that the isolates of variant S. Baildon clustered with those from a foodborne salmonellosis outbreak that occurred in a similar time frame. Cases were primarily from Pennsylvania, and one individual lived within the wastewater treatment catchment area. This study highlights the effectiveness of domestic sewage testing as a proactive public health strategy to track and respond to infectious disease outbreaks.

Genomic characterization and cluster analysis of Carbapenem-resistant Klebsiella pneumoniae

To investigate the genomic features and perform cluster analysis of Carbapenem-resistant Klebsiella pneumoniae (CRKP) to provide an experimental basis for guiding the prevention and treatment of CRKP infections.A retrospective case-cohort study was conducted on 19 non-redundant CRKP strains isolated from the Tenth Affiliated Hospital of Southern Medical University between January and June 2023. Whole genome sequencing (WGS) and multilocus sequence typing (MLST) were performed to compare genomic features and analyze the resistance genes and homology of the strains.The results showed that the 19 CRKP strains were isolated from 8 different clinical departments, mainly from respiratory specimens. The whole genome sequencing revealed that the genomic lengths of CRKP ranged from 4.90 to 5.85 Mbp, with contigs N50 values>20 kb for each genome. The median overall GC content was 57.0% (50.4%-57.1%). Comparative genomic analysis identified three regions with high genomic variability. WGS detected 32 resistance genes across 11 categories. All 19 strains carried carbapenem resistance genes (blaKPC-2 and blaOXA-48), blaTEM-1B extended-spectrum β-lactamase resistance genes, qnrS1 quinolone resistance gene, and fosA fosfomycin resistance gene, with each strain carrying only one carbapenemase gene. The detection rate of blaKPC-2 was 94.7% (18/19). MLST identified three sequence types: ST11, ST437 and ST147, with ST11 being predominant (89.5%, 17/19). Clustering analysis based on acquired resistance genes revealed three clonal transmission patterns among strains 72 and 90, and strains 88, 84, 66 and 79.In conclusion, CRKP strains carry multiple resistance genes, and clustering analysis indicating that nosocomial clonal transmission is closely related to acquired resistance genes. The ST11-blaKPC-2 type strain is the predominant clone. Strengthened surveillance and effective control strategies are necessary to reduce nosocomial transmission of CRKP.

Epidemiology of Staphylococcus aureus food isolates: Comparison of conventional methods with whole genome sequencing typing methods

A variety of methods exists for typing bacteria. However, guidelines for the application and interpretation of typing tools in epidemiologic investigations of Staphylococcus aureus are lacking. This study aimed to identify appropriate typing methods for S. aureus population studies and outbreak investigation. We compared pulsed-field gel electrophoresis (PFGE), seven loci multi-locus sequence typing (MLST), core genome MLST (cgMLST), core single nucleotide polymorphism (cSNP), and enterotoxin (se/SE) profiles on 351 S. aureus isolates. The discriminatory power, concordance, and congruence of typing results were assessed. cgMLST, cSNP, and PFGE yielded the highest discrimination value, followed by se/SE typing and MLST. The best concordance of results was found between cgMLST and cSNP, while the best congruence was observed for cgMLST and cSNP with all methods, followed by PFGE with MLST. The strengths and weaknesses of each method are highlighted. For population structure, cgMLST and cSNP performed better than PFGE and MLST in terms of resolution of clusters and in phylogenetic inference. Enterotoxin profiles matched with MLST groups, suggesting the use of se/SE typing to predict MLST results. For the retrospective analysis of 31 outbreaks, all methods performed almost equally to discriminate epidemiologically related strains and can be used to unambiguously distinguish outbreak strains.

High prevalence of the recently identified clonal lineage ST1299/CT3109 vanA among vancomycin-resistant Enterococcus faecium strains isolated from municipal wastewater.

Previously, we demonstrated that the majority of vancomycin-resistant Enterococcus faecium (VREfm) strains from in-patients of the University Hospital Erlangen, Germany, belonged to only three clonal lineages, namely ST117/CT71 vanB and two novel ST1299 vanA lineages classified as CT3109 and CT1903. The goal of the current study was (i) to investigate whether VREfm is also detectable in wastewater of the city of Erlangen, (ii) to identify their molecular features, and (iii) to clarify whether VREfm could arise from the community of the city of Erlangen or can be (directly) connected to nosocomial infections in the hospital setting. From April to May 2023, a total of 244 VREfm strains from raw wastewater of the city of Erlangen were analyzed by core genome multilocus sequence typing (cgMLST). Moreover, 20 of them were further investigated for single nucleotide polymorphisms (SNPs). The molecular characterization of the wastewater VREfm strains revealed a high prevalence (27.9%) of the recently identified clonal lineage ST1299/CT3109 vanA, which is mainly characterized by the presence of the tetracycline-resistance determinant tet(M) and the virulence genes pilA and prpA. The SNPs analysis revealed the presence of two major clusters, namely cluster I (≤65 SNPs), which included well-known hospital-adapted vanB clonal lineages such as ST117/CT71 and ST80/CT1065 and cluster II (≤70 SNPs), which were mainly characterized by the lineage ST1299/CT3109 vanA. Based on the concomitant resistance to vancomycin and tetracycline, we propose that ST1299/CT3109 vanA primarily originated and spread outside of hospital settings.IMPORTANCEThis study provides a detailed genomic analysis of vancomycin-resistant Enterococcus faecium (VREfm) strains isolated from municipal wastewater with a particular focus on clonal lineages, antimicrobial resistance, and the presence of virulence genes. The high wastewater prevalence of the recently identified clonal lineage ST1299/CT3109 vanA, which has been previously detected in hospitals, suggests an enormous potential for future spread in Germany.

Corynebacterium pseudotuberculosis: Whole genome sequencing reveals unforeseen and relevant genetic diversity in this pathogen.

Corynebacterium pseudotuberculosis (CPS) is an important bacterial animal pathogen. CPS causes chronic, debilitating and currently incurable infectious diseases affecting a wide range of livestock and wild herbivores including camelids worldwide. Belonging to the Corynebacterium diphtheriae complex, this pathogen can also infect humans. The classical characterization of CPS is typically based on the testing of nitrate reductase activity, separating the two biovars Equi and Ovis. However, more refined resolutions are required to unravel routes of infection. This was realized in our study by generating and analyzing whole genome sequencing (WGS) data. Using newly created core genome multilocus sequence typing (cgMLST) profiles we were the first to discover isolates grouping in a cluster adjacent to clusters formed by CPS biovar Equi isolates. This novel cluster includes CPS isolates from alpacas, llamas, camels and dromedaries, which are characterized by a lack of nitrate reductase activity as encountered in biovar Ovis. This is of special interest for molecular epidemiology. Nevertheless, these isolates bear the genes of the nitrate locus, which are characteristic of biovar Equi isolates. However, sequence analysis of the genes narG and narH of the nitrate locus revealed indels leading to frameshifts and inactivity of the enzymes involved in nitrate reduction. Interestingly, one CPS isolate originating from another lama with an insertion in the MFS transporter (narT) is adjacent to a cluster formed by ovine CPS isolates biovar Equi. Based on this knowledge, the combination of biochemical and PCR based molecular biological nitrate reductase detection can be used for a fast and uncomplicated classification of isolates in routine diagnostics in order to check the origin of camelid CPS isolates. Further analysis revealed that partial sequencing of the ABC transporter substrate binding protein (CP258_RS07935) is a powerful tool to assign the biovars and the novel genomovar.

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.

Bacteriological and Serological Investigation of Leptospirosis in Dogs and Pigs in Palau.

Leptospirosis is a zoonotic disease caused by the pathogenic spirochaetes of the genus Leptospira. It is a public health concern in the Pacific Islands and is considered endemic in Palau. However, information on the genotypes and serotypes of causative Leptospira spp. in the country is limited. In this study, we isolated leptospires and detected antileptospiral antibodies in dogs and pigs. The isolates were characterized using a serological method and whole-genome sequencing. Leptospira interrogans was isolated from five of the 20 symptomatic dogs and one of the 58 healthy pigs. Their serogroups were identified as Icterohaemorrhagiae and Pyrogenes; however, the serogroup of one isolate could not be determined. Anti-Leptospira antibodies were detected in 14.4% (26/181) of the dogs and 20% (10/50) of the pigs. The reactive serogroups in dogs and pigs were almost identical, except for the Panama serogroup. Core genome multilocus sequence typing revealed that five of the six core genome sequence types (cgSTs) were newly identified in this study. The cgSTs from the serogroup Icterohaemorrhagiae isolates belonged to the same group as the Copenhageni and Icterohaemorrhagiae serovars isolated in other countries, whereas no similar cgSTs were identified in the Pyrogenes or unidentified serogroup strains. We demonstrated a high incidence of canine and porcine leptospirosis and identified new L. interrogans genotypes (cgSTs) circulating in Palau. Further investigations are needed to determine whether dogs and pigs serve as maintenance hosts for newly identified L. interrogans genotypes and whether they pose a risk of leptospirosis transmission to humans.

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.

Genetic Diversity in Salmonella enterica in Outbreaks of Foodborne and Zoonotic Origin in the USA in 2006-2017.

Whole genome sequencing is replacing traditional laboratory surveillance methods as the primary tool to track and characterize clusters and outbreaks of the foodborne and zoonotic pathogen Salmonella enterica (S. enterica). In this study, 438 S. enterica isolates representing 35 serovars and 13 broad vehicle categories from one hundred epidemiologically confirmed outbreaks were evaluated for genetic variation to develop epidemiologically relevant interpretation guidelines for Salmonella disease cluster detection. The Illumina sequences were analyzed by core genome multi-locus sequence typing (cgMLST) and screened for antimicrobial resistance (AR) determinants and plasmids. Ninety-three of the one hundred outbreaks exhibited a close allele range (less than 10 allele differences with a subset closer than 5). The remaining seven outbreaks showed increased variation, of which three were considered polyclonal. A total of 16 and 28 outbreaks, respectively, showed variations in the AR and plasmid profiles. The serovars Newport and I 4,[5],12:i:-, as well as the zoonotic and poultry product vehicles, were overrepresented among the outbreaks, showing increased variation. A close allele range in cgMLST profiles can be considered a reliable proxy for epidemiological relatedness for the vast majority of S. enterica outbreak investigations. Variations associated with mobile elements happen relatively frequently during outbreaks and could be reflective of changing selective pressures.

Epidemiological pattern and genomic insights into multidrug-resistant ST491 Acinetobacter baumannii BD20 isolated from an infected wound in Bangladesh: Concerning co-occurrence of three classes of beta lactamase genes

ObjectiveMultidrug-resistant (MDR) Acinetobacter baumannii is a major issue within healthcare facilities in Bangladesh due to its frequent association with hospital-acquired infections. In this study we report on a carbapenem-resistant draft genome sequence of an A. baumannii BD20 sample isolated from an infected wound in Bangladesh. MethodsA. baumannii BD20 was isolated from an infected burn wound. Whole-genome sequencing was carried out and annotated using PGAP and Prokka. Sequence type, antimicrobial resistance genes, virulence factor genes, and metal resistance genes were investigated. Core genome multilocus sequence typing–based phylogenomic analysis between A. baumannii BD20 and 213 A. baumannii strains retrieved from the NCBI GenBank database was performed using the BacWGSTdb 2.0 server. ResultsA. baumannii BD20 (MLST 491) was resistant to all the antibiotics tested, except for colistin and polymyxin B. Along with many other antibiotic resistance genes, the isolate harbored three classes of beta lactamase–producing genes: blaGES-11 (class A), blaOXA-69 (class D), blaADC-10 (class C), and blaADC-11 (class C). Additionally, the strain carried several virulence genes and metal resistance determinants, which may contribute to its increased virulence. Core genome MLST–based phylogenomic analysis revealed that A. baumannii BD20 was closely related to another ST491 strain isolated from Singapore. ConclusionsThe findings of this study underscore the growing challenge of MDR A. baumannii, emphasizing the need for vigilant surveillance and infection-control measures in healthcare settings in order to address these emerging threats effectively.

Can genomics and meteorology predict outbreaks of legionellosis in urban settings?

Legionella pneumophila is ubiquitous and sporadically infects humans causing Legionnaire's disease (LD). Globally, reported cases of LD have risen fourfold from 2000 to 2014. In 2016, Sydney, Australia was the epicenter of an outbreak caused by L. pneumophila serogroup 1 (Lpsg1). Whole-genome sequencing was instrumental in identifying the causal clone which was found in multiple locations across the city. This study examined the epidemiology of Lpsg1 in an urban environment, assessed typing schemes to classify resident clones, and investigated the association between local climate variables and LD outbreaks. Of 223 local Lpsg1 isolates, we identified dominant clones with one clone isolated from patients in high frequency during outbreak investigations. The core genome multi-locus sequence typing scheme was the most reliable in identifying this Lpsg1 clone. While an increase in humidity and rainfall was found to coincide with a rise in LD cases, the incidence of the major L. pneumophila outbreak clone did not link to weather phenomena. These findings demonstrated the role of high-resolution typing and weather context assessment in determining source attribution for LD outbreaks in urban settings, particularly when clinical isolates remain scarce.IMPORTANCEWe investigated the genomic and meteorological influences of infections caused by Legionella pneumophila in Sydney, Australia. Our study contributes to a knowledge gap of factors that drive outbreaks of legionellosis compared to sporadic infections in urban settings. In such cases, clinical isolates can be rare, and thus, other data are needed to inform decision-making around control measures. The study revealed that core genome multi-locus sequence typing is a reliable and adaptable technique when investigating Lpsg1 outbreaks. In Sydney, the genomic profile of Lpsg1 was dominated by a single clone, which was linked to numerous community cases over a period of 40 years. Interestingly, the peak in legionellosis cases during Autumn was not associated with this prevalent outbreak clone. Incorporating meteorological data with Lpsg1 genomics can support risk assessment strategies for legionellosis in urban environments, and this approach may be relevant for other densely populated regions globally.

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.