ABSTRACT
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.
Subject(s)
Genome, Bacterial , Haemophilus influenzae , Multilocus Sequence Typing , Phylogeny , Haemophilus influenzae/genetics , Haemophilus influenzae/classification , Multilocus Sequence Typing/methods , Humans , Haemophilus Infections/microbiology , Genetic VariationABSTRACT
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.
Subject(s)
DNA Barcoding, Taxonomic , Genome, Bacterial , Multilocus Sequence Typing , Pneumococcal Infections , Streptococcus pneumoniae , Streptococcus pneumoniae/genetics , Streptococcus pneumoniae/classification , Streptococcus pneumoniae/isolation & purification , Multilocus Sequence Typing/methods , Humans , DNA Barcoding, Taxonomic/methods , Pneumococcal Infections/microbiology , Pneumococcal Infections/epidemiology , Phylogeny , Gene Library , Whole Genome Sequencing/methodsABSTRACT
Among genes present in all group A streptococci (GAS), those encoding M-fibril and T-pilus proteins display the highest levels of sequence diversity, giving rise to the two primary serological typing schemes historically used to define strain. A new genotyping scheme for the pilin adhesin and backbone genes is developed and, when combined with emm typing, provides an account of the global GAS strain population. Cluster analysis based on nucleotide sequence similarity assigns most T-serotypes to discrete pilin backbone sequence clusters, yet the established T-types correspond to only half the clusters. The major pilin adhesin and backbone sequence clusters yield 98 unique combinations, defined as "pilin types." Numerous horizontal transfer events that involve pilin or emm genes generate extensive antigenic and functional diversity on the bacterial cell surface and lead to the emergence of new strains. Inferred pilin genotypes applied to a meta-analysis of global population-based collections of pharyngitis and impetigo isolates reveal highly significant associations between pilin genotypes and GAS infection at distinct ecological niches, consistent with a role for pilin gene products in adaptive evolution. Integration of emm and pilin typing into open-access online tools (pubmlst.org) ensures broad utility for end-users wanting to determine the architecture of M-fibril and T-pilus genes from genome assemblies.IMPORTANCEPrecision in defining the variant forms of infectious agents is critical to understanding their population biology and the epidemiology of associated diseases. Group A Streptococcus (GAS) is a global pathogen that causes a wide range of diseases and displays a highly diverse cell surface due to the antigenic heterogeneity of M-fibril and T-pilus proteins which also act as virulence factors of varied functions. emm genotyping is well-established and highly utilized, but there is no counterpart for pilin genes. A global GAS collection provides the basis for a comprehensive pilin typing scheme, and online tools for determining emm and pilin genotypes are developed. Application of these tools reveals the expansion of structural-functional diversity among GAS via horizontal gene transfer, as evidenced by unique combinations of surface protein genes. Pilin and emm genotype correlations with superficial throat vs skin infection provide new insights on the molecular determinants underlying key ecological and epidemiological trends.
Subject(s)
Genetic Variation , Genotype , Streptococcus pyogenes , Streptococcus pyogenes/genetics , Streptococcus pyogenes/classification , Humans , Recombination, Genetic , Bacterial Outer Membrane Proteins/genetics , Fimbriae Proteins/genetics , Gene Transfer, Horizontal , Antigens, Bacterial/genetics , Streptococcal Infections/microbiology , Streptococcal Infections/epidemiology , Impetigo/microbiology , Impetigo/epidemiology , Pharyngitis/microbiology , Fimbriae, Bacterial/genetics , Carrier ProteinsABSTRACT
BACKGROUND: Invasive meningococcal isolates in South Africa have in previous years (<2008) been characterized by serogroup B, C, W and Y lineages over time, with penicillin intermediate resistance (peni) at 6%. We describe the population structure and genomic markers of peni among invasive meningococcal isolates in South Africa, 2016-2021. METHODS: Meningococcal isolates were collected through national, laboratory-based invasive meningococcal disease (IMD) surveillance. Phenotypic antimicrobial susceptibility testing and whole-genome sequencing were performed, and the mechanism of reduced penicillin susceptibility was assessed in silico. RESULTS: Of 585 IMD cases reported during the study period, culture and PCR-based capsular group was determined for 477/585 (82%); and 241/477 (51%) were sequenced. Predominant serogroups included NmB (210/477; 44%), NmW (116/477; 24%), NmY (96/477; 20%) and NmC (48/477; 10%). Predominant clonal complexes (CC) were CC41/44 in NmB (27/113; 24%), CC11 in NmW (46/56; 82%), CC167 in NmY (23/44; 53%), and CC865 in NmC (9/24; 38%). Peni was detected in 16% (42/262) of isolates, and was due to the presence of a penA mosaic, with the majority harboring penA7, penA9 or penA14. CONCLUSION: IMD lineages circulating in South Africa were consistent with those circulating prior to 2008, however peni was higher than previously reported, and occurred in a variety of lineages.
ABSTRACT
Recombination of short DNA fragments via horizontal gene transfer (HGT) can introduce beneficial alleles, create genomic disharmony through negative epistasis, and create adaptive gene combinations through positive epistasis. For non-core (accessory) genes, the negative epistatic cost is likely to be minimal because the incoming genes have not co-evolved with the recipient genome and are frequently observed as tightly linked cassettes with major effects. By contrast, interspecific recombination in the core genome is expected to be rare because disruptive allelic replacement is likely to introduce negative epistasis. Why then is homologous recombination common in the core of bacterial genomes? To understand this enigma, we take advantage of an exceptional model system, the common enteric pathogens Campylobacter jejuni and C. coli that are known for very high magnitude interspecies gene flow in the core genome. As expected, HGT does indeed disrupt co-adapted allele pairings, indirect evidence of negative epistasis. However, multiple HGT events enable recovery of the genome's co-adaption between introgressing alleles, even in core metabolism genes (e.g., formate dehydrogenase). These findings demonstrate that, even for complex traits, genetic coalitions can be decoupled, transferred, and independently reinstated in a new genetic background-facilitating transition between fitness peaks. In this example, the two-step recombinational process is associated with C. coli that are adapted to the agricultural niche.IMPORTANCEGenetic exchange among bacteria shapes the microbial world. From the acquisition of antimicrobial resistance genes to fundamental questions about the nature of bacterial species, this powerful evolutionary force has preoccupied scientists for decades. However, the mixing of genes between species rests on a paradox: 0n one hand, promoting adaptation by conferring novel functionality; on the other, potentially introducing disharmonious gene combinations (negative epistasis) that will be selected against. Taking an interdisciplinary approach to analyze natural populations of the enteric bacteria Campylobacter, an ideal example of long-range admixture, we demonstrate that genes can independently transfer across species boundaries and rejoin in functional networks in a recipient genome. The positive impact of two-gene interactions appears to be adaptive by expanding metabolic capacity and facilitating niche shifts through interspecific hybridization. This challenges conventional ideas and highlights the possibility of multiple-step evolution of multi-gene traits by interspecific introgression.
Subject(s)
Campylobacter coli , Campylobacter jejuni , Epistasis, Genetic , Gene Transfer, Horizontal , Genome, Bacterial , Recombination, Genetic , Campylobacter jejuni/genetics , Campylobacter coli/genetics , Evolution, Molecular , Adaptation, Physiological/genetics , Adaptation, Biological/geneticsABSTRACT
Respiratory syncytial virus (RSV) is the leading cause of hospitalisation for respiratory infection in young children. RSV disease severity is known to be age-dependent and highest in young infants, but other correlates of severity, particularly the presence of additional respiratory pathogens, are less well understood. In this study, nasopharyngeal swabs were collected from two cohorts of RSV-positive infants <12 months in Spain, the UK, and the Netherlands during 2017-20. We show, using targeted metagenomic sequencing of >100 pathogens, including all common respiratory viruses and bacteria, from samples collected from 433 infants, that burden of additional viruses is common (111/433, 26%) but only modestly correlates with RSV disease severity. In contrast, there is strong evidence in both cohorts and across age groups that presence of Haemophilus bacteria (194/433, 45%) is associated with higher severity, including much higher rates of hospitalisation (odds ratio 4.25, 95% CI 2.03-9.31). There is no evidence for association between higher severity and other detected bacteria, and no difference in severity between RSV genotypes. Our findings reveal the genomic diversity of additional pathogens during RSV infection in infants, and provide an evidence base for future causal investigations of the impact of co-infection on RSV disease severity.
Subject(s)
Coinfection , Respiratory Syncytial Virus Infections , Respiratory Syncytial Virus, Human , Respiratory Tract Infections , Infant , Child , Humans , Child, Preschool , Respiratory Syncytial Virus, Human/genetics , Respiratory Syncytial Virus Infections/diagnosis , Respiratory Syncytial Virus Infections/epidemiology , HospitalizationABSTRACT
Introduction: Non-baumannii Acinetobacter species are increasingly isolated in the clinical setting and the environment. The aim of the present study was to analyze a genome database of 837 Acinetobacter spp. isolates, which included 798 non-baumannii Acinetobacter genomes, in order to define the concordance of classification and discriminatory power of 7-gene MLST, 53-gene MLST, and single-nucleotide polymorphism (SNPs) phylogenies. Methods: Phylogenies were performed on Pasteur Multilocus Sequence Typing (MLST) or ribosomal Multilocus Sequence Typing (rMLST) concatenated alleles, or SNPs extracted from core genome alignment. Results: The Pasteur MLST scheme was able to identify and genotype 72 species in the Acinetobacter genus, with classification results concordant with the ribosomal MLST scheme. The discriminatory power and genotyping reliability of the Pasteur MLST scheme were assessed in comparison to genome-wide SNP phylogeny on 535 non-baumannii Acinetobacter genomes assigned to Acinetobacter pittii, Acinetobacter nosocomialis, Acinetobacter seifertii, and Acinetobacter lactucae (heterotypic synonym of Acinetobacter dijkshoorniae), which were the most clinically relevant non-baumannii species of the A. baumannii group. The Pasteur MLST and SNP phylogenies were congruent at Robinson-Fould and Matching cluster tests and grouped genomes into four and three clusters in A. pittii, respectively, and one each in A. seifertii. Furthermore, A. lactucae genomes were grouped into one cluster within A. pittii genomes. The SNP phylogeny of A. nosocomialis genomes showed a heterogeneous population and did not correspond to the Pasteur MLST phylogeny, which identified two recombinant clusters. The antimicrobial resistance genes belonging to at least three different antimicrobial classes were identified in 91 isolates assigned to 17 distinct species in the Acinetobacter genus. Moreover, the presence of a class D oxacillinase, which is a naturally occurring enzyme in several Acinetobacter species, was found in 503 isolates assigned to 35 Acinetobacter species. Conclusion: In conclusion, Pasteur MLST phylogeny of non-baumannii Acinetobacter isolates coupled with in silico detection of antimicrobial resistance makes it important to study the population structure and epidemiology of Acinetobacter spp. isolates.
ABSTRACT
Acinetobacter baumannii is an opportunistic pathogen responsible for healthcare-associated infections (HAIs) and outbreaks. Antimicrobial resistance mechanisms and virulence factors allow it to survive and spread in the hospital environment. However, the molecular mechanisms of these traits and their association with international clones are frequently unknown in low- and middle-income countries. Here, we analyze the phenotype and genotype of seventy-six HAIs and outbreak-causing A. baumannii isolates from a Mexican hospital over ten years, with special attention to the carbapenem resistome and biofilm formation. The isolates belonged to the global international clone (IC) 2 and the Latin America endemic IC5 and were predominantly extensively drug-resistant (XDR). Oxacillinases were identified as a common source of carbapenem resistance. We noted the presence of the blaOXA-143-like family (not previously described in Mexico), the blaOXA-72 and the blaOXA-398 found in both ICs. A low prevalence of efflux pump overexpression activity associated with carbapenem resistance was observed. Finally, strong biofilm formation was found, and significant biofilm-related genes were identified, including bfmRS, csuA/BABCDE, pgaABCD and ompA. This study provides a comprehensive profile of the carbapenem resistome of A. baumannii isolates belonging to the same pulse type, along with their significant biofilm formation capacity. Furthermore, it contributes to a better understanding of their role in the recurrence of infection and the endemicity of these isolates in a Mexican hospital.
ABSTRACT
Neisseria gonorrhoeae is a significant threat to global health with an estimated incidence of over 80 million cases each year and high levels of antimicrobial resistance. The gonococcal ß-lactamase plasmid, pbla, carries the TEM ß-lactamase, which requires only one or two amino acid changes to become an extended-spectrum ß-lactamase (ESBL); this would render last resort treatments for gonorrhoea ineffective. Although pbla is not mobile, it can be transferred by the conjugative plasmid, pConj, found in N. gonorrhoeae. Seven variants of pbla have been described previously, but little is known about their frequency or distribution in the gonococcal population. We characterised sequences of pbla variants and devised a typing scheme, Ng_pblaST that allows their identification from whole genome short-read sequences. We implemented Ng_pblaST to assess the distribution of pbla variants in 15 532 gonococcal isolates. This demonstrated that only three pbla variants commonly circulate in gonococci, which together account for >99â% of sequences. The pbla variants carry different TEM alleles and are prevalent in distinct gonococcal lineages. Analysis of 2758 pbla-containing isolates revealed the co-occurrence of pbla with certain pConj types, indicating co-operativity between pbla and pConj variants in the spread of plasmid-mediated AMR in N. gonorrhoeae. Understanding the variation and distribution of pbla is essential for monitoring and predicting the spread of plasmid-mediated ß-lactam resistance in N. gonorrhoeae.
Subject(s)
Gonorrhea , Neisseria gonorrhoeae , Humans , Neisseria gonorrhoeae/genetics , beta-Lactamases/genetics , Alleles , Microbial Sensitivity Tests , Plasmids/genetics , Gonorrhea/geneticsABSTRACT
BACKGROUND: The Invasive Respiratory Infection Surveillance (IRIS) Consortium was established to assess the impact of the COVID-19 pandemic on invasive diseases caused by Streptococcus pneumoniae, Haemophilus influenzae, Neisseria meningitidis, and Streptococcus agalactiae. We aimed to analyse the incidence and distribution of these diseases during the first 2 years of the COVID-19 pandemic compared to the 2 years preceding the pandemic. METHODS: For this prospective analysis, laboratories in 30 countries and territories representing five continents submitted surveillance data from Jan 1, 2018, to Jan 2, 2022, to private projects within databases in PubMLST. The impact of COVID-19 containment measures on the overall number of cases was analysed, and changes in disease distributions by patient age and serotype or group were examined. Interrupted time-series analyses were done to quantify the impact of pandemic response measures and their relaxation on disease rates, and autoregressive integrated moving average models were used to estimate effect sizes and forecast counterfactual trends by hemisphere. FINDINGS: Overall, 116 841 cases were analysed: 76 481 in 2018-19, before the pandemic, and 40 360 in 2020-21, during the pandemic. During the pandemic there was a significant reduction in the risk of disease caused by S pneumoniae (risk ratio 0·47; 95% CI 0·40-0·55), H influenzae (0·51; 0·40-0·66) and N meningitidis (0·26; 0·21-0·31), while no significant changes were observed for S agalactiae (1·02; 0·75-1·40), which is not transmitted via the respiratory route. No major changes in the distribution of cases were observed when stratified by patient age or serotype or group. An estimated 36 289 (95% prediction interval 17 145-55 434) cases of invasive bacterial disease were averted during the first 2 years of the pandemic among IRIS-participating countries and territories. INTERPRETATION: COVID-19 containment measures were associated with a sustained decrease in the incidence of invasive disease caused by S pneumoniae, H influenzae, and N meningitidis during the first 2 years of the pandemic, but cases began to increase in some countries towards the end of 2021 as pandemic restrictions were lifted. These IRIS data provide a better understanding of microbial transmission, will inform vaccine development and implementation, and can contribute to health-care service planning and provision of policies. FUNDING: Wellcome Trust, NIHR Oxford Biomedical Research Centre, Spanish Ministry of Science and Innovation, Korea Disease Control and Prevention Agency, Torsten Söderberg Foundation, Stockholm County Council, Swedish Research Council, German Federal Ministry of Health, Robert Koch Institute, Pfizer, Merck, and the Greek National Public Health Organization.
Subject(s)
Bacterial Infections , COVID-19 , Neisseria meningitidis , Humans , Pandemics , COVID-19/epidemiology , Streptococcus pneumoniae , Haemophilus influenzaeABSTRACT
Clostridioides difficile remains a key cause of healthcare-associated infection, with multidrug-resistant (MDR) lineages causing high-mortality (≥20%) outbreaks. Cephalosporin treatment is a long-established risk factor, and antimicrobial stewardship is a key control. A mechanism underlying raised cephalosporin MICs has not been identified in C. difficile, but among other species, this is often acquired via amino acid substitutions in cell wall transpeptidases (penicillin binding proteins [PBPs]). Here, we investigated five C. difficile transpeptidases (PBP1 to PBP5) for recent substitutions, associated cephalosporin MICs, and co-occurrence with fluoroquinolone resistance. Previously published genome assemblies (n = 7,096) were obtained, representing 16 geographically widespread lineages, including healthcare-associated ST1(027). Recent amino acid substitutions were found within PBP1 (n = 50) and PBP3 (n = 48), ranging from 1 to 10 substitutions per genome. ß-Lactam MICs were measured for closely related pairs of wild-type and PBP-substituted isolates separated by 20 to 273 single nucleotide polymorphisms (SNPs). Recombination-corrected phylogenies were constructed to date substitution acquisition. Key substitutions such as PBP3 V497L and PBP1 T674I/N/V emerged independently across multiple lineages. They were associated with extremely high cephalosporin MICs; 1 to 4 doubling dilutions >wild-type, up to 1,506 µg/mL. Substitution patterns varied by lineage and clade, showed geographic structure, and occurred post-1990, coincident with the gyrA and/or gyrB substitutions conferring fluoroquinolone resistance. In conclusion, recent PBP1 and PBP3 substitutions are associated with raised cephalosporin MICs in C. difficile. Their co-occurrence with fluoroquinolone resistance hinders attempts to understand the relative importance of these drugs in the dissemination of epidemic lineages. Further controlled studies of cephalosporin and fluoroquinolone stewardship are needed to determine their relative effectiveness in outbreak control. IMPORTANCE Fluoroquinolone and cephalosporin use in healthcare settings has triggered outbreaks of high-mortality, multidrug-resistant C. difficile infection. Here, we identify a mechanism associated with raised cephalosporin MICs in C. difficile comprising amino acid substitutions in two cell wall transpeptidase enzymes (penicillin binding proteins). The higher the number of substitutions, the greater the impact on phenotype. Dated phylogenies revealed that substitutions associated with raised cephalosporin and fluoroquinolone MICs were co-acquired immediately before clinically important outbreak strains emerged. PBP substitutions were geographically structured within genetic lineages, suggesting adaptation to local antimicrobial prescribing. Antimicrobial stewardship of cephalosporins and fluoroquinolones is an effective means of C. difficile outbreak control. Genetic changes associated with raised MIC may impart a "fitness cost" after antibiotic withdrawal. Our study therefore identifies a mechanism that may explain the contribution of cephalosporin stewardship to resolving outbreak conditions. However, due to the co-occurrence of raised cephalosporin MICs and fluoroquinolone resistance, further work is needed to determine the relative importance of each.
Subject(s)
Clostridioides difficile , Peptidyl Transferases , Fluoroquinolones/pharmacology , Penicillin-Binding Proteins/genetics , Clostridioides , Anti-Bacterial Agents/pharmacology , Cephalosporins/pharmacology , Monobactams/pharmacology , Microbial Sensitivity TestsABSTRACT
Core genome multilocus sequence typing (cgMLST) employs a strategy where the set of orthologous genes common to all members of a group of organisms are used for phylogenetic analysis of the group members. The Bacillus cereus group consists of species with pathogenicity towards insect species as well as warm-blooded animals including humans. While B. cereus is an opportunistic pathogen linked to a range of human disease conditions, including emesis and diarrhoea, Bacillus thuringiensis is an entomopathogenic species with toxicity toward insect larvae, and therefore used as a biological pesticide worldwide. Bacillus anthracis is a classical obligate pathogen causing anthrax, an acute lethal condition in herbivores as well as humans, and which is endemic in many parts of the world. The group also includes a range of additional species, and B. cereus group bacteria have been subject to analysis with a wide variety of phylogenetic typing systems. Here we present, based on analyses of 173 complete genomes from B. cereus group species available in public databases, the identification of a set of 1568 core genes which were used to create a core genome multilocus typing scheme for the group which is implemented in the PubMLST system as an open online database freely available to the community. The new cgMLST system provides unprecedented resolution over existing phylogenetic analysis schemes covering the B. cereus group.
Subject(s)
Bacillus anthracis , Bacillus , Animals , Humans , Multilocus Sequence Typing , Bacillus cereus/genetics , Phylogeny , Bacillus anthracis/genetics , Genome, Bacterial/geneticsABSTRACT
Streptococcus mitis is a common oral commensal and an opportunistic pathogen that causes bacteremia and infective endocarditis; however, the species has received little attention compared to other pathogenic streptococcal species. Effective and easy-to-use molecular typing tools are essential for understanding bacterial population diversity and biology, but schemes specific for S. mitis are not currently available. We therefore developed a multilocus sequence typing (MLST) scheme and defined sequence clusters or lineages of S. mitis using a comprehensive global data set of 322 genomes (148 publicly available and 174 newly sequenced). We used internal 450-bp sequence fragments of seven housekeeping genes (accA, gki, hom, oppC, patB, rlmN, and tsf) to define the MLST scheme and derived the global S. mitis sequence clusters using the PopPUNK clustering algorithm. We identified an initial set of 259 sequence types (STs) and 258 global sequence clusters. The schemes showed high concordance (100%), capturing extensive S. mitis diversity with strains assigned to multiple unique STs and global sequence clusters. The tools also identified extensive within- and between-host S. mitis genetic diversity among isolates sampled from a cohort of healthy individuals, together with potential transmission events, supported by both phylogeny and pairwise single nucleotide polymorphism (SNP) distances. Our novel molecular typing and strain clustering schemes for S. mitis allow for the integration of new strain data, are electronically portable at the PubMLST database (https://pubmlst.org/smitis), and offer a standardized approach to understanding the population structure of S. mitis. These robust tools will enable new insights into the epidemiology of S. mitis colonization, disease and transmission.
Subject(s)
Streptococcus mitis , Streptococcus , Humans , Multilocus Sequence Typing , Streptococcus mitis/genetics , Streptococcus/genetics , Cluster Analysis , PhylogenyABSTRACT
Bacterial genomics is making an increasing contribution to the fields of medicine and public health microbiology. Consequently, accurate species identification of bacterial genomes is an important task, particularly as the number of genomes stored in online databases increases rapidly and new species are frequently discovered. Existing database entries require regular re-evaluation to ensure that species annotations are consistent with the latest species definitions. We have developed an automated method for bacterial species identification that is an extension of ribosomal multilocus sequence typing (rMLST). The method calculates an 'rMLST nucleotide identity' (rMLST-NI) based on the nucleotides present in the protein-encoding ribosomal genes derived from bacterial genomes. rMLST-NI was used to validate the species annotations of 11839 publicly available Klebsiella and Raoultella genomes based on a comparison with a library of type strain genomes. rMLST-NI was compared with two whole-genome average nucleotide identity methods (OrthoANIu and FastANI) and the k-mer based Kleborate software. The results of the four methods agreed across a dataset of 11839 bacterial genomes and identified a small number of entries (n=89) with species annotations that required updating. The rMLST-NI method was 3.5 times faster than Kleborate, 4.5 times faster than FastANI and 1600 times faster than OrthoANIu. rMLST-NI represents a fast and generic method for species identification using type strains as a reference.
Subject(s)
Klebsiella , Nucleotides , Genome, Bacterial/genetics , Genomics/methods , Klebsiella/genetics , Multilocus Sequence Typing/methodsABSTRACT
Brucellosis poses a significant burden to human and animal health worldwide. Robust and harmonized molecular epidemiological approaches and population studies that include routine disease screening are needed to efficiently track the origin and spread of Brucella strains. Core genome multilocus sequence typing (cgMLST) is a powerful genotyping system commonly used to delineate pathogen transmission routes for disease surveillance and control. Except for Brucella melitensis, cgMLST schemes for Brucella species are currently not established. Here, we describe a novel cgMLST scheme that covers multiple Brucella species. We first determined the phylogenetic breadth of the genus using 612 Brucella genomes. We selected 1,764 genes that were particularly well conserved and typeable in at least 98% of these genomes. We tested the new scheme on 600 genomes and found high agreement with the whole-genome-based single nucleotide polymorphism (SNP) analysis. Next, we applied the scheme to reanalyze the genome of Brucella strains from epidemiologically linked outbreaks. We demonstrated the applicability of the new scheme for high-resolution typing required in outbreak investigations as previously reported with whole-genome SNP methods. We also used the novel scheme to define the global population structure of the genus using 1,322 Brucella genomes. Finally, we demonstrated the possibility of tracing distribution of Brucella strains by performing cluster analysis of cgMLST profiles and found nearly identical cgMLST profiles in different countries. Our results show that sequencing depth of more than 40-fold is optimal for allele calling with this scheme. In summary, this study describes a novel Brucella-wide cgMLST scheme that is applicable in Brucella molecular epidemiology and helps in accurately tracking and thus controlling the sources of infection. The scheme is publicly accessible and should represent a valuable resource for laboratories with limited computational resources and bioinformatics expertise.
Subject(s)
Brucella melitensis , Genome, Bacterial , Animals , Brucella melitensis/genetics , Genome, Bacterial/genetics , Humans , Molecular Epidemiology/methods , Multilocus Sequence Typing/methods , PhylogenyABSTRACT
The genus Bordetella includes bacteria that are found in the environment and/or associated with humans and other animals. A few closely related species, including Bordetella pertussis, are human pathogens that cause diseases such as whooping cough. Here, we present a large database of Bordetella isolates and genomes and develop genotyping systems for the genus and for the B. pertussis clade. To generate the database, we merge previously existing databases from Oxford University and Institut Pasteur, import genomes from public repositories, and add 83 newly sequenced B. bronchiseptica genomes. The public database currently includes 2582 Bordetella isolates and their provenance data, and 2085 genomes ( https://bigsdb.pasteur.fr/bordetella/ ). We use core-genome multilocus sequence typing (cgMLST) to develop genotyping systems for the whole genus and for B. pertussis, as well as specific schemes to define antigenic, virulence and macrolide resistance profiles. Phylogenetic analyses allow us to redefine evolutionary relationships among known Bordetella species, and to propose potential new species. Our database provides an expandable resource for genotyping of environmental and clinical Bordetella isolates, thus facilitating evolutionary and epidemiological research on whooping cough and other Bordetella infections.
Subject(s)
Whooping Cough , Animals , Anti-Bacterial Agents , Biodiversity , Bordetella pertussis/genetics , Drug Resistance, Bacterial , Genomics , Humans , Macrolides , Phylogeny , Whooping Cough/epidemiologyABSTRACT
Sublineages (SLs) within microbial species can differ widely in their ecology and pathogenicity, and their precise definition is important in basic research and for industrial or public health applications. Widely accepted strategies to define SLs are currently missing, which confuses communication in population biology and epidemiological surveillance. Here, we propose a broadly applicable genomic classification and nomenclature approach for bacterial strains, using the prominent public health threat Klebsiella pneumoniae as a model. Based on a 629-gene core genome multilocus sequence typing (cgMLST) scheme, we devised a dual barcoding system that combines multilevel single linkage (MLSL) clustering and life identification numbers (LINs). Phylogenetic and clustering analyses of >7,000 genome sequences captured population structure discontinuities, which were used to guide the definition of 10 infraspecific genetic dissimilarity thresholds. The widely used 7-gene multilocus sequence typing (MLST) nomenclature was mapped onto MLSL SLs (threshold: 190 allelic mismatches) and clonal group (threshold: 43) identifiers for backwards nomenclature compatibility. The taxonomy is publicly accessible through a community-curated platform (https://bigsdb.pasteur.fr/klebsiella), which also enables external users' genomic sequences identification. The proposed strain taxonomy combines two phylogenetically informative barcode systems that provide full stability (LIN codes) and nomenclatural continuity with previous nomenclature (MLSL). This species-specific dual barcoding strategy for the genomic taxonomy of microbial strains is broadly applicable and should contribute to unify global and cross-sector collaborative knowledge on the emergence and microevolution of bacterial pathogens.
Subject(s)
Genome, Bacterial , Klebsiella pneumoniae , Genomics , Genotype , Klebsiella pneumoniae/genetics , Multilocus Sequence Typing , PhylogenyABSTRACT
Horizontal gene transfer (HGT) can allow traits that have evolved in one bacterial species to transfer to another. This has potential to rapidly promote new adaptive trajectories such as zoonotic transfer or antimicrobial resistance. However, for this to occur requires gaps to align in barriers to recombination within a given time frame. Chief among these barriers is the physical separation of species with distinct ecologies in separate niches. Within the genus Campylobacter, there are species with divergent ecologies, from rarely isolated single-host specialists to multihost generalist species that are among the most common global causes of human bacterial gastroenteritis. Here, by characterizing these contrasting ecologies, we can quantify HGT among sympatric and allopatric species in natural populations. Analyzing recipient and donor population ancestry among genomes from 30 Campylobacter species, we show that cohabitation in the same host can lead to a six-fold increase in HGT between species. This accounts for up to 30% of all SNPs within a given species and identifies highly recombinogenic genes with functions including host adaptation and antimicrobial resistance. As described in some animal and plant species, ecological factors are a major evolutionary force for speciation in bacteria and changes to the host landscape can promote partial convergence of distinct species through HGT.
Subject(s)
Anti-Infective Agents , Campylobacter , Animals , Bacteria/genetics , Biological Evolution , Campylobacter/genetics , Gene Transfer, Horizontal , PhylogenyABSTRACT
Mycobacterium bovis (M. bovis) is a causative agent of bovine tuberculosis, a significant source of morbidity and mortality in the global cattle industry. The Randomised Badger Culling Trial was a field experiment carried out between 1998 and 2005 in the South West of England. As part of this trial, M. bovis isolates were collected from contemporaneous and overlapping populations of badgers and cattle within ten defined trial areas. We combined whole genome sequences from 1,442 isolates with location and cattle movement data, identifying transmission clusters and inferred rates and routes of transmission of M. bovis. Most trial areas contained a single transmission cluster that had been established shortly before sampling, often contemporaneous with the expansion of bovine tuberculosis in the 1980s. The estimated rate of transmission from badger to cattle was approximately two times higher than from cattle to badger, and the rate of within-species transmission considerably exceeded these for both species. We identified long distance transmission events linked to cattle movement, recurrence of herd breakdown by infection within the same transmission clusters and superspreader events driven by cattle but not badgers. Overall, our data suggests that the transmission clusters in different parts of South West England that are still evident today were established by long-distance seeding events involving cattle movement, not by recrudescence from a long-established wildlife reservoir. Clusters are maintained primarily by within-species transmission, with less frequent spill-over both from badger to cattle and cattle to badger.
Subject(s)
Disease Reservoirs/microbiology , Mustelidae/microbiology , Mycobacterium bovis/isolation & purification , Tuberculosis, Bovine/transmission , Animals , Cattle , Clinical Trials, Veterinary as Topic , England/epidemiology , Random Allocation , Tuberculosis, Bovine/epidemiology , Tuberculosis, Bovine/microbiologyABSTRACT
Clostridium perfringens is a spore-forming anaerobic pathogen responsible for a variety of histotoxic and intestinal infections in humans and animals. High-resolution genotyping aiming to identify bacteria at strain level has become increasingly important in modern microbiology to understand pathogen transmission pathways and to tackle infection sources. This study aimed at establishing a publicly available genome-wide multilocus sequence-typing (MLST) scheme for C. perfringens. A total of 1,431 highly conserved core genes (1.34 megabases; 50% of the reference genome genes) were indexed for a core genome-based MLST (cgMLST) scheme for C. perfringens. The scheme was applied to 282 ecologically and geographically diverse genomes, showing that the genotyping results of cgMLST were highly congruent with the core genome-based single-nucleotide-polymorphism typing in terms of resolution and tree topology. In addition, the cgMLST provided a greater discrimination than classical MLST methods for C. perfringens. The usability of the scheme for outbreak analysis was confirmed by reinvestigating published outbreaks of C. perfringens-associated infections in the United States and the United Kingdom. In summary, a publicly available scheme and an allele nomenclature database for genomic typing of C. perfringens have been established and can be used for broad-based and standardized epidemiological studies. IMPORTANCE Global epidemiological surveillance of bacterial pathogens is enhanced by the availability of standard tools and sharing of typing data. The use of whole-genome sequencing has opened the possibility for high-resolution characterization of bacterial strains down to the clonal and subclonal levels. Core genome multilocus sequence typing is a robust system that uses highly conserved core genes for deep genotyping. The method has been successfully and widely used to describe the epidemiology of various bacterial species. Nevertheless, a cgMLST typing scheme for Clostridium perfringens is currently not publicly available. In this study, we (i) developed a cgMLST typing scheme for C. perfringens, (ii) evaluated the performance of the scheme on different sets of C. perfringens genomes from different hosts and geographic regions as well as from different outbreak situations, and, finally, (iii) made this scheme publicly available supported by an allele nomenclature database for global and standard genomic typing.