Global genomic epidemiology of Clostridioides difficile

Clostridioides difficile gilt als Haupterreger von nosokomial auftretender, antibiotikaassoziierter Diarrhö. Die globale Ausbreitung des Erregers sowie direkte Transmissionswege wurden mit Hilfe der Ganzgenomsequenzierung intensiv untersucht, allerdings nur auf einem begrenzten Datensatz. Die auf der Softwareplattform EnteroBase (http://enterobase.warwick.ac.uk) verfügbare Datenbank von C. difficile Genomen beinhaltete zum Zeitpunkt der Analysen 13.515 Genome. EnteroBase ermöglicht zudem das einheitliche Prozessieren dieser Sequenzdaten. Anhand eines Kerngenom-MLST (cgMLST) Schemas werden diese typisiert und durch ihre paarweisen Übereinstimmungen in den cgMLST Allelprofilen in hierarchische Cluster eingeteilt. Die Ergebnisse dieser Arbeit demonstrieren, dass EnteroBase mit der Einteilung der Genome in hierarchische HC150 Cluster eine einheitliche Typisierungsmethode von C. difficile Isolaten bietet und Genome von Isolaten, die in Publikationen Pandemien zugeordnet wurden, in HC10 Cluster fallen. Der erstmals durchgeführte, quantitative Vergleich mit der standardmäßig zur Detektion von Transmissionsketten verwendeten SNP-Analyse zeigte, dass anhand der cgMLST-Allelprofile vergleichbare genomische Unterschiede zwischen Isolaten erzielt wurden. Eyre et al. stellten fest, dass Isolate mit einer genomischen Distanz von ≤2 SNPs mit 95 %iger Wahrscheinlichkeit einer Transmissionskette angehören. So konnten anhand von HC2 Clustern, die Isolate mit genomischen Distanzen ≤2 cgMLST Allelunterschieden zusammenfassen, verschiedene epidemiologische Erkenntnisse erzielt werden, wie zum Beispiel die retrospektive Aufdeckung von Transmissionswegen in einem Netzwerk von Krankenhäusern. Für nahe genomische Verwandtschaften zwischen epidemiologisch nicht zusammenhängenden Isolaten schien der Wert einer genomischen Distanz von ≤2 allerdings nicht praktikabel, da die Isolate große Unterschiede in ihrem akzessorischem Gengehalt zeigten. In der vorliegenden Arbeit konnte anhand der C. difficile Datenbank in EnteroBase ein zuvor noch nicht erfasster umfangreicher Einblick in die Populationsstruktur des pathogenen Bakteriums gewonnen werden. Der hier erbrachte Beweis, dass anhand der bioinformatischen Werkzeuge in EnteroBase C. difficile Isolate typisiert und Transmissionswege aufgedeckt werden können, ermöglicht auch Wissenschaftlern ohne bioinformatischem Hintergrund in Zukunft entsprechende Analysen in einem globalen Kontext auf standardisierte Weise durchzuführen.Clostridioides difficile is the primary cause of nosocomial, antibiotic-associated diarrhea. Local outbreaks and global spread of this pathogen have been previously investigated by whole-genome sequencing, but was only possible for a limited number of isolates. The extensive database of C. difficile genomes available on the software platform EnteroBase (http://enterobase.warwick.ac.uk) contained 13.515 genomes at the time of analysis. The implemented bioinformatics tools process the sequencing data in a standardized way and to type them using the core-genome multilocus sequence typing (cgMLST) scheme. Subsequently, entries are sorted into hierarchical clusters based on their distances in their cgMLST allelic profiles. This work demonstrates, that by clustering genomes into hierarchical clusters HC150, EnteroBase provides a standardized typing method for C. difficile isolates. Furthermore, genomes of isolates that have been assigned to pandemics fall into HC10 clusters. The quantitative comparison between SNP- and cgMLST-analysis, which was performed for the first time here, resulted in comparable pairwise genomic distances between isolates. Eyre et al. found that isolates with a genomic distance of ≤2 SNPs have a 95% probability to be part of a transmission chain. This value was also applied to core-genome allelic differences. The hierarchical clustering in EnteroBase combines isolates with a chainwise genomic distance of ≤2 core-genome allelic differences into one HC2 cluster. Thus, HC2 clusters were successfully used to answer different epidemiological questions, like the retrospective detection of transmission chains in a network of hospitals. However, it seemed that a distance of ≤2 for close genomic relationships was not applicable to isolates that were not epidemiologically associated, as these isolates showed large differences in their accessory gene content. In the present work, the C. difficile database in EnteroBase was used to gain a previously unexplored insight into the population structure of the pathogenic bacterium. The evidence provided in this work demonstrated that the bioinformatics tools in EnteroBase can be applied to type C. difficile isolates and to unravel transmission chains. This enables non-bioinformaticians to perform corresponding analyses in a global context and standardized manner in the future

Bacterial genome sequencing tracks the housefly-associated dispersal of fluoroquinolone- and cephalosporin-resistant Escherichia coli from a pig farm

The regular use of antimicrobials in livestock production selects for antimicrobial resistance. The potential impact of this practice on human health needs to be studied in more detail, including the role of the environment for the persistence and transmission of antimicrobial-resistant bacteria. During an investigation of a pig farm and its surroundings in Brandenburg, Germany, we detected abundant cephalosporin- and fluoroquinolone-resistant Escherichia coli in pig faeces, sedimented dust, and house flies (Musca domestica). Genome sequencing of E. coli isolates revealed large phylogenetic diversity and plasmid-borne extended-spectrum beta lactamase (ESBL) genes CTX-M-1 in multiple strains. [Correction added on 28 February 2023, after first online publication: In the preceding sentence, ‘and TEM-1’ was previously included but has been deleted in this version.] Close genomic relationships indicated frequent transmission of antimicrobial-resistant E. coli between pigs from different herds and across buildings of the farm and suggested dust and flies as vectors for dissemination of faecal pathogens. Strikingly, we repeatedly recovered E. coli from flies collected up to 2 km away from the source, whose genome sequences were identical or closely related to those from pig faeces isolates, indicating the fly-associated transport of diverse ESBL-producing E. coli from the pig farm into urban habitation areas. The observed proximity of contaminated flies to human households poses a risk of transmission of antimicrobial-resistant enteric pathogens from livestock to man

Agricultural fertilization with poultry manure results in persistent environmental contamination with the pathogen Clostridioides difficile

During a field experiment applying broiler manure for fertilization of agricultural land, we detected viable Clostridioides (also known as Clostridium) difficile in broiler faeces, manure, dust and fertilized soil. A large diversity of toxigenic C. difficile isolates was recovered, including PCR ribotypes common from human disease. Genomic relatedness of C. difficile isolates from dust and from soil, recovered more than 2 years after fertilization, traced their origins to the specific chicken farm that had delivered the manure. We present evidence of long-term contamination of agricultural soil with manure-derived C. difficile and demonstrate the potential for airborne dispersal of C. difficile through dust emissions during manure application. Clostridioides genome sequences virtually identical to those from manure had been recovered from chicken meat and from human infections in previous studies, suggesting broiler-associated C. difficile are capable of zoonotic transmission

Agricultural fertilization with poultry manure results in persistent environmental contamination with the pathogen Clostridioides difficile

During a field experiment applying broiler manure for fertilization of agricultural land, we detected viable Clostridioides (also known as Clostridium) difficile in broiler faeces, manure, dust and fertilized soil. A large diversity of toxigenic C. difficile isolates was recovered, including PCR ribotypes common from human disease. Genomic relatedness of C. difficile isolates from dust and from soil, recovered more than 2 years after fertilization, traced their origins to the specific chicken farm that had delivered the manure. We present evidence of long-term contamination of agricultural soil with manure-derived C. difficile and demonstrate the potential for airborne dispersal of C. difficile through dust emissions during manure application. Clostridioides genome sequences virtually identical to those from manure had been recovered from chicken meat and from human infections in previous studies, suggesting broiler-associated C. difficile are capable of zoonotic transmission

A publicly accessible database for Clostridioides difficile genome sequences supports tracing of transmission chains and epidemics

Clostridioides difficile is the primary infectious cause of antibiotic-associated diarrhea. Local transmissions and international outbreaks of this pathogen have been previously elucidated by bacterial whole-genome sequencing, but comparative genomic analyses at the global scale were hampered by the lack of specific bioinformatic tools. Here we introduce a publicly accessible database within EnteroBase (http://enterobase.warwick.ac.uk) that automatically retrieves and assembles C. difficile short-reads from the public domain, and calls alleles for core-genome multilocus sequence typing (cgMLST). We demonstrate that comparable levels of resolution and precision are attained by EnteroBase cgMLST and single-nucleotide polymorphism analysis. EnteroBase currently contains 18 254 quality-controlled C. difficile genomes, which have been assigned to hierarchical sets of single-linkage clusters by cgMLST distances. This hierarchical clustering is used to identify and name populations of C. difficile at all epidemiological levels, from recent transmission chains through to epidemic and endemic strains. Moreover, it puts newly collected isolates into phylogenetic and epidemiological context by identifying related strains among all previously published genome data. For example, HC2 clusters (i.e. chains of genomes with pairwise distances of up to two cgMLST alleles) were statistically associated with specific hospitals (P<10−4) or single wards (P=0.01) within hospitals, indicating they represented local transmission clusters. We also detected several HC2 clusters spanning more than one hospital that by retrospective epidemiological analysis were confirmed to be associated with inter-hospital patient transfers. In contrast, clustering at level HC150 correlated with k-mer-based classification and was largely compatible with PCR ribotyping, thus enabling comparisons to earlier surveillance data. EnteroBase enables contextual interpretation of a growing collection of assembled, quality-controlled C. difficile genome sequences and their associated metadata. Hierarchical clustering rapidly identifies database entries that are related at multiple levels of genetic distance, facilitating communication among researchers, clinicians and public-health officials who are combatting disease caused by C. difficile

Agricultural fertilization with poultry manure results in persistent environmental contamination with the pathogen Clostridioides difficile

During a field experiment applying broiler manure for fertilization of agricultural land, we detected viable Clostridioides (also known as Clostridium) difficile in broiler faeces, manure, dust and fertilized soil. A large diversity of toxigenic C. difficile isolates was recovered, including PCR ribotypes common from human disease. Genomic relatedness of C. difficile isolates from dust and from soil, recovered more than 2 years after fertilization, traced their origins to the specific chicken farm that had delivered the manure. We present evidence of long-term contamination of agricultural soil with manure-derived C. difficile and demonstrate the potential for airborne dispersal of C. difficile through dust emissions during manure application. Clostridioides genome sequences virtually identical to those from manure had been recovered from chicken meat and from human infections in previous studies, suggesting broiler-associated C. difficile are capable of zoonotic transmission

Hospital outbreak due to Clostridium difficile ribotype 018 (RT018) in Southern Germany

Clostridium (Clostridioides) difficile is the main cause of nosocomial diarrhoea. Ribotype 018 (RT018) has been recognized as the predominant strain responsible for C. difficile infection (CDI) in Italy, whereas in most other European countries only sporadic RT018 cases occur. Between August and October 2015, a suspected C. difficile outbreak at two associated hospitals in Southern Germany was investigated by comprehensive molecular typing. Surprisingly, RT018 was detected in 9/82 CDI patients, which has never been described before in a German outbreak. Phenotypic analysis revealed fluoroquinolone and macrolide resistance. Genetic subtyping using multiple-locus variable-number tandem-repeat analysis (MLVA) and whole genome sequencing (WGS) was performed and outbreak isolates were directly compared to sporadic German RT018 isolates and to epidemic ones from Milan, Northern Italy. Molecular typing confirmed a hospital outbreak with closely related RT018 isolates. Both, MLVA and WGS revealed high similarity of outbreak strains with epidemic isolates from Italy, but low similarity to other German isolates. Comparison between both typing strategies showed that ribotyping in combination with MLVA was appropriate to identify related isolates and clonal complexes, whereas WGS provided a better discrimination with more detailed information about the phylogenetic relationship of isolates. This is the first hospital outbreak in Germany presumably caused by cross-national transmission of an Italian epidemic RT018 strain

A publicly accessible database for genome sequences supports tracing of transmission chains and epidemics.

Clostridioides difficile is the primary infectious cause of antibiotic-associated diarrhea. Local transmissions and international outbreaks of this pathogen have been previously elucidated by bacterial whole-genome sequencing, but comparative genomic analyses at the global scale were hampered by the lack of specific bioinformatic tools. Here we introduce a publicly accessible database within EnteroBase (http://enterobase.warwick.ac.uk) that automatically retrieves and assembles C. difficile short-reads from the public domain, and calls alleles for core-genome multilocus sequence typing (cgMLST). We demonstrate that comparable levels of resolution and precision are attained by EnteroBase cgMLST and single-nucleotide polymorphism analysis. EnteroBase currently contains 18 254 quality-controlled C. difficile genomes, which have been assigned to hierarchical sets of single-linkage clusters by cgMLST distances. This hierarchical clustering is used to identify and name populations of C. difficile at all epidemiological levels, from recent transmission chains through to epidemic and endemic strains. Moreover, it puts newly collected isolates into phylogenetic and epidemiological context by identifying related strains among all previously published genome data. For example, HC2 clusters (i.e. chains of genomes with pairwise distances of up to two cgMLST alleles) were statistically associated with specific hospitals (P<10-4) or single wards (P=0.01) within hospitals, indicating they represented local transmission clusters. We also detected several HC2 clusters spanning more than one hospital that by retrospective epidemiological analysis were confirmed to be associated with inter-hospital patient transfers. In contrast, clustering at level HC150 correlated with k-mer-based classification and was largely compatible with PCR ribotyping, thus enabling comparisons to earlier surveillance data. EnteroBase enables contextual interpretation of a growing collection of assembled, quality-controlled C. difficile genome sequences and their associated metadata. Hierarchical clustering rapidly identifies database entries that are related at multiple levels of genetic distance, facilitating communication among researchers, clinicians and public-health officials who are combatting disease caused by C. difficile