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Cronobacter strains harboring CRISPR-Cas systems are important foodborne pathogens that cause serious neonatal infections. CRISPR typing is a new molecular subtyping method to track the sources of pathogenic bacterial outbreaks and shows a promise in typing Cronobacter, however, this molecular typing procedure using routine PCR method has not been established. Therefore, the purpose of this study was to establish such methodology, 257 isolates of Cronobacter sakazakii, C. malonaticus, and C. dublinensis were used to verify the feasibility of the method. Results showed that 161 C. sakazakii strains could be divided into 129 CRISPR types (CTs), among which CT15 (n = 7) was the most prevalent CT followed by CT6 (n = 4). Further, 65 C. malonaticus strains were divided into 42 CTs and CT23 (n = 8) was the most prevalent followed by CT2, CT3, and CT13 (n = 4). Finally, 31 C. dublinensis strains belonged to 31 CTs. There was also a relationship among CT, sequence type (ST), food types, and serotype. Compared to multi-locus sequence typing (MLST), this new molecular method has greater power to distinguish similar strains and had better accordance with whole genome sequence typing (WGST). More importantly, some lineages were found to harbor conserved ancestral spacers ahead of their divergent specific spacer sequences; this can be exploited to infer the divergent evolution of Cronobacter and provide phylogenetic information reflecting common origins. Compared to WGST, CRISPR typing method is simpler and more affordable, it could be used to identify sources of Cronobacter food-borne outbreaks, from clinical cases to food sources and the production sites.

Cronobacter strains harboring CRISPR-Cas systems are important foodborne pathogens that cause serious neonatal infections. CRISPR typing is a new molecular subtyping method to track the sources of pathogenic bacterial outbreaks and shows a promise in typing Cronobacter , however, this molecular typing procedure using routine PCR method has not been established. Therefore, the purpose of this study was to establish such methodology, 257 isolates of Cronobacter sakazakii , C. malonaticus , and C. dublinensis were used to verify the feasibility of the method. Results showed that 161 C. sakazakii strains could be divided into 129 CRISPR types (CTs), among which CT15 (n = 7) was the most prevalent CT followed by CT6 (n = 4). Further, 65 C. malonaticus strains were divided into 42 CTs and CT23 (n = 8) was the most prevalent followed by CT2, CT3, and CT13 (n = 4). Finally, 31 C. dublinensis strains belonged to 31 CTs. There was also a relationship among CT, sequence type (ST), food types, and serotype. Compared to multi-locus sequence typing (MLST), this new molecular method has greater power to distinguish similar strains and had better accordance with whole genome sequence typing (WGST). More importantly, some lineages were found to harbor conserved ancestral spacers ahead of their divergent specific spacer sequences; this can be exploited to infer the divergent evolution of Cronobacter and provide phylogenetic information reflecting common origins. Compared to WGST, CRISPR typing method is simpler and more affordable, it could be used to identify sources of Cronobacter food-borne outbreaks, from clinical cases to food sources and the production sites. [ABSTRACT FROM AUTHOR]

Background. Candida auris, a multidrug-resistant yeast that causes invasive infections, was first described in 2009 in Japan and has since been reported from several countries. Methods. To understand the global emergence and epidemiology of C. auris, we obtained isolates from 54 patients with C. auris infection from Pakistan, India, South Africa, and Venezuela during 2012·015 and the type specimen from Japan. Patient information was available for 41 of the isolates. We conducted antifungal susceptibility testing and whole-genome sequencing (WGS). Results. Available clinical information revealed that 41% of patients had diabetes mellitus, 51% had undergone recent surgery, 73% had a central venous catheter, and 41% were receiving systemic antifungal therapy when C. auris was isolated. The median time from admission to infection was 19 days (interquartile range, 9·6 days), 61% of patients had bloodstream infection, and 59% died. Using stringent break points, 93% of isolates were resistant to fluconazole, 35% to amphotericin B, and 7% to echinocandins; 41% were resistant to 2 antifungal classes and 4% were resistant to 3 classes. WGS demonstrated that isolates were grouped into unique clades by geographic region. Clades were separated by thousands of single-nucleotide polymorphisms, but within each clade isolates were clonal. Different mutations in ERG11 were associated with azole resistance in each geographic clade. Conclusions. C. auris is an emerging healthcare-associated pathogen associated with high mortality. Treatment options are limited, due to antifungal resistance. WGS analysis suggests nearly simultaneous, and recent, independent emergence of different clonal populations on 3 continents. Risk factors and transmission mechanisms need to be elucidated to guide control measures. [ABSTRACT FROM AUTHOR]

Next-generation sequencing enables use of whole-genome sequence typing (WGST) as a viable and discriminatory tool for genotyping and molecular epidemiologic analysis. We used WGST to confirm the linkage of a cluster of Coccidioides immitis isolates from 3 patients who received organ transplants from a single donor who later had positive test results for coccidioidomycosis. Isolates from the 3 patients were nearly genetically identical (a total of 3 single-nucleotide polymorphisms identified among them), thereby demonstrating direct descent of the 3 isolates from an original isolate. We used WGST to demonstrate the genotypic relatedness of C. immitis isolates that were also epidemiologically linked. Thus, WGST offers unique benefits to public health for investigation of clusters considered to be linked to a single source.

Multi isolate whole genome sequencing (WGS) and typing for outbreak investigations has become a reality in the post-genomics era. We applied this technology to strains from Escherichia coli O157:H7 outbreaks. These include isolates from seven North America outbreaks, as well as multiple isolates from the same patient and from different infected individuals in the same household. Customized high-resolution bioinformatics sequence typing strategies were developed to assess the core genome and mobilome plasticity. Sequence typing was performed using an in-house single nucleotide polymorphism (SNP) discovery and validation pipeline. Discriminatory power becomes of particular importance for the investigation of isolates from outbreaks in which macrogenomic techniques such as pulse-field gel electrophoresis or multiple locus variable number tandem repeat analysis do not differentiate closely related organisms. We also characterized differences in the phage inventory, allowing us to identify plasticity among outbreak strains that is not detectable at the core genome level. Our comprehensive analysis of the mobilome identified multiple plasmids that have not previously been associated with this lineage. Applied phylogenomics approaches provide strong molecular evidence for exceptionally little heterogeneity of strains within outbreaks and demonstrate the value of intra-cluster comparisons, rather than basing the analysis on archetypal reference strains. Next generation sequencing and whole genome typing strategies provide the technological foundation for genomic epidemiology outbreak investigation utilizing its significantly higher sample throughput, cost efficiency, and phylogenetic relatedness accuracy. These phylogenomics approaches have major public health relevance in translating information from the sequence-based survey to support timely and informed countermeasures. Polymorphisms identified in this work offer robust phylogenetic signals that index both short- and long-term evolution and can complement currently employed typing schemes for outbreak ex- and inclusion, diagnostics, surveillance, and forensic studies. [ABSTRACT FROM AUTHOR]

Introduction: Salmonella enterica serovar Typhimurium ST313 is an invasive and phylogenetically distinct lineage present in sub-Saharan Africa. We report the presence of S. Typhimurium ST313 from patients in the Democratic Republic of Congo and Nigeria. Methodology: Eighteen S. Typhimurium ST313 isolates were characterized by antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). Additionally, six of the isolates were characterized by whole genome sequence typing (WGST). The presence of a putative virulence determinant was examined in 177 Salmonella isolates belonging to 57 different serovars. Results: All S. Typhimurium ST313 isolates harbored resistant genes encoded by blaTEM1b, catA1, strA/B, sul1, and dfrA1. Additionally, aac(6')1aa gene was detected. Phylogenetic analyses revealed close genetic relationships among Congolese and Nigerian isolates from both blood and stool. Comparative genomic analyses identified a putative virulence fragment (ST313-TD) unique to S. Typhimurium ST313 and S. Dublin. Conclusion: We showed in a limited number of isolates that S. Typhimurium ST313 is a prevalent sequence-type causing gastrointestinal diseases and septicemia in patients from Nigeria and DRC. We found three distinct phylogenetic clusters based on the origin of isolation suggesting some spatial evolution. Comparative genomics showed an interesting putative virulence fragment (ST313-TD) unique to S. Typhimurium ST313 and invasive S. Dublin. [ABSTRACT FROM AUTHOR]