Recent Outbreaks of Shigellosis in California Caused by Two Distinct Populations of Shigella sonnei with either Increased Virulence or Fluoroquinolone Resistance.

Shigella sonnei has caused unusually large outbreaks of shigellosis in California in 2014 and 2015. Preliminary data indicated the involvement of two distinct bacterial populations, one from San Diego and San Joaquin (SDi/SJo) and one from the San Francisco (SFr) Bay area. Whole-genome analysis and antibiotic susceptibility testing of 68 outbreak and archival isolates of S. sonnei were performed to investigate the microbiological factors related to these outbreaks. Both SDi/SJo and SFr populations, as well as almost all of the archival S. sonnei isolates belonged to sequence type 152 (ST152). Genome-wide single nucleotide polymorphism (SNP) analysis clustered the majority of California (CA) isolates to an earlier described lineage III. Isolates in the SDi/SJo population had a novel lambdoid bacteriophage carrying genes encoding Shiga toxin (STX) that were most closely related to that found in Escherichia coli O104:H4. However, the STX genes (stx1A and stx1B) from this novel phage had sequences most similar to the phages from Shigella flexneri and S. dysenteriae. The isolates in the SFr population were resistant to ciprofloxacin due to point mutations in gyrA and parC genes and were related to the fluoroquinolone-resistant S. sonnei clade within lineage III that originated in South Asia. The emergence of a highly virulent S. sonnei strain and introduction of a fluoroquinolone-resistant strain reflect the changing traits of this pathogen in California. An enhanced monitoring is advocated for early detection of future outbreaks caused by such strains. IMPORTANCE Shigellosis is an acute diarrheal disease causing nearly half a million infections, 6,000 hospitalizations, and 70 deaths annually in the United States. S. sonnei caused two unusually large outbreaks in 2014 and 2015 in California. We used whole-genome sequencing to understand the pathogenic potential of bacteria involved in these outbreaks. Our results suggest the persistence of a local S. sonnei SDi/SJo clone in California since at least 2008. Recently, a derivative of the original clone acquired the ability to produce Shiga toxin (STX) via exchanges of bacteriophages with other bacteria. STX production is connected with more severe disease, including bloody diarrhea. A second population of S. sonnei that caused an outbreak in the San Francisco area was resistant to fluoroquinolones and showed evidence of connection to a fluoroquinolone-resistant lineage from South Asia. These emerging trends in S. sonnei populations in California must be monitored for future risks of the spread of increasingly virulent and resistant clones

A hybridization target enrichment approach for pathogen genomics

ImportanceEmerging infectious diseases require continuous pathogen monitoring. Rapid clinical diagnosis by nucleic acid amplification is limited to a small number of targets and may miss target detection due to new mutations in clinical isolates. Whole-genome sequencing (WGS) identifies genome-wide variations that may be used to determine a pathogen's drug resistance patterns and phylogenetically characterize isolates to track disease origin and transmission. WGS is typically performed using DNA isolated from cultured clinical isolates. Culturing clinical specimens increases turn-around time and may not be possible for fastidious bacteria. To overcome some of these limitations, direct sequencing of clinical specimens has been attempted using expensive capture probes to enrich the entire genomes of target pathogens. We present a method to produce a cost-effective, time-efficient, and large-scale synthesis of probes for whole-genome enrichment. We envision that our method can be used for direct clinical sequencing of a wide range of microbial pathogens for genomic epidemiology

A hybridization target enrichment approach for pathogen genomics

ABSTRACT Genomic epidemiology uses pathogens’ whole-genome sequences to understand and manage the spread of infectious diseases. Whole-genome data can be used to monitor outbreaks and cluster formation, identify cross-community transmissions, and characterize drug resistance and immune evasion. Typically, bacteria are cultured from clinical samples to obtain DNA for sequencing to generate whole-genome data. However, culture-independent diagnostic methods are utilized for some fastidious bacteria for better diagnostic yield and rapid pathogen genomics. Whole-genome enrichment (WGE) using targeted DNA sequencing enables direct sequencing of clinical samples without having to culture pathogens. However, the cost of capture probes (“baits”) limits the utility of this method for large-scale genomic epidemiology. We developed a cost-effective method named Circular Nucleic acid Enrichment Reagent synthesis (CNERs) to generate whole-genome enrichment probes. We demonstrated the method by producing probes for Mycobacterium tuberculosis, which we used to enrich M. tuberculosis DNA that had been spiked at concentrations as low as 0.01% and 100 genome copies against a human DNA background to 1,225-fold and 4,636-fold. Furthermore, we enriched DNA from different M. tuberculosis lineages and M. bovis and demonstrated the utility of the WGE-CNERs data for lineage identification and drug-resistance characterization using an established pipeline. The CNERs method for whole-genome enrichment will be a valuable tool for the genomic epidemiology of emerging and difficult-to-grow pathogens. IMPORTANCE Emerging infectious diseases require continuous pathogen monitoring. Rapid clinical diagnosis by nucleic acid amplification is limited to a small number of targets and may miss target detection due to new mutations in clinical isolates. Whole-genome sequencing (WGS) identifies genome-wide variations that may be used to determine a pathogen’s drug resistance patterns and phylogenetically characterize isolates to track disease origin and transmission. WGS is typically performed using DNA isolated from cultured clinical isolates. Culturing clinical specimens increases turn-around time and may not be possible for fastidious bacteria. To overcome some of these limitations, direct sequencing of clinical specimens has been attempted using expensive capture probes to enrich the entire genomes of target pathogens. We present a method to produce a cost-effective, time-efficient, and large-scale synthesis of probes for whole-genome enrichment. We envision that our method can be used for direct clinical sequencing of a wide range of microbial pathogens for genomic epidemiology

Convergent In Vivo and In Vitro Selection of Ceftazidime Resistance Mutations at Position 167 of CTX-M-3 β-Lactamase in Hypermutable Escherichia coli Strains▿

We report on a novel CTX-M extended-spectrum β-lactamase (ESBL), designated CTX-M-42, with enhanced activity toward ceftazidime. CTX-M-42 was identified in a hypermutable Escherichia coli nosocomial isolate (isolate Irk2320) and is a Pro167Thr amino acid substitution variant of CTX-M-3. By molecular typing of ESBL-producing E. coli strains previously isolated in the same hospital ward, we were able to identify a putative progenitor (strain Irk1224) of Irk2320, which had a mutator phenotype and harbored the CTX-M-3 β-lactamase. To reproduce the natural evolution of CTX-M-3, we selected for ceftazidime resistance mutations in blaCTX-M-3 gene in vitro both in clinical isolate Irk1224 and in laboratory-derived hypermutable (mutD5) strain GM2995. These experiments yielded CTX-M-3Pro167Ser and CTX-M-3Asn136Lys mutants which conferred higher levels of resistance to ceftazidime than to cefotaxime. CTX-M-3Asn136Lys had a level of low activity toward ampicillin, which may explain its absence from clinical isolates. We conclude that the selection of CTX-M-42 could have occurred in vivo following treatment with ceftazidime and was likely facilitated by the hypermutable background