DNA methylation by three Type I restriction modification systems of Escherichia coli does not influence gene regulation of the host bacterium.

DNA methylation is a common epigenetic mark that influences transcriptional regulation, and therefore cellular phenotype, across all domains of life. In particular, both orphan methyltransferases and those from phasevariable restriction modification systems (RMSs) have been co-opted to regulate virulence epigenetically in many bacteria. We now show that three distinct non-phasevariable Type I RMSs in Escherichia coli have no measurable impact on gene expression, in vivo virulence, or any of 1190 in vitro growth phenotypes. We demonstrated this using both Type I RMS knockout mutants as well as heterologous installation of Type I RMSs into two E. coli strains. These data provide three clear and currently rare examples of restriction modification systems that have no impact on their host organism's gene regulation. This leads to the possibility that other such nonregulatory methylation systems may exist, broadening our view of the potential role that RMSs may play in bacterial evolution.

Sequencing of E. coli strain UTI89 on multiple sequencing platforms.

OBJECTIVES: The availability of matched sequencing data for the same sample across different sequencing platforms is a necessity for validation and effective comparison of sequencing platforms. A commonly sequenced sample is the lab-adapted MG1655 strain of Escherichia coli; however, this strain is not fully representative of more complex and dynamic genomes of pathogenic E. coli strains. DATA DESCRIPTION: We present six new sequencing data sets for another E. coli strain, UTI89, which is an extraintestinal pathogenic strain isolated from a patient suffering from a urinary tract infection. We now provide matched whole genome sequencing data generated using the PacBio RSII, Oxford Nanopore MinION R9.4, Ion Torrent, ABI SOLiD, and Illumina NextSeq sequencers. Together with other publically available datasets, UTI89 has a nearly complete suite of data generated on most second- and third-generation sequencers. These data can be used as an additional validation set for new sequencing technologies and analytical methods. More than being another E. coli strain, however, UTI89 is pathogenic, with a 10% larger genome, additional pathogenicity islands, and a large plasmid, features that are common among other naturally occurring and disease-causing E. coli isolates. These data therefore provide a more medically relevant test set for development of algorithms.

Group B Streptococcus Infections Caused by Improper Sourcing and Handling of Fish for Raw Consumption, Singapore, 2015-2016.

We assessed microbial safety and quality of raw fish sold in Singapore during 2015-2016 to complement epidemiologic findings for an outbreak of infection with group B Streptococcus serotype III sequence type (ST) 283 associated with raw fish consumption. Fish-associated group B Streptococcus ST283 strains included strains nearly identical (0-2 single-nucleotide polymorphisms) with the human outbreak strain, as well as strains in another distinct ST283 clade (57-71 single-nucleotide polymorphisms). Our investigations highlight the risk for contamination of freshwater fish (which are handled and distributed separately from saltwater fish sold as sashimi) and the need for improved hygienic handling of all fish for raw consumption. These results have led to updated policy and guidelines regarding the sale of ready-to-eat raw fish dishes in Singapore.

Complete Genome Sequence of the Uropathogenic Escherichia coli Strain NU14.

Escherichia coli is the most common bacterium causing urinary tract infections in humans. We report here the complete genome sequence of the uropathogenic Escherichia coli strain NU14, a clinical pyelonephritis isolate used for studying pathogenesis.

2015 Epidemic of Severe Streptococcus agalactiae Sequence Type 283 Infections in Singapore Associated With the Consumption of Raw Freshwater Fish: A Detailed Analysis of Clinical, Epidemiological, and Bacterial Sequencing Data.

BACKGROUND: Streptococcus agalactiae (group B Streptococcus [GBS]) has not been described as a foodborne pathogen. However, in 2015, a large outbreak of severe invasive sequence type (ST) 283 GBS infections in adults epidemiologically linked to the consumption of raw freshwater fish occurred in Singapore. We attempted to determine the scale of the outbreak, define the clinical spectrum of disease, and link the outbreak to contaminated fish. METHODS: Time-series analysis was performed on microbiology laboratory data. Food handlers and fishmongers were screened for enteric carriage of GBS. A retrospective cohort study was conducted to assess differences in demographic and clinical characteristics of patients with invasive ST283 and non-ST283 infections. Whole-genome sequencing was performed on human and fish ST283 isolates from Singapore, Thailand, and Hong Kong. RESULTS: The outbreak was estimated to have started in late January 2015. Within the study cohort of 408 patients, ST283 accounted for 35.8% of cases. Patients with ST283 infection were younger and had fewer comorbidities but were more likely to develop meningoencephalitis, septic arthritis, and spinal infection. Of 82 food handlers and fishmongers screened, none carried ST283. Culture of 43 fish samples yielded 13 ST283-positive samples. Phylogenomic analysis of 161 ST283 isolates from humans and fish revealed they formed a tight clade distinguished by 93 single-nucleotide polymorphisms. CONCLUSIONS: ST283 is a zoonotic GBS clone associated with farmed freshwater fish, capable of causing severe disease in humans. It caused a large foodborne outbreak in Singapore and poses both a regional and potentially more widespread threat.

Complete Genome Sequence of the Original Escherichia coli Isolate, Strain NCTC86.

Escherichia coli is the most well-studied bacterium and a common colonizer of the lower mammalian gastrointestinal tract. We report here the complete genome sequence of the original Escherichia coli isolate, strain NCTC86, which was described by Theodor Escherich, for whom the genus is named.

Brighter Fluorescent Derivatives of UTI89 Utilizing a Monomeric vGFP.

Fluorescent proteins, especially green fluorescent protein (GFP), have been instrumental in understanding urinary tract infection pathogenesis by uropathogenic Escherichia coli (UPEC). We have used a recently developed GFP variant, vsfGFP-9, to create new plasmid- and chromosome-based GFP derivatives of the UPEC strain UTI89. The vsfGFP-9 strains are nearly 10× brighter with no in vitro growth or in vivo virulence defects compared to previously reported GFP-expressing UTI89 strains. The chromosomal vsfGFP-9 strain is equivalent to the wild type UTI89 during in vivo UTI, while both plasmid GFP constructs have an equivalent virulence defect compared to non-plasmid carrying UTI89. These new vsfGFP-9 expressing strains should be useful for further studies of the pathogenesis of UTI89, and similar strategies can be used to create improved fluorescent derivatives of other UPEC strains.

Application and Optimization of relE as a Negative Selection Marker for Making Definitive Genetic Constructs in Uropathogenic Escherichia coli.

Studies of Uropathogenic Escherichia coli (UPEC) pathogenesis have relied heavily on genetic manipulation to understand virulence factors. We applied a recently reported positive-negative selection system to create a series of unmarked, scarless FimH mutants that show identical phenotypes to previously reported marked FimH mutants; these are now improved versions useful for definitive assignment of phenotypes to FimH mutations. We also increased the efficiency of this system by designing new primer sites, which should further improve the efficiency and convenience of using negative selection in UTI89, other UPEC, and other Enterobacteriaceae.

Complete Genome Sequence of Streptococcus agalactiae Serotype III, Multilocus Sequence Type 283 Strain SG-M1.

Streptococcus agalactiae (group B Streptococcus) is a common commensal strain in the human gastrointestinal tract that can also cause invasive disease in humans and other animals. We report here the complete genome sequence of S. agalactiae SG-M1, a serotype III, multilocus sequence type 283 strain, isolated from a Singaporean patient suffering from meningitis.

Complete Genome Sequence of Uropathogenic Escherichia coli Strain CI5.

Escherichia coli represents the primary etiological agent responsible for urinary tract infections, one of the most common infections in humans. We report here the complete genome sequence of uropathogenic Escherichia coli strain CI5, a clinical pyelonephritis isolate used for studying pathogenesis.