Significance: Escherichia coli sequence type 131 (ST131) is a globally disseminated multidrug-resistant clone associated with human urinary tract and bloodstream infections. Here, we have used genome sequencing to map the temporal and spatial relationship of a large collection of E. coli ST131 strains isolated from six distinct geographical regions across the world. We show that E. coli ST131 strains are distinct from other extraintestinal pathogenic E. coli and arose from a single progenitor strain prior to the year 2000.

Recently, a CTX-M-15 extended-spectrum β-lactamase (ESBL)-producing Escherichia coli O25b-ST131 clone, belonging to the B2 phylogenetic group and with a high virulence potential, has been reported all over the world, representing a major public health problem. The present study was carried out to develop a rapid and simple detection assay that identifies members of this clone. A total of 627 E. coli isolates of which 373 produced an ESBL, collected across four continents, were screened using a O25b-ST131 clone allele-specific PCR for the pabB gene. One hundred and forty-three ESBL isolates were found positive with the assay. These isolates were all of O25b type and, when studied by multilocus sequence typing (25 cases), were all of ST131. The O25b-ST131 clone was found to produce ESBLs other than CTX-M-15, specifically CTX-M-2, -3, -14, -27, -32 and -61 as well as TEM-24. This clone represents 3% of non-ESBL B2 isolates originating from urinary tract infections in Paris. We have developed a PCR-based assay that easily identifies a clone with high likelihood of producing ESBLs, including CTX-M-15.

Escherichia coli strains causing urinary tract infection (UTI) are increasingly recognized as belonging to specific clones. E. coli clone O25b:H4-ST131 has recently emerged globally as a leading multi-drug resistant pathogen causing urinary tract and bloodstream infections in hospitals and the community. While most molecular studies to date examine the mechanisms conferring multi-drug resistance in E. coli ST131, relatively little is known about their virulence potential. Here we examined E. coli ST131 clinical isolates from two geographically diverse collections, one representing the major pathogenic lineages causing UTI across the United Kingdom and a second representing UTI isolates from patients presenting at two large hospitals in Australia. We determined a draft genome sequence for one representative isolate, E. coli EC958, which produced CTX-M-15 extended-spectrum β-lactamase, CMY-23 type AmpC cephalosporinase and was resistant to ciprofloxacin. Comparative genome analysis indicated that EC958 encodes virulence genes commonly associated with uropathogenic E. coli (UPEC). The genome sequence of EC958 revealed a transposon insertion in the fimB gene encoding the activator of type 1 fimbriae, an important UPEC bladder colonization factor. We identified the same fimB transposon insertion in 59% of the ST131 UK isolates, as well as 71% of ST131 isolates from Australia, suggesting this mutation is common among E. coli ST131 strains. Insertional inactivation of fimB resulted in a phenotype resembling a slower off-to-on switching for type 1 fimbriae. Type 1 fimbriae expression could still be induced in fimB-null isolates; this correlated strongly with adherence to and invasion of human bladder cells and bladder colonisation in a mouse UTI model. We conclude that E. coli ST131 is a geographically widespread, antibiotic resistant clone that has the capacity to produce numerous virulence factors associated with UTI.

Escherichia coli ST131 is a globally disseminated, multidrug resistant clone responsible for a high proportion of urinary tract and bloodstream infections. The rapid emergence and successful spread of E. coli ST131 is strongly associated with antibiotic resistance; however, this phenotype alone is unlikely to explain its dominance amongst multidrug resistant uropathogens circulating worldwide in hospitals and the community. Thus, a greater understanding of the molecular mechanisms that underpin the fitness of E. coli ST131 is required. In this study, we employed hyper-saturated transposon mutagenesis in combination with multiplexed transposon directed insertion-site sequencing to define the essential genes required for in vitro growth and the serum resistome (i.e. genes required for resistance to human serum) of E. coli EC958, a representative of the predominant E. coli ST131 clonal lineage. We identified 315 essential genes in E. coli EC958, 231 (73%) of which were also essential in E. coli K-12. The serum resistome comprised 56 genes, the majority of which encode membrane proteins or factors involved in lipopolysaccharide (LPS) biosynthesis. Targeted mutagenesis confirmed a role in serum resistance for 46 (82%) of these genes. The murein lipoprotein Lpp, along with two lipid A-core biosynthesis enzymes WaaP and WaaG, were most strongly associated with serum resistance. While LPS was the main resistance mechanism defined for E. coli EC958 in serum, the enterobacterial common antigen and colanic acid also impacted on this phenotype. Our analysis also identified a novel function for two genes, hyxA and hyxR, as minor regulators of O-antigen chain length. This study offers novel insight into the genetic make-up of E. coli ST131, and provides a framework for future research on E. coli and other Gram-negative pathogens to define their essential gene repertoire and to dissect the molecular mechanisms that enable them to survive in the bloodstream and cause disease. The emergence and rapid dissemination of new bacterial pathogens presents multiple challenges to healthcare systems, including the need for rapid detection, precise diagnostics, effective transmission control and effective treatment. E. coli ST131 is an example of a recently emerged multidrug resistant pathogen that is capable of causing urinary tract and bloodstream infections with limited available treatment options. In order to increase our molecular understanding of E. coli ST131, we developed a high-throughput transposon mutagenesis system in combination with next generation sequencing to test every gene for its essential role in growth and for its contribution to serum resistance. We identified 315 essential genes, 270 of which were conserved among all currently available complete E. coli genomes. Fifty-six genes that define the serum resistome of E. coli ST131 were identified, including genes encoding membrane proteins, proteins involved in LPS biosynthesis, regulators and several novel proteins with previously unknown function. This study therefore provides an inventory of essential and serum resistance genes that could form a framework for the future development of targeted therapeutics to prevent disease caused by multidrug-resistant E. coli ST131 strains.

Multilocus sequence typing (MLST) has been used to characterize diverse pathogens, including uropathogenic Escherichia coli (UPEC). There has been significant interest in the contribution of the O25b:H4-ST131 lineage to UPEC disease, as these isolates are often highly virulent and exhibit multidrug resistance. To reveal the wider impact of sequence type (ST) 131, we have examined its contribution to the overall population structure of UPEC isolates that were not selected on the basis of virulence or antibiotic resistance. Three hundred UPEC isolates were recovered from community and hospital urine samples examined by clinical microbiology laboratories in the Northwest region of England in June 2007 and June 2009. They were characterized by susceptibility profiling, MLST and virulence gene PCR. PFGE was used to examine isolates from key clones. The most common lineage was ST73 (16.6%) followed by ST131 (13.3%), ST69 (9%), ST95 (6.3%), ST10 (4.3%) and ST127 (3.6%). ST131 isolates were significantly more likely to exhibit high levels of antibiotic resistance (35% being CTX-M-15 PCR positive) and those of ST127 were the most widely susceptible but carried the highest number of virulence genes. Only when the CTX-M-15-O25b-positive strains were examined was a high level of virulence observed for ST131 isolates. PFGE indicated ongoing local evolution in ST131. ST131 isolates are well established in the wider UPEC population. This clone is still evolving and we further support suggestions that it represents a real threat to health. We suggest that ST127 is a recently emerged, community-associated, virulent clone that warrants further study.

The commercial probiotic Streptococcus salivarius strain K12 is the prototype of those S. salivarius strains that are the most strongly inhibitory in a standardized test of streptococcal bacteriocin production and has been shown to produce the 2,368-Da salivaricin A2 (SalA2) and the 2,740-Da salivaricin B (SboB) lantibiotics. The previously uncharacterized SboB belongs to the type AII class of lantibiotic bacteriocins and is encoded by an eight-gene cluster. The genetic loci encoding SalA2 and SboB in strain K12 have been fully characterized and are localized to nearly adjacent sites on pSsal-K12, a 190-kb megaplasmid. Of 61 strongly inhibitory strains of S. salivarius , 19 (31%) were positive for the sboB structural gene. All but one (strain NR) of these 19 strains were also positive for salA2 , and in each of these cases of double positivity, the two loci were separated by fewer than 10 kb. This is the first report of a single streptococcus strain producing two distinct lantibiotics.