Global dissemination of a multidrug resistant Escherichia coli clone.

Escherichia coli sequence type 131 (ST131) is a globally disseminated, multidrug resistant (MDR) 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 several factors, including resistance to fluoroquinolones, high virulence gene content, the possession of the type 1 fimbriae FimH30 allele, and the production of the CTX-M-15 extended spectrum ß-lactamase (ESBL). Here, we used genome sequencing to examine the molecular epidemiology of a collection of E. coli ST131 strains isolated from six distinct geographical locations across the world spanning 2000-2011. The global phylogeny of E. coli ST131, determined from whole-genome sequence data, revealed a single lineage of E. coli ST131 distinct from other extraintestinal E. coli strains within the B2 phylogroup. Three closely related E. coli ST131 sublineages were identified, with little association to geographic origin. The majority of single-nucleotide variants associated with each of the sublineages were due to recombination in regions adjacent to mobile genetic elements (MGEs). The most prevalent sublineage of ST131 strains was characterized by fluoroquinolone resistance, and a distinct virulence factor and MGE profile. Four different variants of the CTX-M ESBL-resistance gene were identified in our ST131 strains, with acquisition of CTX-M-15 representing a defining feature of a discrete but geographically dispersed ST131 sublineage. This study confirms the global dispersal of a single E. coli ST131 clone and demonstrates the role of MGEs and recombination in the evolution of this important MDR pathogen.

Hospital-wide Eradication of a Nosocomial Legionella pneumophila Serogroup 1 Outbreak.

BACKGROUND: Two proven nosocomial cases of Legionella pneumonia occurred at the Wesley Hospital (Brisbane, Australia) in May 2013. To trace the epidemiology of these cases, whole genome sequence analysis was performed on Legionella pneumophila isolates from the infected patients, prospective isolates collected from the hospital water distribution system (WDS), and retrospective patient isolates available from the Wesley Hospital and other local hospitals. METHODS: Legionella pneumophila serogroup 1 isolates were cultured from patient sputum (n = 3), endobronchial washings (n = 3), pleural fluid (n = 1), and the Wesley Hospital WDS (n = 39). Whole genome sequencing and de novo assembly allowed comparison with the L. pneumophila Paris reference strain to infer phylogenetic and epidemiological relationships. Rapid disinfection of the hospital WDS with a chlorinated, alkaline detergent and subsequent superchlorination followed by maintenance of residual free chlorine, combined with removal of redundant plumbing, was instituted. RESULTS: The 2011 and 2013 L. pneumophila patient isolates were serogroup 1 and closely related to all 2013 hospital water isolates based on single nucleotide polymorphisms and mobile genetic element profiles, suggesting a single L. pneumophila population as the source of nosocomial infection. The L. pneumophila population has evolved to comprise 3 clonal variants, each associated with different parts of the hospital WDS. CONCLUSIONS: This study provides an exemplar for the use of clinical and genomic epidemiological methods together with a program of rapid, effective remedial biofilm, plumbing and water treatment to characterize and eliminate a L. pneumophila population responsible for nosocomial infections.

Molecular analysis of asymptomatic bacteriuria Escherichia coli strain VR50 reveals adaptation to the urinary tract by gene acquisition.

Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli responsible for >80% of all cases. One extreme of UTI is asymptomatic bacteriuria (ABU), which occurs as an asymptomatic carrier state that resembles commensalism. To understand the evolution and molecular mechanisms that underpin ABU, the genome of the ABU E. coli strain VR50 was sequenced. Analysis of the complete genome indicated that it most resembles E. coli K-12, with the addition of a 94-kb genomic island (GI-VR50-pheV), eight prophages, and multiple plasmids. GI-VR50-pheV has a mosaic structure and contains genes encoding a number of UTI-associated virulence factors, namely, Afa (afimbrial adhesin), two autotransporter proteins (Ag43 and Sat), and aerobactin. We demonstrated that the presence of this island in VR50 confers its ability to colonize the murine bladder, as a VR50 mutant with GI-VR50-pheV deleted was attenuated in a mouse model of UTI in vivo. We established that Afa is the island-encoded factor responsible for this phenotype using two independent deletion (Afa operon and AfaE adhesin) mutants. E. coli VR50afa and VR50afaE displayed significantly decreased ability to adhere to human bladder epithelial cells. In the mouse model of UTI, VR50afa and VR50afaE displayed reduced bladder colonization compared to wild-type VR50, similar to the colonization level of the GI-VR50-pheV mutant. Our study suggests that E. coli VR50 is a commensal-like strain that has acquired fitness factors that facilitate colonization of the human bladder.

Third-generation cephalosporin resistance conferred by a chromosomally encoded blaCMY-23 gene in the Escherichia coli ST131 reference strain EC958.

OBJECTIVES: Escherichia coli ST131 is a globally disseminated MDR clone originally identified due to its association with the blaCTX-M-15 gene encoding an ESBL. It is thus assumed that blaCTX-M-15 is the major determinant for resistance to ß-lactam antibiotics in this clone. The complete sequence of EC958, a reference strain for E. coli ST131, revealed that it contains a chromosomally located blaCMY-23 gene with an upstream ISEcp1 element as well as several additional plasmid-encoded ß-lactamase genes. Here, we examined the genetic context of the blaCMY-23 element in EC958 and other E. coli ST131 strains and investigated the contribution of blaCMY-23 to EC958 resistance to a range of ß-lactam antibiotics. METHODS: The genetic context of blaCMY-23 and its associated mobile elements was determined by PCR and sequencing. Antibiotic susceptibility testing was performed using Etests. The activity of the blaCMY-23 promoter was assessed using lacZ reporter assays. Mutations were generated using λ-Red-recombination. RESULTS: The genetic structure of the ISEcp1-IS5-blaCMY-23 mobile element was determined and localized within the betU gene on the chromosome of EC958 and five other E. coli ST131 strains. The transcription of blaCMY-23, driven by a previously defined promoter within ISEcp1, was significantly higher than other ß-lactamase genes and could be induced by cefotaxime. Deletion of the blaCMY-23 gene resulted in enhanced susceptibility to cefoxitin, cefotaxime and ceftazidime. CONCLUSIONS: This is the first known report to demonstrate the chromosomal location of blaCMY-23 in E. coli ST131. In EC958, CMY-23 plays a major role in resistance to third-generation cephalosporins and cephamycins.

uPEPperoni: an online tool for upstream open reading frame location and analysis of transcript conservation.

BACKGROUND: Several small open reading frames located within the 5' untranslated regions of mRNAs have recently been shown to be translated. In humans, about 50% of mRNAs contain at least one upstream open reading frame representing a large resource of coding potential. We propose that some upstream open reading frames encode peptides that are functional and contribute to proteome complexity in humans and other organisms. We use the term uPEPs to describe peptides encoded by upstream open reading frames. RESULTS: We have developed an online tool, termed uPEPperoni, to facilitate the identification of putative bioactive peptides. uPEPperoni detects conserved upstream open reading frames in eukaryotic transcripts by comparing query nucleotide sequences against mRNA sequences within the NCBI RefSeq database. The algorithm first locates the main coding sequence and then searches for open reading frames 5' to the main start codon which are subsequently analysed for conservation. uPEPperoni also determines the substitution frequency for both the upstream open reading frames and the main coding sequence. In addition, the uPEPperoni tool produces sequence identity heatmaps which allow rapid visual inspection of conserved regions in paired mRNAs. CONCLUSIONS: uPEPperoni features user-nominated settings including, nucleotide match/mismatch, gap penalties, Ka/Ks ratios and output mode. The heatmap output shows levels of identity between any two sequences and provides easy recognition of conserved regions. Furthermore, this web tool allows comparison of evolutionary pressures acting on the upstream open reading frame against other regions of the mRNA. Additionally, the heatmap web applet can also be used to visualise the degree of conservation in any pair of sequences. uPEPperoni is freely available on an interactive web server at http://upep-scmb.biosci.uq.edu.au.

Engineering [Ln(DPA)3] 3- binding sites in proteins: a widely applicable method for tagging proteins with lanthanide ions.

Paramagnetic relaxation enhancements from unpaired electrons observed in nuclear magnetic resonance (NMR) spectra present powerful long-range distance restraints. The most frequently used paramagnetic tags, however, are tethered to the protein via disulfide bonds, requiring proteins with single cysteine residues for covalent attachment. Here we present a straightforward strategy to tag proteins site-specifically with paramagnetic lanthanides without a tether and independent of cysteine residues. It relies on preferential binding of the complex between three dipicolinic acid molecules (DPA) and a lanthanide ion (Ln(3+)), [Ln(DPA)(3)](3-), to a pair of positively charged amino acids whose charges are not compensated by negatively charged residues nearby. This situation rarely occurs in wild-type proteins, allowing the creation of specific binding sites simply by introduction of positively charged residues that are positioned far from glutamate or aspartate residues. The concept is demonstrated with the hnRNPLL RRM1 domain. In addition, we show that histidine- and arginine-tags present binding sites for [Ln(DPA)(3)](3-).

Tunable paramagnetic relaxation enhancements by [Gd(DPA)(3)] (3-) for protein structure analysis.

Paramagnetic relaxation enhancements (PRE) present a powerful source of structural information in nuclear magnetic resonance (NMR) studies of proteins and protein-ligand complexes. In contrast to conventional PRE reagents that are covalently attached to the protein, the complex between gadolinium and three dipicolinic acid (DPA) molecules, [Gd(DPA)(3)](3-), can bind to proteins in a non-covalent yet site-specific manner. This offers straightforward access to PREs that can be scaled by using different ratios of [Gd(DPA)(3)](3-) to protein, allowing quantitative distance measurements for nuclear spins within about 15 A of the Gd(3+) ion. Such data accurately define the metal position relative to the protein, greatly enhancing the interpretation of pseudocontact shifts induced by [Ln(DPA)(3)](3-) complexes of paramagnetic lanthanide (Ln(3+)) ions other than gadolinium. As an example we studied the quaternary structure of the homodimeric GCN4 leucine zipper.

Integrating multiple genomic technologies to investigate an outbreak of carbapenemase-producing Enterobacter hormaechei.

Carbapenem-resistant Enterobacteriaceae (CRE) represent an urgent threat to human health. Here we report the application of several complementary whole-genome sequencing (WGS) technologies to characterise a hospital outbreak of blaIMP-4 carbapenemase-producing E. hormaechei. Using Illumina sequencing, we determined that all outbreak strains were sequence type 90 (ST90) and near-identical. Comparison to publicly available data linked all outbreak isolates to a 2013 isolate from the same ward, suggesting an environmental source in the hospital. Using Pacific Biosciences sequencing, we resolved the complete context of the blaIMP-4 gene on a large IncHI2 plasmid carried by all IMP-4-producing strains across different hospitals. Shotgun metagenomic sequencing of environmental samples also found evidence of ST90 E. hormaechei and the IncHI2 plasmid within the hospital plumbing. Finally, Oxford Nanopore sequencing rapidly resolved the true relationship of subsequent isolates to the initial outbreak. Overall, our strategic application of three WGS technologies provided an in-depth analysis of the outbreak.

Stability of active prophages in industrial Lactococcus lactis strains in the presence of heat, acid, osmotic, oxidative and antibiotic stressors.

Lactococcus lactis is a starter bacterium commonly used in cheese making where it has an important role in acid-mediated curd formation as well as the development of flavour compounds. Industrial L. lactis strains can harbour one or more inducible prophages which when induced can affect cell growth and possibly lead to cell lysis. This is undesirable during growth and fermentation, but can beneficially lead to faster release of enzymes during cheese ripening. Lactococci can encounter multiple stress inducing conditions during the production of cheese, such as low and high temperatures, low pH, high osmotic pressure and long-term incubation. In this study, we tested the effect of these industrial stressors on prophage induction in two cheese making L. lactis subsp. cremoris strains (ASCC890049 and ASCC890310) as well as the laboratory strain L. lactis MG1363. Firstly, in order to identify inducible prophages in these strains we exposed them to the prophage inducing chemical mitomycin C (MMC) for 1 and 2h and then subjected the total genomic DNA to next-generation Illumina sequencing. Mapping of sequence reads back to the genome sequences revealed regions which contained a much higher fold coverage indicating DNA replication. These regions were amplified by up to 332-fold per cell (relative to the control tufA gene) and were identified as having similarities to different subgroups of P335 phages including MG-5, TP901-1, ul36.k1, bIL286, TP712 and BK5-T. Next, quantitative PCR was used to confirm the strong induction of prophages by MMC and then determine the copy number of the inducible prophages following exposure to various growth inhibitory levels of HCl, lactic acid, high temperature, NaCl, hydrogen peroxide and bacitracin. With the exception of a slight induction (2 to 4-fold) with hydrogen peroxide and long-term incubation after 21days in one industrial strain, none of the other stressors induced prophage DNA replication. These findings show that the repression system that maintains prophages in the dormant state in cheese making lactococcal strains is very tight and that several stressors encountered singularly are not predicted to be major inducers of prophage activation.

Sequential Acquisition of Virulence and Fluoroquinolone Resistance Has Shaped the Evolution of Escherichia coli ST131.

UNLABELLED: Escherichia coli ST131 is the most frequently isolated fluoroquinolone-resistant (FQR) E. coli clone worldwide and a major cause of urinary tract and bloodstream infections. Although originally identified through its association with the CTX-M-15 extended-spectrum ß-lactamase resistance gene, global genomic epidemiology studies have failed to resolve the geographical and temporal origin of the ST131 ancestor. Here, we developed a framework for the reanalysis of publically available genomes from different countries and used this data set to reconstruct the evolutionary steps that led to the emergence of FQR ST131. Using Bayesian estimation, we show that point mutations in chromosomal genes that confer FQR coincide with the first clinical use of fluoroquinolone in 1986 and illustrate the impact of this pivotal event on the rapid population expansion of ST131 worldwide from an apparent origin in North America. Furthermore, we identify virulence factor acquisition events that predate the development of FQR, suggesting that the gain of virulence-associated genes followed by the tandem development of antibiotic resistance primed the successful global dissemination of ST131. IMPORTANCE: Escherichia coli sequence type 131 (ST131) is a recently emerged and globally disseminated multidrug-resistant clone frequently associated with human urinary tract and bloodstream infections. In this study, we have used two large publically available genomic data sets to define a number of critical steps in the evolution of this important pathogen. We show that resistance to fluoroquinolones, a class of broad-spectrum antibiotic used extensively in human medicine and veterinary practice, developed in ST131 soon after the introduction of these antibiotics in the United States, most likely in North America. We also mapped the acquisition of several fitness and virulence determinants by ST131 and demonstrate these events occurred prior to the development of fluoroquinolone resistance. Thus, ST131 has emerged by stealth, first acquiring genes associated with an increased capacity to cause human infection, and then gaining a resistance armory that has driven its massive population expansion across the globe.

Draft Genome Sequence of Pseudomonas fluorescens SRM1, an Isolate from Spoiled Raw Milk.

Pseudomonas fluorescens is considered a major milk spoilage organism due to its psychrotrophic nature and ability to produce heat-stable proteases and lipases. Here, we report the draft genome and annotation of P. fluorescens SRM1 isolated from spoiled raw milk and the presence of an operon encoding spoilage enzymes.

Molecular Characterization of the Multidrug Resistant Escherichia coli ST131 Clone.

Escherichia coli ST131 is a recently emerged and globally disseminated multidrug resistant clone associated with urinary tract and bloodstream infections in both community and clinical settings. The most common group of ST131 strains are defined by resistance to fluoroquinolones and possession of the type 1 fimbriae fimH30 allele. Here we provide an update on our recent work describing the globally epidemiology of ST131. We review the phylogeny of ST131 based on whole genome sequence data and highlight the important role of recombination in the evolution of this clonal lineage. We also summarize our findings on the virulence of the ST131 reference strain EC958, and highlight the use of transposon directed insertion-site sequencing to define genes associated with serum resistance and essential features of its large antibiotic resistance plasmid pEC958.

Molecular characterization of a multidrug resistance IncF plasmid from the globally disseminated Escherichia coli ST131 clone.

Escherichia coli sequence type 131 (E. coli ST131) is a recently emerged and globally disseminated multidrug resistant clone associated with urinary tract and bloodstream infections. Plasmids represent a major vehicle for the carriage of antibiotic resistance genes in E. coli ST131. In this study, we determined the complete sequence and performed a comprehensive annotation of pEC958, an IncF plasmid from the E. coli ST131 reference strain EC958. Plasmid pEC958 is 135.6 kb in size, harbours two replicons (RepFIA and RepFII) and contains 12 antibiotic resistance genes (including the blaCTX-M-15 gene). We also carried out hyper-saturated transposon mutagenesis and multiplexed transposon directed insertion-site sequencing (TraDIS) to investigate the biology of pEC958. TraDIS data showed that while only the RepFII replicon was required for pEC958 replication, the RepFIA replicon contains genes essential for its partitioning. Thus, our data provides direct evidence that the RepFIA and RepFII replicons in pEC958 cooperate to ensure their stable inheritance. The gene encoding the antitoxin component (ccdA) of the post-segregational killing system CcdAB was also protected from mutagenesis, demonstrating this system is active. Sequence comparison with a global collection of ST131 strains suggest that IncF represents the most common type of plasmid in this clone, and underscores the need to understand its evolution and contribution to the spread of antibiotic resistance genes in E. coli ST131.

Lineage-Specific Methyltransferases Define the Methylome of the Globally Disseminated Escherichia coli ST131 Clone.

UNLABELLED: Escherichia coli sequence type 131 (ST131) is a clone of uropathogenic E. coli that has emerged rapidly and disseminated globally in both clinical and community settings. Members of the ST131 lineage from across the globe have been comprehensively characterized in terms of antibiotic resistance, virulence potential, and pathogenicity, but to date nothing is known about the methylome of these important human pathogens. Here we used single-molecule real-time (SMRT) PacBio sequencing to determine the methylome of E. coli EC958, the most-well-characterized completely sequenced ST131 strain. Our analysis of 52,081 methylated adenines in the genome of EC958 discovered three (m6)A methylation motifs that have not been described previously. Subsequent SMRT sequencing of isogenic knockout mutants identified the two type I methyltransferases (MTases) and one type IIG MTase responsible for (m6)A methylation of novel recognition sites. Although both type I sites were rare, the type IIG sites accounted for more than 12% of all methylated adenines in EC958. Analysis of the distribution of MTase genes across 95 ST131 genomes revealed their prevalence is highly conserved within the ST131 lineage, with most variation due to the presence or absence of mobile genetic elements on which individual MTase genes are located. IMPORTANCE: DNA modification plays a crucial role in bacterial regulation. Despite several examples demonstrating the role of methyltransferase (MTase) enzymes in bacterial virulence, investigation of this phenomenon on a whole-genome scale has remained elusive until now. Here we used single-molecule real-time (SMRT) sequencing to determine the first complete methylome of a strain from the multidrug-resistant E. coli sequence type 131 (ST131) lineage. By interrogating the methylome computationally and with further SMRT sequencing of isogenic mutants representing previously uncharacterized MTase genes, we defined the target sequences of three novel ST131-specific MTases and determined the genomic distribution of all MTase target sequences. Using a large collection of 95 previously sequenced ST131 genomes, we identified mobile genetic elements as a major factor driving diversity in DNA methylation patterns. Overall, our analysis highlights the potential for DNA methylation to dramatically influence gene regulation at the transcriptional level within a well-defined E. coli clone.

Transfer of scarlet fever-associated elements into the group A Streptococcus M1T1 clone.

The group A Streptococcus (GAS) M1T1 clone emerged in the 1980s as a leading cause of epidemic invasive infections worldwide, including necrotizing fasciitis and toxic shock syndrome. Horizontal transfer of mobile genetic elements has played a central role in the evolution of the M1T1 clone, with bacteriophage-encoded determinants DNase Sda1 and superantigen SpeA2 contributing to enhanced virulence and colonization respectively. Outbreaks of scarlet fever in Hong Kong and China in 2011, caused primarily by emm12 GAS, led to our investigation of the next most common cause of scarlet fever, emm1 GAS. Genomic analysis of 18 emm1 isolates from Hong Kong and 16 emm1 isolates from mainland China revealed the presence of mobile genetic elements associated with the expansion of emm12 scarlet fever clones in the M1T1 genomic background. These mobile genetic elements confer expression of superantigens SSA and SpeC, and resistance to tetracycline, erythromycin and clindamycin. Horizontal transfer of mobile DNA conferring multi-drug resistance and expression of a new superantigen repertoire in the M1T1 clone should trigger heightened public health awareness for the global dissemination of these genetic elements.

The complete genome sequence of Escherichia coli EC958: a high quality reference sequence for the globally disseminated multidrug resistant E. coli O25b:H4-ST131 clone.

Escherichia coli ST131 is now recognised as a leading contributor to urinary tract and bloodstream infections in both community and clinical settings. Here we present the complete, annotated genome of E. coli EC958, which was isolated from the urine of a patient presenting with a urinary tract infection in the Northwest region of England and represents the most well characterised ST131 strain. Sequencing was carried out using the Pacific Biosciences platform, which provided sufficient depth and read-length to produce a complete genome without the need for other technologies. The discovery of spurious contigs within the assembly that correspond to site-specific inversions in the tail fibre regions of prophages demonstrates the potential for this technology to reveal dynamic evolutionary mechanisms. E. coli EC958 belongs to the major subgroup of ST131 strains that produce the CTX-M-15 extended spectrum ß-lactamase, are fluoroquinolone resistant and encode the fimH30 type 1 fimbrial adhesin. This subgroup includes the Indian strain NA114 and the North American strain JJ1886. A comparison of the genomes of EC958, JJ1886 and NA114 revealed that differences in the arrangement of genomic islands, prophages and other repetitive elements in the NA114 genome are not biologically relevant and are due to misassembly. The availability of a high quality uropathogenic E. coli ST131 genome provides a reference for understanding this multidrug resistant pathogen and will facilitate novel functional, comparative and clinical studies of the E. coli ST131 clonal lineage.

Numbat: an interactive software tool for fitting Deltachi-tensors to molecular coordinates using pseudocontact shifts.

Pseudocontact shift (PCS) effects induced by a paramagnetic lanthanide bound to a protein have become increasingly popular in NMR spectroscopy as they yield a complementary set of orientational and long-range structural restraints. PCS are a manifestation of the chi-tensor anisotropy, the Deltachi-tensor, which in turn can be determined from the PCS. Once the Deltachi-tensor has been determined, PCS become powerful long-range restraints for the study of protein structure and protein-ligand complexes. Here we present the newly developed package Numbat (New User-friendly Method Built for Automatic Deltachi-Tensor determination). With a Graphical User Interface (GUI) that allows a high degree of interactivity, Numbat is specifically designed for the computation of the complete set of Deltachi-tensor parameters (including shape, location and orientation with respect to the protein) from a set of experimentally measured PCS and the protein structure coordinates. Use of the program for Linux and Windows operating systems is illustrated by building a model of the complex between the E. coli DNA polymerase III subunits epsilon186 and theta using PCS.