The serum resistome of a globally disseminated multidrug resistant uropathogenic Escherichia coli Clone

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.

Chaperone-usher fimbriae of Escherichia coli

Chaperone-usher (CU) fimbriae are adhesive surface organelles common to many Gram-negative bacteria. Escherichia coli genomes contain a large variety of characterised and putative CU fimbrial operons, however, the classification and annotation of individual loci remains problematic. Here we describe a classification model based on usher phylogeny and genomic locus position to categorise the CU fimbrial types of E. coli. Using the BLASTp algorithm, an iterative usher protein search was performed to identify CU fimbrial operons from 35 E. coli (and one Escherichia fergusonnii) genomes representing different pathogenic and phylogenic lineages, as well as 132 Escherichia spp. plasmids. A total of 458 CU fimbrial operons were identified, which represent 38 distinct fimbrial types based on genomic locus position and usher phylogeny. The majority of fimbrial operon types occupied a specific locus position on the E. coli chromosome; exceptions were associated with mobile genetic elements. A group of core-associated E. coli CU fimbriae were defined and include the Type 1, Yad, Yeh, Yfc, Mat, F9 and Ybg fimbriae. These genes were present as intact or disrupted operons at the same genetic locus in almost all genomes examined. Evaluation of the distribution and prevalence of CU fimbrial types among different pathogenic and phylogenic groups provides an overview of group specific fimbrial profiles and insight into the ancestry and evolution of CU fimbriae in E. coli.

Molecular characterization of the EhaG and UpaG trimeric autotransporter proteins from pathogenic Escherichia coli

Trimeric autotransporter proteins (TAAs) are important virulence factors of many Gram-negative bacterial pathogens. A common feature of most TAAs is the ability to mediate adherence to eukaryotic cells or extracellular matrix (ECM) proteins via a cell surface-exposed passenger domain. Here we describe the characterization of EhaG, a TAA identified from enterohemorrhagic Escherichia coli (EHEC) O157:H7. EhaG is a positional orthologue of the recently characterized UpaG TAA from uropathogenic E. coli (UPEC). Similarly to UpaG, EhaG localized at the bacterial cell surface and promoted cell aggregation, biofilm formation, and adherence to a range of ECM proteins. However, the two orthologues display differential cellular binding: EhaG mediates specific adhesion to colorectal epithelial cells while UpaG promotes specific binding to bladder epithelial cells. The EhaG and UpaG TAAs contain extensive sequence divergence in their respective passenger domains that could account for these differences. Indeed, sequence analyses of UpaG and EhaG homologues from several E. coli genomes revealed grouping of the proteins in clades almost exclusively represented by distinct E. coli pathotypes. The expression of EhaG (in EHEC) and UpaG (in UPEC) was also investigated and shown to be significantly enhanced in an hns isogenic mutant, suggesting that H-NS acts as a negative regulator of both TAAs. Thus, while the EhaG and UpaG TAAs contain some conserved binding and regulatory features, they also possess important differences that correlate with the distinct pathogenic lifestyles of EHEC and UPEC.

Characterisation of a cell wall-anchored protein of Staphylococcus saprophyticus associated with linoleic acid resistance

The Gram-positive bacterium Staphylococcus saprophyticus is the second most frequent causative agent of community-acquired urinary tract infections (UTI), accounting for up to 20% of cases. A common feature of staphylococci is colonisation of the human skin. This involves survival against innate immune defenses including antibacterial unsaturated free fatty acids such as linoleic acid which act by disrupting bacterial cell membranes. Indeed, S. saprophyticus UTI is usually preceded by perineal skin colonisation.

Molecular analysis of type 3 fimbrial genes from Escherichia coli, Klebsiella and Citrobacter species

Background Catheter-associated urinary tract infection (CAUTI) is the most common nosocomial infection in the United States and is caused by a range of uropathogens. Biofilm formation by uropathogens that cause CAUTI is often mediated by cell surface structures such as fimbriae. In this study, we characterised the genes encoding type 3 fimbriae from CAUTI strains of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri and Citrobacter freundii. Results Phylogenetic analysis of the type 3 fimbrial genes (mrkABCD) from 39 strains revealed they clustered into five distinct clades (A-E) ranging from one to twenty-three members. The majority of sequences grouped in clade A, which was represented by the mrk gene cluster from the genome sequenced K. pneumoniae MGH78578. The E. coli and K. pneumoniae mrkABCD gene sequences clustered together in two distinct clades, supporting previous evidence for the occurrence of inter-genera lateral gene transfer. All of the strains examined caused type 3 fimbriae mediated agglutination of tannic acid treated human erythrocytes despite sequence variation in the mrkD-encoding adhesin gene. Type 3 fimbriae deletion mutants were constructed in 13 representative strains and were used to demonstrate a direct role for type 3 fimbriae in biofilm formation. Conclusions The expression of functional type 3 fimbriae is common to many Gram-negative pathogens that cause CAUTI and is strongly associated with biofilm growth. Our data provides additional evidence for the spread of type 3 fimbrial genes by lateral gene transfer. Further work is now required to substantiate the clade structure reported here by examining more strains as well as other bacterial genera that make type 3 fimbriae and cause CAUTI.

Expression and crystallization of SeDsbA, SeDsbL and SeSrgA from Salmonella enterica serovar Typhimurium

Pathogens require protein-folding enzymes to produce functional virulence determinants. These foldases include the Dsb family of proteins, which catalyze oxidative folding in bacteria. Bacterial disulfide catalytic processes have been well characterized in Escherichia coli K-12 and these mechanisms have been extrapolated to other organisms. However, recent research indicates that the K-12 complement of Dsb proteins is not common to all bacteria. Importantly, many pathogenic bacteria have an extended arsenal of Dsb catalysts that is linked to their virulence. To help to elucidate the process of oxidative folding in pathogens containing a wide repertoire of Dsb proteins, Salmonella enterica serovar Typhimurium has been focused on. This Gram-negative bacterium contains three DsbA proteins: SeDsbA, SeDsbL and SeSrgA. Here, the expression, purification, crystallization and preliminary diffraction analysis of these three proteins are reported. SeDsbA, SeDsbL and SeSrgA crystals diffracted to resolution limits of 1.55, 1.57 and 2.6 Å and belonged to space groups P21, P21212 and C2, respectively.

UpaG, a new member of the Trimeric Autotransporter family of Adhesins in Uropathogenic Escherichia coli

The ability of Escherichia coli to colonize both intestinal and extraintestinal sites is driven by the presence of specific virulence factors, among which are the autotransporter (AT) proteins. Members of the trimeric AT adhesin family are important virulence factors for several gram-negative pathogens and mediate adherence to eukaryotic cells and extracellular matrix (ECM) proteins. In this study, we characterized a new trimeric AT adhesin (UpaG) from uropathogenic E. coli (UPEC). Molecular analysis of UpaG revealed that it is translocated to the cell surface and adopts a multimeric conformation. We demonstrated that UpaG is able to promote cell aggregation and biofilm formation on abiotic surfaces in CFT073 and various UPEC strains. In addition, UpaG expression resulted in the adhesion of CFT073 to human bladder epithelial cells, with specific affinity to fibronectin and laminin. Prevalence analysis revealed that upaG is strongly associated with E. coli strains from the B2 and D phylogenetic groups, while deletion of upaG had no significant effect on the ability of CFT073 to colonize the mouse urinary tract. Thus, UpaG is a novel trimeric AT adhesin from E. coli that mediates aggregation, biofilm formation, and adhesion to various ECM proteins.

The special branch : governing mentalities through alternative-site placement

"…one should try to locate power at the extreme points of its exercise, where it is always less legal in character." (Foucault, 1980, p.97) Studies of schooling practices as techniques deriving from a particular art of governing that Foucault (2003b) called ‘governmentality’ have shown how psychopathologising discourses work to construct particular student-subjects and legitimise various practices of exclusion (Gram, 2007b). Here I extend this work to consider the use of alternative-site placement as an intensification in response to governmentality being put ‘at risk’. Governing ‘at a distance’ conjures an illusion of individual freedom which relies on the production of subjects who ‘choose’ to make choices that are consistent with the aspirations of government. In this chapter, it is argued that the designation of a child as ‘disorderly’ legitimises the intrusion of state into the private domain of the family via the Trojan horse of early intervention. This is enabled by the psy-sciences, whose technologies and aims amount to the moral retraining of ‘improper’ future-citizens who, in choosing to choose otherwise, threaten to make visible invisible relations of power. Alternative-site placement in special schools running intensive behaviour modification programs allows for a ‘redoubled insistence’ (Ewald, 1992) of these norms and limits that a ‘disorderly’ child threatens to transgress.

Sortase A : an ideal target for anti-virulence drug development

Sortase A is a membrane enzyme responsible for the anchoring of surface-exposed proteins to the cell wall envelope of Gram-positive bacteria. As a well-studied member of the sortase subfamily catalysing the cell wall anchoring of important virulence factors to the surface of staphylococci, enterococci and streptococci, sortase A plays a critical role in Gram-positive bacterial pathogenesis. It is thus considered a promising target for the development of new anti-infective drugs that aim to interfere with important Gram-positive virulence mechanisms, such as adhesion to host tissues, evasion of host defences, and biofilm formation. The additional properties of sortase A as an enzyme that is not required for Gram-positive bacterial growth or viability and is conveniently located on the cell membrane making it more accessible to inhibitor targeting, constitute additional reasons reinforcing the view that sortase A is an ideal target for anti-virulence drug development. Many inhibitors of sortase A have been identified to date using high-throughput or in silico screening of compound libraries (synthetic or natural), and while many have proved useful tools for probing the action model of the enzyme, several are also promising candidates for the development into potent inhibitors. This review is focused on the most promising sortase A inhibitor compounds that are currently in development as leads towards a new class of anti-infective drugs that are urgently needed to help combat the alarming increase in antimicrobial resistance.

A Boundary-Oriented Chinese Segmentation Method Using NGram Mutual Information

This paper describes our participation in the Chinese word segmentation task of CIPS-SIGHAN 2010. We implemented an n-gram mutual information (NGMI) based segmentation algorithm with the mixed-up features from unsupervised, supervised and dictionarybased segmentation methods. This algorithm is also combined with a simple strategy for out-of-vocabulary (OOV) word recognition. The evaluation for both open and closed training shows encouraging results of our system. The results for OOV word recognition in closed training evaluation were however found unsatisfactory.

Promiscuity, pheromones and pathogenicity : why all Enterococci are not created equal

Enterococcus faecalis is a Gram-positive, coccus shaped, lactic acid bacterium, with demonstrated ubiquity across multiple anatomical sites. Enterococcus faecalis isolates have been isolated from clinical samples as the etiological agent in patients with overt infections, and from body sites previously thought to be sterile but absent of signs and symptoms of infection. E. faecalis is implicated in both human health and disease, recognized as a commensal, a probiotic and an opportunistic multiply resistant pathogen. E. faecalis has emerged as a key pathogen in nosocomial infections. E. faecalis is well equipped to avert recognition by host cell immune mediators. Antigenic cell wall components including lipotechoic acids are concealed from immune detection by capsular polysaccharides produced by some strains. Thereby preventing complement activation, the pro-inflammatory response, opsonisation and phagocytosis. E. faecalis also produces a suite of enzymes including gelatinase and cytolysin, which aid in both virulence and host immune evasion. The ability of enterococci to form biofilms in vivo further increases virulence, whilst simultaneously preventing detection by host cells. E. faecalis exhibits high levels of both intrinsic and acquired antimicrobial resistance. The mobility of the E. faecalis genome is a significant contributor to antimicrobial resistance, with this species also transferring resistance to other Gram-positive bacteria. Whilst E. faecalis is of increasing concern in nosocomial infections, its role as a member of the endogenous microbiota cannot be underestimated. As a commensal and probiotic, E. faecalis plays an integral role in modulating the immune response, and in providing endogenous antimicrobial activity to enhance exclusion or inhibition of opportunistic pathogens in certain anatomical niches. In this chapter we will review possible mediators of enterococcal transition from commensal microbe to opportunistic pathogen, considering isolates obtained from patients diagnosed with pathogenic infections and those obtained from asymptomatic patients.

FixVegas

From blocked drains to damaged street signs, rubbish or graffiti, FixVegas allows users to send fix-o-gram requests directly to Brisbane City Council. Users FixVegas snap a photo of the problem and the fix-o-gram is submitted to Council along with the location information of the issue that is being reported.

Exploration of the fundamental equilibrium behaviour of calcium exchange with weak acid cation resins

This study evaluated the complexity of calcium ion exchange with sodium exchanged weak acid cation resin (DOW MAC-3). Exchange equilibria recorded for a range of different solution normalities revealed profiles which were represented by conventional “L” or “H” type isotherms at low values of equilibrium concentration (Ce) of calcium ions, plus a superimposed region of increasing calcium uptake was observed at high Ce values. The loading of calcium ions was determined to be ca. 53.5 to 58.7 g/kg of resin when modelling only the sorption curve created at low Ce values,which exhibited a well-defined plateau. The calculated calcium ion loading capacity for DOWMAC-3 resin appeared to correlate with the manufacturer's recommendation. The phenomenon of super equivalent ion exchange (SEIX) was observed when the “driving force” for the exchange process was increased in excess of 2.25 mmol calcium ions per gram of resin in the starting solution. This latter event was explained in terms of displacement of sodium ions from sodium hydroxide solution which remained in the resin bead following the initial conversion of the as supplied “H+” exchanged resin sites to the “Na+” version required for softening studies. Evidence for hydrolysis of a small fraction of the sites on the sodium exchanged resin surface was noted. The importance of carefully choosing experimental parameters was discussed especially in relation to application of the Langmuir–Vageler expression. This latter model which compared the ratio of the initial calcium ion concentration in solution to resin mass, versus final equilibrium loading of the calcium ions on the resin; was discovered to be an excellent means of identifying the progress of the calcium–sodium ion exchange process. Moreover, the Langmuir–Vageler model facilitated standardization of various calcium–sodium ion exchange experiments which allowed systematic experimental design.

The genetics and structure of the O-specific polysaccharide of Yersinia pseudotuberculosis serotype O:10 and its relationship with Escherichia coli O111 and Salmonella enterica O35

The O-specific polysaccharide (OPS) is a variable constituent of the lipopolysaccharide of Gram-negative bacteria. The polymorphic nature of OPSs within a species is usually first defined serologically, and the current serotyping scheme for Yersinia pseudotuberculosis consists of 21 O serotypes of which 15 have been characterized genetically and structurally. Here, we present the structure and DNA sequence of Y. pseudotuberculosis O:10 OPS. The O unit consists of one residue each of d-galactopyranose, N-acetyl-d-galactosamine (2-amino-2-deoxy-d-galactopyranose) and d-glucopyranose in the backbone, with two colitose (3,6-dideoxy-l-xylo-hexopyranose) side-branch residues. This structure is very similar to that shared by Escherichia coli O111 and Salmonella enterica O35. The gene cluster sequences of these serotypes, however, have only low levels of similarity to that of Y. pseudotuberculosis O:10, although there is significant conservation of gene order. Within Y. pseudotuberculosis, the O10 structure is most closely related to the O:6 and O:7 structures.

Infectious complications during therapy of acute leukaemia in Saudi Arabia

In 40 febrile neutropenic episodes during the induction and consolidation chemotherapy of acute leukaemia in Riyadh, 51% of organisms causing septicaemia were gram-negative, 26% gram-positive, 8% anaerobes and 15% fungi. In 21 (52%) febrile episodes there were pulmonary infiltrates; of the 12 where aetiology was known, six were due to fungi. Pulmonary infiltrates progressed to adult respiratory distress syndrome and death in nine instances. There was no significant occurrence of parasitic and tropical infections. The results show that the pattern of infections, during therapy of acute leukaemia in developing countries, may have important differences when compared with western centres. Empiric amphotericin B may need to be introduced at an earlier stage in patients with persistent fever or progressive pulmonary infiltrates.