Stress-Induced Catch-Bonds to Enhance Bacterial Adhesion.

Physical forces have a profound influence on bacterial cell physiology and disease. A striking example is the formation of catch-bonds that reinforce under mechanical stress. While mannose-binding by the Escherichia coli FimH adhesin has long been the only thoroughly studied microbial catch-bond, it has recently become clear that proteins from other species, such as staphylococci, are also engaged in such stress-dependent interactions.

SILAC-based comparative analysis of pathogenic Escherichia coli secretomes.

Comparative studies of pathogenic bacteria and their non-pathogenic counterparts has led to the discovery of important virulence factors thereby generating insight into mechanisms of pathogenesis. Protein-based antigens for vaccine development are primarily selected among unique virulence-related factors produced by the pathogen of interest. However, recent work indicates that proteins that are not unique to the pathogen but instead selectively expressed compared to its non-pathogenic counterpart could also be vaccine candidates or targets for drug development. Modern methods in quantitative proteome analysis have the potential to discover both classes of proteins and hence form an important tool for discovering therapeutic targets. Adherent-invasive Escherichia coli (AIEC) and Enterotoxigenic E. coli (ETEC) are pathogenic variants of E. coli which cause intestinal disease in humans. AIEC is associated with Crohn's disease (CD), a chronic inflammatory condition of the gastrointestinal tract whereas ETEC is the major cause of human diarrhea which affects hundreds of millions annually. In spite of the disease burden associated with these pathogens, effective vaccines conferring long-term protection are still needed. In order to identify proteins with therapeutic potential, we have used mass spectrometry-based Stable Isotope Labeling with Amino acids in Cell culture (SILAC) quantitative proteomics method which allows us to compare the proteomes of pathogenic strains to commensal E. coli. In this study, we grew the pathogenic strains ETEC H10407, AIEC LF82 and the non-pathogenic reference strain E. coli K-12 MG1655 in parallel and used SILAC to compare protein levels in OMVs and culture supernatant. We have identified well-known virulence factors from both AIEC and ETEC, thus validating our experimental approach. In addition we find proteins that are not unique to the pathogenic strains but expressed at levels different from the commensal strain, including the colonization factor YghJ and the surface adhesin antigen 43, which is involved in pathogenesis of other Gram-negative bacteria. The described method provides a framework for further understanding E. coli pathogenesis but can also be applied to interrogate relative protein expression levels of other pathogens that have non-pathogenic counterparts thereby facilitating the discovery of new vaccine targets.

Did I pick the right colony? Pitfalls in the study of regulation of the phase variable antigen 43 adhesin.

Ag43 is an abundant outer membrane autotransporter adhesin present in most commensal and pathogenic Escherichia coli. Expression of the agn43 gene is characterized by a regulated reversible switch or phase variation between the agn43 ON and agn43 OFF states. Although the agn43 regulatory switch leads to a heterogeneous population of ON and OFF bacteria, studies of Ag43 seldom consider potential biases associated with phase variation. We monitored agn43 ON/OFF phase-variation status genetically and phenotypically and we show that the use of populations with random agn43 ON or OFF status could result in misleading conclusions about Ag43 function or regulation. In particular, we demonstrate that Lrp and MqsR, previously identified as agn43 regulators, do not regulate agn43 expression or ON/OFF switch frequency. We also show that biofilm formation in dynamic flow conditions does not influence agn43 ON/OFF switching but physically selects aggregating agn43 ON cells. This indicates that misinterpretation is possible when studying gene expression within biofilms. Finally, we provide evidence that ignoring the initial agn43 ON/OFF status of the E. coli populations studied is likely to bias analyses of phenotypes associated with other E. coli adhesins. This study therefore emphasizes the importance of monitoring Ag43 phase variation and indicates that caution is required when interpreting experiments using strains that are neither deleted for agn43 nor carefully assessed for agn43 ON/OFF status.

Orthopaedic-implant infections by Escherichia coli: molecular and phenotypic analysis of the causative strains.

OBJECTIVES: Little is known about Escherichia coli Orthopaedic Implant Infections (OII) pathogenesis. Thus, we compared 30 clinical strains isolated in this context with 30 clinical strains of faecal origin, in order to identify phenotypic and genetic features related to E. coli OII. METHODS: Phylogenetic analysis and detection of 19 virulence genes were performed by PCR. Ability to form biofilm was studied using the crystal violet reference method and the innovative BioFilm Ring Test(®). RESULTS: Most of the OII isolates (56.7%) belonged to the virulence-associated phylogenetic group B2, but did not present a specific set of virulence factors. S fimbriae was the only adhesin significantly associated with OII isolates. Isolates varied greatly in their ability to form biofilm but OII isolates did not produce significantly more biofilm in vitro than isolates of faecal origin, whatever the method used. CONCLUSIONS: Neither a specific pathogenic signature nor an increased ability to form biofilm in vitro was detected in E. coli strains isolated from OII. Nevertheless, genetic properties of these isolates could provide a clue to their origin. Hence, we found that virulence factors of uropathogenic strains and urological disorders were frequently detected among our OII cohort.

Preparation of highly luminescent mannose-gold nanodots for detection and inhibition of growth of Escherichia coli.

In this paper, we describe a novel, simple, and convenient method for preparing water-soluble biofunctional gold nanodots (Au NDs) for the sensitive and selective detection of Escherichia coli (E. coli) and the inhibition of its growth. We obtained luminescent mannose-capped Au NDs (Man-Au NDs) from as-prepared 2.9-nm Au nanoparticles (Au NPs) and 29,29'-dithio bis(3',6',9',12',15',18'-hexaoxa-nonacosyl α-D-mannopyranoside) (Man-RSSR-Man). To obtain improved quantum yield (>20%), luminescent Man-Au NDs (1.8 nm) were prepared from Au NPs (0.47 µM) and Man-RSSR-Man (2.5 mM) in the presence of sodium borohydride (NaBH(4); 1.0 mM). The highly luminescent properties of Man-Au NDs prepared by the NaBH(4)-assisted method were characterized by UV-vis absorption, photoluminescence, and X-ray photoelectron spectroscopies. The results supported the high-density coverage of the NDs surface by Man-RS ligands. Multivalent interactions between Man-Au NDs and FimH proteins located on the bacterial pili of E. coli resulted in the formation of aggregated cell clusters. After concentrating this agglutinative E. coli from a large-volume cell solution (5 mL), Man-Au NDs were displaced by mannose (100 mM) and stabilized by Man-RSSR-Man (5 mM). Monitoring the luminescence of Man-Au NDs allowed the detection of E. coli at levels as low as 150 CFU/mL. Man-Au NDs were also found to be efficient antibacterial agents, selectively inhibiting the growth of E. coli through Man-Au ND-induced agglutination. Our small-diameter Man-Au NDs, which provided an ultra high ligand density (local concentration) of mannose units for multivalent interactions with E. coli, have great potential for use as an antibacterial agent in other applications.

Characterization of IcmF of the type VI secretion system in an avian pathogenic Escherichia coli (APEC) strain

The intracellular multiplication factor (IcmF) protein is a component of the recently described typeVI secretion system (T6SS). IcmF has been shown to be required for intra-macrophage replicationand inhibition of phagosome–lysosome fusion in Legionella pneumophila. In Vibrio cholerae it is involved in motility, adherence and conjugation. Given that we previously reported that two T6SSgenes (hcp and clpV) contribute to the pathogenesis of a septicaemic strain (SEPT362) of avian pathogenic Escherichia coli (APEC), we investigated the function of IcmF in this strain. Further elucidation of the virulence mechanisms of APEC is important because this pathogen isresponsible for financial losses in the poultry industry, and is closely related to human extraintestinal pathogenic E. coli (ExPEC) strains, representing a potential zoonotic risk, as well asserving as a reservoir of virulence genes. Here we show that an APEC icmF mutant has decreased adherence to and invasion of epithelial cells, as well as decreased intra-macrophagesurvival. The icmF mutant is also defective for biofilm formation on abiotic surfaces. Additionally, expression of the flagella operon is decreased in the icmF mutant, leading to decreased motility.The combination of these phenotypes culminates in this mutant being altered for infection in chicks. These results suggest that IcmF in APEC may play a role in disease, and potentially also inthe epidemiological spread of this pathogen through enhancement of biofilm formation.

[Genetic mechanisms of Salmonella enteritidis biodiversity and clinical features of salmonellosis].

AIM: To assess prevalence of fragments of Escherichia coli pathogenicity islands in Salmonella enteritidis strains as well as to study clinical signs of disease caused by these strains in adults. MATERIALS AND METHODS: Ninety-six patients with salmonellosis were studied. Ninety strains of S. enteritidis were isolated and tested by PCR for the presence of genes associated with pathogenicity islands of E. coli: hlyA, hlyB, sfaG, and sfaA. RESULTS: It was determined that DNA fragments homologous to pathogenicity islands of E. coli were present in 87 (96.7%) of S. enteritidis clinical isolates. Disease caused by Salmonella strains which possess only sfaG was mostly mild--7 (33.3%), whereas strains which had sfaG with fragments of hlyA and/or hlyB caused severe disease--7 (50%). sfaA fragments were found mostly in combination with other genes. In such cases the disease was mostly severe--6 (42.8%). CONCLUSION: Correlation between presence of E. coli pathogenicity islands in Salmonella spp., their antibiotic resistance and severity of infection was established.

Role of P-fimbrial-mediated adherence in pyelonephritis and persistence of uropathogenic Escherichia coli (UPEC) in the mammalian kidney.

P fimbria, a mannose-resistant adhesin of uropathogenic Escherichia coli (UPEC), has been shown to be associated with acute pyelonephritis. The pap gene cluster encodes the proteins required for P-fimbrial biogenesis, including papG, which encodes the tip adhesin. The three most studied PapG molecular variants, which are shown to bind distinct isoreceptors, are PapGI, -II, and -III. PapGII preferentially binds globoside, or GbO4, a glycolipid isoreceptor of the human kidney. Studies using different animal models of ascending urinary tract infection (UTI) have demonstrated a variable role for P fimbriae, and specifically PapGII-mediated adherence, in renal colonization. The disparities in the results obtained from those studies are likely to be attributed to the differences in animal models and UPEC strains utilized. One explanation that is discussed in detail is the contribution of multiple fimbriae of UPEC that potentially mediate adherence to the mammalian kidney. Overall, P fimbriae appear to play some role in mediating adherence to uroepithelial cells in vivo and establishing an inflammatory response during renal colonization, thus contributing to kidney damage during acute pyelonephritis. To verify that P fimbriae contribute to the pathogenesis of UPEC during ascending UTI (and in particular acute pyelonephritis), future studies should be conducted to satisfy fully all three tenets of the molecular Koch's postulates, including complementation of a mutated allele.

Classification of temporal profiles of F4+ E. coli shedding and faecal dry matter in experimental post-weaning diarrhoea of pigs.

Enterotoxigenic F4+ Escherichia coli can colonize the intestine of pigs and cause diarrhoea. Our primary goal was to find a discriminant rule to discriminate between F4+ E. coli shedding profiles as this may reflect differences in the infectiousness of pigs. Our secondary goal was to find a discriminant rule to discriminate between diarrhoeic and non-diarrhoeic pigs. Repeated measurements (bacterial shedding and percentage dry matter of faeces) were taken of 74 weaned pigs that were infected experimentally with F4+ E. coli. These measurements were summarized into two new variables by means of a principal components analysis. Discriminant rules were derived based on these summary variables by fitting a mixture of normal distributions. Finally, the association between the classifications (as derived from the discriminant rules) and the occurrence in the pigs of the F4 receptor, an adhesion site for F4+ E. coli, was studied. We found that only the classification based on bacterial shedding allowed us to distinguish two significantly different groups of pigs (high and low shedders). Presence of the F4 receptor was associated strongly with pigs being high shedders.

Asymptomatic bacteriuria Escherichia coli strains: adhesins, growth and competition.

Urinary tract infections (UTIs) affect millions of people each year. Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU) in humans. Persons affected by ABU may carry a particular E. coli strain for extended periods of time without any symptoms. In contrast to uropathogenic E. coli (UPEC) that cause symptomatic UTI, very little is known about the mechanisms by which these strains colonize the urinary tract. Here, we have investigated the growth characteristics in human urine as well as adhesin repertoire of nine ABU strains; the ability of ABU strains to compete against the UPEC strain CFT073 was also studied. The different ABU strains displayed a wide variety of the measured characteristics. Half of the ABU strains displayed functional type 1 fimbriae while only one expressed functional P fimbriae. A good correlation between the growth rate of a particular strain and the survival of the strain in competition against CFT073 was observed. Our results support the notion that for strains with reduced capacity to express fimbriae, the ability to grow fast in human urine becomes crucial for colonization of the urinary tract.

Clonal analysis reveals high rate of structural mutations in fimbrial adhesins of extraintestinal pathogenic Escherichia coli.

Type 1 fimbriae of Escherichia coli mediate mannose-specific adhesion to host epithelial surfaces and consist of a major, antigenically variable pilin subunit, FimA, and a minor, structurally conserved adhesive subunit, FimH, located on the fimbrial tip. We have analysed the variability of fimA and fimH in strains of vaginal and other origin that belong to one of the most prominent clonal groups of extraintestinal pathogenic E. coli, comprised of O1:K1-, O2:K1- and O18:K1-based serotypes. Multiple locus sequence typing (MLST) of this group revealed that the strains have identical (at all but one nucleotide position) eight housekeeping loci around the genome and belong to the ST95 complex defined by the publicly available E. coli MLST database. Multiple highly diverse fimA alleles have been introduced into the ST95 clonal complex via horizontal transfer, at a frequency comparable to that of genes defining the major O- and H-antigens. However, no further significant FimA diversification has occurred via point mutation after the transfers. In contrast, while fimH alleles also move horizontally (along with the fimA loci), they acquire point amino acid replacements at a higher rate than either housekeeping genes or fimA. These FimH mutations enhance binding to monomannose receptors and bacterial tropism for human vaginal epithelium. A similar pattern of rapid within-clonal structural evolution of the adhesive, but not pilin, subunit is also seen, respectively, in papG and papA alleles of the di-galactose-specific P-fimbriae. Thus, while structurally diverse pilin subunits of E. coli fimbriae are under selective pressure for frequent horizontal transfer between clones, the adhesive subunits of extraintestinal E. coli are under strong positive selection (Dn/Ds > 1 for fimH and papG) for functionally adaptive amino acid replacements.

Characterization of fimbriae extracts from porcine enterotoxigenic Escherichia coli strains carrying F6 (987P) antigen.

Fimbrial extracts from porcine enterotoxigenic Escherichia coli (ETEC) strains carrying F6 (987P) intestinal colonization factor antigen wereobtained using the thermal shock method. The extracts were analyzed by SDSPAGE and immunoblotting using different fimbriae-specific antisera. Two major protein bands with molecular masses of 17.5 and 21.9 kDa were detected. The 21.9-kDa band was identified as the major subunit of F6 fimbrial antigen in strains of serogroups O9 and O141. The 17.5-kDa band was associated with porcine strains of serogroups O9 and O20.

Transferencia de genes plasmídicos de resistencia a antibióticos mediante conjugación entre cepas de Escherichia coli provenientes de procesos diarréicosde niños menores a 3 años de edad. Hospital Materno Infantil de la ciudad de La Paz / Transfer of plasmid genes of resistance to antibiotics through conjugation between strains of escherichia coli from processes diarréicos de smaller children than 3 years of edad. Hospital Maternal and Child of the city of La Paz

Escherichia coli es el agente etiológico enterobacteriano más común causante de enfermedades diarreicas en lapoblación infantil. Por este motivo se estudió la transferencia por conjugación de genes plasmídicos de resistencia a antibióticos de bacterias E. coli provenientes de heces de niños menores de 3 años con procesos diarreicos que asistieron al laboratorio clínico delHospital materno infantil durante abril y diciembre del 2003. Se analizó el antibiograma de 44 cepas de E. colique presentó altos porcentajes de resistencia (Amoxicilina 95.55%, cotrimoxazol 68.88%, cloranfenicol51.11%). Por otra parte, las asociaciones más comunes de antibióticos fueron amoxicilina-cotrimoxazol (65%), y amoxicilina-cotrimoxazol-cloranfenicol (47,73%). Los experimentos de conjugación presentaron frecuencias entre 10-5 a 1 0-7, siendo de mayor valor para latransferencia de amoxicilina y menor para la amikacina. Los porcentajes de resistencia y las frecuencias deconjugación demuestran que la mayoría de los genes de resistencia son capaces de ser transferidos por conjugación. Lo que implica probablemente que muchosde estos genes están localizados en elementos genéticos como plásmidos y transposones.

A surface-exposed DraD protein of uropathogenic Escherichia coli bearing Dr fimbriae may be expressed and secreted independently from DraC usher and DraE adhesin.

The dra gene cluster, expressed by uropathogenic Escherichia coli strains, determines bacterial attachment and invasion. The Dr fimbrial structures formed at the bacterial cell surface are composed of DraE subunits. The Dr fimbriae-coding cluster contains six open reading frames--draA, draB, draC, draD, draP and draE--among which the draE gene encodes the structural fimbrial subunit DraE. Very little is known about E. coli surface expression of the draD gene product. The expression of DraD and its role in the biogenesis of Dr fimbriae were determined by constructing mutants in the dra operon and by immunoblot and immunofluorescence experiments. In this study, DraD was found to be a surface-exposed protein. The expression of DraD was independent of the DraC usher and DraE fimbrial subunits. Polymerization of DraE fimbrial subunits into fimbrial structures did not require expression of the DraD protein.

Receptors for Escherichia coli adhesins in the genitourinary tract in a non-human primate.

OBJECTIVE: To study the expression of receptors allowing adhesin-mediated binding of Escherichia coli to urogenital tissues ranging from the kidney to the vagina in cynomolgus monkeys using an in situ assay. MATERIAL AND METHODS: Receptors specific for four relevant adhesins were investigated: PapG and PrsG of P-fimbriae binding to gal-alpha(1-4)gal glycosphingolipids (preferentially globoside and the Forssman antigen, respectively): and two variants of FimH of type 1 fimbriae, one binding to monomannose/trimannose and the other to trimannose only. To ascertain the specificity of the observed bindings we used adhesion inhibition by receptor analogues as well as E. coli adhesin knockout mutants. RESULTS: The distributions of PapG and FimH receptors in monkey tissues showed great similarities to available data in humans. Whilst monomannose receptors were expressed on the surface epithelium in both the monkey bladder and ureter, trimannose receptors were not. The different distribution of FimH isoreceptors and the heterogeneity of FimH adhesin variants among E. coli may explain contradictory earlier findings in type 1 fimbriae-mediated adhesion to the human bladder and to renal tissues. We also found evidence of a hitherto unknown type of host-aggressor interaction on vaginal and urethral mucosa, which was not discovered until type 1 fimbriae had been eliminated. CONCLUSIONS: A precise molecular fit between host receptors and bacterial lectins is important in infectious pathogenesis. We conclude that urinary tract infection in the cynomolgus monkey is a relevant model of the human disease because of the similarity in the expression of receptors for E. coli adhesins on epithelial surfaces in the two species.

Mortality in murine peritonitis correlates with increased Escherichia coli adherence to the intestinal mucosa.

During peritonitis, bacterial adherence is the initial step in a series of events that include mucosal infection, bacterial translocation, organ dysfunction, and death. Adherent Escherichia coli levels increase in response to stress. This study was designed to assess the adherence of E. coli to the cecal mucosa after cecal ligation and puncture (CLP) of increasing severity and to determine whether a relationship exists between adherence of bacteria and mortality. Sham surgery, sterile peritonitis (thioglycollate administration), lethal CLP (18-gauge double-puncture), and nonlethal CLP (23-gauge single-puncture) were performed on Swiss Webster mice and compared with normal mice or before CLP (time 0). Specimens of bowel tissue were harvested, and serial log dilutions of homogenized specimens or bowel contents were plated and cultured on media selective for determination of individual bacterial species. Low levels of E. coli and Proteus mirabilis adhered to the mucosa of unmanipulated controls; however, adherence of both species increased significantly by 18 hours after both lethal and nonlethal CLP. After 18 hours, adherent E. coli levels increased by greater than 5 x 10(6)-fold compared to unmanipulated controls, whereas P. mirabilis levels decreased. After nonlethal CLP, adherent P. mirabilis increased 3 x 10(6)-fold compared to unmanipulated animals, whereas E. coli levels did not increase after 24 hours. Sterile peritonitis had little effect on bacterial adherence. Higher levels of adherent E. coli in the cecum correlate with the increased mortality observed after lethal CLP. Higher levels of adherent P. mirabilis appear to prevent the overgrowth of adherent E. coli following nonlethal CLP. Our data indicate that E. coli plays a key role in mortality from polymicrobial peritonitis and that Proteus may be antagonistic to E. coli in murine peritonitis.

Distribution of afaE adhesins in Escherichia coli isolated from Japanese patients with urinary tract infection.

PURPOSE: Afimbrial adhesin is known to be one of the most prevalent virulence factors in uropathogenic Escherichia coli. A recent report showed that the new subtype afaE8 predominated in afa positive isolates from patients with pyelonephritis (55.6%), suggesting that this subtype may be an important factor in ascending urinary tract infections. MATERIALS AND METHODS: A total of 457 E. coli strains consisting, of 194, 76 and 107 isolates from patients with cystitis, pyelonephritis and prostatitis, respectively, and 80 isolates from the rectal flora of healthy individuals were subjected to polymerase chain reaction to determine the afa operon as well as afaE subtypes. RESULTS: We identified 32 afa positive isolates of 377 strains (8.5%) and 2 of 80 strains (2.5%) from urinary tract infection isolates and normal flora, respectively. When afaE subtypes were determined, the afaE3 subtype predominated in afa positive isolates from cystitis (64.7%), pyelonephritis (66.7%) and prostatitis (50%). However, the afaE8 subtype was absent from urinary tract infection isolates, while only 1 isolate from the stool of a healthy adult harbored this subtype. CONCLUSIONS: Our data show that the afaE3 subtype predominated in pyelonephritis as well as in other urinary tract infections, indicating that the afa gene may be important in urinary tract infection. However, the distribution of afaE subtypes may be diverse in different areas of the world.

Ampicillin-resistant Escherichia coli in gestational pyelonephritis: increased occurrence and association with the colonization factor Dr adhesin.

The pattern of ampicillin resistance and possible association with virulence factors of 78 Escherichia coli isolates taken from 78 pregnant women with pyelonephritis were evaluated. The current incidence of ampicillin resistance among pyelonephritis isolates (46%) was significantly higher than that reported in 1985 (22%). Resistance was found more frequently during the first (60%) and third (53%) trimesters than during the second trimester (33%). Of all dra(+) E. coli isolates, 75% were ampicillin resistant, whereas dra(+) isolates of O75 serotype E. coli accounted for 87% of ampicillin-resistant strains. The significant increase of ampicillin resistance among gestational pyelonephritis E. coli and the association with the dra gene cluster encoding colonization and invasive capacity may warrant further study involving obstetric and neonate wards, with the latter being at the higher risk for potential problems.

Immunocytochemistry of the AfaE adhesin and AfaD invasin produced by pathogenic Escherichia coli strains during interaction of the bacteria with HeLa cells by high-resolution scanning electron microscopy.

We used a recent scanning electron microscope equipped with field emission gun and highly sensitive detectors to develop a fast and simple protocol for double immunogold staining using 10- and 15-nm gold particles. We used this approach to analyse the afimbrial adhesive sheath produced by pathogenic Escherichia coli interacting with the surface of epithelial cells. We demonstrated that AfaE adhesin and AfaD invasin were exposed at the bacterial surface during the interaction. This method could be easily and widely extended to the study of the early invasion process of many bacterial and viral pathogens, by immunocytochemical probing.

Identification of S, F1C and three PapG fimbrial adhesins in uropathogenic Escherichia coli by polymerase chain reaction.

S and F1C fimbrial adhesins often expressed by uropathogenic Escherichia coli are genetically homologous. A multiply primed polymerase chain reaction (PCR) was developed for discriminating the S (sfa) and F1C (foc) fimbrial operons. A total of 270 uropathogenic E coli strains and 80 fecal isolates were examined. PCR specifically detected the sfa and foc alleles in 105 (93%) of 113 sfa/foc+ strains by DNA hybridization. Furthermore, 87% of sfa+ uropathogenic E. coli simultaneously possessed the genes encoding the class III P fimbrial adhesin (prsG(J96)), alpha-hemolysin and cytotoxic necrotizing factor 1. Statistical analysis showed the class II P fimbrial adhesin (papG(IA2)) and F1C fimbria to be associated with high relative virulence in pyelonephritis and cystitis, respectively. The multiply primed PCR developed should be useful for assessing the contribution of the S and F1C fimbriae in the pathogenesis of urinary tract infections.