Molecular genetic testing does not improve the detection of fluoroquinolone resistance before transrectal prostate biopsy.

Background: Fluoroquinolone-resistant (FQR) Escherichia coli (E. coli) causes transrectal prostate biopsy infections. We seek to further identify fluoroquinolones resistance by the incorporation of genetic profiling to influence antibiotic selection for transrectal prostate biopsy and whether the addition of this genetic testing could improve the prediction of FQR detection at the time of biopsy. Materials and methods: In this prospective observational cohort study, rectal swabs were collected within 30 days of an upcoming prostate biopsy. These swabs were sent for phenotypic and genotypic assessment to predict FQR on the day of the biopsy. Phenotype: Specimens were inoculated onto MacConkey agar containing ciprofloxacin using standard culture techniques to determine FQR status. Genotype: We compared cultures to polymerase chain reaction (PCR) sequence typing (E.coli- ST131/H30/ST69) and bacterial plasmids (gyrA, qnrQ, and qnrS). The presence of FQR on this testing was compared to the second rectal swab collected just before biopsy (2 hours after ciprofloxacin prophylaxis), which served as the gold standard for FQR. Results: Overall, the FQR rate was 23.6%. The bacterial plasmids (qnr) were present in 54.1% of samples, and multidrug-resistant E. coli ST131 was present in 12.5% of samples. In comparison, phenotypic assessment using rectal culture had a better prediction for the presence of FQR as compared to genotypic testing [area under the curve (AUC) = 0.85 in phenotype arm vs. AUC = 0.45 in genotype arm]. Conclusion: We detected a high prevalence of FQR genes in the rectum, but the addition of PCR-based genotyping did not improve the prediction of culture-based FQR at the time of biopsy.

Inactivation of Transcriptional Regulators during Within-Household Evolution of Escherichia coli.

We analyzed the within-household evolution of two household-associated Escherichia coli strains from pandemic clonal group ST131-H30, using isolates recovered from five individuals within two families, each of which had a distinct strain. Family 1's strain was represented by a urine isolate from the index patient (older sister) with recurrent cystitis and a blood isolate from her younger sister with fatal urosepsis. Family 2's strain was represented by a urine isolate from the index patient (father) with pyelonephritis and renal abscesses, blood and kidney drainage isolates from the daughter with emphysematous pyelonephritis, and urine and fecal isolates from the mother with cystitis. Collectively, the several variants of each family's strain had accumulated a total of 8 (family 1) and 39 (family 2) point mutations; no two isolates were identical. Of the 47 total mutations, 36 resulted in amino acid changes or truncation of coded proteins. Fourteen such mutations (39%) targeted genes encoding transcriptional regulators, and 9 (25%) involved DNA-binding transcription factors (TFs), which significantly exceeded the relative contribution of TF genes to the isolates' genomes (∼6%). At least one-half of the transcriptional regulator mutations were inactivating, based on phenotypic and/or transcriptional analysis. In particular, inactivating mutations in the global regulator LrhA (repressor of type 1 fimbriae and flagella) occurred in the blood isolates from both households and increased the virulence of E. coli strains in a murine sepsis model. The results indicate that E. coli undergoes adaptive evolution between and/or within hosts, generating subpopulations with distinctive phenotypes and virulence potential.IMPORTANCE The clonal evolution of bacterial strains associated with interhost transmission is poorly understood. We characterized the genome sequences of clonal descendants of two Escherichia coli strains, recovered at different time points from multiple individuals within two households who had different types of urinary tract infection. We found evidence that the E. coli strains underwent extensive mutational diversification between and within these individuals, driven disproportionately by inactivation of transcriptional regulators. In urosepsis isolates, the mutations observed in the global regulator LrhA increased bacterial virulence in a murine sepsis model. Our findings help in understanding the adaptive dynamics and strategies of E. coli during short-term natural evolution.

Prevalence and persistence of Escherichia coli in the airways of cystic fibrosis patients - an unrecognized CF pathogen?

Cystic fibrosis (CF) patients suffer from chronic recurrent bacterial airway infections, which eventually lead to reduced life expectancy. Escherichia coli has not been considered as a CF pathogen. A total of 176 patients were observed over 5.6 years on average from 2002 to 2009 in two CF centers in Muenster, Germany. Sputum and throat swab cultures were screened for E. coli. E. coli isolates were analyzed for clinical microbiologic characteristics as well as strain identity, clonal distribution and phenotypic variability. In 45 patients (25.6%) E. coli was cultured at least once, mostly at medium to high bacterial load and primarily from patients less than 5 and older than 8 years. In 19 patients (10.8%) the same E. coli strain was isolated at least 3 times within a period of more than 6 months, with a mean persistence of 29 months. Multi-locus sequence typing revealed a distinctively strong association of CF E. coli with the B2 major clonal group. During persistence, long-term colonizing strains exhibited phenotypic variability known for typical CF pathogens such as surface capsule overproduction and changes in colony size or hemolytic activity. E. coli was occasionally or persistently isolated in a quarter of CF patients, mostly in very young or older patients. The relatively high bacterial load of E. coli colonization, the distinct association with the highly virulent extra-intestinal B2 clonal group and phenotypic variability in the long-term colonizing strains suggests a previously unrecognized clinical significance of E. coli as a CF pathogen.

Abrupt emergence of a single dominant multidrug-resistant strain of Escherichia coli.

BACKGROUND: Fluoroquinolone-resistant Escherichia coli are increasingly prevalent. Their clonal origins--potentially critical for control efforts--remain undefined. METHODS: Antimicrobial resistance profiles and fine clonal structure were determined for 236 diverse-source historical (1967-2009) E. coli isolates representing sequence type ST131 and 853 recent (2010-2011) consecutive E. coli isolates from 5 clinical laboratories in Seattle, Washington, and Minneapolis, Minnesota. Clonal structure was resolved based on fimH sequence (fimbrial adhesin gene: H subclone assignments), multilocus sequence typing, gyrA and parC sequence (fluoroquinolone resistance-determining loci), and pulsed-field gel electrophoresis. RESULTS: Of the recent fluoroquinolone-resistant clinical isolates, 52% represented a single ST131 subclonal lineage, H30, which expanded abruptly after 2000. This subclone had a unique and conserved gyrA/parC allele combination, supporting its tight clonality. Unlike other ST131 subclones, H30 was significantly associated with fluoroquinolone resistance and was the most prevalent subclone among current E. coli clinical isolates, overall (10.4%) and within every resistance category (11%-52%). CONCLUSIONS: Most current fluoroquinolone-resistant E. coli clinical isolates, and the largest share of multidrug-resistant isolates, represent a highly clonal subgroup that likely originated from a single rapidly expanded and disseminated ST131 strain. Focused attention to this strain will be required to control the fluoroquinolone and multidrug-resistant E. coli epidemic.

Evolution of Salmonella enterica virulence via point mutations in the fimbrial adhesin.

Whereas the majority of pathogenic Salmonella serovars are capable of infecting many different animal species, typically producing a self-limited gastroenteritis, serovars with narrow host-specificity exhibit increased virulence and their infections frequently result in fatal systemic diseases. In our study, a genetic and functional analysis of the mannose-specific type 1 fimbrial adhesin FimH from a variety of serovars of Salmonella enterica revealed that specific mutant variants of FimH are common in host-adapted (systemically invasive) serovars. We have found that while the low-binding shear-dependent phenotype of the adhesin is preserved in broad host-range (usually systemically non-invasive) Salmonella, the majority of host-adapted serovars express FimH variants with one of two alternative phenotypes: a significantly increased binding to mannose (as in S. Typhi, S. Paratyphi C, S. Dublin and some isolates of S. Choleraesuis), or complete loss of the mannose-binding activity (as in S. Paratyphi B, S. Choleraesuis and S. Gallinarum). The functional diversification of FimH in host-adapted Salmonella results from recently acquired structural mutations. Many of the mutations are of a convergent nature indicative of strong positive selection. The high-binding phenotype of FimH that leads to increased bacterial adhesiveness to and invasiveness of epithelial cells and macrophages usually precedes acquisition of the non-binding phenotype. Collectively these observations suggest that activation or inactivation of mannose-specific adhesive properties in different systemically invasive serovars of Salmonella reflects their dynamic trajectories of adaptation to a life style in specific hosts. In conclusion, our study demonstrates that point mutations are the target of positive selection and, in addition to horizontal gene transfer and genome degradation events, can contribute to the differential pathoadaptive evolution of Salmonella.

Single nucleotide polypmorphisms of fimH associated with adherence and biofilm formation by serovars of Salmonella enterica.

Type 1 fimbriae produced by serovars of Salmonella are characterized by their ability to agglutinate guinea pig erythrocytes in the absence of d-mannose but not in its presence. The FimH protein is the adhesin that mediates this reaction; it is distinct from the major fimbrial protei.n (FimA) that composes the fimbrial shaft. Avian-adapted serovars of Salmonella produce non-haemagglutinating fimbriae that have been reported to mediate adherence to avian cells. A single amino acid substitution is present in the FimH adhesin of these strains compared to that of a Typhimurium isolate. Also, previous studies have shown that single nucleotide polymorphisms in two strains of the Typhimurium fimH alter the binding specificity. We therefore investigated the allelic variation of fimH from a range of serotypes (both host-adapted and non-host-adapted) and isolates of Salmonella. Most FimH adhesins mediated the mannose-sensitive haemagglutination of guinea pig erythrocytes, but many did not facilitate adherence to HEp-2 cells. A small number of isolates also produced fimbriae but did not mediate adherence to either cell type. Transformants possessing cloned fimH genes exhibited a number of different substitutions within the predicted amino acid sequence of the FimH polypeptide. No identical FimH amino sequence was found between strains that adhere to erythrocytes and/or HEp-2 cells and those produced by non-adherent strains. FimH-mediated adherence to HEp-2 cells was invariably associated with the ability to form biofilms on mannosylated bovine serum albumin.

The bacterial fimbrial tip acts as a mechanical force sensor.

There is increasing evidence that the catch bond mechanism, where binding becomes stronger under tensile force, is a common property among non-covalent interactions between biological molecules that are exposed to mechanical force in vivo. Here, by using the multi-protein tip complex of the mannose-binding type 1 fimbriae of Escherichia coli, we show how the entire quaternary structure of the adhesive organella is adapted to facilitate binding under mechanically dynamic conditions induced by flow. The fimbrial tip mediates shear-dependent adhesion of bacteria to uroepithelial cells and demonstrates force-enhanced interaction with mannose in single molecule force spectroscopy experiments. The mannose-binding, lectin domain of the apex-positioned adhesive protein FimH is docked to the anchoring pilin domain in a distinct hooked manner. The hooked conformation is highly stable in molecular dynamics simulations under no force conditions but permits an easy separation of the domains upon application of an external tensile force, allowing the lectin domain to switch from a low- to a high-affinity state. The conformation between the FimH pilin domain and the following FimG subunit of the tip is open and stable even when tensile force is applied, providing an extended lever arm for the hook unhinging under shear. Finally, the conformation between FimG and FimF subunits is highly flexible even in the absence of tensile force, conferring to the FimH adhesin an exploratory function and high binding rates. The fimbrial tip of type 1 Escherichia coli is optimized to have a dual functionality: flexible exploration and force sensing. Comparison to other structures suggests that this property is common in unrelated bacterial and eukaryotic adhesive complexes that must function in dynamic conditions.

Anomalously increased lifetimes of biological complexes at zero force due to the protein-water interface.

A number of biological bonds show dramatically increased lifetimes at zero-force conditions, compared to lifetimes when even a small tensile force is applied to the ligand. The discrepancy is so great that it cannot be explained by the traditional receptor-ligand binding models. This generic phenomenon is rationalized here by considering the interaction of water with the receptor-ligand complex. It is argued that the water-protein interaction creates an energy barrier that prevents the ligand unbinding in the absence of the force. The properties of the interaction are such that even application of a relatively low force results in a dramatic drop of the bond lifetime due to the alteration of the water-receptor and water-ligand interaction network. The phenomenon is described by the presence of a second shallow interaction energy minimum for the bound ligand followed by a wide receptor-ligand dissociation barrier. The general analysis is applied quantitatively to the actin-myosin system, which demonstrates the gigantic drop of the bond lifetime at small forces and catch behavior (an increase in the lifetime) at moderate forces. The base hypothesis proposed to explain the small-force abnormal drop in the bond lifetime suggests that the majority of biological bonds may exhibit this phenomenon irrespectively whether they behave as slip or catch-slip bonds.

The cysteine bond in the Escherichia coli FimH adhesin is critical for adhesion under flow conditions.

Cysteine bonds are found near the ligand-binding sites of a wide range of microbial adhesive proteins, including the FimH adhesin of Escherichia coli. We show here that removal of the cysteine bond in the mannose-binding domain of FimH did not affect FimH-mannose binding under static or low shear conditions (< or = 0.2 dyne cm(-2)). However, the adhesion level was substantially decreased under increased fluid flow. Under intermediate shear (2 dynes cm(-2)), the ON-rate of bacterial attachment was significantly decreased for disulphide-free mutants. Molecular dynamics simulations demonstrated that the lower ON-rate of cysteine bond-free FimH could be due to destabilization of the mannose-free binding pocket of FimH. In contrast, mutant and wild-type FimH had similar conformation when bound to mannose, explaining their similar binding strength to mannose under intermediate shear. The stabilizing effect of mannose on disulphide-free FimH was also confirmed by protection of the FimH from thermal and chemical inactivation in the presence of mannose. However, this stabilizing effect could not protect the integrity of FimH structure under high shear (> 20 dynes cm(-2)), where lack of the disulphide significantly increased adhesion OFF-rates. Thus, the cysteine bonds in bacterial adhesins could be adapted to enable bacteria to bind target surfaces under increased shear conditions.

Differential stability and trade-off effects of pathoadaptive mutations in the Escherichia coli FimH adhesin.

FimH is the tip adhesin of mannose-specific type 1 fimbriae of Escherichia coli, which are critical to the pathogenesis of urinary tract infections. Point FimH mutations increasing monomannose (1M)-specific uroepithelial adhesion are commonly found in uropathogenic strains of E. coli. Here, we demonstrate the emergence of a mixed population of clonally identical E. coli strains in the urine of a patient with acute cystitis, where half of the isolates carried a glycine-to-arginine substitution at position 66 of the mature FimH. The R66 mutation induced an unusually strong 1M-binding phenotype and a 20-fold advantage in mouse bladder colonization. However, E. coli strains carrying FimH-R66, but not the parental FimH-G66, had disappeared from the patient's rectal and urine samples collected from 29 to 44 days later, demonstrating within-host instability of the R66 mutation. No FimH variants with R66 were identified in a large (>600 strains) sequence database of fimH-positive E. coli strains. However, several strains carrying genes encoding FimH with either S66 or C66 mutations appeared to be relatively stable in the E. coli population. Relative to FimH-R66, the FimH-S66 and FimH-C66 variants mediated only moderate increases in 1M binding but preserved the ability to enhance binding under flow-induced shear conditions. In contrast, FimH-R66 completely lost shear-enhanced binding properties, with bacterial adhesion being inhibited by shear forces and lacking a rolling mode of binding. These functional trade-offs may determine the natural populational instability of this mutation or other pathoadaptive FimH mutations that confer dramatic increases in 1M binding strength.

Selection for functional diversity drives accumulation of point mutations in Dr adhesins of Escherichia coli.

Immune escape is considered to be the driving force behind structural variability of major antigens on the surface of bacterial pathogens, such as fimbriae. In the Dr family of Escherichia coli adhesins, structural and adhesive functions are carried out by the same subunit. Dr adhesins have been shown to bind decay-accelerating factor (DAF), collagen IV, and carcinoembryonic antigen-related cell adhesion molecules (CEACAMs). We show that genes encoding Dr adhesins from 100 E. coli strains form eight structural groups with a high level of amino acid sequence diversity between them. However, genes comprising each group differ from each other by only a small number of point mutations. Out of 66 polymorphisms identified within the groups, only three were synonymous mutations, indicating strong positive selection for amino acid replacements. Functional analysis of intragroup variants comprising the Dr haemagglutinin (DraE) group revealed that the point mutations result in distinctly different binding phenotypes, with a tendency of increased affinity to DAF, decreased sensitivity of DAF binding to inhibition by chloramphenicol, and loss of binding capability to collagen, CEACAM3 and CEACAM6. Thus, variability by point mutation of major antigenic proteins on the bacterial surface can be a signature of selection for functional modification.

The two-pathway model for the catch-slip transition in biological adhesion.

Some recently studied biological noncovalent bonds have shown increased lifetime when stretched by mechanical force. In each case these counterintuitive "catch-bonds" have transitioned into ordinary "slip-bonds" that become increasingly shorter lived as the tensile force on the bond is further increased. We describe analytically how these results are supported by a physical model whereby the ligand escapes the receptor binding site via two alternative routes, a catch-pathway that is opposed by the applied force and a slip-pathway that is promoted by force. The model predicts under what conditions and at what critical force the catch-to-slip transition would be observed, as well as the degree to which the bond lifetime is enhanced at the critical force. The model is applied to four experimentally studied systems taken from the literature, involving the binding of P- and L-selectins to sialyl Lewis(X) oligosaccharide-containing ligands. Good quantitative fit to the experimental data is obtained, both for experiments with a constant force and for experiments where the force increases linearly with time.

Identification of amino acids in the Dr adhesin required for binding to decay-accelerating factor.

Members of the Dr family of adhesins of Escherichia coli recognize as a receptor the Dr(a) blood-group antigen present on the complement regulatory and signalling molecule, decay-accelerating factor (DAF). One member of this family, the Dr haemagglutinin, also binds to a second receptor, type IV collagen. Structure/function information regarding these adhesins has been limited and domains directly involved in the interaction with DAF have not been determined. We devised a strategy to identify amino acids in the Dr haemagglutinin that are specifically involved in the interaction with DAF. The gene encoding the adhesive subunit, draE, was subjected to random mutagenesis and used to complement a strain defective for its expression. The resulting mutants were enriched and screened to obtain those that do not bind to DAF, but retain binding to type IV collagen. Individual amino acid changes at positions 10, 63, 65, 75, 77, 79 and 131 of the mature DraE sequence significantly reduced the ability of the DraE adhesin to bind DAF, but not collagen. Over half of the mutants obtained had substitutions within amino acids 63-81. Analysis of predicted structures of DraE suggest that these proximal residues may cluster to form a binding domain for DAF.