Hydrogen peroxide stimulates uropathogenic Escherichia coli strains to cellulose production.

Reactive oxygen intermediates, such as hydrogen peroxide, are toxic molecules produced by immune cells in response to bacterial invasion into the host. Bacteria try to protect themselves against the immune system through specific properties such as biofilm formation. This phenomenon occurs also during urinary tract infections. Cellulose is an important factor of Escherichia coli biofilm and contributes to building a protective shield around bacterial cells upon the host immune response. In this study, we aimed to analyze the effect of hydrogen peroxide on the production of this biofilm component. To achieve this goal, 25 clinical E. coli strains isolated from patients with urinary tract infections were used. These bacterial strains were characterized based on their growth characteristics, their ability to form biofilm and their capacity to produce cellulose upon exposure to sub-lethal concentrations of hydrogen peroxide growth, and the biofilm formation of these strains was analyzed. Our results revealed that the analyzed uropathogenic E. coli strains slightly, but significantly, reduced growth and biofilm production upon hydrogen peroxide treatment. However, when separating these strains regarding their ability to produce cellulose, we found that general biofilm production was reduced but cellulose expression was induced upon peroxide treatment. This finding contributes to a better understanding of how bacterial biofilm formation is triggered and provides interesting insights into how uropathogenic E. coli protect themselves in an inhospitable environment.

Enterococci populations of a metropolitan river after an extreme flood event: prevalence, persistence and virulence determinants.

We investigated the prevalence, persistence and virulence determinants of enterococci populations in water samples collected over three rounds following an extreme flood event in a metropolitan river. Enterococci (n = 482) were typed using the high resolution biochemical fingerprinting method (PhP typing) and grouped into common (C) or single (S) biochemical phenotypes (BPTs). In all, 23 C-BPTs (72.6% of isolates) were found across the sites. A representative isolate of each C-BPT was identified to the species level and tested for the presence of seven virulence genes (VGs), biofilm formation and resistance to 14 antibiotics. The enterococci concentrations in samples collected during the first two rounds were above national recreational water guidelines. By round three, enterococci concentrations decreased significantly (P < 0.05). However, 11 C-BPTs (55.5% of isolates) persisted across all sampling rounds. E. casseliflavus and E. mundtii were the most common enterococci populations comprising of >57% of all isolates. Ten of the 11 most dominant C-BPTs were resistant to multiple antibiotics and harboured one or more VGs. The high prevalence of antibiotic resistance and VGs among enterococci isolates in this catchment not only provides them with niche advantages but also poses a risk to public health.

Assessing the population dynamics of Escherichia coli in a metropolitan river after an extreme flood event.

We investigated Escherichia coli populations in a metropolitan river after an extreme flood event. Between nine and 15 of the 23 selected sites along the river were sampled fortnightly over three rounds. In all, 307 E. coli were typed using the PhP typing method and were grouped into common (C) or single (S) biochemical phenotypes (BPTs). A representative from each of the 31 identified C-BPTs was tested for 58 virulence genes (VGs) associated with intestinal and extra-intestinal E. coli, resistance to 22 antibiotics, production of biofilm and cytotoxicity to Vero cells. The number of E. coli in the first sampling round was significantly (P < 0.01) higher than subsequent rounds, whereas the number of VGs was significantly (P < 0.05) higher in isolates from the last sampling round when compared to previous rounds. Comparison of the C-BPTs with an existing database from wastewater treatment plants (WWTPs) in the same catchment showed that 40.6% of the river isolates were identical to the WWTP isolates. The relatively high number of VGs and antibiotic resistance among the C-BPTs suggests possessing and retaining these genes may provide niche advantages for those naturalised and/or persistent E. coli populations which may pose a health risk to the community.

A comparative study of the clonal diversity and virulence characteristics of uropathogenic Escherichia coli isolated from Australian and Turkish (Turkey) children and adults with urinary tract infections.

Introduction: The virulence-associated gene (VAG) repertoire and clonal organization of uropathogenic Escherichia coli (UPEC) strains is influenced by host demographic, geographic locale, and the setting of urinary tract infection (UTI). Nevertheless, a direct comparison of these features among Australian and Turkish UPEC remains unexplored. Accordingly, this study investigated the clonal composition and virulence characteristics of a collection of UPEC isolated from Australian and Turkish UTI patients. Methods: A total of 715 UPEC strains isolated from Australian (n=361) and Turkish (n=354) children and adults with hospital (HA)- and community-acquired (CA)-UTIs were included in this study. Typing of the strains using RAPD-PCR and PhPlate fingerprinting grouped all strains into 25 clonal groups (CGs). CG representatives were phylogrouped and screened for the presence of 18 VAGs associated with extraintestinal pathogenic E. coli. Results: Turkish UPEC strains were characterized by high clonal diversity and predominance of the phylogroup D, while few distinct clonal groups with phylogenetic group B2 backgrounds dominated among the Australian strains. Twelve identical CGs were shared between ≥1 patient group from either country. Australian strains, particularly those isolated from children with HA-UTI, showed higher virulence potential than their Turkish counterparts, carrying significantly more genes associated with adhesion, iron acquisition and capsule biosynthesis. Conclusions: This study identified identical CGs of UPEC causing HA- and CA-UTIs among Australian and Turkish UTI patients. These CGs frequently carried VAGs associated with adhesion, iron acquisition, immune evasion, and toxin production, which may contribute to their ability to disseminate internationally and to cause UTI.

Absence of Curli in Soil-Persistent Escherichia coli Is Mediated by a C-di-GMP Signaling Defect and Suggests Evidence of Biofilm-Independent Niche Specialization.

Escherichia coli is commonly viewed as a gastrointestinal commensal or pathogen although an increasing body of evidence suggests that it can persist in non-host environments as well. Curli are a major component of biofilm in many enteric bacteria including E. coli and are important for adherence to different biotic and abiotic surfaces. In this study we investigated curli production in a unique collection of soil-persistent E. coli isolates and examined the role of curli formation in environmental persistence. Although most soil-persistent E. coli were curli-positive, 10% of isolates were curli-negative (17 out of 170). Curli-producing E. coli (COB583, COB585, and BW25113) displayed significantly more attachment to quartz sand than the curli-negative strains. Long-term soil survival experiments indicated that curli production was not required for long-term survival in live soil (over 110 days), as a curli-negative mutant BW25113ΔcsgB had similar survival compared to wild type BW25113. Mutations in two genes associated with c-di-GMP metabolism, dgcE and pdeR, correlated with loss of curli in eight soil-persistent strains, although this did not significantly impair their survival in soil compared to curli-positive strains. Overall, the data indicate that curli-deficient and biofilm-defective strains, that also have a defect in attachment to quartz sand, are able to reside in soil for long periods of time thus pointing to the possibility that niches may exist in the soil that can support long-term survival independently of biofilm formation.

Interactions of uroseptic Escherichia coli with renal (A-498) and gastrointestinal (HT-29) cell lines.

We investigated the ability of Escherichia coli isolated from septic patients with urinary tract infection (UTI) to translocate through the gastrointestinal (GI) tract of the same patients using cell-culture models. Forty-seven hospitalized patients with urosepsis were included in this study. E. coli was isolated from their urine and blood (total 94 isolates) and investigated for genetic relatedness and interaction with the cell lines A-498 and HT-29. An initial comparison of the strains isolated from urine and blood showed that 44 out of 47 patients (94 %) had identical strains in their blood and urine. The blood isolates adhered to both cell lines, although their rate of adherence to A-498 cells was significantly higher than that to HT-29 cells (5.8±3.8 per cell vs 2.8±1.9; P<0.0001). The rate of translocation in A-498 cells was also significantly higher after 120 min (8.7×10(5) vs 2.9×10(5); P = 0.0006). Three non-identical blood isolates were unable to translocate in HT-29 cells, indicating that host immune factors might be more important than bacterial ability to translocate the GI epithelium in these patients. Our data showed that blood isolates from uroseptic patients are able to adhere to and translocate through both cell lines. This suggests that E. coli in patients with UTI may translocate from either the GI tract or the urinary tract, hence questioning the assumption that the urinary tract is the only source of septicaemia in these patients.

Decoctions from Citrus reticulata Blanco seeds protect the uroepithelium against Escherichia coli invasion.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional usage suggests Citrus reticulata Blanco seeds have beneficial effects against infection. The purpose of this study was to investigate the effect of Citrus reticulata on the uroepithelium and to determine the mechanisms responsible for protection against urinary tract infection (UTI). MATERIALS AND METHODS: Human bladder cell lines T24 and 5637 were employed in a cell culture infection model to determine the effects of Citrus reticulata treatment on Escherichia coli adherence and invasion of the uroepithelium. ß1 integrin and caveolin-1 mRNA expression was assessed using RT real-time PCR. ß1 integrin protein expression was confirmed by Western Blot. The effect of Citrus reticulata on bacteria was investigated using antibacterial sensitivity, yeast agglutination and biofilm assays. RESULTS: Citrus reticulata treatment decreased ß1 integrin expression and reduced bacterial invasion while adhesion of uroepithelial cells was not affected. Caveolin-1 expression was not influenced either and Citrus reticulata did neither exhibit any direct antimicrobial effect nor interfered with type 1 fimbriae binding. CONCLUSIONS: Our results show that Citrus reticulata has a protective effect on the uroepithelium as seen by reduced bacterial invasion of uroepithelial cells. These properties suggest that seeds from Citrus reticulata may have therapeutic potential in preventing UTI.

Antimicrobial resistance and distribution of sul genes and integron-associated intI genes among uropathogenic Escherichia coli in Queensland, Australia.

We studied 137 uropathogenic Escherichia coli (UPEC) isolates from hospitalized adult patients (Queensland, Australia) for their resistance to 17 antimicrobial agents using the calibrated dichotomous sensitivity method and the presence of class I, II and III integron-associated integrase (intI) genes, including functional class II intI2, as well as the presence of sul1, sul2 and sul3 genes, using PCR. Randomly amplified polymorphic DNA PCR, a high-resolution biochemical-fingerprinting method (PhP) and phylogenetic grouping were also used to identify the clonality of the sulphonamide-resistant isolates. One hundred and twenty (87.6 %) isolates were resistant to one or more of the tested antimicrobial drugs, with the highest resistance (70.1 %) observed against sulphafurazole (96 isolates). Of these, 84 (87.5 %) contained one or more sul alleles, with sul1 being the most common allele [occurring in 69 (72 %) isolates]. Only 38 of 69 (55.1 %) strains carrying the sul1 gene were positive for class I integrase. Our results indicate a high prevalence of sulphafurazole-resistant UPEC strains belonging to different clones among patients with urinary tract infection in Queensland, Australia. We also conclude that these strains carry predominantly a sul1 gene that is not commonly associated with the presence of class I integrase, indicating that it may be carried on either a bacterial chromosome or other genetic elements.

Population structure and uropathogenic virulence-associated genes of faecal Escherichia coli from healthy young and elderly adults.

We investigated the population structures of faecal Escherichia coli in 30 healthy young adults (13 males and 17 females) aged between 20 and 45 years and 29 elderly adults (14 females and 15 males) aged between 65 and 77 years. In all, 1566 strains were typed with the PhPlate system and grouped into biochemical phenotypes (BPTs). Strains with shared BPTs were further typed using randomly amplified polymorphic DNA analysis. Forty-four per cent of the strains were shared between two or more age and gender groups. Elders had a significantly higher (P<0.001) number of BPTs (mean±standard error 3.3±0.27) than younger groups (1.82±0.27). Phylogenetic affiliation and virulence-associated genes (VAGs) of the strains showed that more than 80 % of the strains belonging to dominant types belonged to phylogroups B2 and D. Amongst dominant BPTs, phylogenetic group A was significantly associated with females (P<0.0001), and elders were more likely to carry group D (P<0.0124). Elderly males had a higher prevalence of VAGs than young males (P<0.0001) and young females (P<0.0005). We conclude that there is a lower prevalence of E. coli with uropathogenic properties in healthy young adults than in elders.