Specific selection for virulent urinary tract infectious Escherichia coli strains during catheter-associated biofilm formation

Biofilm-associated bacterial infections have a major impact on artificial implants such as urinary catheters, often with devastating consequences. The capacity of a microorganism to form a biofilm on a surface depends on the nature of the surface and its conditioning. When a urinary catheter is exposed to urine, various components adsorb onto the surface and form a conditioning film, which becomes the real interface where microbial interaction takes place. It follows that the material constituting the catheter determines the composition of the conditioning film, which in turn influences which microorganisms can attach. Urinary tract infectious (UTI) Escherichia coli range in pathogenicity and the damage they cause--from benign asymptomatic bacteriuria (ABU) strains, which inflict no or few problems to the host, to uropathogenic E. coli (UPEC) strains, which are virulent and often cause severe symptoms and complications. We have found that whereas ABU strains produce better biofilms on polystyrene and glass, UPEC strains have a clear competitive advantage during biofilm growth on catheter surfaces. Our results indicate that some silicone and silicone-latex catheters actually select for and promote biofilm formation of the most virulent group of UTI E. coli strains, hardly a desirable situation for the catheterized patient.     

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.     

Prevalence of virulence genes in Escherichia coli strains isolated from Romanian adult urinary tract infection cases

A total of 78 E. coli strains isolated from adults with different types of urinary tract infections were screened by polymerase chain reaction for prevalence of genetic regions coding for virulence factors. The targeted genetic determinants were those coding for type 1 fimbriae ( fimH ), pili associated with pyelonephritis ( pap ), S and F1C fimbriae ( sfa and foc ), afimbrial adhesins ( afa ), hemolysin ( hly ), cytotoxic necrotizing factor ( cnf ), aerobactin ( aer ). Among the studied strains, the prevalence of genes coding for fimbrial adhesive systems was 86 %, 36%, and 23% for fimH, pap , and sfa/foc , respectively. The operons coding for Afa afimbrial adhesins were identified in 14% of strains. The hly and cnf genes coding for toxins were amplified in 23% and 13% of strains, respectively. A prevalence of 54% was found for the aer gene. The various combinations of detected genes were designated as virulence patterns. The strains isolated from the hospitalized patients displayed a greater number of virulence genes and a diversity of gene associations compared to the strains isolated from the ambulatory subjects. A rapid assessment of the bacterial pathogenicity characteristics may contribute to a better medical approach of the patients with urinary tract infections.     

Escherichia coli outer membrane protease OmpT confers resistance to urinary cationic peptides

Escherichia coli OmpT, located in the outer membrane, has been characterized as a plasminogen activator, with the ability to hydrolyze protamine and block its entry. In this investigation, a complex of low molecular weight cationic peptides purified from human urine by a combination of membrane ultrafiltration and weak cation exchange chromatography was characterized. The impact of OmpT on E. coli resistance to urinary cationic peptides was investigated by testing ompT knockout strains. The ompT mutants were more susceptible to urinary cationic peptides than ompT⁺ strains, and this difference was abolished by complementation of the mutants with pUC19 carrying the ompT gene. The urinary protease inhibitor ulinastatin greatly decreased the resistance of the ompT⁺ strains. Overall, the data indicate that OmpT may help E. coli persist longer in the urinary tract by enabling it to resist the antimicrobial activity of urinary cationic peptides.     

Rapid immunodetection of Escherichia coli

A filtration flow-through design was used to develop the rapid immunodetection of Escherichia coli. Polyclonal anti-E. coli IgG was conjugated to small, 0.8 Blue latex beads. Cells were mixed with conjugated beads in the presence of anti-E. coli monoclonal IgM. The suspension was then filtered through a 5 nitrocellulose membrane. The cell-containing complexes were effectively collected on the filter, forming a blue spot. The method produced reliable detection of E. coli at a concentration of 10⁵ cells ml^–1, which is a current benchmark figure for urinary tract infection (UTI) diagnosis.     

Mutator phenotype confers advantage in Escherichia coli chronic urinary tract infection pathogenesis

It has been suggested that mutator phenotype could be associated with an increase in virulence, but to date experimental evidences are lacking. Epidemiological studies have revealed that urinary tract infection isolates encompass the highest proportion of mutator strains within the Escherichia coli species. Using the uropathogenic strain CFT073 and its mutS- mutator mutant, we show that the mutator strain is selected in vitro in urine and in the late stages of infection in a mouse model having urinary tract infection. Thus, we report that, under specific conditions, i.e., urinary tract infection, the mutator phenotype may confer an advantage in pathogenesis.     

Biofilm formation by Acinetobacter baumannii strains isolated from urinary tract infection and urinary catheters

Systemic infections in avian species caused by avian pathogenic Escherichia coli (APEC) are economically devastating to poultry industries worldwide. To unravel factors possibly involved in APEC pathogenicity, suppression subtractive hybridization was applied, leading to the identification of a putative APEC autotransporter adhesin gene aatA in our previous study. In this study, pathogenic mechanism of AatA was further determined. A deletion mutant of aatA was constructed in the APEC DE205B, which results in the reduced capacity to adhere to DF-1 cells, defective virulence in vivo, and decreased colonization capacity in lung during the systemic infection compared with the wild-type strain. Furthermore, these capacities were restored in the complementation strains. These results indicated that AatA makes a significant contribution to APEC virulence through bacterial adherence to host tissues in vivo and in vitro. In addition, aggregation assays for strain AAEC189 expressing aatA indicated that AatA mediates cell aggregation and settling of cells. However, this cell aggregation is blocked by Type I fimbriae. This study illustrates the first examination of the role of AatA in aggregation and systemic infection.     

An in vitro urinary tract catheter system to investigate biofilm development in catheter-associated urinary tract infections

Biofilm development in urinary tract catheters is an often underestimated problem. However, this form of infection leads to high mortality rates and causes significant costs in health care. Therefore, it is important to analyze these biofilms and establish avoiding strategies. In this study a continuous flow-through system for the cultivation of biofilms under catheter-associated urinary tract infection conditions was established and validated. The in vitro urinary tract catheter system implies the composition of urine (artificial urine medium), the mean volume of urine of adults (1 mL min^-1), the frequently used silicone catheter (foley silicon catheter) as well as the infection with uropathogenic microorganisms like Pseudomonas aeruginosa. Three clinical isolates from urine of catheterized patients were chosen due to their ability to form biofilms, their mobility and their cell surface hydrophobicity. As reference strain P. aeruginosa PA14 has been used. Characteristic parameters as biofilm thickness, specific biofilm growth rate and substrate consumption were observed. Biofilm thicknesses varied from 105 ± 16 μm up to 246 ± 67 μm for the different isolates. The specific biofilm growth rate could be determined with a non invasive optical biomass sensor. This sensor allows online monitoring of the biofilm growth in the progress of the cultivation.     

156例尿路感染大肠埃希菌耐药性分析

目的探讨大肠埃希菌尿路感染的临床发病特点及对抗生素的耐药情况。方法对2001年1月至2005年12月尿路感染患者尿培养分离出的156株大肠埃希菌进行耐药性分析,用纸片扩散法表型确证试验检测ESBLs。结果大肠埃希菌耐药率最低的抗菌药物是亚胺培南、美罗培南、头孢哌酮/舒巴坦、氧哌嗪青霉素/他唑巴坦,分别为1.28%、1.92%、3.21%、5.13%;对氨苄西林、氟喹诺酮类、庆大霉素、复方新诺明耐药率均>70%。产ESBLs的大肠埃希菌对亚胺培南、美罗培南、头孢哌酮/舒巴坦、氧哌嗪青霉素/他唑巴坦的耐药性均<10%,对氨苄西林、头孢菌素类、氟喹诺酮类均表现出很强的耐药性。结论治疗大肠埃希菌感染时,需根据药敏结果选用碳青霉烯类、β-内酰胺类/β-内酰胺酶抑制剂等。     

Biofilm formation and interactions of bacterial strains found in wastewater treatment systems

Biofilm formation and adherence properties of 13 bacterial strains commonly found in wastewater treatment systems were studied in pure and mixed cultures using a crystal violet microtiter plate assay. Four different culture media were used, wastewater, acetate medium, glucose medium and diluted nutrient broth. The medium composition strongly affected biofilm formation. All strains were able to form pure culture biofilms within 24 h in at least one of the tested culture media and three strains were able to form biofilm in all four culture media, namely Acinetobacter calcoaceticus ATCC 23055, Comamonas denitrificans 123 and Pseudomonas aeruginosa MBL 0199. The adherence properties assessed were initial adherence, cell surface hydrophobicity, and production of amyloid fibers and extracellular polymeric substances. The growth of dual-strain biofilms showed that five organisms formed biofilm with all 13 strains while seven formed no or only weak biofilm when cocultured. In dual-strain cultures, strains with different properties were able to complement each other, giving synergistic effects. Strongest biofilm formation was observed when a mixture of all 13 bacteria were grown together. These results on attachment and biofilm formation can serve as a tool for the design of tailored systems for the degradation of municipal and industrial wastewater.     

Isolation and characterisation of dog uropathogenic Escherichia coli strains and their fimbriae

A number of Escherichia coli strains have been isolated from dogs with urinary tract infections. These strains have been characterised with respect to their O, K, H, and fimbrial antigens, colicin production, antibiotic resistance, plasmid content and their ability to haemagglutinate erythrocytes from various species. Crossed immunoelectrophoresis of fimbrial extracts, as well as the reaction of partly purified fimbriae of a number of these strains with monoclonal antibodies revealed homology or a strong crossereaction with an F12 fimbrial subunit protein of human uropathogenic E. coli strains. Unlike human F12 fimbriae producing strains, the dog isolates did agglutinate dog erythrocytes in the presence of D-mannose but not human erythrocytes, indicating that the adhesin carried by these strains is different from the adhesin on fimbriae of human uropathogenic E. coli. Similar indications were obtained from experiments with latex beads coated with the receptor for P-fimbriae. These beads were agglutinated by Escherichia coli strains from human urinary tract infections, but not by the dog isolates described here. Preliminary adhesion experiments of human and dog Escherichia coli to human bladder epithelial and canine kidney epithelial cells also showed differences in adhesion depending on the origin of the strain tested.     

Pathogenic characteristics of Escherichia coli strains in cases of asymptomatic bacteriuria and symptomatic urinary tract infections

The aim of this study was to examine if E. coli isolated from asymptomatic bacteriuria differed in pathogenic features from strains isolated from symptomatic infections of urinary tract. In this study 130 strains of E. coli isolated from women having asymptomatic bacteriuria and 112 strains isolated from patients with symptoms of urinary tract infection were examined. It was shown that E. coli isolated from patients with symptomatic urinary tract infection showed the more frequently ability to cause mannose-resistant haemagglutination of human erythrocytes, resistance to bactericidal activity of serum and haemolytic properties than those isolated from asymptomatic bacteriuria. These strains showed also the higher ability to adhere to Vero cells in tissue culture. Among E. coli strains isolated from persons with asymptomatic bacteriuria the pathogenic features were most frequently found in strains from healthy women and the most rarely in isolated from diabetic women.     

Biofilm formation by avian Escherichia coli in relation to media, source and phylogeny

AIMS: To assess the abilities of 105 avian pathogenic Escherichia coli (APEC) and 103 avian faecal commensal E. coli (AFEC) to form biofilms on a plastic surface and to investigate the possible association of biofilm formation with the phylotype of these isolates. METHODS AND RESULTS: Biofilm production was assessed in 96-well microtitre plates using three different media, namely, M63 minimal medium supplemented with glucose and casamino acids, brain-heart infusion broth, and diluted tryptic soy broth. Avian E. coli are highly variable in their ability to form biofilms. In fact, no strain produced a strong biofilm in all three types of media; however, most (75.7% AFEC and 55.2% APEC) were able to form a moderate or strong biofilm in at least one medium. Biofilm formation in APEC seems to be mostly limited to nutrient deplete media; whereas, AFEC are able to form biofilms in both nutrient deplete and replete media. Also, biofilm formation in E. coli from phylogenetic groups B2, D and B1 was induced by nutrient deplete conditions; whereas, biofilm formation by members of phylogenetic group A was strongest in a rich medium. CONCLUSIONS: Biofilm formation by APEC and phylotypes B2, D and B1 is induced by nutrient deplete conditions, while AFEC are able to form biofilms in both nutrient rich and deplete media. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to investigate biofilm formation by a large sample of avian E. coli isolates, and it provides insight into the conditions that induce biofilm formation in relation to the source (APEC or AFEC) and phylogenetic group (A, B1, B2 and D) of an isolate.     

Biofilm formation by ica-positive and ica-negative strains of Staphylococcus epidermidis in vitro

Staphylococcus epidermidis is a clinically important opportunistic pathogen that forms biofilm infections on nearly all types of indwelling medical devices. The biofilm forming capability of S. epidermidis has been linked to the presence of the ica operon in the genome, and the amount of biofilm formation measured by the crystal violet (CV) adherence assay. Six S. epidermidis strains were characterized for their ica status using PCR, and their biofilm forming ability over 6 days, using the CV assay and a flow cell system. Ica-negative strains characterized as ‘negative for biofilm formation’ based on the CV assay were demonstrated to form strongly attached biofilms after 6 days. However, the biofilms were not as extensive as the ica-positive strains. It was concluded that ica is not required for biofilm formation, nor is the 24-h CV assay generalizable for predicting the 6-day biofilm-forming ability for all S. epidermidis strains.     

Biofilm formation by a fimbriae-deficient mutant of Actinobacillus actinomycetemcomitans

Actinobacillus actinomycetemcomitans strain 310-TR produces fimbriae and forms a tight biofilm in broth cultures, without turbid growth. The fimbriae-deficient mutant 310-DF, constructed in this study, was grown as a relatively fragile biofilm at the bottom of a culture vessel. Scanning electron microscopy revealed that on glass coverslips, 310-TR formed tight and spherical microcolonies, while 310-DF produced looser ones. These findings suggest that fimbriae are not essential for the surface-adherent growth but are required for enhancing cell-to-surface and cell-to-cell interactions to stabilize the biofilm. Treatment of the 310-DF biofilm with either sodium metaperiodate or DNase resulted in significant desorption of cells from glass surfaces, indicating that both carbohydrate residues and DNA molecules present on the cell surface are also involved in the biofilm formation.