Horizontal transfer of nonconjugative plasmids in a colony biofilm of Escherichia coli

We tested the possibility of nonconjugative lateral DNA transfer in a colony biofilm of mixed Escherichia coli strains. By simply coculturing a plasmid-free F(-) strain and another F(-) strain harboring a nonconjugative plasmid in a colony biofilm on antibiotic-free agar media, transformed cells were produced within 24-48 h at the frequency of 10(-10)-10(-9) per recipient cell. PCR analysis of the transformed cells demonstrated the occurrence of lateral plasmid transfer. These cells survived until at least day 7 under antibiotic-free conditions. Liquid cultures of the same strains in Luria-Bertani broth produced no or few transformants, suggesting the importance of colony-biofilm formation for plasmid transfer. This is a novel line of evidence indicating that nonconjugative, nonviral horizontal gene transfer can occur between E. coli cells.     

Characterization of typical and atypical enteroaggregative Escherichia coli in Kagoshima,Japan: biofilm formation and acid resistance

EAEC is increasingly recognized as an emerging enteric pathogen. Typical EAEC expressing the AggR regulon have been proven to be an important cause of childhood diarrhea in industrialized countries as well as in the developing world, while atypical EAEC without this regulon have not been thoroughly investigated. To investigate the bacteriological characteristics of EAEC, including both typical and atypical strains in Kagoshima, Japan, 2417 E. coli strains from Japanese children with diarrhea were screened by a quantitative biofilm assay to detect possible EAEC strains, resulting in the identification of 102 (4.2%) of these strains by the HEp‐2 cell adherence test. Virulence gene patterns, PFGE analysis and O‐serogrouping demonstrated the heterogeneity of the EAEC. The EAEC strains were classified into two groups: typical EAEC with aggR (74.5%, 76/102) and atypical EAEC without aggR (25.5%, 26/102). There was no significant difference between the typical EAEC strains (median OD₅₇₀= 0.73) and the atypical strains (median OD₅₇₀= 0.61) in biofilm formation (P= 0.17). Incidences of resistance against ampicillin, cefotaxime and tetracycline were significantly higher in the typical EAEC strains than the atypical EAEC strains (84.2% vs. 53.8%, 36.8% vs. 7.7% and 93.4% vs. 73.1%, respectively, P < 0.05). The typical EAEC strains showed significantly higher resistance ratios against HCl and lactate than the atypical strains (94.7% vs. 61.5% and 92.1% vs. 57.7%, respectively, P < 0.001). To investigate the pathogenicity of not only typical but also atypical EAEC, further bacteriological and epidemiologic studies including atypical EAEC are needed.     

Measurement of biofilm formation by clinical isolates of Escherichia coli is method-dependent

Aims:  In this study, we have evaluated the impact of methodological approaches in the determination of biofilm formation by four clinical isolates of Escherichia coli in static assays.
Methods and Results:  The assays were performed in microtitre plates with two minimal and two enriched broths, with one- or two-steps protocol, and using three different mathematical formulas to quantify adherent bacteria. Different biofilm formation patterns were found depending on the E. coli strain, culture medium and reading optical density on one- and two-steps protocol. Strong or moderate biofilm formation occurred mostly in minimal media. The mathematical formulas used to quantify biofilm formation also gave different results and bacterial growth rate should be taken into account to quantify biofilm.
Conclusions:  Escherichia coli forms biofilms on static assays in a method-dependent fashion, depending on strain, and it is strongly modulated by culture conditions.
Significance and Impact of the Study:  As verified in the studied E. coli strains, biofilm formation by any organism should be cautiously interpreted, considering all variables in the experimental settings.     

Polysorbates prevent biofilm formation and pathogenesis of Escherichia coli O104:H4

Escherichia coli biotype O104:H4 recently caused the deadliest E. coli outbreak ever reported. Based on prior results, it was hypothesized that compounds inhibiting biofilm formation by O104:H4 would reduce its pathogenesis. The nonionic surfactants polysorbate 80 (PS80) and polysorbate 20 (PS20) were found to reduce biofilms by ≥ 90% at submicromolar concentrations and elicited nearly complete dispersal of preformed biofilms. PS80 did not significantly impact in vivo colonization in a mouse infection model; however, mice treated with PS80 exhibited almost no intestinal inflammation or tissue damage while untreated mice exhibited robust pathology. As PS20 and PS80 are classified as ‘Generally Recognized as Safe’ (GRAS) compounds by the Food and Drug Administration (FDA), these compounds have clinical potential to treat future O104:H4 outbreaks.     

The effect of surface charge property on Escherichia coli initial adhesion and subsequent biofilm formation

Polyethylene (PE) sheets were modified by radiation-induced graft polymerization (RIGP) of an epoxy-group containing monomer glycidyl methacrylate (GMA). The epoxy group of GMA was opened by introducing sodium sulfite (SS) and diethylamine (DEA) as representatives of negatively and positively charged functional groups, respectively. These modified surfaces by RIGP, termed GMA, SS, and DEA sheets, were investigated to elucidate their effects on initial adhesion and subsequent biofilm formation of Escherichia coli. Initial adhesion test revealed that E. coli density and viability were governed by sheet surface electrostatic property: E. coli cell density on the DEA sheet was 23 times higher than that on the SS sheet after 8 h incubation. The viability of E. coli cells dramatically decreased after contact with the DEA sheet, but remained high on the SS sheet. E. coli biofilm structure on the DEA sheet was dense, homogeneous, and uniform, with biomass higher than that of the GMA and SS sheets by factors of 14.0 and 37.5, respectively. On the contrary, biofilm structure on the SS sheet was sparse, heterogeneous, and mushroom-shaped. More than 40% of E. coli biofilm on the DEA sheet was retained under a high liquid shear force condition (5,000 s(-1)), whereas 97% and 100% of biofilms on the GMA and SS sheets were sloughed, indicating that E. coli biofilm robustness depends on surface charge property of the substratum. This suggests that substratum surface fabrication by RIGP may enhance or suppress biofilm formation, a finding with potentially important practical implications.     

Interaction of Escherichia coli and its culture supernatant with Vibrio vulnificus during biofilm formation

Vibrio vulnificus is a foodborne pathogen causing septicemia with high mortality rate. In this study, we explored how Escherichia coli, one of the commensal bacteria in the human gastrointestinal tract, can interact with V. vulnificus. Our study results show that the amount of biofilm produced by V. vulnificus was reduced in the presence of E. coli ATCC 35218, although the growth of V. vulnificus L-180 remained unaffected. We also detected an antibiofilm effect of E. coli culture supernatant against V. vulnificus, which could not be reduced even after heat treatment. These findings indicate that E. coli and its culture supernatant may be suitable to prevent biofilm formation by V. vulnificus. By contrast, live cells of V. vulnificus could reduce the amount of preformed E. coli biofilm, but its culture supernatant could not. This suggests that the cell-associated factors contribute toward reduction in E. coli biofilm. Therefore, we speculate that ingestion of an infectious dose of V. vulnificus might induce dislodging of the commensal bacteria from the intestinal epithelia and thus can colonize to initiate the infection.     

Inhibition of Escherichia coli and Proteus mirabilis adhesion and biofilm formation on medical grade silicone surface

Silicone has been utilized extensively for biomedical devices due to its excellent biocompatibility and biodurability properties. However, its surface is easily colonized by bacteria which will increase the probability of nosocomial infection. In the present work, a hydrophilic antimicrobial carboxymethyl chitosan (CMCS) layer has been grafted on medical grade silicone surface pre-treated with polydopamine (PDA). The increase in hydrophilicity was confirmed from contact angle measurement. Bacterial adhesion tests showed that the PDA-CMCS coating reduced the adhesion of Escherichia coli and Proteus mirabilis by ≥ 90%. The anti-adhesion property was preserved even after the aging of the functionalized surfaces for 21 days in phosphate-buffered saline (PBS), and also after autoclaving at 121°C for 20 min. Both E. coli and P. mirabilis readily form biofilms on the pristine surface under static and flow conditions but with the PDA-CMCS layer, biofilm formation is inhibited. The flow experiments indicated that it is more difficult to inhibit biofilm formation by the highly motile P. mirabilis as compared to E. coli. No significant cytotoxicity of the modified substrates was observed with 3T3 fibroblasts.     

The influence of biofilm structure and total interaction energy on Escherichia coli retention by Pseudomonas aeruginosa biofilm

The retention of a surrogate pathogenic bacterium, Escherichia coli^T , in Pseudomonas aeruginosa biofilms (with various EPS excreting capacities) was investigated using a laboratory flow cell system. The structural characteristics of the biofilm, as well as the quantity of E. coli^T retained in the biofilm, were assessed using confocal laser scanning microscopy coupled with image analysis. In addition, the total interaction energy between E. coli^T and the P. aeruginosa biofilm was computed with the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, which provided an additional context to explain the pathogen interaction in aquatic biofilms. The correlations between the quantity of detained E. coli^T cells and the structural characteristics of the biofilm were analysed and the results indicated that the heterogeneity of the biofilm could create a quiescent zone leading to temporary retention of E. coli^T within the biofilm. Overall, this study provided insights toward understanding the retention of pathogenic bacteria in environmental biofilms.     

Attachment and biofilm formation on stainless steel by Escherichia coli O157:H7 as affected by curli production

AIMS: The aim of this study was to determine the role of curli in attachment and biofilm formation by Escherichia coli O157:H7 on stainless steel. METHODS AND RESULTS: Three curli-deficient strains (43895-, 43894- and E0018-) and three curli over-producing strains (43895+, 43894+ and E0018+) of E. coli O157:H7 were studied. Stainless steel coupons (SSC) were immersed in cell suspensions of each strain for 24 h at 4 degrees C. The number of cells attached to SSC was determined. To determine the ability of attached cells to form biofilm, SSC were immersed in 10% of tryptic soya broth up to 6 days at 22 degrees C. Curli-deficient and curli-producing strains did not differ in their ability to attach to SSC, but only curli-producing strains formed biofilms. CONCLUSIONS: Curli production by E. coli O157:H7 does not affect attachment of cells on stainless steel but curli-producing strains are better able to form biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: Curli production by E. coli O157:H7 enhances its ability to form biofilm on stainless steel, thereby potentially resulting in increased difficulty in removing or killing cells by routine cleaning and sanitizing procedures used in food-processing plants.     

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.     

Mechanisms of deletion formation in Escherichia coli plasmids. II. Deletions mediated by short direct repeats.

Summary A set of plasmids containing 42, 21 and 13 bp direct repeats was used to analyze the effect of repeat length on the frequencies of deletion formation and the structure of the deleted derivatives of different recombination-deficient Escherichia coli strains. Agarose gel electrophoresis of plasmid DNA demonstrated that the formation of deletions in these plasmids was associated with dimerization of plasmid DNA. Restriction analysis of the dimers showed that deletions at short direct repeats arose non-conservatively, that is, the formation of a deletion in one monomeric plasmid unit was not associated with a duplication in the other. Mutations in the recA, recF, recJ and recO genes had no marked effect on either the frequencies of deletion formation or the structure of dimers. In contrast, recB recC mutations greatly increased the frequencies of deletion formation, 6-fold for 42 bp, and 115-fold for 21 by direct repeats. Conversion of DNA replication to the rolling circle mode in a recB recC strain, resulting in the formation of double-stranded ends, is suggested as the stimulatory effector.     

Role of type 1 fimbriae and mannose in the development of Escherichia coli K12 biofilm: from initial cell adhesion to biofilm formation

The influence of type 1 fimbriae, mannose-sensitive structures, on biofilm development and maturation has been examined by the use of three isogenic Escherichia coli K12 strains: wild type, fimbriated, and non-fimbriated. Experiments with the three strains were done in minimal medium or Luria–Bertani broth supplemented with different concentrations of d-mannose. The investigation consisted of: (1) characterizing the bacterial surface of the three strains with respect to hydrophilicity and surface charge, (2) investigating the effect of type 1 fimbriae on bacterial adhesion rate and reversibility of initial adhesion on glass surfaces, and (3) verifying the role of type 1 fimbriae and exopolysaccharides (EPS) in biofilm maturation. The results suggest that type 1 fimbriae are not required for the initial bacterial adhesion on glass surfaces as the non-fimbriated cells had higher adhesion rates and irreversible deposition. Type 1 fimbriae, however, are critical for subsequent biofilm development. It was hypothesized that in the biofilm maturation step, the cells synthesize mannose-rich EPS, which functions as a ‘conditioning film’ that can be recognized by the type 1 fimbriae.     

Iron-limited condition modulates biofilm formation and interaction with human epithelial cells of enteroaggregative Escherichia coli (EAEC)

Aims:  The aim of this study was to investigate the influence of low iron availability on biofilm formation and adherence to HEp-2 cells of enteroaggregative Escherichia coli (EAEC) strains isolated from diarrhoea cases.
Methods and Results:  The ability of EAEC to form biofilm on a plastic surface was evaluated quantitatively and qualitatively after 3 and 18 h of incubation of strains with or without the iron chelator 2,2-dipyridyl. When submitted to low iron conditions, prototype EAEC 042 strain showed a decrease in biofilm formation. Conversely, an increase in biofilm formation was observed for the clinical EAEC strains cultured in restricted iron condition. Moreover, the reduction of iron concentration inhibited the aggregative adherence to HEp-2 cells of all EAEC strains tested. However, all effects promoted by iron chelation were suppressed by thiourea.
Conclusions:  Low iron availability may modulate biofilm formation and adhesive properties of EAEC strains to HEp-2 cells.
Significance and Impact of the Study:  The data obtained in this study provide useful insights on the influence of low iron conditions possibly associated with redox stress on the pathogenesis of EAEC strains.