Andreas Reisner

Development and maturation of Escherichia coli K-12 biofilms

The development and maturation of E. coli biofilms in flow‐chambers was investigated. We found that the presence of transfer constitutive IncF plasmids induced biofilm development forming structures resembling those reported for Pseudomonas aeruginosa. The development occurred in a step‐wise process: (i) attachment of cells to the substratum, (ii) clonal growth and microcolony formation, and (iii) differentiation into expanding structures rising 70–100 µm into the water phase. The first two steps were the same in the plasmid‐carrying and plasmid‐free strains, whereas the third step only occurred in conjugation pilus proficient plasmid‐carrying strains. The final shapes of the expanding structures in the mature biofilm seem to be determined by the pilus configuration, as various mutants affected in the processing and activity of the transfer pili displayed differently structured biofilms. We further provide evidence that flagella, type 1 fimbriae, curli and Ag43 are all dispensable for the observed biofilm maturation. In addition, our results indicate that cell‐to‐cell signalling mediated by autoinducer 2 (AI‐2) is not required for differentiation of E. coli within a biofilm community. We suggest on the basis of these results that E. coli K‐12 biofilm development and maturation is dependent on cell‐cell adhesion factors, which may act as inducers of self‐assembly processes that result in differently structured biofilms depending on the adhesive properties on the cell surface.

In Vitro Biofilm Formation of Commensal and Pathogenic Escherichia coli Strains: Impact of Environmental and Genetic Factors

Our understanding of Escherichia coli biofilm formation in vitro is based on studies of laboratory K-12 strains grown in standard media. However, pathogenic E. coli isolates differ substantially in their genetic repertoire from E. coli K-12 and are subject to heterogeneous environmental conditions. In this study, in vitro biofilm formation of 331 nondomesticated E. coli strains isolated from healthy (n = 105) and diarrhea-afflicted children (n = 68), bacteremia patients (n = 90), and male patients with urinary tract infections (n = 68) was monitored using a variety of growth conditions and compared to in vitro biofilm formation of prototypic pathogenic and laboratory strains. Our results revealed remarkable variation among the capacities of diverse E. coli isolates to form biofilms in vitro. Notably, we could not identify an association of increased biofilm formation in vitro with a specific strain collection that represented pathogenic E. coli strains. Instead, analysis of biofilm data revealed a significant dependence on growth medium composition (P < 0.05). Poor correlation between biofilm formation in the various media suggests that diverse E. coli isolates respond very differently to changing environmental conditions. The data demonstrate that prevalence and expression of three factors known to strongly promote biofilm formation in E. coli K-12 (F-like conjugative pili, aggregative adherence fimbriae, and curli) cannot adequately account for the increased biofilm formation of nondomesticated E. coli isolates in vitro. This study highlights the complexity of genetic and environmental effectors of the biofilm phenotype within the species E. coli.

Novel Roles for the AIDA Adhesin from Diarrheagenic Escherichia coli: Cell Aggregation and Biofilm Formation

Diarrhea-causing Escherichia coli strains are responsible for numerous cases of gastrointestinal disease and constitute a serious health problem throughout the world. The ability to recognize and attach to host intestinal surfaces is an essential step in the pathogenesis of such strains. AIDA is a potent bacterial adhesin associated with some diarrheagenic E. coli strains. AIDA mediates bacterial attachment to a broad variety of human and other mammalian cells. It is a surface-displayed autotransporter protein and belongs to the selected group of bacterial glycoproteins; only the glycosylated form binds to mammalian cells. Here, we show that AIDA possesses self-association characteristics and can mediate autoaggregation of E. coli cells. We demonstrate that intercellular AIDA-AIDA interaction is responsible for bacterial autoaggregation. Interestingly, AIDA-expressing cells can interact with antigen 43 (Ag43)-expressing cells, which is indicative of an intercellular AIDA-Ag43 interaction. Additionally, AIDA expression dramatically enhances biofilm formation by E. coli on abiotic surfaces in flow chambers.

Synergistic Effects in Mixed Escherichia coli Biofilms: Conjugative Plasmid Transfer Drives Biofilm Expansion

Bacterial biofilms, often composed of multiple species and genetically distinct strains, develop under complex influences of cell-cell interactions. Although detailed knowledge about the mechanisms underlying formation of single-species laboratory biofilms has emerged, little is known about the pathways governing development of more complex heterogeneous communities. In this study, we established a laboratory model where biofilm-stimulating effects due to interactions between genetically diverse strains of Escherichia coli were monitored. Synergistic induction of biofilm formation resulting from the cocultivation of 403 undomesticated E. coli strains with a characterized E. coli K-12 strain was detected at a significant frequency. The survey suggests that different mechanisms underlie the observed stimulation, yet synergistic development of biofilm within the subset of E. coli isolates (n = 56) exhibiting the strongest effects was most often linked to conjugative transmission of natural plasmids carried by the E. coli isolates (70%). Thus, the capacity of an isolate to promote the biofilm through cocultivation was (i) transferable to the K-12 strain, (ii) was linked with the acquisition of conjugation genes present initially in the isolate, and (iii) was inhibited through the presence in the cocultured K-12 strain of a related conjugative plasmid, presumably due to surface exclusion functions. Synergistic effects of cocultivation of pairs of natural isolates were also observed, demonstrating that biofilm promotion in this system is not dependent on the laboratory strain and that the described model system could provide relevant insights on mechanisms of biofilm development in natural E. coli populations.

Biofilm formation of Klebsiella pneumoniae on urethral catheters requires either type 1 or type 3 fimbriae

Urinary catheters are standard medical devices utilized in both hospital and nursing home settings, but are associated with a high frequency of catheter-associated urinary tract infections (CAUTI). In particular, biofilm formation on the catheter surface by uropathogens such as Klebsiella pneumoniae causes severe problems. Here we demonstrate that type 1 and type 3 fimbriae expressed by K. pneumoniae enhance biofilm formation on urinary catheters in a catheterized bladder model that mirrors the physico-chemical conditions present in catheterized patients. Furthermore, we show that both fimbrial types are able to functionally compensate for each other during biofilm formation on urinary catheters. In situ monitoring of fimbrial expression revealed that neither of the two fimbrial types is expressed when cells are grown planktonically. Interestingly, during biofilm formation on catheters, both fimbrial types are expressed, suggesting that they are both important in promoting biofilm formation on catheters. Additionally, transformed into and expressed by a nonfimbriated Escherichia coli strain, both fimbrial types significantly increased biofilm formation on catheters compared with the wild-type E. coli strain. The widespread occurrence of the two fimbrial types in different species of pathogenic bacteria stresses the need for further assessment of their role during urinary tract infections.

Type 1 Fimbriae Contribute to Catheter-Associated Urinary Tract Infections Caused by Escherichia coli

Biofilm formation on catheters is thought to contribute to persistence of catheter-associated urinary tract infections (CAUTI), which represent the most frequent nosocomial infections. Knowledge of genetic factors for catheter colonization is limited, since their role has not been assessed using physicochemical conditions prevailing in a catheterized human bladder. The current study aimed to combine data from a dynamic catheterized bladder model in vitro with in vivo expression analysis for understanding molecular factors relevant for CAUTI caused by Escherichia coli. By application of the in vitro model that mirrors the physicochemical environment during human infection, we found that an E. coli K-12 mutant defective in type 1 fimbriae, but not isogenic mutants lacking flagella or antigen 43, was outcompeted by the wild-type strain during prolonged catheter colonization. The importance of type 1 fimbriae for catheter colonization was verified using a fimA mutant of uropathogenic E. coli strain CFT073 with human and artificial urine. Orientation of the invertible element (IE) controlling type 1 fimbrial expression in bacterial populations harvested from the colonized catheterized bladder in vitro suggested that the vast majority of catheter-colonizing cells (up to 88%) express type 1 fimbriae. Analysis of IE orientation in E. coli populations harvested from patient catheters revealed that a median level of ∼73% of cells from nine samples have switched on type 1 fimbrial expression. This study supports the utility of the dynamic catheterized bladder model for analyzing catheter colonization factors and highlights a role for type 1 fimbriae during CAUTI.

In situ monitoring of IncF plasmid transfer on semi-solid agar surfaces reveals a limited invasion of plasmids in recipient colonies

Highlights ► We monitored extent of IncF plasmid R1 spread during agar surface matings in situ. ► A zygotic induction strategy was used to visualize fluorescent transconjugants. ► Plasmids did not spread beyond the first five recipient cell layers adjacent to the donor cells. ► Derepressed IncF plasmid R1drd19 reduces swarming ability.

Effect of oxygen and growth medium on in vitro biofilm formation by Escherichia coli

The effects of oxygen availability on in vitro biofilm formation by an Escherichia coli K-12 strain and 13 clinical E. coli strains were compared. All E. coli strains were capable of forming monospecies biofilm on polystyrene in aerobic media. The K-12 strain produced biofilm in both aerobic glucose minimal medium (ABTG), and aerobic trypticase soy broth (TSB) whereas the majority of the clinical strains produced significant biofilm only in aerobic TSB (9 of 13). In anaerobic media, E. coli K-12 and 9 of the 13 clinical strains were capable of forming biofilm in vitro . Only three clinical strains formed biofilm in anaerobic TSB whereas six clinical strains produced detectable biofilm in anaerobic ABTG. None of the strains tested were capable of forming biofilm in both anaerobic ABTG and anaerobic TSB. Strains that were good biofilm formers in aerobic ABTG also produced the highest amount of biofilm in anaerobic ABTG ( R 2 = 0.90). Image analysis revealed notable differences in architecture for biofilms grown in the presence and in the absence of oxygen. In aerobic ABTG, the biofilm was dominated by tall, mushroom-shaped microcolonies with pores and channels whereas biofilm in anaerobic ABTG was thinner and less heterogeneous, resulting in reduced maximum thickness and biovolume. Analysis of phospholipid fatty acid (PLFA) profiles from E. coli K-12 and three clinical strains did not reveal a specific pattern associated with the biofilm phenotypes. Interestingly, the clinical E. coli strains adjusted their PLFA composition much more than did E. coli K-12 in response to changes in growth regimens. Collectively, the results indicate that oxygen availability may affect E. coli biofilm formation in minimal and complex media. The results confirm that E. coli K-12 and some clinical E. coli strains are capable of forming in vitro biofilm under anaerobic conditions. However, the data also suggest that this attribute is highly strain dependent and may vary significantly among clinical isolates.

Common Requirement for the Relaxosome of Plasmid R1 in Multiple Activities of the Conjugative Type IV Secretion System

Macromolecular transport by bacterial type IV secretion systems involves regulated uptake of (nucleo)protein complexes by the cell envelope-spanning transport channel. A coupling protein receptor is believed to recognize the specific proteins destined for transfer, but the steps initiating their translocation remain unknown. Here, we investigate the contribution of a complex of transfer initiation proteins, the relaxosome, of plasmid R1 to translocation of competing transferable substrates from mobilizable plasmids ColE1 and CloDF13 or the bacteriophage R17. We found that not only does the R1 translocation machinery engage the R1 relaxosome during conjugative self-transfer and during infection by R17 phage but it is also activated by its cognate relaxosome to mediate the export of an alternative plasmid. Transporter activity was optimized by the R1 relaxosome even when this complex itself could not be transferred, i.e., when the N-terminal activation domain (amino acids 1 to 992 [N1-992]) of TraI was present without the C-terminal conjugative helicase domain. We propose that the functional dependence of the transfer machinery on the R1 relaxosome for initiating translocation ensures that dissemination of heterologous plasmids does not occur at the expense of self-transfer.

Cloning, expression and characterization of a new 2-Cl-propionic acid ester hydrolase from B. subtilis

Abstract Applying an agar plate assay, a novel gene encoding an esterase from Bacillus subtilis Est4B was cloned and highly overexpressed in E. coli . The protein sequence revealed high homology to the srf D gene, which encodes the fourth open reading frame, a putative thioesterase gene from the surfactin synthetase gene cluster. It was almost identical to an unpublished sequence from a putative surfactin synthetase cluster from B. subtilis A13. The enzyme (Est4B1) was produced in E. coli , purified to homogeneity and crystallized. Computational prediction of the protein fold showed high structural similarity of Est4B1 to haloperoxidases and dehalogenases and much lower similarity to lipases and esterases. However, by primary sequence analysis we found a typical esterase/thioesterase/lipase motif. Biocatalytic activity on several model esterase substrates was detected and quantified. This is the first time enzymatic activity could be shown for this type of independent putative thioesterases and this enzyme may serve as a model protein to solve the structure of this type of hydrolytic enzymes, which is commonly found in gene clusters of non-ribosomal peptide synthetases.