Biofilm formation - what we can learn from recent developments.

Although biofilms have been observed early in the history of microbial research, their impact has only recently been fully recognized. Biofilm infections, which contribute to up to 80% of human microbial infections, are associated with common human disorders, such as diabetes mellitus and poor dental hygiene, but also with medical implants. The associated chronic infections such as wound infections, dental caries and periodontitis significantly enhance morbidity, affect quality of life and can aid development of follow-up diseases such as cancer. Biofilm infections remain challenging to treat and antibiotic monotherapy is often insufficient, although some rediscovered traditional compounds have shown surprising efficiency. Innovative anti-biofilm strategies include application of anti-biofilm small molecules, intrinsic or external stimulation of production of reactive molecules, utilization of materials with antimicrobial properties and dispersion of biofilms by digestion of the extracellular matrix, also in combination with physical biofilm breakdown. Although basic principles of biofilm formation have been deciphered, the molecular understanding of the formation and structural organization of various types of biofilms has just begun to emerge. Basic studies of biofilm physiology have also resulted in an unexpected discovery of cyclic dinucleotide second messengers that are involved in interkingdom crosstalk via specific mammalian receptors. These findings even open up new venues for exploring novel anti-biofilm strategies.

First clinical isolation report of Shewanella algae from the stools of a patient with acute enteritis in Spain.

We report a case of acute enteritis caused by Shewanella algae in a cirrhotic patient. Biochemical identification systems revealed to be insufficient to identify the Shewanella isolate at the species level, thus requiring 16S rRNA and gyrB partial gene sequencing. Even if co-infection by Clostridium difficile could not be ruled out, this is, to our knowledge, the first report of acute enteritis caused by Shewanella algae in Europe.

Amyloid formation: functional friend or fearful foe?

Amyloid formation has been most studied in the context of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease, as well as in amyloidosis. However, it is becoming increasingly clear that amyloid is also present in the healthy setting; for example nontoxic amyloid formation is important for melanin synthesis and in innate immunity. Furthermore, bacteria have mechanisms to produce functional amyloid structures with important roles in bacterial physiology and interaction with host cells. Here, we will discuss some novel aspects of fibril-forming proteins in humans and bacteria. First, the amyloid-forming properties of the antimicrobial peptide human defensin 6 (HD6) will be considered. Intriguingly, unlike other antimicrobial peptides, HD6 does not kill bacteria. However, recent data show that HD6 can form amyloid structures at the gut mucosa with strong affinity for bacterial surfaces. These so-called nanonets block bacterial invasion by entangling the bacteria in net-like structures. Next, the role of functional amyloid fibrils in human semen will be discussed. These fibrils were discovered through their property to enhance HIV infection but they may also have other yet unknown functions. Finally, the role of amyloid formation in bacteria will be reviewed. The recent finding that bacteria can make amyloid in a controlled fashion without toxic effects is of particular interest and may have implications for human disease. The role of amyloid in health and disease is beginning to be unravelled, and here, we will review some of the most recent findings in this exciting area.

Escherichia coli: an old friend with new tidings.

Escherichia coli is one of the most-studied microorganisms worldwide but its characteristics are continually changing. Extraintestinal E. coli infections, such as urinary tract infections and neonatal sepsis, represent a huge public health problem. They are caused mainly by specialized extraintestinal pathogenic E. coli (ExPEC) strains that can innocuously colonize human hosts but can also cause disease upon entering a normally sterile body site. The virulence capability of such strains is determined by a combination of distinctive accessory traits, called virulence factors, in conjunction with their distinctive phylogenetic background. It is conceivable that by developing interventions against the most successful ExPEC lineages or their key virulence/colonization factors the associated burden of disease and health care costs could foreseeably be reduced in the future. On the other hand, one important problem worldwide is the increase of antimicrobial resistance shown by bacteria. As underscored in the last WHO global report, within a wide range of infectious agents including E. coli, antimicrobial resistance has reached an extremely worrisome situation that 'threatens the achievements of modern medicine'. In the present review, an update of the knowledge about the pathogenicity, antimicrobial resistance and clinical aspects of this 'old friend' was presented.

Biofilm infections, their resilience to therapy and innovative treatment strategies.

Biofilm formation of microorganisms causes persistent tissue and foreign body infections resistant to treatment with antimicrobial agents. Up to 80% of human bacterial infections are biofilm associated; such infections are most frequently caused by Staphylococcus epidermidis, Pseudomonas aeruginosa, Staphylococcus aureus and Enterobacteria such as Escherichia coli. The accurate diagnosis of biofilm infections is often difficult, which prevents the appropriate choice of treatment. As biofilm infections significantly contribute to patient morbidity and substantial healthcare costs, novel strategies to treat these infections are urgently required. Nucleotide second messengers, c-di-GMP, (p)ppGpp and potentially c-di-AMP, are major regulators of biofilm formation and associated antibiotic tolerance. Consequently, different components of these signalling networks might be appropriate targets for antibiofilm therapy in combination with antibiotic treatment strategies. In addition, cyclic di-nucleotides are microbial-associated molecular patterns with an almost universal presence. Their conserved structures sensed by the eukaryotic host have a widespread effect on the immune system. Thus, cyclic di-nucleotides are also potential immunotherapeutic agents to treat antibiotic-resistant bacterial infections.

Human cathelicidin peptide LL37 inhibits both attachment capability and biofilm formation of Staphylococcus epidermidis.

AIMS: The aim of this work was to investigate the possible effect of human cathelicidin antimicrobial peptide LL37 on biofilm formation of Staphylococcus epidermidis, a major causative agent of indwelling device-related infections. METHODS AND RESULTS: We performed initial attachment assay and biofilm formation solid surface assay in microtitre plates, as well as growth experiment in liquid medium using laboratory strain Staph. epidermidis ATCC35984. We found that already a low concentration of the peptide LL37 (1 mg l(-1)) significantly decreased both the attachment of bacteria to the surface and also the biofilm mass. No growth inhibition was observed even at 16 mg l(-1) concentration of LL37, indicating a direct effect of the peptide on biofilm production. CONCLUSIONS: As biofilm protects bacteria during infections in humans and allows their survival in a hostile environment, inhibition of biofilm formation by LL37 may have a key role to prevent bacterial colonization on indwelling devices. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings suggest that this host defence factor can be a potential candidate in prevention and treatment strategies of Staph. epidermidis infections in humans.

Salicylic acid-based poly(anhydride esters) for control of biofilm formation in Salmonella enterica serovar Typhimurium.

AIMS: Bacterial biofilms generally are more resistant to stresses as compared with free planktonic cells. Therefore, the discovery of antimicrobial stress factors that have strong inhibitory effects on bacterial biofilm formation would have great impact on the food, personal care, and medical industries. METHODS AND RESULTS: Salicylate-based poly(anhydride esters) (PAE) have previously been shown to inhibit biofilm formation, possibly by affecting surface attachment. Our research evaluated the effect of salicylate-based PAE on biofilm-forming Salmonella enterica serovar Typhimurium. To remove factors associated with surface physical and chemical parameters, we utilized a strain that forms biofilms at the air-liquid interface. Surface properties can influence biofilm characteristics, so the lack of attachment to a solid surface eliminates those constraints. The results indicate that the salicylic acid-based polymers do interfere with biofilm formation, as a clear difference was seen between bacterial strains that form biofilms at the air-liquid interface (top-forming) and those that form at the surface-liquid interface (bottom-forming). CONCLUSION: These results lead to the conclusion that the polymers may not interfere with attachment; rather, the polymers likely affect another mechanism essential for biofilm formation in Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: Biofilm formation can be prevented through controlled release of nature-derived antimicrobials formulated into polymer systems.

Aeromonas-Acanthamoeba interaction and early shift to a viable but nonculturable state of Aeromonas by Acanthamoeba.

AIMS: To investigate the hypothesis that amoeba may comprise a significant environmental reservoir for Aeromonas, Acanthamoeba-Aeromonas interaction experiments were performed. METHODS AND RESULTS: Acanthamoeba were grown in monoculture and co-cultures with three different species of Aeromonas. Survival, invasion and viable but nonculturable state experiments were performed. We showed that at a low initial bacterial cell density, growth of Aeromonas spp. was inhibited by Acanthamoeba castellanii, while A. castellanii growth was unaffected. In contrast, a high initial bacterial cell density, Aeromonas hydrophila AEW44 and Aeromonas veronii biovar sobria AEW104 suppressed the growth of A. castellanii. Fluorescent and phase-contrast microscopic observations of GFP tagged Aer. hydrophila AEW44 demonstrated that the bacterial cells aggregated on A. castellanii cells after 15 min of incubation and internalized. Aeromonas hydrophila AEW44 cells were found to be actively moving. Interestingly, Aer. hydrophila AEW44 cells shifted more rapidly to a viable but nonculturable form when co-cultured with A. castellanii than in monoculture. CONCLUSIONS: We demonstrated that Aeromonas spp. are able to interact with and to infect the protozoan A. castellanii under laboratory conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Free-living amoeba might play a role as reservoir for Aeromonas, and thus may increase the transmission of Aeromonas by acting as a vehicle.

Impact of biofilm matrix components on interaction of commensal Escherichia coli with the gastrointestinal cell line HT-29.

Commensal Escherichia coli form biofilms at body temperature by expressing the extracellular matrix components curli fimbriae and cellulose. The role of curli fimbriae and cellulose in the interaction of commensal E. coli with the intestinal epithelial cell line HT-29 was investigated. Expression of curli fimbriae by the typical commensal isolate E. coli TOB1 caused adherence and internalization of the bacteria and triggered IL-8 production in HT-29 cells. In particular, induction of IL-8 production was complex and involved curli-bound flagellin. While cellulose alone had no effect on the interaction of TOB1 with HT-29 cells, co-expression of cellulose with curli fimbriae decreased adherence to, internalization and IL-8 induction of HT-29 cells. Investigation of a panel of commensal isolates showed a partial correlation between expression of curli fimbriae and enhanced internalization and IL-8 production. In addition, a high immunostimulatory flagellin was identified. Thus, the consequences of expression of extracellular matrix components on commensal bacterial-host interactions are complex.

Characterization of the rdar morphotype, a multicellular behaviour in Enterobacteriaceae.

The rdar morphotype, a multicellular behaviour of Salmonella enterica and Escherichia coli is characterized by the expression of the adhesive extracellular matrix components cellulose and curli fimbriae. The response regulator CsgD, which transcriptionally activates the biosynthesis of the exopolysaccharide cellulose and curli, also transforms cell physiology to the multicellular state. However, the only role of CsgD in cellulose biosynthesis is the activation of AdrA, a GGDEF domain protein that mediates production of the allosteric activator cyclic-di-(3'-5')guanylic acid (c-di-GMP). In S. enterica serovar Typhimurium a regulatory network consisting of 19 GGDEF/EAL domain-containing proteins tightly controls the concentration of c-di-GMP. c-di-GMP not only regulates the expression of cellulose, but also stimulates expression of adhesive curli and represses various modes of motility. Functions of characterized GGDEF and EAL domain proteins, as well as database searches, point to a global role for c-di-GMP as a novel secondary messenger that regulates a variety of cellular functions in response to diverse environmental stimuli already in the deepest roots of the prokaryotes.

European-wide distribution of Pseudomonas aeruginosa clone C.

A collection of 77 epidemiologically unrelated Pseudomonas aeruginosa isolates was screened for the occurrence of clone C isolates by the appearance of characteristic SpeI fragment patterns obtained by pulsed-field gel electrophoresis. Three strains with a clone C characteristic SpeI fragment pattern were found which also harbored the clone C-specific plasmid either in the free form or chromosomally integrated. Genomic islands were detected in the new clone C strains, as in already characterized clone C strains. Clone C not only infected cystic fibrosis patients throughout Europe, but was also found in the UK as an isolate in urinary tract infections and in peritoneal dialysis fluid, in addition to an otitis media isolate. Therefore, P. aeruginosa clone C is widely distributed in Europe, with a broad pathogenic potential.

Oxygen tension and nutrient starvation are major signals that regulate agfD promoter activity and expression of the multicellular morphotype in Salmonella typhimurium.

Expression of multicellular behaviour (rdar morphotype) is a characteristic of wild-type Salmonella typhimurium strains. The key target for the regulation of rdar morphotype expression is the agfD promoter. The regulation of two rdar morphotypes, regulated and semi-constitutive (the latter differs from the former by the insertion of A after position -17), by various environmental conditions was studied using transcriptional fusions to the regulated and semi-constitutive agfD promoters by Western blot analysis and phenotypic analysis of the rdar morphotype. AgfD promoter activities were strongly dependent on oxygen tension. Expression maxima were observed in rich medium under microaerophilic conditions and in minimal medium under aerobic conditions. The regulated rdar morphotype was only expressed under conditions of maximal promoter activity. Glucose did not influence rdar morphotype expression, and the two promoters showed no consistent response to pH. In the stationary phase of growth, nitrogen and phosphate depletion were found to be signals that switch on the agfD promoters. In the logarithmic phase of growth, ethanol was the stress signal that enhanced rdar morphotype expression. The results indicate that, although the regulated and semi-constitutive agfD promoters are key factors in the grade of expression of the multicellular behaviour, common signals such as oxygen tension, depletion of nutrients and ethanol vary their levels of expression significantly.

The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix.

Production of cellulose has been thought to be restricted to a few bacterial species such as the model organism Acetobacter xylinus. We show by enzymatic analysis and mass spectrometry that, besides thin aggregative fimbriae, the second component of the extracellular matrix of the multicellular morphotype (rdar) of Salmonella typhimurium and Escherichia coli is cellulose. The bcsA, bcsB, bcsZ and bcsC genes responsible for cellulose biosynthesis are not regulated by AgfD, the positive transcriptional regulator of the rdar morphotype. Transcription of the bcs genes was not co-expressed with the rdar morphotype under any of the environmental conditions examined. However, cellulose biosynthesis was turned on by the sole expression of adrA, a gene encoding a putative transmembrane protein regulated by agfD, indicating a novel pathway for the activation of cellulose synthesis. The co-expression of cellulose and thin aggregative fimbriae leads to the formation of a highly hydrophobic network with tightly packed cells aligned in parallel in a rigid matrix. As the production of cellulose would now appear to be a property widely distributed among bacteria, the function of the cellulose polymer in bacteria will have to be considered in a new light.

AgfD, the checkpoint of multicellular and aggregative behaviour in Salmonella typhimurium regulates at least two independent pathways.

The regulatory programme of multicellular behaviour in Salmonella typhimurium is determined by mutations in the agfD promoter. AgfD has already been identified to regulate the extracellular matrix associated with the multicellular morphotype composed of thin aggregative fimbriae (agf). To detect additional components contributing to the multicellular morphotype in S. typhimurium, we constructed a mutant in agfD, the positive transcriptional regulator of the agfBA(C) operon encoding for fimbrial subunit proteins. The agfD mutant lacked any form of multicellular behaviour as shown by analysis at the macroscopic and microscopic level. In contrast, the agfBA mutant unable to form thin aggregative fimbriae still maintained long-range intercellular adhesion. Promoter and expression analysis revealed that the genes downstream of agfD agfEFG most likely did not contribute to the remaining aggregative behaviour. Screening of transcriptional fusions for agfD dependency uncovered adrA, a homologue of yaiC in Escherichia coli. Environmental factors regulating adrA correspond to the regulation of thin aggregative fimbriae. AdrA is a putative transmembrane protein with a C-terminal GGDEF domain of unknown function although it is present in over 50 bacterial proteins. AdrA mutant cells, which still formed thin aggregative fimbriae with all binding characteristics, exhibited community behaviour but, unlike the wild type, lacked long-range intercellular adhesion. An agfBA adrA double mutant behaved like the agfD mutant. Therefore, it was concluded that agfD regulates at least two independent pathways contributing to the multicellular morphotype in S. typhimurium.

Flagella modulate the multicellular behavior of Salmonella typhimurium on the community level.

Salmonella typhimurium strains MAE52 and MAE32 display a constitutive multicellular morphotype mediated by the expression of the agfD operon. In those strains, the role of flagella in the formation of various modes of multicellular behavior was investigated. Flagella were not required for the formation of the multicellular morphotype (rdar) on plates. However, visual examination showed that the global behavior of the bacterial community on air-liquid, surface-liquid or cell-cell-liquid interfaces changed in the absence of flagella. No differences in the local cell-cell interactions were observed at the microscopic level. Using Western blot analysis, no co-regulation of flagella and thin aggregative fimbriae, an extracellular component of the multicellular morphotype, was observed either on plates or in standing culture. In a mutant lacking flagella and thin aggregative fimbriae, the contribution of the latter to the multicellular morphotype was dominant. We concluded that independently regulated genes can act in an additive fashion to confer a pronounced multicellular behavior.

Identification of a gene cluster, czr, involved in cadmium and zinc resistance in Pseudomonas aeruginosa.

Pseudomonas aeruginosa CMG103 was isolated from a metal-polluted river in Pakistan and displayed a high level of Zn and Cd resistance. An omega-Km transposon mutant of strain CMG103, which showed a substantial decrease in resistance to Zn and Cd, was obtained. A 12.8 kb region determining Zn and Cd resistance in strain CM103 was cloned by complementing the mutant strain, and its nt sequence was determined. Five genes, czrSRCBA, involved in Zn and Cd resistance, were identified. The predicted gene products of czrCBA show a significant similarity with the proteins encoded by the plasmid borne metal resistant determinants czc, cnr and ncc of Ralstonia strains, which determine a chemiosmotic cation-antiporter efflux system. The predicted CzrS and CzrR proteins show a significant similarity to the sensor and regulatory protein, respectively, of two component regulatory systems, such as CopS/CopR and PcoS/PcoR involved in the regulation of plasmid-borne Cu-resistant determinants, and CzcS/CzcR involved in the regulation of czc. The cloned czr region contained downstream of czrCBA additional ORFs whose predicted gene products are similar to proteins involved in catabolism of aromatic compounds. DNA-DNA hybridization indicated strong conservation of czr in other environmental P. aeruginosa isolates and in the P. aeruginosa type strain PAO1, a clinical isolate. This was confirmed by a comparison of the sequence of the CMG103 czr region with the currently available genome sequence of strain PAO1. A high sequence identity (till 99% at the nt level) and organizatory conservation of the czr region of CMG103 was found in PAO1 as well regarding coding sequences as intervening sequences between ORFs. The czr locus was localized between coordinates 2400 and 2550 kb on the physical map of the chromosome of PAO1.

Regulation of Pseudomonas aeruginosa hemF and hemN by the dual action of the redox response regulators Anr and Dnr.

The oxidative decarboxylation of coproporphyrinogen III catalysed by an oxygen-dependent oxidase (HemF) and an oxygen-independent dehydrogenase (HemN) is one of the key regulatory points of haem biosynthesis in Pseudomonas aeruginosa. To investigate the oxygen-dependent regulation of hemF and hemN, the corresponding genes were cloned from the P. aeruginosa chromosome. Recognition sequences for the Fnr-type transcriptional regulator Anr were detected -44.5 bp from the 5' end of the hemF mRNA transcript and at an optimal distance of -41.5 bp with respect to the transcriptional start of hemN. An approximately 10-fold anaerobic induction of hemN gene expression was mediated by the dual action of Anr and a second Fnr-type regulator, Dnr. Regulation by both proteins required the Anr recognition sequence. Surprisingly, aerobic expression of hemN was dependent only on Anr. An anr mutant did not contain detectable amounts of hemN mRNA and accumulated coproporphyrin III both aerobically and anaerobically, indicating the importance of HemN for aerobic and anaerobic haem formation. Mutation of hemN and hemF did not abolish aerobic or anaerobic growth, indicating the existence of an additional HemN-type enzyme, which was termed HemZ. Expression of hemF was induced approximately 20-fold during anaerobic growth and, as was found for hemN, both Anr and Dnr were required for anaerobic induction. Paradoxically, oxygen is necessary for HemF catalysis, suggesting the existence of an additional physiological function for the P. aeruginosa HemF protein.