Histophilus somni biofilm formation in cardiopulmonary tissue of the bovine host following respiratory challenge

Biofilms form in a variety of host sites following infection with many bacterial species. However, the study of biofilms in a host is hindered due to the lack of protocols for the proper experimental investigation of biofilms in vivo. Histophilus somni is an agent of respiratory and systemic diseases in bovines, and readily forms biofilms in vitro. In the present study the capability of H. somni to form biofilms in cardiopulmonary tissue following experimental respiratory infection in the bovine host was examined by light microscopy, transmission electron microscopy, immunoelectron microscopy of ultrathin cryosections, scanning electron microscopy of freeze-fractured samples, and fluorescent in situ hybridization. Biofilms were evident and most prominent in the myocardium, and were associated with a large amount of amorphous extracellular material. Furthermore, Pasteurella multocida was often cultured with H. somni from heart and lung samples. Transposon mutagenesis of H. somni strain 2336 resulted in the generation of mutants that expressed more or less biofilm than the parent strain. Six mutants deficient in biofilm formation had an insertion in the gene encoding for a homolog of filamentous haemagglutinin (FHA), predicted to be involved in attachment. Thus, this investigation demonstrated that H. somni is capable of forming a biofilm in its natural host, that such a biofilm may be capable of harboring other bovine respiratory disease pathogens, and that the genes responsible for biofilm formation can be identified by transposon mutagenesis.     

Complete genome sequence of Haemophilus somnus (Histophilus somni) strain 129Pt and comparison to Haemophilus ducreyi 35000HP and Haemophilus influenzae Rd

Haemophilus somnus can be either a commensal of bovine mucosal surfaces or an opportunistic pathogen. Pathogenic strains of H. somnus are a significant cause of systemic disease in cattle. We report the genome sequence of H. somnus 129Pt, a nonpathogenic commensal preputial isolate, and the results of a genome-wide comparative analysis of H. somnus 129Pt, Haemophilus influenzae Rd, and Haemophilus ducreyi 35000HP. We found unique genes in H. somnus 129Pt involved in lipooligosaccharide biosynthesis, carbohydrate uptake and metabolism, cation transport, amino acid metabolism, ubiquinone and menaquinone biosynthesis, cell surface adhesion, biosynthesis of cofactors, energy metabolism, and electron transport. There were also many genes in common among the three organisms. Our comparative analyses of H. somnus 129Pt, H. influenzae Rd, and H. ducreyi 35000HP revealed similarities and differences in the numbers and compositions of genes involved in metabolism, host colonization, and persistence. These results lay a foundation for research on the host specificities and niche preferences of these organisms. Future comparisons between H. somnus 129Pt and virulent strains will aid in the development of protective strategies and vaccines to protect cattle against H. somnus disease.     

Histophilus somni Stimulates Expression of Antiviral Proteins and Inhibits BRSV Replication in Bovine Respiratory Epithelial Cells

Our previous studies showed that bovine respiratory syncytial virus (BRSV) followed by Histophilus somni causes more severe bovine respiratory disease and a more permeable alveolar barrier in vitro than either agent alone. However, microarray analysis revealed the treatment of bovine alveolar type 2 (BAT2) epithelial cells with H. somni concentrated culture supernatant (CCS) stimulated up-regulation of four antiviral protein genes as compared with BRSV infection or dual treatment. This suggested that inhibition of viral infection, rather than synergy, may occur if the bacterial infection occurred before the viral infection. Viperin (or radical S-adenosyl methionine domain containing 2—RSAD2) and ISG15 (IFN-stimulated gene 15—ubiquitin-like modifier) were most up-regulated. CCS dose and time course for up-regulation of viperin protein levels were determined in treated bovine turbinate (BT) upper respiratory cells and BAT2 lower respiratory cells by Western blotting. Treatment of BAT2 cells with H. somni culture supernatant before BRSV infection dramatically reduced viral replication as determined by qRT PCR, supporting the hypothesis that the bacterial infection may inhibit viral infection. Studies of the role of the two known H. somni cytotoxins showed that viperin protein expression was induced by endotoxin (lipooligosaccharide) but not by IbpA, which mediates alveolar permeability and H. somni invasion. A naturally occurring IbpA negative asymptomatic carrier strain of H. somni (129Pt) does not cause BAT2 cell retraction or permeability of alveolar cell monolayers, so lacks virulence in vitro. To investigate initial steps of pathogenesis, we showed that strain 129Pt attached to BT cells and induced a strong viperin response in vitro. Thus colonization of the bovine upper respiratory tract with an asymptomatic carrier strain lacking virulence may decrease viral infection and the subsequent enhancement of bacterial respiratory infection in vivo.     

Low metabolic activity of biofilm formed by Enterococcus faecalis isolated from healthy humans and wild mallards (Anas platyrhynchos)

It is widely known that Enterococcus faecalis virulence is related to its biofilm formation. Although Enterococci are common commensal organisms of the gastrointestinal tract, the difference between commensal and pathogen strains remain unclear. In this study, we compare the biochemical profile of the biofilms formed by two groups of medical and two groups of commensal strains. The medical strains were isolated as pathogens from infections of urinary tract and other infections (wounds, pus and bedsores), and the commensal strains were taken from faeces of healthy volunteers and faeces of wild mallards (Anas platyrhynchos) living in an urban environment. The properties of biofilms formed by medical and commensal strains differed significantly. Commensal strains showed lower metabolic activity and glucose uptake and higher biofilm biomass than the medical ones. Consistent with glucose uptake experiments, we found that the glucose dehydrogenase gene was more expressed in medical strains. These results indicate that higher metabolic activity and lower protein concentration of E. faecalis cells within biofilms are formed during infections.     

Structural analysis of the lipooligosaccharide-derived oligosaccharide of Histophilus somni (Haemophilus somnus) strain 8025

Previous structural studies in our laboratory on lipooligosaccharide (LOS) inner core oligosaccharide (OS) had identified structures from several strains of Histophilus (Haemophilus) somni (738, 2336, 1P, 129Pt). Recently a type strain 8025 was proposed for this species and we therefore sought to determine the core OS structure of this H. somni strain. Core OS was isolated by standard methods from Westphal purified LOS. Structural information was established by a combination of monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structure for the core OS was determined on the basis of the combined data from these experiments: [carbohydrates: see text]. The structure determined contains aspects of other Histophilus somni core OS structures, such as the beta-Gal attached at the 2-position of Hep II (2336), PEtn only at the 6-position of Hep II (738, 129Pt) and a lactose extension from Hep I (1P). Since genetic manipulation has been achieved with this strain, the identification of the core OS structure will enable experiments designed to identify the role of glycosyltransferases involved in LOS biosynthesis.     

High prevalence of biofilm synergy among bacterial soil isolates in cocultures indicates bacterial interspecific cooperation

Biofilms that form on roots, litter and soil particles typically contain multiple bacterial species. Currently, little is known about multispecies biofilm interactions and few studies have been based on environmental isolates. Here, the prevalence of synergistic effects in biofilm formation among seven different soil isolates, cocultured in combinations of four species, was investigated. We observed greater biofilm biomass production in 63% of the four-species culture combinations tested than in biofilm formed by single-species cultures, demonstrating a high prevalence of synergism in multispecies biofilm formation. One four-species consortium, composed of Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus, exhibited strong synergy in biofilm formation and was selected for further study. Of the four strains, X. retroflexus was the only one capable of forming abundant biofilm in isolation, under the in vitro conditions investigated. In accordance, strain-specific quantitative PCR revealed that X. retroflexus was predominant within the four-species consortium (>97% of total biofilm cell number). Despite low relative abundance of all the remaining strains, all were indispensable for the strong synergistic effect to occur within the four-species biofilm. Moreover, absolute individual strain cell numbers were significantly enhanced when compared with those of single-species biofilms, indicating that all the individual strains benefit from inclusion in the multispecies community. Our results show a high prevalence of synergy in biofilm formation in multispecies consortia isolated from a natural bacterial habitat and suggest that interspecific cooperation occurs.     

Antimicrobial activity of tigecycline alone or in combination with rifampin against Staphylococcus epidermidis in biofilm

Staphylococcus epidermidis is a commensal inhabitant of the healthy human skin, but in the recent years, it has been recognized as a nosocomial pathogen especially in immunocompromised patients. The pathogenesis of S. epidermidis is thought to be based on its capacity to form biofilms on the surface of medical devices, where bacterial cells may persist, protected from host defence and antimicrobial agents. Rifampin has been shown to be one of the most active antimicrobial agents in the eradication of the staphylococcal biofilm. However, this antibiotic should not be used in monotherapy. Therefore, one of the objectives of our research was to study the efficacy of the tigecycline/rifampin combination against methicillin-resistant S. epidermidis embedded in biofilms. Of the 80 clinically significant S. epidermidis isolates, 75 strains possess the ability to form a biofilm. These bacteria formed the biofilm via ica-dependent mechanisms. However, other biofilm-associated genes, including aap (encoding accumulation-associated protein) and bhp (coding cell wall-associated protein), were present in 85 and 29 % of isolates, respectively. The biofilm structures of S. epidermidis strains were also analyzed in confocal laser scanning microscopy (CLSM) and the obtained image demonstrated differences in their architecture. In vitro studies showed that the MIC value for tigecycline against S. epidermidis growing in the biofilm ranged from 0.125 to 2 μg/mL. Tigecycline in combination with rifampin demonstrated higher activity against bacteria embedded in biofilms than tigecycline alone.     

A new biofilm-associated colicin with increased efficiency against biofilm bacteria

Formation of bacterial biofilm communities leads to profound physiological modifications and increased physical and metabolic exchanges between bacteria. It was previously shown that bioactive molecules produced within the biofilm environment contribute to bacterial interactions. Here we describe new pore-forming colicin R, specifically produced in biofilms formed by the natural isolate Escherichia coli ROAR029 but that cannot be detected under planktonic culture conditions. We demonstrate that an increased SOS stress response within mature biofilms induces SOS-dependent colicin R expression. We provide evidence that colicin R displays increased activity against E. coli strains that have a reduced lipopolysaccharide length, such as the pathogenic enteroaggregative E. coli LF82 clinical isolate, therefore pointing to lipopolysaccharide size as an important determinant for resistance to colicins. We show that colicin R toxicity toward E. coli LF82 is increased under biofilm conditions compared with planktonic susceptibility and that release of colicin R confers a strong competitive advantage in mixed biofilms by rapidly outcompeting sensitive neighboring bacteria. This work identifies the first biofilm-associated colicin that preferentially targets biofilm bacteria. Furthermore, it indicates that the study of antagonistic molecules produced in biofilm and multispecies contexts could reveal unsuspected, ecologically relevant bacterial interactions influencing population dynamics in natural environments.     

Biofilm production, a marker of pathogenic potential of colonizing and commensal staphylococci

Biofilm is one of the known virulence factors of staphylococci, a human and animal pathogen and commensal. Some of the strains become invasive under favorable conditions while others do not cause disease. Early detection and management of potentially pathogenic staphylococci is the essential step to prevent device-associated infections. There is also a need to evaluate one simple method for the detection of potential pathogens. Hence this study was planned to study the difference in potential of commensal, colonizing and invasive strains of staphylococci to produce biofilm. We used one qualitative (Congo red agar) and one quantitative (microtiter plate) method for detection of biofilm production and evaluated the sensitivity and specificity of Congo red agar method by using microtiter plate method as a gold standard. We consecutively enrolled staphylococcal strains isolated from peripheral intravenous device (IVD), venous blood, site of IVD insertion and nasal mucosa of patients admitted to pediatric ward with peripheral intravenous devices in place for more than 48 h. Total 100 invasive, 50 colonizing and 50 commensal isolates were studied. Of 100 invasive isolates 74% (74/100) were biofilm positive while only 68% (34/50) colonizing and 32% (16/50) commensal isolates were biofilm positive. The difference in biofilm production by commensal, colonizing and invasive strains was statistically significant (p<0.0001). Sensitivity and specificity of Congo red agar test for detection of biofilm producers were 90.63% and 90.79% for Staphylococcus aureus and 75.86% and 96.88% respectively for coagulase negative staphylococci. CRA is a method that could be used to determine whether an isolate has the potential for biofilm production or not.     

Construction of a high-efficiency shuttle vector for Histophilus somni

The genetic manipulation of Histophilus somni is limited due to its high-fidelity restriction-modification system. The broad host-range shuttle plasmid pLS88 is capable of transforming some strains of H. somni, but is an inefficient vector. We have constructed an improved version of pLS88, pNS3K, that transforms H. somni strain 2336 100-fold more efficiently than its predecessor. The transformation efficiency was further increased when pNS3K was isolated from H. somni and retransformed into the same strain. As proof of principle, the lipooligosaccharide biosynthesis gene lob-2A was cloned into pNS3K and expressed in H. somni strain 129Pt, which lacks this gene. Thus, pNS3K is a useful shuttle vector for H. somni and a potential vector for genetic manipulation of this bacterium.     

Strain-specific colonization patterns and serum modulation of multi-species oral biofilm development

Periodontitis results from an ecological shift in the composition of subgingival biofilms. Subgingival community maturation is modulated by inter-organismal interactions and the relationship of communities with the host. In an effort to better understand this process, we evaluated biofilm formation, with oral commensal species, by three strains of the subgingivally prevalent microorganism Fusobacterium nucleatum and four strains of the periodontopathogen Porphyromonas gingivalis. We also tested the effect of serum, which resembles gingival exudates, on subgingival biofilms. Biofilms were allowed to develop in flow cells using salivary medium. We found that although not all strains of F. nucleatum were able to grow in mono-species biofilms, forming a community with health-associated partners Actinomyces oris and Veillonella parvula promoted biofilm growth of all F. nucleatum strains. Strains of P. gingivalis also showed variable ability to form mono-species biofilms. P. gingivalis W50 and W83 did not form biofilms, while ATCC 33277 and 381 formed biofilm structures, but only strain ATCC 33277 grew over time. Unlike the enhanced growth of F. nucleatum with the two health-associated species, no strain of P. gingivalis grew in three-species communities with A. oris and V. parvula. However, addition of F. nucleatum facilitated growth of P. gingivalis ATCC 33277 with health-associated partners. Importantly, serum negatively affected the adhesion of F. nucleatum, while it favored biofilm growth by P. gingivalis. This work highlights strain specificity in subgingival biofilm formation. Environmental factors such as serum alter the colonization patterns of oral microorganisms and could impact subgingival biofilms by selectively promoting pathogenic species.     

Biofilm formation on human airway epithelia by encapsulated Neisseria meningitidis serogroup B

Neisseria meningitidis is the etiologic agent of meningococcal meningitis. We compared 48-h biofilm formation by N. meningitidis serogroup B strains NMB, MC58, C311 and isogenic mutants defective in capsule formation on SV-40 transformed human bronchial epithelial (HBE) cells in a flow cell. We demonstrated that strains NMB and NMB siaA-D were defective in biofilm formation over glass, and there was a partial rescue of biofilm growth for strain NMB on collagen-coated coverslips at 48 h. We demonstrated all three serogroup B strains form biofilms of statistically equivalent average height on HBE cells as their isogenic capsular mutants. Strain NMB also formed a biofilm of statistically equivalent biomass as the NMB siaA-D mutant on HBE cells at 6 and 48 h. These biofilms are significantly larger than biofilms formed over glass or collagen. Verification that strain NMB expressed capsule in biofilms on HBE cells was demonstrated by staining with 2.2.B, a monoclonal antibody with specificity for the serogroup B capsule. ELISA analysis demonstrated that strains MC58 and C311 also produced capsules during biofilm growth. These findings suggest that encapsulated meningococci can form biofilms on epithelial cells suggesting that biofilm formation may play a role in nasopharyngeal colonization.     

Commensal Interactions in a Dual-Species Biofilm Exposed to Mixed Organic Compounds

There is limited knowledge of interspecies interactions in biofilm communities. In this study, Pseudomonas sp. strain GJ1, a 2-chloroethanol (2-CE)-degrading organism, and Pseudomonas putida DMP1, a p-cresol-degrading organism, produced distinct biofilms in response to model mixed waste streams composed of 2-CE and various p-cresol concentrations. The two organisms maintained a commensal relationship, with DMP1 mitigating the inhibitory effects of p-cresol on GJ1. A triple-labeling technique compatible with confocal microscopy was used to investigate the influence of toxicant concentrations on biofilm morphology, species distribution, and exopolysaccharide production. Single-species biofilms of GJ1 shifted from loosely associated cell clusters connected by exopolysaccharide to densely packed structures as the p-cresol concentrations increased, and biofilm formation was severely inhibited at high p-cresol concentrations. In contrast, GJ1 was abundant when associated with DMP1 in a dual-species biofilm at all p-cresol concentrations, although at high p-cresol concentrations it was present only in regions of the biofilm where it was surrounded by DMP1. Evidence in support of a commensal relationship between DMP1 and GJ1 was obtained by comparing GJ1-DMP1 biofilms with dual-species biofilms containing GJ1 and Escherichia coli ATCC 33456, an adhesive strain that does not mineralize p-cresol. Additionally, the data indicated that only tower-like cell structures in the GJ1-DMP1 biofilm produced exopolysaccharide, in contrast to the uniform distribution of EPS in the single-species GJ1 biofilm.     

A Novel Protein-Deamidating Enzyme from Chryseobacterium proteolyticum sp. nov., a Newly Isolated Bacterium from Soil

A novel protein-deamidating enzyme, which has potential for industrial applications, was purified from the culture supernatant of Chryseobacterium proteolyticum strain 9670^T isolated from rice field soil in Tsukuba, Japan. The deamidating activities on carboxybenzoxy (Cbz)-Gln-Gly and caseins and protease activity were produced synchronously by the isolate. Both deamidating activities were eluted as identical peaks separated from several proteases by phenyl-Sepharose chromatography of the culture supernatant. The enzyme catalyzed the deamidation of native caseins with no protease and transglutaminase activities. Phenotypic characterization and DNA analyses of the isolate were performed to determine its taxonomy. Physiological and biochemical characteristics, 16S rRNA gene sequence analysis, and DNA-DNA relatedness data indicated that the isolate should be placed as a new species belonging to the genus Chryseobacterium. The isolate showed no growth on MacConkey agar and produced acid from sucrose. The levels of DNA-DNA relatedness between the isolate and other related strains were less than 17%. The name Chryseobacterium proteolyticum is proposed for the new species; strain 9670 is the type strain (=FERM P-17664).     

Comparative analyses of two cryptic plasmids from Haemophilus somnus (Histophilus somni)

Haemophilus somnus is an opportunistic bacterial pathogen capable of causing pneumonia, septicemia, and other systemic infections in bovines. An H. somnus isolate from bovine abortion (strain 649) was found to carry a approximately 1.3 kb plasmid (pHS649) that contained partial homology to two previously sequenced Haemophilus/Histophilus plasmids by BLAST analyses. Sequence analysis of pHS649 identified a putative RepA protein with 48% similarity to the RepA protein of Escherichia coli plasmid pKL1. A approximately 5 kb plasmid (pHS129) from H. somnus preputial isolate 129Pt was also sequenced and found to encode two copies of a putative RepB protein. Whereas pHS649 stably replicated in E. coli DH5alpha, pHS129 did not. Genetic relatedness and possible replication mechanisms of these plasmids are described.     

Isolation and Characterization of Strains CVO and FWKO B, Two Novel Nitrate-Reducing, Sulfide-Oxidizing Bacteria Isolated from Oil Field Brine

Bacterial strains CVO and FWKO B were isolated from produced brine at the Coleville oil field in Saskatchewan, Canada. Both strains are obligate chemolithotrophs, with hydrogen, formate, and sulfide serving as the only known energy sources for FWKO B, whereas sulfide and elemental sulfur are the only known electron donors for CVO. Neither strain uses thiosulfate as an energy source. Both strains are microaerophiles (1% O₂). In addition, CVO grows by denitrification of nitrate or nitrite whereas FWKO B reduces nitrate only to nitrite. Elemental sulfur is the sole product of sulfide oxidation by FWKO B, while CVO produces either elemental sulfur or sulfate, depending on the initial concentration of sulfide. Both strains are capable of growth under strictly autotrophic conditions, but CVO uses acetate as well as CO₂ as its sole carbon source. Neither strain reduces sulfate; however, FWKO B reduces sulfur and displays chemolithoautotrophic growth in the presence of elemental sulfur, hydrogen, and CO₂. Both strains grow at temperatures between 5 and 40°C. CVO is capable of growth at NaCl concentrations as high as 7%. The present 16s rRNA analysis suggests that both strains are members of the epsilon subdivision of the division Proteobacteria, with CVO most closely related to Thiomicrospira denitrifcans and FWKO B most closely related to members of the genus Arcobacter. The isolation of these two novel chemolithotrophic sulfur bacteria from oil field brine suggests the presence of a subterranean sulfur cycle driven entirely by hydrogen, carbon dioxide, and nitrate.     

Pili contribute to biofilm formation in vitro in Mycobacterium tuberculosis

Organized bacterial communities, or biofilms, provide an important reservoir for persistent cells that are inaccessible or tolerant to antibiotics. Curli pili are cell-surface structures produced by certain bacteria and have been implicated in biofilm formation in these species. In order to determine whether these structures, which were suggested to be encoded by the Rv3312A (mtp) gene, have a similar role in Mycobacterium tuberculosis, we generated a Δmtp mutant and a mtp-complemented strain of a clinical isolate of M. tuberculosis and analyzed these strains for their ability to produce pili in comparison to the wild-type strain. Phenotypic analysis by transmission electron microscopy proved the essentiality of mtp for piliation in M. tuberculosis. We then compared biofilm formation of the derived strains in detergent-free Sauton’s media. Biofilm mass was quantified spectrophotometrically using crystal violet. Furthermore, we examined mtp gene expression by quantitative real-time PCR in wild-type cells grown under biofilm versus planktonic growth conditions. We found a 68.4 % reduction in biofilm mass in the mutant compared to the wild-type strain (P = 0.002). Complementation of the mutant resulted in a restoration of the wild-type biofilm phenotype (P = 0.022). We, however, found no significant difference between mtp expression in cells of the biofilm to those growing planktonically. Our findings highlight a crucial, but non-specific, role of pili in the biofilm lifestyle of M. tuberculosis and indicate that they may represent an important target for the development of therapeutics to attenuate biofilm formation, thereby potentially reducing persistence.     

Signaling-mediated cross-talk modulates swarming and biofilm formation in a coral pathogen Serratia marcescens

Interactions within microbial communities associated with marine holobionts contribute importantly to the health of these symbiotic organisms formed by invertebrates, dinoflagellates and bacteria. However, mechanisms that control invertebrate-associated microbiota are not yet fully understood. Hydrophobic compounds that were isolated from surfaces of asymptomatic corals inhibited biofilm formation by the white pox pathogen Serratia marcescens PDL100, indicating that signals capable of affecting the associated microbiota are produced in situ. However, neither the origin nor structures of these signals are currently known. A functional survey of bacteria recovered from coral mucus and from cultures of the dinoflagellate Symbiodinium spp. revealed that they could alter swarming and biofilm formation in S. marcescens. As swarming and biofilm formation are inversely regulated, the ability of some native α-proteobacteria to affect both behaviors suggests that the α-proteobacterial signal(s) target a global regulatory switch controlling the behaviors in the pathogen. Isolates of Marinobacter sp. inhibited both biofilm formation and swarming in S. marcescens PDL100, without affecting growth of the coral pathogen, indicative of the production of multiple inhibitors, likely targeting lower level regulatory genes or functions. A multi-species cocktail containing these strains inhibited progression of a disease caused by S. marcescens in a model polyp Aiptasia pallida. An α-proteobacterial isolate 44B9 had a similar effect. Even though ∼4% of native holobiont-associated bacteria produced compounds capable of triggering responses in well-characterized N-acyl homoserine lactone (AHL) biosensors, there was no strong correlation between the production of AHL-like signals and disruption of biofilms or swarming in S. marcescens.     

The Enterococcal Surface Protein, Esp, Is Involved in Enterococcus faecalis Biofilm Formation

The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalis isolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on the E. faecalis isolate, insertional mutagenesis of esp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboring esp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.