You are here: Home Research Themes Biofilms


Article Reference Biological roles of nontypeable Haemophilus influenzae type IV pilus proteins encoded by the pil and com operons.
We previously demonstrated that one or more products of the genes in the pil and com gene clusters of the opportunistic human respiratory pathogen nontypeable Haemophilus influenzae (NTHI) are required for type IV pilus (Tfp) biogenesis and function. Here, we have now demonstrated that the pilABCD and comABCDEF gene clusters are operons and that the product of each gene is essential for normal pilus function. Mutants with nonpolar deletions in each of the 10 pil and com genes had an adherence defect when primary human airway cells were used as the target. These mutants were also diminished in their ability to form a biofilm in vitro and, additionally, were deficient in natural transformation. Collectively, our data demonstrate that the product of each gene within these operons is required for the normal biogenesis and/or function of NTHI Tfp. Based on the similarity of PilA to other type IV pilins, we further predicted that the product of the pilA gene would be the major pilin subunit. Toward that end, we also demonstrated by immunogold labeling and mass spectrometry that PilA is indeed the majority type IV pilin protein expressed by NTHI. These new observations set the stage for experiments designed to dissect the function of each of the proteins encoded by genes within the pil and com gene clusters. The ability to characterize individual proteins with vital roles in NTHI colonization or pathogenesis has the potential to reduce the burden of NTHI-induced diseases through development of a Tfp-derived vaccine or a pilus-directed therapeutic.
Article Reference Regulation of virulence gene expression resulting from Streptococcus pneumoniae and nontypeable Haemophilus influenzae interactions in chronic disease.
Chronic rhinosinusitis (CRS) is a common inflammatory disease of the sinonasal cavity mediated, in part, by polymicrobial communities of bacteria. Recent molecular studies have confirmed the importance of Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) in CRS. Here, we hypothesize that interaction between S. pneumoniae and NTHi mixed-species communities cause a change in bacterial virulence gene expression. We examined CRS as a model human disease to validate these polymicrobial interactions. Clinical strains of S. pneumoniae and NTHi were grown in mono- and co-culture in a standard biofilm assay. Reverse transcriptase real-time PCR (RTqPCR) was used to measure gene expression of key virulence factors. To validate these results, we investigated the presence of the bacterial RNA transcripts in excised human tissue from patients with CRS. Consequences of physical or chemical interactions between microbes were also investigated. Transcription of NTHi type IV pili was only expressed in co-culture in vitro, and expression could be detected ex vivo in diseased tissue. S. pneumoniae pyruvate oxidase was up-regulated in co-culture, while pneumolysin and pneumococcal adherence factor A were down-regulated. These results were confirmed in excised human CRS tissue. Gene expression was differentially regulated by physical contact and secreted factors. Overall, these data suggest that interactions between H. influenzae and S. pneumoniae involve physical and chemical mechanisms that influence virulence gene expression of mixed-species biofilm communities present in chronically diseased human tissue. These results extend previous studies of population-level virulence and provide novel insight into the importance of S. pneumoniae and NTHi in CRS.
Article Reference Haemophilus influenzae in children with cystic fibrosis: antimicrobial susceptibility, molecular epidemiology, distribution of adhesins and biofilm formation.
Haemophilus influenzae commonly infects the respiratory tract of patients with cystic fibrosis (CF), early in childhood. In this investigation, 79 H. influenzae isolates were recovered from the respiratory secretions of 64 CF patients (median age: 5 years) included in a 5-year follow-up study. Fifteen of the 64 patients contributed two or more H. influenzae isolates overtime. Serotyping, antibiotic susceptibility testing, genotyping, detection of both hmwA and hia adhesin genes and hypermutable strains was carried out. Biofilm formation ability was investigated. Most strains (72/79, 91.2%) were nonencapsulated or nontypeable (NTHi). Resistance to ampicillin (13.9%) and imipenem (17.7%) was the most detected. Few isolates (2.5%) exhibited the hypermutable phenotype. The NTHi strains showed 55 different genotypes, but 19 clusters of closely related strains were identified. Nine clusters included strains that cross-colonised several patients over a long-time period (mean: 3.7 years). Most patients with sequential isolates harboured strains genetically unrelated, but persistent colonisation with the same clone was observed in 37.5% of patients. Over 45% of NTHi strains contained hmwA-related sequences, 26.3%, hia, 8.3% both hmwA and hia, while 19.4% lacked both. A significant association was found between occurrence of an adhesive gene (irrespective of which) and both persistence (P<0.0001) and long-term cross-colonisation (P<0.0001). Mean biofilm level formed by the persistent strains was found significantly increased compared to non-persistent ones (P<0.0001). Hia-positive strains produced significantly more biofilm than hmwA-carrying strains (P<0.01). Although a high turnover of NTHi strains in FC patients was observed, distinct clones with increased capacity of persistence or cross-colonisation occurred.
Article Reference RbsB (NTHI_0632) mediates quorum signal uptake in nontypeable Haemophilus influenzae strain 86-028NP.
Nontypeable Haemophilus influenzae (NTHI) is a respiratory commensal and opportunistic pathogen, which persists within biofilms on airway mucosal surfaces. For many species, biofilm formation is impacted by quorum signalling. Our prior work shows that production of autoinducer-2 (AI-2) promotes biofilm development and persistence for NTHI 86-028NP. NTHI 86-028NP encodes an ABC transporter annotated as a ribose transport system that includes a protein (RbsB) with similarity to the Escherichia coli LsrB and Aggregatibacter actinomycetemcomitans RbsB proteins that bind AI-2. In this study, inactivation of rbsB significantly reduced uptake of AI-2 and the AI-2 precursor dihydroxypentanedione (DPD) by NTHI 86-028NP. Moreover, DPD uptake was not competitively inhibited by ribose or other pentose sugars. Transcript levels of rbsB increased in response to DPD and as bacteria approached stationary-phase growth. The NTHI 86-028NP rbsB mutant also formed biofilms with significantly reduced thickness and total biomass and reduced surface phosphorylcholine, similar to a luxS mutant. Infection studies revealed that loss of rbsB impaired bacterial persistence in the chinchilla middle ear, similar to our previous results with luxS mutants. Based on these data, we conclude that in NTHI 86-028NP, RbsB is a LuxS/AI-2 regulated protein that is required for uptake of and response to AI-2.
Article Reference Divergent mechanisms for passive pneumococcal resistance to β-lactam antibiotics in the presence of Haemophilus influenzae.
Otitis media, for which antibiotic treatment failure is increasingly common, is a leading pediatric public health problem.
Article Reference Mechanisms of bacterial resistance to antibiotics in infections of COPD patients.
A key characteristic of airway inflammation in chronic obstructive pulmonary disease (COPD) is the persistent presence of bacteria in the lower airways. The most commonly isolated bacteria in the lower respiratory tract of COPD patients are nontypeable Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae, with growing evidence of the significance of Pseudomonas aeruginosa infections in severe COPD disease. This review focuses on the antibiotic resistant mechanisms associated with the gram-negative bacteria H. influenzae and M. catarrhalis and comparison with P. aeruginosa infection because of the recent evidence of its significance in patients with severe COPD disease. These mechanisms of resistance to β-lactams in H. influenzae and M. catarrhalis are mostly associated with serine β-lactamases of class A type, whereas P. aeruginosa strains exhibit a much broader repertoire with class A-D type mechanisms. Other mechanisms of antibiotic resistance include membrane permeability, efflux pump systems and mutations in antimicrobial targets. Antimicrobial resistance within biofilm matrices appears to be different to the mechanisms observed when the bacteria are in the planktonic state. P. aeruginosa exhibits a more numerous and diverse range of antimicrobial resistance mechanisms in comparison to M. catarrhalis and H. influenzae. The recognition that P. aeruginosa is associated with exacerbations in patients with more severe COPD and that turnover in infecting strains is detected (unlike in cystic fibrosis patients), then further investigation is required to better understand the contribution of antimicrobial resistance and other virulence mechanisms to poor clinical outcomes to improve therapeutic approaches.
Article Reference Coinfection with Haemophilus influenzae promotes pneumococcal biofilm formation during experimental otitis media and impedes the progression of pneumococcal disease.
Otitis media is an extremely common pediatric infection and is mostly caused by bacteria that are carried within the nasopharyngeal microbiota. It is clear that most otitis media cases involve simultaneous infection with multiple agents.
Article Reference Microbial interactions in the respiratory tract.
Upper respiratory tract infections are caused by the synergistic and antagonistic interactions between upper respiratory tract viruses and 3 predominant bacterial pathogens: Streptococcus pneumoniae, nontypeable Haemophilus influenzae (NTHi), and Moraxella catarrhalis, which are members of the commensal flora of the nasopharynx. For many bacterial pathogens, colonization of host mucosal surfaces is a first and necessary step in the infectious process. S. pneumoniae and H. influenzae have intricate interactions in the nasopharynx. The host innate immune response may influence these interactions and therefore influence the composition of the colonizing flora and the invading bacteria. S. pneumoniae, nontypeable H. influenzae, and M. catarrhalis can behave as opportunistic pathogens of the middle ear when conditions are optimal. Chronic otitis media (OM) and recurrent OM include a biofilm component. Each of the 3 predominant pathogens of OM can form a biofilm and have been shown to comprise biofilms present on middle ear mucosa specimens recovered from children with recurrent or chronic OM. Some of these characterized biofilms are of mixed bacterial etiology, suggesting that progress made on single-microbe directed strategies for treatment and/or prevention of OM, although highly encouraging, are likely to be inadequate. A significantly greater understanding about microbial physiology is required as it relates to the involvement of biofilms in OM, to identify points in the natural course of the disease that are perhaps more amenable to treatment strategies, as well as to identify biofilm-relevant antigenic targets that would be helpful in the rational design of vaccines to prevent OM.
Article Reference Otitis media: viruses, bacteria, biofilms and vaccines.
Otitis media typically presents as either acute otitis media (AOM), with symptoms including fever, otalgia, otorrhoea or irritability and short duration; or as otitis media with effusion (OME), which is often asymptomatic and characterised by accumulation of fluid in the middle ear. Diagnostic certainty of otitis media is challenging, given the young age of patients and variability of symptoms. Otitis media predominantly occurs as coincident to viral upper respiratory tract infections and/or bacterial infections. Common viruses that cause upper respiratory tract infection are frequently associated with AOM and new-onset OME. These include respiratory syncytial virus, rhinovirus, adenovirus, parainfluenza and coronavirus. Predominant bacteria that cause otitis media are Streptococcus pneumoniae, Moraxella catarrhalis, and non-typeable Haemophilus influenzae. Antibiotic therapy does not significantly benefit most patients with AOM, but long-term prophylactic antibiotic therapy can reduce the risk of otitis media recurrence among children at high risk. In Australia, 84% of AOM is treated with antibiotic therapy, which contributes to development of antibiotic resistance. Vaccine development is a key future direction for reducing the world burden of otitis media, but requires polymicrobial formulation and ongoing monitoring and modification to ensure sustained reduction in disease burden.
Article Reference LuxS promotes biofilm maturation and persistence of nontypeable haemophilus influenzae in vivo via modulation of lipooligosaccharides on the bacterial surface.
Nontypeable Haemophilus influenzae (NTHI) is an extremely common airway commensal which can cause opportunistic infections that are usually localized to airway mucosal surfaces. During many of these infections, NTHI forms biofilm communities that promote persistence in vivo. For many bacterial species, density-dependent quorum-signaling networks can affect biofilm formation and/or maturation. Mutation of luxS, a determinant of the autoinducer 2 (AI-2) quorum signal pathway, increases NTHI virulence in the chinchilla model for otitis media infections. For example, bacterial counts in middle-ear fluids and the severity of the host inflammatory response were increased in luxS mutants compared with parental strains. As these phenotypes are consistent with those that we have observed for biofilm-defective NTHI mutants, we hypothesized that luxS may affect NTHI biofilms. A luxS mutant was generated using the well-characterized NTHI 86-028NP strain and tested to determine the effects of the mutation on biofilm phenotypes in vitro and bacterial persistence and disease severity during experimental otitis media. Quantitation of the biofilm structure by confocal microscopy and COMSTAT analysis revealed significantly reduced biomass for NTHI 86-028NP luxS biofilms, which was restored by a soluble mediator in NTHI 86-028NP supernatants. Analysis of lipooligosaccharide moieties using an enzyme-linked immunosorbent assay and immunoblotting showed decreased levels of biofilm-associated glycoforms in the NTHI 86-028NP luxS strain. Infection studies showed that NTHI 86-028NP luxS had a significant persistence defect in vivo during chronic otitis media infection. Based on these data, we concluded that a luxS-dependent soluble mediator modulates the composition of the NTHI lipooligosaccharides, resulting in effects on biofilm maturation and bacterial persistence in vivo.
Article Reference Intercellular adhesion and biocide resistance in nontypeable Haemophilus influenzae biofilms.
Respiratory infections caused by nontypeable Haemophilus influenzae (NTHi) are a major medical problem. Evidence suggests that the ability to form biofilms on mucosal surfaces may play a role in NTHi pathogenesis. However, the factors that contribute to NTHi biofilm cohesion remain largely unknown. In this study we investigated the biofilm growth and detachment phenotypes of eight NTHi clinical strains in vitro. We found that the majority of strains produced biofilms within 6h when cultured statically in tubes. Biofilm formation was inhibited when culture medium was supplemented with proteinase K or DNase I. Both enzymes also caused significant detachment of pre-formed NTHi biofilms. These findings indicate that both proteinaceous adhesins and extracellular DNA contribute to NTHi biofilm cohesion. Treatment of NTHi biofilms cultured in centrifugal filter devices with DNase I, but not with proteinase K, caused a significant decrease in fluid convection through the biofilms. These results suggest that extracellular DNA is the major volumetric component of the NTHi biofilm matrix. Mechanical or enzymatic disruption of NTHi biofilms cultured in microtiter plates significantly increased their sensitivity to killing by SDS, cetylpyridinium chloride, chlorhexidine gluconate, povidone iodine and sodium hypochlorite. These findings indicate that biocide resistance in NTHi biofilms is mediated to a large part by the cohesive and protective properties of the biofilm matrix. Understanding the mechanisms of biofilm cohesion and biocide resistance in NTHi biofilms may lead to new methods for treating NTHi-associated infections.
Article Reference Nanoscale structural and mechanical properties of nontypeable Haemophilus influenzae biofilms.
Nontypeable Haemophilus influenzae (NTHI) bacteria are commensals in the human nasopharynx, as well as pathogens associated with a spectrum of acute and chronic infections. Two important factors that influence NTHI pathogenicity are their ability to adhere to human tissue and their ability to form biofilms. Extracellular polymeric substances (EPS) and bacterial appendages such as pili critically influence cell adhesion and intercellular cohesion during biofilm formation. Structural components in the outer cell membrane, such as lipopolysaccharides, also play a fundamental role in infection of the host organism. In spite of their importance, these pathogenic factors are not yet well characterized at the nanoscale. Here, atomic force microscopy (AFM) was used in aqueous environments to visualize structural details, including probable Hif-type pili, of live NTHI bacteria at the early stages of biofilm formation. Using single-molecule AFM-based spectroscopy, the molecular elasticities of lipooligosaccharides present on NTHI cell surfaces were analyzed and compared between two strains (PittEE and PittGG) with very different pathogenicity profiles. Furthermore, the stiffness of single cells of both strains was measured and subsequently their turgor pressure was estimated.
Article Reference Formation of biofilm by Haemophilus influenzae isolated from pediatric intractable otitis media.
The aims of this study are to evaluate biofilm formation by nontypeable Haemophilus influenzae (NTHi) isolated from children with acute otitis media (AOM) and its relation with clinical outcome of the disease.
Article Reference Survival of bacterial biofilms within neutrophil extracellular traps promotes nontypeable Haemophilus influenzae persistence in the chinchilla model for otitis media.
Nontypeable Haemophilus influenzae (NTHi) is a leading cause of acute and chronic otitis media, which are a major public health problem worldwide. The persistence of NTHi during chronic and recurrent otitis media infections involves multicellular biofilm communities formed within the middle-ear chamber. Bacterial biofilms resist immune clearance and antibiotic therapy due in part to encasement within a polymeric matrix. In this study, the contribution of biofilms to bacterial persistence in vivo and composition of the NTHi biofilm matrix during experimental otitis media were investigated. The presence of biofilms within the chinchilla middle-ear chamber was significantly correlated with increased bacterial load in middle-ear effusions and tissue. Examination of thin sections revealed polymorphonuclear cells within a DNA lattice containing elastase and histones, which is consistent with the definition of neutrophil extracellular traps. Viable multicellular biofilm communities with biofilm phenotypes were found within the DNA lattice throughout the biofilm. Further, NTHi was resistant to both phagocytic and extracellular neutrophil killing in vitro by means of lipooligosaccharide moieties that promote biofilm formation. These data support the conclusion that NTHi subverts neutrophil extracellular traps to persist in vivo. These data also indicate that a more inclusive definition for biofilms may be warranted.
Article Reference Diminished ICAM-1 expression and impaired pulmonary clearance of nontypeable Haemophilus influenzae in a mouse model of chronic obstructive pulmonary disease/emphysema.
The airways of patients with chronic obstructive pulmonary disease (COPD) are continually colonized with bacterial opportunists like nontypeable Haemophilus influenzae (NTHi), and a wealth of evidence indicates that changes in bacterial populations within the lung can influence the severity of COPD. In this study, we used a murine model for COPD/emphysema to test the hypothesis that COPD affects pulmonary clearance. Mice were treated with a pulmonary bolus of elastase, and as reported previously, the lungs of these mice were pathologically similar to those with COPD/emphysema at approximately 1 month posttreatment. Pulmonary clearance of NTHi was significantly impaired in elastase-treated versus mock-treated mice. While histopathologic analysis revealed minimal differences in localized lung inflammation between the two groups, lower levels of intercellular adhesion molecule 1 (ICAM-1) were observed for the airway epithelial surface of elastase-treated mice than for those of control mice. Following infection, elastase-treated mice had lung pathology consistent with pneumonia for as long as 72 h postinfection, whereas at the same time point, mock-treated mice had cleared NTHi and showed little apparent pathology. Large aggregates of bacteria were observed within damaged lung tissue of the elastase-treated mice, whereas sparse individual bacteria were observed in lungs of mock-treated mice at the same time point postinfection. Additional infection studies showed that NTHi mutants with biofilm defects were less persistent in the elastase-treated mice than the parent strain. These findings establish a model for COPD-related infections and support the hypotheses that ICAM-1 promotes clearance of NTHi. Furthermore, the data indicate that NTHi may form biofilms within the context of COPD-related infections.
Article Reference Antimicrobial effect of fluoroquinolones for the eradication of nontypeable Haemophilus influenzae isolates within biofilms.
Biofilms can be defined as communities of microorganisms attached to a surface. Those bacterial biofilms cause serious problems, such as antibiotic resistance and medical device-related infections. Nontypeable Haemophilus influenzae (NTHi) is an important pathogen in respiratory infections, as it forms biofilms both in vitro and in vivo such as human middle ear. Recent reports indicate that otitis media, paranasal sinusitis and lower respiratory tract infections caused by Haemophilus influenzae have become more difficult to treat with oral antibiotic therapy. However, there has been no attention given to antibiotic eradication of NTHi biofilm. To investigate the antimicrobial effect of various antibiotics against NTHi biofilm formation, we conducted the following comparative study using both beta-lactamase-negative ampicillin (AMP)-susceptible (BLNAS) and AMP-resistant (BLNAR) NTHi strains. In a microtiter biofilm assay, both levofloxacin and gatifloxacin, of the fluoroquinolone antibiotic group, significantly inhibited biofilm formation by BLNAS and BLNAR NTHi in a dose-dependent fashion compared to ampicillin of the penicillin antibiotic group, cefotaxime of the cephalosporin antibiotic group, and both erythromycin and clarithromycin of the macrolide antibiotic group. Furthermore, in flow cell chamber studies, confocal laser scanning microscopy counted survival bacteria in mature biofilm had been treated with gatifloxacin, ampicillin, cefotaxime and erythromycin. Only gatifloxacin completely killed the BLNAR NTHi isolates within biofilms without regard to the thickness of biofilm formation. The results of this study suggest that fluoroquinolones potentially have a role in therapy against diseases caused by both BLNAS and BLNAR NTHi isolates within biofilms.
Article Reference Haemophilus influenzae biofilms: hypothesis or fact?
Many publications state that nontypeable Haemophilus influenzae (NTHi) produces biofilms. Here, we review many of the publications that have led to acceptance by some that NTHi expresses a biofilm-specific phenotype as a distinct part of its life cycle. Biofilm formation was originally invoked to explain the failure to culture NTHi from middle-ear effusions, recalcitrance to antibiotics and its pathogenic behaviour. We argue that the current evidence for NTHi biofilm formation in vitro and in vivo is inconclusive. We consider that NTHi biofilm is hypothesis not fact, and although it might yet prove to be correct, there has been little or no consideration of alternative interpretations for the in vitro and in vivo observations. Uncritical acceptance of a distinctive NTHi biofilm phenotype has the potential to mislead and could confuse and compromise research efforts aimed at improving management and prevention of NTHi diseases of the human respiratory tract.
Article Reference Subinhibitory concentrations of azithromycin decrease nontypeable Haemophilus influenzae biofilm formation and Diminish established biofilms.
Nontypeable Haemophilus influenzae (NTHi) commonly causes otitis media, chronic bronchitis in emphysema, and early airway infections in cystic fibrosis. Long-term, low-dose azithromycin has been shown to improve clinical outcomes in chronic lung diseases, although the mechanism of action remains unclear. The inhibition of bacterial biofilms by azithromycin has been postulated to be one mechanism mediating these effects. We hypothesized that subinhibitory concentrations of azithromycin would affect NTHi biofilm formation. Laboratory strains of NTHi expressing green fluorescent protein and azithromycin-resistant clinical isolates were grown in flow-cell and static-culture biofilm models. Using a range of concentrations of azithromycin and gentamicin, we measured the degree to which these antibiotics inhibited biofilm formation and persistence. Large biofilms formed over 2 to 4 days in a flow cell, displaying complex structures, including towers and channels. Subinhibitory concentrations of azithromycin significantly decreased biomass and maximal thickness in both forming and established NTHi biofilms. In contrast, subinhibitory concentrations of gentamicin had no effect on biofilm formation. Furthermore, established NTHi biofilms became resistant to gentamicin at concentrations far above the MIC. Biofilm formation of highly resistant clinical NTHi isolates (azithromycin MIC of > 64 microg/ml) was similarly decreased at subinhibitory azithromycin concentrations. Clinically obtainable azithromycin concentrations inhibited biofilms in all but the most highly resistant isolates. These data show that subinhibitory concentrations of azithromycin have antibiofilm properties, provide mechanistic insights, and supply an additional rationale for the use of azithromycin in chronic biofilm infections involving H. influenzae.
Article Reference The PilA protein of non-typeable Haemophilus influenzae plays a role in biofilm formation, adherence to epithelial cells and colonization of the mammalian upper respiratory tract.
We recently described the expression of type IV pili (Tfp) by non-typeable Haemophilus influenzae (NTHI), a common respiratory tract pathogen. Prior to that report, Tfp were not thought to be produced by NTHI as they are not observed on NTHI when grown on chocolate agar or other commonly used growth media. To further characterize growth conditions permissive for the expression of NTHI Tfp, as well as determine their role in colonization and virulence, we transformed an NTHI otitis media isolate with a reporter plasmid containing the lux gene cluster driven by the pilA promoter. Transcription from the pilA promoter was demonstrated under a variety of in vitro growth conditions and, importantly, by ex vivo imaging of luciferase-producing NTHI in infected chinchillas. Luciferase-producing NTHI were also identified within a biofilm formed by NTHI in vivo. We further demonstrated a role for NTHI PilA in adherence to human respiratory epithelial cells, in colonization of the chinchilla respiratory tract as well as a requirement for PilA in biofilm development, both in vitro and in vivo. Collectively, our data demonstrate that NTHI express PilA in vivo, and that PilA plays an important role in the pathogenesis of an upper respiratory tract infection induced by NTHI.
Article Reference Nontypeable Haemophilus influenzae: understanding virulence and commensal behavior.
Haemophilus influenzae is genetically diverse and exists as a near-ubiquitous human commensal or as a pathogen. Invasive type b disease has been almost eliminated in developed countries; however, unencapsulated strains - nontypeable H. influenzae (NTHi) - remain important as causes of respiratory infections. Respiratory tract disease occurs when NTHi adhere to or invade respiratory epithelial cells, initiating one or more of several proinflammatory pathways. Biofilm formation explains many of the observations seen in chronic otitis media and chronic bronchitis. However, NTHi biofilms seem to lack a biofilm-specific polysaccharide in the extracellular matrix, a source of controversy regarding their relevance. Successful commensalism requires dampening of the inflammatory response and evasion of host defenses, accomplished in part through phase variation.