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Virulencia del Haemophilus no tipificable


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Revisión sobre virulencia del Haemophilus no tipificable

Revisión sobre virulencia del Haemophilus no tipificable

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  • 1. Nontypeable Haemophilus influenzae: understanding virulence and commensal behavior Alice L. Erwin1,2 and Arnold L. Smith1 1 Microbial Pathogens Program, Seattle Biomedical Research Institute, 307 Westlake Avenue North, Suite 500, Seattle, WA 98109-5219, USA 2 Present address: Vertex Pharmaceuticals, 130 Waverly Street, Cambridge, MA 02139, USA Haemophilus influenzae is genetically diverse and exists as a near-ubiquitous human commensal or as a path- ogen. Invasive type b disease has been almost elimi- nated in developed countries; however, unencapsulated strains – nontypeable H. influenzae (NTHi) – remain important as causes of respiratory infections. Respirat- ory 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. Why study Haemophilus influenzae? H. influenzae is a human-restricted gram-negative bacterium that is part of the normal nasopharyngeal flora of most humans. Until twenty years ago, the study of this speciesfocusedonH.influenzaeserotype b,a majorbacterial pathogen ofchildren. Sincethe introduction in 1990oftypeb polysaccharide–protein conjugate vaccines, invasive H. influenzae disease has been nearly eliminated in developed countries. H. influenzae that lack capsular polysaccharides are referred to as nontypeable (NTHi). Although NTHi are most commonly associated with asymptomatic colonization, they are also pathogenic. H. influenzae is frequently associ- ated with otitis media, chronic bronchitis, and community- acquired pneumonia, and the strains associated with these mucosal infections are NTHi [1–3]. Colonization by any single NTHi is transitory, with new strains acquired every few months [4]. A major goal of NTHi research today is identification of bacterial factors that influence whether acquisition of an NTHi strain results in disease or in asymp- tomatic colonization. At the time of writing this review, complete or nearly complete genome sequences are available for three strains: the otitis media strains 86-028NP [5] and R2846 (also known as strain 12) [6] and the invasive NTHi strain R2866 (also called strain Int1) [7]. At least ten other strains are reportedly being sequenced, most isolated from middle ear aspirates [8]. Here, we review the current understanding of the interactions between NTHi and host tissue that are thought to lead to disease, and we will summarize the evidence that NTHi strains, including the sequenced strains, differ in these or other aspects of virulence. What is H. influenzae? The species H. influenzae is defined by its nutritional requirements: both b-nicotinamide adenine dinucleotide and heme must be supplied for growth in ordinary labora- tory conditions. Heme is not required for anaerobic growth, but seems to be important for full virulence. Each sequenced NTHi strain has two to four hpg genes which facilitate heme acquisition from hemoglobin or hemo- globin-haptoglobin complexes. Deletion of the three hpg genes from NTHi 86-028NP resulted in a delayed onset of infection in the chinchilla otitis media model, and the infection was also of shorter duration [9]. Are all H. influenzae equally virulent? Encapsulated H. influenzae are unquestionably more virulent than NTHi. Nearly all H. influenzae strains associ- ated with systemic disease possess carbohydrate capsules, and these capsules are essential for virulence in exper- imental infections [10]. For NTHi, several surface struc- tures have been reported to affect virulence, but there is no single feature that is characteristic of all disease-associ- ated strains. It is possible that NTHi strains with similar potential for causing disease might possess different com- binations of virulence-related genes. Further, it is not known whether NTHi strains differ in virulence. A recent study provided strong evidence for a group of Haemophilus isolates with low potential for virulence [11]. In a longitudinal study of 118 patients with chronic obstructive pulmonary disease, Murphy et al. isolated Haemophilus strains from >600 sputum cultures. It was noted that certain isolates initially identified as NTHi (i.e. unencapsulated strains requiring NAD+ and heme) had unusual growth characteristics. Further investigation identified these isolates as meeting all characteristics of H. haemolyticus except that many of them lacked hemolytic activity. Correlation of laboratory Review TRENDS in Microbiology Vol.15 No.8 Corresponding author: Smith, A.L. ( Available online 27 June 2007. 0966-842X/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2007.06.004
  • 2. findings with clinical data led to the recognition that acquisitions of ‘non-hemolytic H. haemolyticus’ were rarely, if ever, associated with exacerbations of symptoms of bronchitis, in comparison to acquisitions of ‘true’ NTHi. Similar strains were isolated from pharyngeal cultures of healthy children, but not from the middle ear of children with otitis media [11]. Multilocus sequence typing (MLST) and 16S RNA sequencing indicated that the H. haemolyticus strains formed a phylogenetic cluster dis- tinct from NTHi. Investigators should not only evaluate the possibility that strains isolated from the airway and identified as NTHi might be H. haemolyticus, but should consider that there might be other distinct subspecies, as yet undiscovered, that differ phenotypically and in viru- lence potential. Most of the studies cited here used well- characterized strains of NTHi that have been typed by MLST and are unlikely to be H. haemolyticus. H. influenzae virulence is host mediated The word virulent is derived from the Latin virulentus (poisonous). For H. influenzae, as for many pathogens, the organism does not produce a true poison or toxin. Disease results from the response of the host to bacterial factors, particularly endotoxin (lipopolysaccharide). Because of their short saccharide chains, the lipopolysaccharides of H. influenzae are often referred to as lipooligosaccharides (LOS), the term that will be used here. The interaction between bacteria and host cells is affected by LOS struc- ture, which varies between strains and also among bac- terial cells within a strain. The initial interaction between NTHi and the host is the adherence of bacteria to the mucus or cells of the upper airway. The nasopharynx contains both stratified squamous epithelium and pseu- dostratified ciliated columnar epithelium typical of the respiratory tract; the latter contain mucus-secreting goblet cells. Submucosal glands also contribute to surface mucus. Subsequent steps might include invasion of respiratory epithelial cells, transcytosis to the sub-epithelial compart- ment, or formation of microcolonies and ultimately a bio- film. These processes, and the host response to them, have been studied using several different NTHi strains that are now recognized to differ in genetic content. A summary of the reported NTHi–host-cell interaction using different cells, including transient transfection systems, is depicted in Figure 1. Primary role of adherence Several H. influenzae adhesins are known (reviewed in [12]). The fimbriae encoded by the hif locus were first characterized in a type b strain, in which they were found to mediate adherence to all cell types found in the respir- atory tract. Not all NTHi contain the hif locus; when it is present it is thought to be most important in the early steps of colonization [13]. Many NTHi, including 86- 028NP and R2846, contain the hmw1 and hmw2 loci, encoding high molecular weight adhesins. HMW1 and HMW2 have different binding specificities. HMW1 tar- gets sialyl-a2,3 hexose [12], which is not prevalent in the upper respiratory tract [14]. This indicates that for hmw- containing strains, other adhesins are more important in the initial colonization. Most NTHi strains lacking hmw possess genes for a different adhesin, Hia, which is similar in sequence to the surface fibrils (Hsf) previously identified in H. influenzae type b [12]. Strain R2866 includes both the hia and hif loci. Other surface struc- tures shown to bind to eukaryotic cells include the outer membrane proteins Hap [15], outer membrane protein (OMP)-2 [16] and OMP-5 [17]; the lipoprotein PCP [18]; a protein associated with colony opacity [19]; and a phos- phorylcholine (ChoP) moiety on LOS [20]. Most of these potential adhesins have substantial interstrain sequence heterogeneity. The hif and hmw loci and the licA gene (required for phosphorylcholine synthesis) are subject to phase-variable expression, mediated by changes in length of tandem repeat tracts [21–23]. Table 1 summarizes the H. influenzae surface structures and the known cognate host-cell ligands. How does respiratory tract colonization occur? After being deposited on respiratory epithelium, NTHi must overcome the innate clearance mechanisms of the upper respiratory tract. Mucociliary clearance and anti- bacterial molecules such as lysozyme, lactoferrin and antimicrobial peptides are all designed to remove infec- tious particles from the airway. Mucus contains the gel- forming mucins, MUC5AC, MUC5B and MUC2; respirat- ory mucins bind fimbriated NTHi [24] and should facilitate bacterial clearance. However, because fimbrial expression is phase-variable, the emergence of nonfimbriated strains that possess other adhesins will facilitate airway coloni- zation. The secretion of IgA protease by NTHi is inferred to facilitate colonization in individuals with S-IgA1 that recognizes surface molecules of NTHi. The gene (iga) encoding IgA1 protease is present in at least 97% of H. influenzae, but seems to be absent from nonpathogenic Haemophilus species, including H. haemolyticus [25,26]. Some NTHi have a second IgA1 protease gene, igaB, the prevalence of which in sputum and middle ear isolates is significantly greater than in ‘carriage’ isolates (P < 0.001, P = 0.011 and P = 0.0014, respectively) [27]. Neither lyso- zyme nor lactoferrin inhibit the growth of NTHi, but the serine protease activity of lactoferrin cleaves NTHi IgA protease and the adhesin Hap [28,29]. This activity might mitigate colonization. Human b defensin-1 had minimal activity toward strain 12 (R2846), whereas the antibacterial activity of the indu- cible b defensin-2 was found to be comparable to b-lactam antibiotics [30]. The susceptibility of strain 2019 to b defensin-2 was increased in a mutant (htrB) with reduced acylation of lipid A [31]. These peptides act by damaging the outer membrane, causing loss of intracellular contents including potassium. The Sap transporter, which is upre- gulated in strain 86-028NP during experimental otitis media, mediates resistance to b defensins [32] and also seems to counteract the cellular potassium loss [33]. A sapA mutant of 86-028NP has markedly attenuated viru- lence in the chinchilla model of otitis media [32]. For the antimicrobial peptide LL-37 (also called hCAP18), the susceptibility of strain H233 was reduced by expression of the ChoP moiety on LOS [34]. 356 Review TRENDS in Microbiology Vol.15 No.8
  • 3. Dampened inflammation required for commensalism? The nature of the initial binding of bacteria to host cells could be the crucial event in virulence. Work in several laboratories, using different strains and cells, indicates that binding to epithelial cells by multiple ligands initiates a proinflammatory response via several pathways (Figure 1). Adherence triggers microvillus formation in respiratory epithelial cells, leading to engulfment of NTHi [35]. The primary ligands seem to be the Toll-like receptors TLR2 and TLR4 and the platelet-activating factor receptor Table 1. H. influenzae adhesins for eukaryotic cells and their cognate ligands H. influenzae structure Cognate eukaryotic ligand(s) Initiates intracellular signaling Commenta Refs Fimbriae (pili) Fibronectin, heparin binding matrix proteins Not known Type b [58,69,70] Heat shock protein TLR-2 and TLR-4 sialylated gangliosides (SO4) Inflammation via MyD88/IRAK/NF-kb NTHi [78] Hsf Vitronectin Not known Type b [69] OMP-2 TLR-2 TNF-a and IL-6 via MyD88 Type b [79,80] OMP-5 CECAM Increased expression of CD105 (less exfoliation) Strain Rd [37] OMP-6 TLR-2 Induction of NF-kB NTHi [81] Peptides from OMP-4, OMP- 6 and PCP TLR-2 Synergizes with IFN-b NTHi [18] LOS ChoP PAFR PtdIns 3K calcium signaling NTHi 2019 [39] Hap Fibronectin, laminin, type IV collagen Not known NTHi N187 [15] a The indication of ‘type b’ or ‘NTHi’ indicates the bacteria in which the interaction was studied; it is not intended to imply that the adhesin or interaction is limited to type b or to NTHi. Figure 1. Intracellular signaling pathways activated by the binding of H. influenzae to the cell surface. The information is extracted from published reports of experiments using macrophages, respiratory epithelial cells and transient transfection systems. The major intracellular signaling molecules are depicted in color. The interaction between LBP, CD14 and LOS occurs before they bind to TLR2. Abbreviations and references: CEACAM, carcinoembryonic antigen-related cell-adhesion molecules [37]; Dectin, b-glucan receptor [71]; EGFR, epidermal growth factor receptor [38]; HB ECM, heparin-binding extracellular matrix proteins [70]; HSP70, heat shock protein70 [75,76]; IKKa/b, IkB kinase; LBP, lipopolysaccharide binding protein [73]; LOS, lipooligosaccharide; MEK3/6, MAPK/Erk kinase; MKP-1, MAP Kinase phosphatase-1; MyD88, myeloid differentiation antigen -88; NF-kB, nuclear factor kB; OMP, outer membrane protein [18,77,79,81]; PI3K, phosphoinositol-3 kinase; PAFR, platelet activating factor receptor [39]; PKC, protein kinase C; SMAD, receptor serine/threonine kinase for direct transport to nucleus; Src, cytoplasmic tyrosine kinase; TAK1, transforming growth factor-b activated kinase; TGF-b, transforming growth factor-b [74]; TLR, toll-like receptors [72]; TRAF, tumor necrosis factor receptor-associated receptor factor. Review TRENDS in Microbiology Vol.15 No.8 357
  • 4. (PAFR). Signaling by these ligands initiates proinflamma- tory signaling through the mitogen activated kinase cas- cade or by way of p38 activation [36]. Treatment of epithelial cells with NTHi or bacterial fractions does not lead to appreciable cell death. However, polarized epi- thelial cells infected with H. influenzae strain Rd KW20 have been noted to exfoliate. This process seems to be inhibited by binding of the bacterial protein OMP-5 to CECAM, triggering the expression of CD105 [37]. Intracellular residence can shield the NTHi from the action of antibodies and antibiotics and could provide the organism a means of avoiding clearance from the respir- atory tract. For intracellular residence to be an effective pathway in the life cycle of NTHi, there must be a mech- anism for dampening or terminating the inflammatory process. Using a transient transfection system, Mikami et al. showed that a lysate of NTHi strain 12 (R2846) contained a factor that activated the epidermal growth factor receptor. Activation of this receptor downregulates the p38-mediated proinflammatory response [38]. A sep- arate series of experiments [20,39] studied the interaction of NTHi strain 2019 with PAFR. Binding of ChoP, which is variably present on NTHi LOS, to the PAFR activates the phosphoinositide 3-kinase (PI3-K); this pathway can initiate anti-inflammatory pathways by way of negative regulation of TLR-2, TLR-4 and TLR-9 [40]. Because PI3-K is activated by NTHi, it is expected that inflammation would be downregulated early after invasion. ChoP expressed on the LOS of NTHi engages the PAFR through molecular mimicry, and NTHi probably enters the cell in a PAFR-linked pinocytotic vacuole. PAFR is a G-protein coupled receptor and binds to arrestins which target vacuolar contents to clathrin-coated pits or to the endocytic pathway, both seem to occur with NTHi. In addition, b- arrestins uncouple G-protein receptors terminating their signaling. Limiting the inflammatory response is probably essential for NTHi to be a successful commensal. Invasion across respiratory epithelium Adherence of NTHi to respiratory epithelial cells can also result in transcytosis of organisms into the subepithelial compartment. NTHi were found in the bronchial submu- cosa of patients with chronic bronchitis >50 years ago, whereas more recent studies identified NTHi between epithelial cells and in clusters in the submucosa of similar patients [41]. Most often the bacteria are extracellular, but they have been identified in macrophage-like cells [42]. For NTHi strain 2019 to transcytose murine M cells derived from Peyer’s patches, a wild-type LOS structure was required [43]. Once in the submucosa, NTHi can be pro- cessed for immune presentation. As noted, NTHi LOS is involved in multiple aspects of virulence. LOS structure affects the ability of bacteria to adhere effectively to cells and to invade them. The LOS- dependent intracellular signaling that occurs after NTHi enter respiratory epithelial cells seems to be necessary for colonization of human respiratory epithelium. The coloni- zation of human tissue by NTHi was studied using a model in which normal human bronchiolar xenografts are imbedded in severe combined immunodeficient (SCID, nu/nu) mice. Inoculation with as few as 10 colony forming units (CFU) of NTHi strain R3001 (isolated from a patient with cystic fibrosis) results in colonization of the xenograft. Within 48 h, the xenograft lumen contains 107 CFU. By five days, copious mucus is present and the bacterial density is 108 CFU per xenograft. Colonization in this model was found to be affected by LOS structure because mutants of strains R3001 and 2019 in which the acyltransferase gene htrB was inactivated were unable to colonize the respiratory epithelium in the xenograft model [35]. Does NTHi produce biofilms? Several recent studies have evaluated the concept that biofilm production contributes to the pathogenesis of NTHi respiratory infections, particularly those that become chronic such as otitis media with effusion and chronic bronchitis. Biofilms would be expected to protect bacteria from antibiotics and from innate respiratory epithelial clearance mechanisms. In most cases, formation of biofilms is controlled by a regulatory switch [44], and the transition from planktonic to biofilm growth involves production of an extracellular polysaccharide [45]. Although neither of these has been well documented for NTHi, there is evi- dence that NTHi can grow in an aggregate form that is consistent with a biofilm and that this form of growth affects virulence. NTHi consisting of aggregates of bacteria in a matrix containing LOS, outer membrane proteins and IgA have been observed on immortalized human middle ear epithelial cells in vitro [46] and the Calu-3 cell line [47]. When grown for extended periods on Calu-3 cells, the cells become less sensitive to gentamicin and continue to elicit a proinflammatory response [47]. Structures consistent with a biofilm have also been observed on middle ear tissue from chinchillas in experimental infections [48,49] and from children with chronic otitis media undergoing tympanost- omy tube placement [50]. Although an extracellular matrix has been visualized [51], no biofilm-specific exopolysac- charide has been identified. For strain 2019, biofilm-like structures form on respiratory epithelial cells in vitro and, in the chinchilla model of otitis, media contain an extra- cellular polymer that includes sialic acid [49] in addition to LOS. A recent study of strain 86-028NP from chinchilla middle ears identified double-stranded DNA and type IV pilin protein in an extracellular matrix [49]. A study of strain 9274 found that the extracellular matrix contained the outer membrane proteins Hap, HMW1 and HMW2, but that at least four other proteins were detected only in bacterial cells and not in the extracellular matrix [51]. Studies using several NTHi strains have shown that pre- vention of sialic acid incorporation by mutation of the sialic acid activator gene siaB or the appropriate sialyltransfer- ase genes reduces virulence in the chinchilla and gerbil models of otitis media [52,53]. Both NTHi strains 2019 and 86-028NP displayed ChoP expression to a greater extent when grown in biofilm conditions, compared with plank- tonic growth [46], and the increase in ChoP expression in biofilms was associated with decreased endotoxic activity [46]. In strain 86-028NP, mutations in the ChoP transfer- ase gene licD led to a less dense surface growth that did not persist in the middle ear of chinchillas [54]. It is unlikely that LOS structure is the only factor affecting NTHi 358 Review TRENDS in Microbiology Vol.15 No.8
  • 5. biofilm-like structures, as wecA mutants of NTHi strain 2019 do not form the structure and are attenuated in virulence in the chinchilla model [49]. WecA has been characterized in Salmonella enterica as an undecaprenyl- phosphate a-N-acetylglucosaminyltransferase [55]; it has no known role in LOS biosynthesis in NTHi. NTHi strains differ in virulence-related phenotypes Some reports support the idea that differences between strains in virulence-related phenotypes correlate with their clinical sources. Upon infection of H292 cells (a human adenocarcinoma cell line), NTHi strains that per- sist in the lower respiratory tract of patients with chronic bronchitis induced lower levels of IL-6 and IL-8 than non- persisting strains. This was not explained by differences in adherence to the H292 cells, and indicated that the reduced inflammatory potential of certain strains enables them to persist in the lungs [56]. NTHi isolated from the sputum of patients with chronic obstructive pulmonary disease who are undergoing an exacerbation are more adherent to respiratory epithelial cells and induce the secretion of more proinflammatory cytokines than strains causing asympto- matic infection [57]. Although adherence and the ability to induce inflammation are not tightly linked, the obser- vations support the concept that certain in vitro pheno- types reflect virulence. For other virulence-related phenotypes, strains vary widely but without apparent correlation with the clinical source. Bresser et al. reported that certain NTHi from patients with chronic bronchitis showed a high level of adherence to laminin and type I collagen, whereas other strains had a substantially lower level of adherence. There was no correlation between adherence to these extracellu- lar matrix proteins and whether the strains were persist- ent [58]. Phase variation complicates the study of virulence: serum resistance as an example Interpretation of strain comparison data are complicated by the fact that virulence-related phenotypes are often subject to phase variation. As each of a dozen or so phase-variable genes in each strain switches on and off independently, a culture of H. influenzae consists of a mixture of hundreds of different variants that differ in LOS structure or in expres- sion of surface antigens such as fimbriae. Such heterogen- eity permits selection of the derivative most fit for the immediate environment. The practical result is that the phenotype of the laboratory grown culture can differ sub- stantially from that of the same strain when it was in the patient. In particular, phase-variable aspects of LOS struc- ture have a crucial role in several aspects of virulence, including adherence to cells, induction of inflammation and resistance to complement. Encapsulated H. influenzae are usually resistant to the complement-mediated bactericidal activity of pooled serum from normal adults. NTHi are usually much more sensitive, but display a wide range of susceptibility to human serum [59]. The serum resistance of strain R2866 has been studied in some detail [60,61]. Strain R2866 (also known as Int1) was of particular interest because it was isolated from the bloodstream of a child with no known abnormality that would predispose him to bacteremia [7]. Strain R2866 was found to cause bacteremia and meningitis in the infant rat and weanling rat models of invasive disease (unlike any other NTHi strain tested) [7] and to have unusually high resistance to normal adult serum [62]. For this strain, high- level serum resistance depended on expression of a galacto- syltransferase encoded by the phase-variable gene lgtC [61]. Analysis of the tetranucleotide repeat region within the lgtC gene determined that the gene was out of frame (off) in a serum-sensitive variant (R3392) and in frame (on), enabling translation in the serum-resistant parent. Chemical anal- ysis of LOS confirmed that the principal glycoform in R2866 differed from that in R3392 by a single hexose. lgtC-on variants of R3392 with increased serum resistance were recovered after serum selection, confirming the relationship between phase variation and phenotype [60,61]. Several LOS biosynthetic genes in addition to lgtC are known to affect serum resistance; further, the role of different genes in serum resistance differs from strain to strain. lgtC expression (reflected by binding of monoclonal antibody 4C4) had been reported to increase serum resist- ance of other strains, but the effect was much smaller than that seen with strain R2866 [22]. It is well established that sialylation of LOS increases serum resistance [63]. By contrast, expression of ChoP increases the sensitivity of bacteria to human serum, as ChoP binds C-reactive protein, activating complement [64]. These and other fea- tures of LOS structure are subject to phase variation, as described for lgtC. The high-level serum resistance of strain R2866 was maintained after isolation in the clinical laboratory, leading to the hypothesis that the association of this strain with invasive disease was a result of its ability to survive in human blood. In a survey of other NTHi, invasive isolates (from blood or cerebrospinal fluid of apparently normal children) were not more resistant to serum than throat or middle ear isolates [59]. For some of these cases of invasive disease, unrecognized host factors might have predisposed these children to systemic NTHi infection. For other cases, the NTHi strains might be unusually virulent by mechanisms other than serum resistance. It is likely that for some of the invasive strains, phase variants with increased sensitivity to serum could have emerged in the laboratory. Genetic heterogeneity of NTHi It is well established that NTHi are genetically diverse and it is thought that this observation results in part from horizontal genetic exchange. Strains differ in their comp- lement of loci encoding adhesins and IgA proteases, as noted. In some cases, these differences have been sugg- ested to be associated with differences in virulence. Several loci, including the LOS biosynthetic gene lic2B, the adhe- sin gene hmwA, and the heme acquisition genes hemR and hgpB, have been reported to be more prevalent in otitis media isolates than in those recovered from the throat of healthy children [65,66]. In addition, differential hybrid- ization studies identified several loci that were more com- mon in NTHi strains associated with exacerbations of symptoms in patients with chronic obstructive pulmonary disease than in strains from patients not experiencing exacerbations [67]. Review TRENDS in Microbiology Vol.15 No.8 359
  • 6. To consider the possibility that NTHi strains from invasive infections in children were closely related or con- tained common virulence determinants, PCR was used to evaluate the presence of several genetic loci in a collection of 52 clinical isolates [59]. The isolates from invasive disease were found to be as diverse as isolates from otitis media or from healthy children. The publicly available H. influenzae multilocus sequence typing database (http:// currently contains 600 strains, of which 300 are NTHi. There is no obvious phylogenetic clustering of otitis isolates or strains from blood or cere- brospinal fluid. However, few NTHi strains from healthy subjects or from sputum cultures have been typed. It is possible that such isolates might form clusters distinct from the disease-associated strains. Strain 86-028NP was the first NTHi strain to be completely sequenced [5]. Annotation of the sequences of strains R2846 and R2866 (available at http://www.ncbi. identified a core of 1500 open reading frames common to all three NTHi genomes (Figure 2). Nearly all of these are also in the published sequence of H. influenzae Rd KW20 [68]. There are no obvious candidates for otitis-specific determinants, supporting the concept that virulence is multifactorial and can be mediated by different mechanisms in different strains. Differences in host risk factors also need to be considered. Concluding remarks and future perspectives Several bacterial-specific and host-specific processes that are thought to be involved in the pathogenesis of NTHi disease have been described. NTHi strains are genetically and phenotypically diverse. Some of the genes encoding proposed virulence determinants are not present in all disease isolates, indicating that strains can differ in mech- anisms of pathogenesis. It is not yet clear whether NTHi strains differ in their capacity for causing mucosal or systemic infections, or whether host factors are primarily responsible for the outcome of acquisition of a new NTHi strain. The availability of sequenced genomes will facili- tate study of the diversity of NTHi pathogenesis and provide insights into vaccine candidates for NTHi disease. Other unanswered questions (Box 1) also need to be pur- sued to advance our understanding of NTHi disease and carriage. Acknowledgements This work was supported in part by grants AI44002, AI46512 and DC 005833 from the National Institutes of Health (USA). References 1 Brunton, S. (2006) Current face of acute otitis media: microbiology and prevalence resulting from widespread use of heptavalent pneumococcal conjugate vaccine. Clin. Ther. 28, 118–123 2 Apisarnthanarak, A. and Mundy, L.M. (2005) Etiology of community- acquired pneumonia. Clin. Chest Med. 26, 47–55 3 Eldika, N. and Sethi, S. (2006) Role of nontypeable Haemophilus influenzae in exacerbations and progression of chronic obstructive pulmonary disease. Curr. Opin. Pulm. Med. 12, 118–124 4 Faden, H. et al. (1996) Epidemiology of nasopharyngeal colonization with nontypeable Haemophilus influenzae in the first two years of life. Acta Otolaryngol. Suppl. 523, 128–129 5 Harrison, A. et al. (2005) Genomic sequence of an otitis media isolate of nontypeable Haemophilus influenzae: comparative study with H. influenzae serotype d, strain KW20. J. Bacteriol. 187, 4627–4636 6 Barenkamp, S.J. and Leininger, E. (1992) Cloning, expression, and DNA sequence analysis of genes encoding nontypeable Haemophilus influenzae high-molecular-weight surface-exposed proteins related to filamentous hemagglutinin of Bordetella pertussis. Infect. Immun. 60, 1302–1313 7 Nizet, V. et al. (1996) A virulent nonencapsulated Haemophilus influenzae. J. Infect. Dis. 173, 180–186 8 Shen, K. et al. (2005) Identification, distribution, and expression of novel genes in 10 clinical isolates of nontypeable Haemophilus influenzae. Infect. Immun. 73, 3479–3491 9 Morton, D.J. et al. (2004) Reduced severity of middle ear infection caused by nontypeable Haemophilus influenzae lacking the Figure 2. Comparison of the genomes of the three sequenced nontypeable H. influenzae. Open reading frames (ORFs) in the nucleotide sequences of str- ains R2846 and R2866 (available at genomeprjcmd=Retrievedopt=Overviewlist_uids=9621) were identified by Glimmer ( For Rd KW20 and 86-028NP, the published annotated sequences were used [5,68]. Each pair of nucleotide sequences was aligned using BLASTN, and the Artemis Comparison Tool (http:// was used to visualize the alignments. Gaps in alignment were noted, and each gene was categorized as being present in one, two, three or four of the sequenced strains. The results were confirmed by BLASTP comparisons of each predicted amino acid sequence to the sequences of the other strains. Most regions of the genomes were shared among all four genomes, but each strain has several dozen open reading frames that are absent from the other sequenced strains or shared with only one of the sequenced strain. The Venn diagram shows the numbers of shared and strain-specific genes for the three NTHi genomes. Most of the 1522 genes shared by all three NTHi are also in Rd KW20. It is notable that the two otitis strains (R2846 and 86-028NP) differ from each other in gene content to as great an extent as they differ from the invasive strain R2866. Box 1. Outstanding questions What is the minimal gene content that permits nontypeable H. influenzae to initiate an inflammatory response from respira- tory epithelial cells? What bacterial or host factors enable NTHi to persist without eliciting an inflammatory response? 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