Pneumococcal conjugate vaccination and nasopharyngeal acquisition of pneumococcal serotype 19A strains.

CONTEXT: The rapid increase in multiresistant serotype 19A as a cause of invasive and respiratory pneumococcal disease has been associated in time with the widespread implementation of 7-valent pneumococcal conjugate vaccination (PCV-7) in several countries. Because spontaneous fluctuations in time and antibiotic selective pressure may have induced this serotype 19A increase, controlled studies are needed to assess the role of PCV-7. OBJECTIVE: To examine the association of PCV-7 vaccination and nasopharyngeal acquisition of serotype 19A pneumococci, their clonal distribution, and antibiotic susceptibility. DESIGN, SETTING, AND PATIENTS: Post hoc per-protocol completer's analysis as part of a randomized controlled trial of nasopharyngeal Streptococcus pneumoniae carriage enrolling 1003 healthy newborns with follow-up to the age of 24 months in The Netherlands, which has low antibiotic resistance rates. The study was conducted before widespread PCV-7 implementation in infants, between July 7, 2005, and February 14, 2008. Nasopharyngeal swabs were obtained at the age of 6 weeks and at 6, 12, 18, and 24 months. INTERVENTION: Infants were randomly assigned to receive 2 doses of PCV-7 at 2 and 4 months; 2 + 1 doses of PCV-7 at 2, 4, and 11 months; or no dosage (unvaccinated control group). MAIN OUTCOME MEASURE: Cumulative proportion of children with nasopharyngeal acquisition of a new serotype 19A strain from 6 through 24 months of age. RESULTS: Nine hundred forty-eight children completed the study. Fifty-four nasopharyngeal serotype 19A carriage isolates from 318 in the 2-dose group, 66 isolates from 327 in the 2 + 1-dose group, and 33 isolates from 303 in the unvaccinated were collected from 6 weeks through 24 months. The cumulative proportion who tested positive for new nasopharyngeal serotype 19A acquisition from 6 through 24 months of age was significantly higher in those having received the 2 + 1-dose PCV-7 schedule (16.2%; 95% confidence interval [CI], 12.6%-20.6%) vs those who were unvaccinated (9.2%; 95% CI, 6.5%-13.0%; relative risk [RR], 1.75; 95% CI, 1.14-2.70) but not after a 2-dose schedule (13.2%; 95% CI, 9.9%-17.4%; RR, 1.43; 95% CI, 0.91-2.25). There were 28 different sequence types identified, including 6 new types. The proportion of children with new 19A acquisition who had used antibiotics in the last 6 months (18.7%) did not differ among groups. Five isolates were penicillin-intermediate susceptible and another 3 were nonsusceptible to erythromycin and azithromycin, all in the vaccine groups. CONCLUSION: A 2 + 1-dose PCV-7 schedule was associated with an increase in serotype 19A nasopharyngeal acquisition compared with unvaccinated controls. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00189020.

Invasive pneumococcal disease among adults: associations among serotypes, disease characteristics, and outcome.

BACKGROUND: The Streptococcus pneumoniae polysaccharide capsule may be related to invasive pneumococcal disease (IPD) course. METHODS: We performed a retrospective cohort study with nationally representative surveillance data from 1075 hospitalized patients with IPD from the Netherlands from 1 June 2004 through 31 May 2006 in the prevaccination era. Serotypes were grouped according to invasive disease potential, rate of the most serious clinical syndromes of meningitis and bacteremia without focus, and case-fatality rates. Multivariable logistic regression analysis was performed to obtain odds ratios adjusted for baseline confounders for the association of serotypes and these outcomes, using the serotypes with the lowest rates as reference. RESULTS: IPD caused by serogroups with low invasive disease potential concerned meningitis or bacteremia without focus in 22% of cases, and 74% of patients had an underlying comorbidity. For highly invasive serogroups these figures were 10% (P < .01) and 56% (P < .01). Individual serotypes varied in the relative rate by which they caused meningitis or bacteremia without focus. Compared with the reference group composed of serotypes 1, 5, 7F, 15B, 20, and 33F, the group of serotypes 3, 19F, 23A, 16F, 6B, 9N, and 18C was associated with increased case-fatality rates (group adjusted odds ratio, 2.6; 95% confidence interval, 1.5-4.7). CONCLUSIONS: The serotype appeared to be independently associated with IPD severity in adults, which indicates that careful monitoring of IPD after implementation of conjugate vaccines is necessary.

Portrait of a leading vaccinologist: dedicated to meningitis and respiratory tract infections.

Since the early days of Jenner, vaccination against infectious agents appeared a very efficacious way for preventing millions of deaths and patients with serious sequelae. However, only limited success has been obtained in eradicating infectious diseases. Smallpox is the only human infectious disease that has been eradicated so far by vaccination, and eradication of polio is in its endgame. Data from the WHO in 2003 show that 2.5 million children under five still die from vaccine preventable diseases, but many more die from the major microbial killers in the world, i.e. HIV/AIDS, malaria and tuberculosis for which there are no effective vaccines. Very serious morbidity is the consequence of many infectious diseases often leading to disablement for a lifetime. As a junior post-doc with a position in the department of Medical Microbiology at the University of Amsterdam, I was touched by the seriousness of bacterial meningitis. This laboratory is the National Reference Center for Bacterial Meningitis in the Netherlands. It receives more than 80% of all isolates from meningitis patients in the country and has access to patient information for epidemiological research. This environment triggered me to use my experience as a biochemist with a PhD on composition, structure and functioning of the outer membrane of Escherichia coli to move from basic microbiology research to research on pathogenic bacteria, and ultimately prevention of serious bacterial infections.

16th International Pathogenic Neisseria Conference: recent progress towards effective meningococcal disease vaccines.

The report describes developments in meningococcal disease vaccines presented at the 16th International Pathogenic Neisseria Conference, Rotterdam, 7-12 September 2008. Great progress has been made by the Meningitis Vaccine Project to provide an affordable and effective serogroup A conjugate vaccine for use in the meningitis belt of Sub-Saharan Africa. The vaccine has been shown to be safe and to produce excellent immune response in phase 2 clinical trials in India and Africa in the target populations and will be rolled out to the worst affected countries from 2009. This vaccine has the potential to make a huge impact on public health in this region. This conference heard that the use of an epidemic strain-specific outer membrane vesicle (OMV) vaccine in New Zealand has been discontinued. Views for and against this decision were presented. Several MenB vaccines have progressed to clinical evaluation. The most advanced are the Novartis five recombinant protein variants and the Wyeth vaccine based on two factor H binding protein variants. Promising results from both vaccines with genetically-detoxified lipooligosaccharide and overexpressed heterologous antigens, OMV's from Neisseria lactamica and recombinant Opa proteins.

Effect of reduced-dose schedules with 7-valent pneumococcal conjugate vaccine on nasopharyngeal pneumococcal carriage in children: a randomized controlled trial.

CONTEXT: The effects of reduced-dose schedules of 7-valent pneumococcal conjugate vaccine (PCV-7) on pneumococcal carriage in children are largely unknown, although highly relevant in the context of subsequent herd effects. OBJECTIVE: To examine the effects of a 2-dose and 2 + 1-dose PCV-7 schedule on nasopharyngeal pneumococcal carriage in young children compared with controls. DESIGN, SETTING, AND PATIENTS: A randomized controlled trial of nasopharyngeal carriage of Streptococcus pneumoniae enrolling 1003 healthy newborns and 1 of their parents in a general community in The Netherlands, with follow-up to age 24 months and conducted between July 7, 2005, and February 14, 2008. INTERVENTION: Infants were randomly assigned to receive 2 doses of PCV-7 at 2 and 4 months; 2 + 1 doses of PCV-7 at 2, 4, and 11 months; or no dosage (control group). MAIN OUTCOME MEASURE: Vaccine serotype pneumococcal carriage rates in infants in the second year of life. RESULTS: At 12 months, vaccine serotype pneumococcal carriage was significantly decreased after both PCV-7 schedules, with vaccine serotype pneumococcal carriage rates of 25% (95% confidence interval [CI], 20%-30%) and 20% (95% CI, 16%-25%) in the 2-dose and 2 + 1-dose schedule groups, respectively, vs 38% (95% CI, 33%-44%) in the control group (both P < .001). At 18 months, in the 2 + 1-dose schedule group, vaccine serotype pneumococcal carriage had further decreased to 16% (95% CI, 12%-20%) and, at 24 months, to 14% (95% CI, 11%-18%; both P < .001); whereas in the 2-dose schedule group, vaccine serotype pneumococcal carriage had remained stable at 18 months (24%; 95% CI, 20%-29%), but at 24 months had further decreased to 15% (95% CI, 11%-19%; both P < .001). In the control group, vaccine serotype pneumococcal carriage remained around 36% to 38% until 24 months. CONCLUSION: Compared with no pneumococcal vaccination, a 2 + 1-dose and 2-dose schedule of PCV-7 resulted in significant reductions of vaccine serotype pneumococcal carriage in the second year of life. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00189020.

Meningococcal B vaccine development and evaluation of efficacy.

In this commentary we stress that serum bactericidal activity is now internationally accepted as correlate of protection for evaluating new vaccines for epidemic and endemic meningococcal B control, and that the standardization of the assay and the choice of representative strains for each of the vaccine candidates are critical.

A novel phase-variable autotransporter serine protease, AusI, of Neisseria meningitidis.

The sequenced genomes of pathogenic Neisseria meningitidis strains contain up to eight genes putatively encoding autotransporters, which are secreted proteins implicated in virulence. Here, we have characterized one of these genes, designated ausI, which encodes an autotransporter of the serine protease family. It was found to be specific for N. meningitidis and present in 14 out of 20 isolates, although only six of them expressed the gene. We show that expression of the gene is subject to phase variation as a result of a variable number of cytosines in a poly-C tract in the coding region. The open reading frame went out-of-phase at the poly-C tract in seven strains that did not express AusI. In the eighth strain, the open reading frame remained in frame at the poly-C tract, but it was disrupted by a premature stop codon further downstream. In accordance with its assignment as an autotransporter, a secreted AusI passenger domain was released into the extracellular milieu. This release was influenced by another autotransporter, NalP, as different forms of AusI were produced in the presence or absence of NalP. In silico sequence analysis suggested several putative functions for AusI, which, however, could not be confirmed experimentally.

Crystal structure of an Anti-meningococcal subtype P1.4 PorA antibody provides basis for peptide-vaccine design.

In various western countries, subtype P1.4 of Neisseria meningitidis serogroup B causes the greatest incidence of meningococcal disease. To investigate the molecular recognition of this subtype, we crystallised a peptide (P1HVVVNNKVATH(P11)), corresponding to the subtype P1.4 epitope sequence of outer membrane protein PorA, in complex with a Fab fragment of the bactericidal antibody MN20B9.34 directed against this epitope. Structure determination at 1.95 A resolution revealed a unique complex of one P1.4 antigen peptide bound to two identical Fab fragments. One Fab recognises the putative epitope residues in a 2:2 type I beta-turn at residues P5NNKV(P8), whereas the other Fab binds the C-terminal residues of the peptide that we consider a crystallisation artefact. Interestingly, recognition of the P1.4 epitope peptide is mediated almost exclusively through the complementarity-determining regions of the heavy chain. We exploited the observed turn conformation for designing conformationally restricted cyclic peptides for use as a peptide vaccine. The conformational stability of the two peptide designs was assessed by molecular dynamics simulations. Unlike the linear peptide, both cyclic peptides, conjugated to tetanus toxoid as a carrier protein, elicited antibody responses in mice that recognised meningococci of subtype P1.7-2,4. Serum bactericidal assays showed that some, but not all, of the sera induced with the cyclic peptide conjugates could activate the complement system with titres that were very high compared to the titres induced by complete PorA protein in its native conformation administered in outer membrane vesicles.

Combination vaccine against invasive meningococcal B and pneumococcal infections: potential epidemiological and economic impact in the Netherlands.

BACKGROUND: Streptococcus pneumoniae and Neisseria meningitidis group B are among the main causes of invasive bacterial meningitis infections in infants. Worldwide, these diseases lead to significant mortality, morbidity and costs. The societal impact is especially severe since the majority of cases occur in very young infants. A combination vaccine consisting of 9-valent conjugated pneumococcal and meningococcal B components is currently being developed. The aim of this study was to estimate the potential impact and cost effectiveness from the societal perspective of vaccinating infants in The Netherlands with this combination pneumococcal and meningococcal B vaccine versus no vaccination. METHODS: A Markov cycle model was developed using epidemiological and healthcare resource use data from 1996 to 2001. This model was used to project the annual costs, benefits and health gains associated with vaccinating all newborns. The base year for the costing was 2003 and all costs and health effects were discounted at 4%. The results of the analysis are expressed in costs per QALY and both probabilistic and univariate sensitivity analyses were used to identify the robustness of the results. RESULTS: Annually, an average of 755 cases of invasive pneumococcal and meningococcal B infection occurred in infants aged 0-10 years in The Netherlands. Introduction of the combination vaccine would prevent 201 cases of meningococcal B meningitis and 165 cases of invasive pneumococcal disease per year. Additionally, 3410 cases of pneumococcal pneumonia and 46,350 cases of otitis media would be prevented. Vaccination would save 35 lives per year and prevent 71 cases of severe sequelae. This translates into 860 life-years gained, or 1128 QALYs gained. Alongside these health gains, vaccination would prevent euro 17,681,370 of direct medical and indirect costs attributable to meningococcal and pneumococcal infections in The Netherlands. Depending on vaccine price, cost effectiveness varied from euro 3160 (vaccine price per dose euro 20) to euro 32,170 (vaccine price euro 60 per dose) per QALY. Base-case cost effectiveness (vaccine price euro 40) was euro 17,700 per QALY. The model was most sensitive to changes in incidence, vaccine price and duration of protective efficacy. CONCLUSION: Our results suggest that the introduction of a combination meningococcal B and pneumococcal vaccine into the Dutch infant vaccination programme is potentially cost effective compared with no vaccination.

Immunogenicity of peptide-vaccine candidates predicted by molecular dynamics simulations.

We present an in silico, structure-based approach for design and evaluation of conformationally restricted peptide-vaccines. In particular, we designed four cyclic peptides of ten or 11 residues mimicking the crystallographically observed beta-turn conformation of a predicted immunodominant loop of PorA from Neisseria meningitidis. Conformational correctness and stability of the peptide designs, as evaluated by molecular dynamics simulations, correctly predicted the immunogenicity of the peptides. We observed a peptide-induced functional antibody response that, remarkably, exceeded the response induced by the native protein in outer membrane vesicles, without losing specificity for related strains. The presented approach offers tools for a priori design and selection of peptide-vaccine candidates with full biological activity. This approach could be widely applicable: to outer membrane proteins of Gram-negative bacteria, and to other epitopes in a large range of pathogens.

Effect of seven-valent pneumococcal conjugate vaccine on Staphylococcus aureus colonisation in a randomised controlled trial.

BACKGROUND: Heptavalent pneumococcal conjugate vaccine (PCV7) shifts nasopharyngeal colonisation with vaccine serotype pneumococci towards nonvaccine serotypes. Because of the reported negative association of vaccine serotype pneumococci and Staphylococcus aureus in the nasopharynx, we explored the effect of PCV7 on nasopharyngeal colonisation with S. aureus in children and parents. METHODOLOGY/PRINCIPAL FINDINGS: This study was part of a randomised controlled trial on the effect of PCV7 on pneumococcal carriage, enrolling healthy newborns who were randomly assigned (1:1:1) to receive PCV7 (1) at 2 and 4 months of age (2) at 2, 4 and 11 months or (3) no PCV7 (controls). Nasopharyngeal colonisation of S. aureus was a planned secondary outcome. Nasopharyngeal swabs were obtained from all children over a 2-year period with 6-months interval and from one parent at the child's age of 12 and 24 months and cultured for Streptococcus pneumoniae and S. aureus. Between July 2005 and February 2006, 1005 children were enrolled and received either 2-doses of PCV7 (n = 336), 2+1-doses (336) or no dose (n = 333) before PCV7 implementation in the Dutch national immunization program. S. aureus colonisation had doubled in children in the 2+1-dose group at 12 months of age compared with unvaccinated controls (10.1% versus 5.0%; p = 0.019). A negative association for co-colonisation of S. pneumoniae and S. aureus was observed for both vaccine serotype (adjusted odds ratio (aOR) 0.53, 95% confidence interval (CI) 0.38-0.74) and nonvaccine serotype pneumococci (aOR 0.67, 95% CI 0.52-0.88). CONCLUSIONS/SIGNIFICANCE: PCV7 induces a temporary increase in S. aureus colonisation in children around 12 months of age after a 2+1-dose PCV7 schedule. The potential clinical consequences are unknown and monitoring is warranted. TRIAL REGISTRATION: ClinicalTrials.gov NCT00189020.

Comparability of antibody response to a booster dose of 7-valent pneumococcal conjugate vaccine in infants primed with either 2 or 3 doses.

In this cohort study we compared IgG antibody levels between infants immunized with 7-valent CRM197-conjugated pneumococcal vaccine (PCV-7) at 2, 4 and 11 months and at 2, 3, 4 and 11 months of age, as measured by double adsorption ELISA. Pre- and post-booster levels following the 2+1- and 3+1-dose schedule were comparable for 5 out of 7 serotypes except for serotypes 6B and 19F. The proportion of children reaching post-booster antibody thresholds were comparable except for 6B (>or=1.0 microg/ml and >or=5.0 microg/ml) and 19F (>or=5.0 microg/ml). Surveillance studies are warranted for vaccine impact on 6B and 19F disease cases after reduced-dose PCV-7 schedules.

Immunogenicity of a combination vaccine containing pneumococcal conjugates and meningococcal PorA OMVs.

The pre-clinical immunogenicity of a combination vaccine containing 13-valent pneumococcal conjugate (13vPnC) vaccine (serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F conjugated to CRM197) and nine-valent meningococcal B PorA vaccine (NonaMen; serosubtypes P1.7,16; P1.5-1,2-2; P1.19,15-1; P1.5-2,10; P1.12-1,13; P1.7-2,4; P1.22,14; P1.7-1,1 and P1.18-1,3,6), and any potential immunological interference between pneumococcal and MenB components of the vaccine were evaluated. NIH mice were immunized twice subcutaneously with the vaccines combined in one syringe, or given individually. Combining 13vPnC vaccine with NonaMen vaccine in one syringe had no negative effect on the induced antibody response against any MenB serosubtypes compared to separate injection of the vaccines, and the anti-pneumococcal antibody responses were enhanced. Furthermore, co-administration of the combination vaccine with a combined diphtheria/tetanus/acellular pertussis/inactivated poliomyelitis vaccine/Haemophilus influenzae type b-TT conjugate (DTaP/IPV-Hib) vaccine to New Zealand white rabbits at a different injection site did not affect the anti-pneumococcal polysaccharide and anti-PorA antibody titres. We conclude that no immunological interference was observed by combined administration of pneumococcal conjugate and meningococcal B vaccines in one syringe.

Characterization of genetic and phenotypic diversity of invasive nontypeable Haemophilus influenzae.

The ability of unencapsulated (nontypeable) Haemophilus influenzae (NTHi) to cause systemic disease in healthy children has been recognized only in the past decade. To determine the extent of similarity among invasive nontypeable isolates, we compared strain R2866 with 16 additional NTHi isolates from blood and spinal fluid, 17 nasopharyngeal or throat isolates from healthy children, and 19 isolates from middle ear aspirates. The strains were evaluated for the presence of several genetic loci that affect bacterial surface structures and for biochemical reactions that are known to differ among H. influenzae strains. Eight strains, including four blood isolates, shared several properties with R2866: they were biotype V (indole and ornithine decarboxylase positive, urease negative), contained sequence from the adhesin gene hia, and lacked a genetic island flanked by the infA and ksgA genes. Multilocus sequence typing showed that most biotype V isolates belonged to the same phylogenetic cluster as strain R2866. When present, the infA-ksgA island contains lipopolysaccharide biosynthetic genes, either lic2B and lic2C or homologs of the losA and losB genes described for Haemophilus ducreyi. The island was found in most nasopharyngeal and otitis isolates but was absent from 40% of invasive isolates. Overall, the 33 hmw-negative isolates were much more likely than hmw-containing isolates to have tryptophanase, ornithine decarboxylase, or lysine decarboxylase activity or to contain the hif genes. We conclude (i) that invasive isolates are genetically and phenotypically diverse and (ii) that certain genetic loci of NTHi are frequently found in association among NTHi strains.

Distribution of environmentally regulated genes of Streptococcus suis serotype 2 among S. suis serotypes and other organisms.

The occurrence of 36 environmentally regulated genes of Streptococcus suis strain 10 among all 35 S. suis serotypes was determined by using hybridization with the amplified genes as probes. In addition, the distribution of these genes among the virulence phenotypes of serotypes 1 and 2 was assessed. Hybridization was also performed with various other streptococcal species and nonstreptococcal bacterial species which may be present in pigs. Interestingly, probe ivs-25/iri-1, similar to agrA and sapR, hybridized only with S. suis serotype 1 and 2 strains with virulent phenotypes and is therefore suitable as a diagnostic parameter. Only one probe was specific for S. suis. This probe's sequence was identical to the epf gene, a putative virulence factor of S. suis. Probe ivs-31 was similar to a virulence factor of S. suis, namely, a gene encoding a fibronectin- and fibrinogen-binding protein. This probe hybridized only with oral streptococci. Nearly half of the probes (45%) hybridized with the oral streptococci (S. oralis, S. milleri, S. sanguis, S. gordonii, and S. mitis) and with Streptococcus pneumoniae. This indicates a close relationship between S. suis, the oral streptococci, and S. pneumoniae with respect to the selected environmentally regulated genes. One probe only hybridized with gram-negative species and therefore seems to be obtained by S. suis from a gram-negative organism by horizontal transfer.

Differences in genetic diversity of nonecapsulated Haemophilus influenzae from various diseases.

Genetic relationships among 80 isolates of nonencapsulated Haemophilus influenzae recovered from different disease types were determined by multilocus enzyme electrophoresis (MEE) at 13 enzyme loci in an attempt to assess the association between multilocus genotype and disease. The isolates were obtained from 15 patients with meningitis, 10 with otitis media, 19 with chronic bronchitis, 20 with cystic fibrosis, and 16 were obtained from healthy carriers. The 80 isolates were assigned to 69 electrophoretic types (ETs) falling into 5 groups. Isolates from each disease entity were represented by a variety of genotypes; however, cluster analysis from a matrix of genetic distances between ETs revealed that the ETs of the otitis media and meningitis isolates were all clustered within a genetic distance of 0.55 (group I). In addition, no genotypes were shared between H. influenzae carrier isolates and isolates from cases of disease, H. influenzae isolates from healthy individuals were distributed significantly differently from those from chronic bronchitis meningitis and otitis media patients. The genetic diversity (H) of carrier strains was greatest, although not statistically different from that of isolates from patients with disease. It was concluded that the genetic distribution of acute disease isolates is not random over the five ET groups, although the genetic diversity within the groups is not different. The effect of bacterial persistence in the host on the genetic diversity of H. influenzae is discussed.

Mucin in middle ear effusions inhibits attachment of Haemophilus influenzae to mucosal epithelial cells.

Although otitis media with effusion is often preceded by an infection of the tympanic cavity, when cultured, many effusions show no culturable bacteria. Based on the hypothesis that the effusion might play a protective role in the course of infection, the influence of this fluid on adhesion of H. influenzae (Hi) type-b strain 770235 and nontypeable H. influenzae (NTHi) strains to buccal epithelial cells was investigated. Effusions were classified as mucoid, seromucoid and serous. Mucoid secretions inhibited adhesion to a significantly greater extent (62%) than did seromucous (52%) and serous effusions (47%) ( P<0.001). The glycoprotein and high-molecular-weight fractions showed similar levels of inhibition. Sialic acid concentration, and, to a lesser extent, protein concentration, correlated with the level of inhibition. Desialylated effusions lost their ability to block bacterial attachment. Thus, middle ear effusion fluid exhibits an inhibitory effect that is due to mucins, which determine viscosity and represent the sialylated high-molecular-weight glycoprotein fraction of the effusion.

Genes of non-typeable Haemophilus influenzae expressed during interaction with human epithelial cell lines.

Non-typeable Haemophilus influenzae may infect the lower respiratory airways of chronic obstructive pulmonary disease patients. We characterized genes of non-typeable H. influenzae expressed during interaction with two human respiratory tract-derived epithelial cell lines. A library of 8000 clones was constructed in H. influenzae Rd (rec1) by cloning chromosomal fragments upstream of a promoterless cat gene. Exposure of this library to NCI-H292 epithelial cell layers in the presence of chloramphenicol (Cam) resulted in survival of bacteria expressing cat. A total of 52 clones were selected that were resistant to Cam in the presence of epithelial cells of cell line NCI-H292. These did not (n = 42) or hardly grow (n = 10) on sBHI plates containing Cam and were sensitive to Cam in cell culture medium alone. All clones, moreover, survived Cam in the presence of Hep2 epithelial cell layers. Sequence analysis showed that four clones contained sequences without homology to Rd or any other sequence, and therefore contained promoters and parts of open reading frames (ORFs) of novel genes. The other 48 clones were homologous to Rd, and characterization was based upon this genome. Six different functional classes were distinguished: (i) metabolic processes; (ii) stress response; (iii) gene expression; (iv) cell envelope biosynthesis; (v) DNA-related processes and cell division; and (vi) ORFs encoding proteins of unknown function. The contribution of identified genes to non-typeable H. influenzae adaptation to the epithelial cell environment is discussed.