Genetic Diversity of Paired Middle-Ear and Pharyngeal Nontypeable Haemophilus influenzae Isolates from Children with Acute Otitis Media

Abstract

Pulsed-field gel electrophoresis was used to determine genetic diversities of multiple nontypeable Haemophilus influenzae isolates from throat and ear specimens of eight children with otitis media. From five children, all ear and throat isolates were identical. The bacterial populations in these specimens showed less diversity than populations in throat isolates of healthy children.Nontypeable Haemophilus influenzae (NTHi) are gram-negative coccobacilli that naturally colonize the pharynges of humans, with carriage rates between 20 and 80% (1,6,8). In addition to colonizing asymptomatically, H. influenzae causes upper and lower respiratory tract infections, such as sinusitis, otitis media (OM), and bronchitis.OM is a common infection of the middle-ear space, experienced by 50 to 85% of children by 3 years of age (3,17). In children, between 30 and 52% of episodes have been attributed to infection with NTHi (4). Previous studies (9,11,13,19) examining single NTHi colonies from each specimen have shown that middle-ear isolates are both genotypically and phenotypically similar to throat isolates obtained from children with OM. Recent studies from our laboratory examining multiple NTHi colonies from each specimen suggest that throat isolates of healthy children attending day care are genetically very diverse; pulsed-field gel electrophoresis (PFGE) analysis found that between 37 and 43% of children were colonized by more than one strain of NTHi (5,16) and that the PFGE patterns of strains varied dramatically from week to week (16).The present study was designed to examine the genetic diversity of colonization and disease-causing NTHi isolates. Simultaneous pharyngeal and middle-ear specimens were collected from six children in Bardstown, KY, and from two children in Pittsburgh, PA, with acute OM; children P6, K8, and K21 had isolates obtained from both ears. A minimum of 5 and no more than 10 H. influenzae isolates were obtained from each specimen. All isolates were suspended in ϳ1 ml of sterile skim milk and stored at Ϫ80°C.Putative H. influenzae isolates were confirmed by growth on brain heart infusion agar (Difco, Detroit, MI) supplemented with X factor (hemin), V factor (NAD), or both (Sigma-Aldrich, St. Louis, MO); by porphyrin testing; and by an isolate's failure to hemolyze horse erythrocytes as determined using the methods of Kilian (12). In addition, each isolate was tested by colony blot analysis with the monoclonal antibody 7F3 for the presence of this epitope of P6 surface antigen (14) and by DNA dot blot hybridization for the immunoglobulin A1 protease gene to distinguish suspected H. influenzae isolates from nonhemolytic Haemophilus haemolyticus (7, 12). All isolates in this study bound to the 7F3 antibody and hybridized with the iga probe, indicating that they are H. influenzae.To examine genetic diversity, all study isolates were characterized by PFGE as previously described (15, 16). The PFGE patterns were first analyzed visually using the criteria of Tenover et al. (18) to determine band differenc...

Antagonistic Pleiotropy in the Bifunctional Surface Protein FadL (OmpP1) during Adaptation of Haemophilus influenzae to Chronic Lung Infection Associated with Chronic Obstructive Pulmonary Disease

“…S4b ). (i) Individual patients were colonized by numerous genetically diverse strains over time, though diversity at any given time point remains unclear ( 24 , 32 , 37 – 40 ). High polyclonality and a high average degree of divergence among CTs contrasts with studies of environmentally acquired opportunistic pathogens, which have typically supported colonization by single CTs followed by subsequent diversification ( 3 , 6 , 41 ).…”

“…First, we are unlikely to have sampled the initial colonizing bacterial isolates; therefore, we did not order the observed genetic variations or determine the most recent common ancestor ( 13 ). Second, we analyzed single isolates from each sampling point, yet we know that NTHi colonization and infection are often polyclonal ( 24 , 32 , 37 – 40 ). Despite these considerations, our results show the utility of comparing the genomes of bacterial isolates collected over time from long-term chronic infections, showing that genetic polymorphisms within clonally related NTHi isolates can help tease apart transmission of strains between subjects and the genetic changes that result in changes in pathogenic traits.…”

Phase variation and host immunity against high molecular weight (HMW) adhesins shape population dynamics of nontypeable Haemophilus influenzae within human hosts

“…3). This result is interesting because during otitis media, the pharyngeal NTHi community, which in healthy individuals commonly supports multiple different NTHi strains, is dominated by the disease-causing isolate that is simultaneously present in the middle ear (Berrens et al, 2007). This would have the same effect as increasing the carrying capacity in our model since a larger portion of the NTHi in the pharynx could be represented by a single strain, thus, allowing that strain to achieve a larger overall population size.…”