J. Christopher Post

Comparative Genomic Analyses of Seventeen Streptococcus pneumoniae Strains: Insights into the Pneumococcal Supragenome

The distributed-genome hypothesis (DGH) states that pathogenic bacteria possess a supragenome that is much larger than the genome of any single bacterium and that these pathogens utilize genetic recombination and a large, noncore set of genes as a means of diversity generation. We sequenced the genomes of eight nasopharyngeal strains of Streptococcus pneumoniae isolated from pediatric patients with upper respiratory symptoms and performed quantitative genomic analyses among these and nine publicly available pneumococcal strains. Coding sequences from all strains were grouped into 3,170 orthologous gene clusters, of which 1,454 (46%) were conserved among all 17 strains. The majority of the gene clusters, 1,716 (54%), were not found in all strains. Genic differences per strain pair ranged from 35 to 629 orthologous clusters, with each strains genome containing between 21 and 32% noncore genes. The distribution of the orthologous clusters per genome for the 17 strains was entered into the finite-supragenome model, which predicted that (i) the S. pneumoniae supragenome contains more than 5,000 orthologous clusters and (ii) 99% of the orthologous clusters ( approximately 3,000) that are represented in the S. pneumoniae population at frequencies of >or=0.1 can be identified if 33 representative genomes are sequenced. These extensive genic diversity data support the DGH and provide a basis for understanding the great differences in clinical phenotype associated with various pneumococcal strains. When these findings are taken together with previous studies that demonstrated the presence of a supragenome for Streptococcus agalactiae and Haemophilus influenzae, it appears that the possession of a distributed genome is a common host interaction strategy.

Direct evidence of bacterial biofilms in otitis media.

Objectives/hypothesis Bacteriologic studies of otitis media with effusion (OME) using highly sensitive techniques of molecular biology such as the polymerase chain reaction have demonstrated that traditional culturing methods are inadequate to detect many viable bacteria present in OME. The presence of pathogens attached to the middle-ear mucosa as a bacterial biofilm, rather than as free-floating organisms in a middle-ear effusion, has previously been suggested to explain these observations. The suggestion has been speculative, however, because no visual evidence of such biofilms on middle-ear mucosa has heretofore been collected. The hypotheses motivating the current study were: 1) biofilms of nontypable Hemophilus influenzae will form on the middle-ear mucosa of chinchillas in an experimental model of OME, 2) these biofilms will exhibit changes in density or structure over time, and 3) biofilms are also present on tympanostomy tubes in children with refractory post-tympanostomy otorrhea. The objective of this study was to collect visual evidence of the formation of bacterial biofilms in these situations. Study design Laboratory study of bacteriology in an animal model and on medical devices removed from pediatric patients. Methods Experimental otitis media was induced in chinchillas by transbullar injection of nontypable H. influenzae. Animals were killed in a time series and the surface of the middle-ear mucosa was examined by scanning electron microscopy (SEM) for the presence of bacterial biofilms. Adult and fetal chinchilla uninfected controls were similarly examined for comparison. In addition, tympanostomy tubes that had been placed in childrens ears to treat OME and removed after onset of refractory otorrhea or other problems were examined by SEM and by confocal scanning laser microscopy for bacterial biofilms, and compared with unused control tubes. Results Bacterial biofilms were visually detected by SEM on the middle-ear mucosa of multiple chinchillas in which H. influenzae otitis media had been induced. Qualitative evaluation indicated that the density and thickness of the biofilm might increase until at least 96 hours after injection. The appearance of the middle-ear mucosa of experimental animals contrasted with that of uninjected control animals. Robust bacterial biofilms were also visually detected on tympanostomy tubes removed from childrens ears for clinical reasons, in contrast with unused control tubes. Conclusions Bacterial biofilms form on the middle-ear mucosa of chinchillas in experimentally induced H. influenzae otitis media and can form on tympanostomy tubes placed in childrens ears. Such biofilms can be directly observed by microscopy. These results reinforce the hypothesis that the bacterial aggregates called biofilms, resistant to treatment by antibiotics and to detection by standard culture techniques, may play a major etiologic role in OME and in one of its frequent complications, post-tympanostomy otorrhea.

Characterization and modeling of the Haemophilus influenzae core and supragenomes based on the complete genomic sequences of Rd and 12 clinical nontypeable strains

BackgroundThe distributed genome hypothesis (DGH) posits that chronic bacterial pathogens utilize polyclonal infection and reassortment of genic characters to ensure persistence in the face of adaptive host defenses. Studies based on random sequencing of multiple strain libraries suggested that free-living bacterial species possess a supragenome that is much larger than the genome of any single bacterium.ResultsWe derived high depth genomic coverage of nine nontypeable Haemophilus influenzae (NTHi) clinical isolates, bringing to 13 the number of sequenced NTHi genomes. Clustering identified 2,786 genes, of which 1,461 were common to all strains, with each of the remaining 1,328 found in a subset of strains; the number of clusters ranged from 1,686 to 1,878 per strain. Genic differences of between 96 and 585 were identified per strain pair. Comparisons of each of the NTHi strains with the Rd strain revealed between 107 and 158 insertions and 100 and 213 deletions per genome. The mean insertion and deletion sizes were 1,356 and 1,020 base-pairs, respectively, with mean maximum insertions and deletions of 26,977 and 37,299 base-pairs. This relatively large number of small rearrangements among strains is in keeping with what is known about the transformation mechanisms in this naturally competent pathogen.ConclusionA finite supragenome model was developed to explain the distribution of genes among strains. The model predicts that the NTHi supragenome contains between 4,425 and 6,052 genes with most uncertainty regarding the number of rare genes, those that have a frequency of <0.1 among strains; collectively, these results support the DGH.

Candidate's Thesis: Direct Evidence of Bacterial Biofilms in Otitis Media†

Objectives/Hypothesis Bacteriologic studies of otitis media with effusion (OME) using highly sensitive techniques of molecular biology such as the polymerase chain reaction have demonstrated that traditional culturing methods are inadequate to detect many viable bacteria present in OME. The presence of pathogens attached to the middle‐ear mucosa as a bacterial biofilm, rather than as free‐floating organisms in a middle‐ear effusion, has previously been suggested to explain these observations. The suggestion has been speculative, however, because no visual evidence of such biofilms on middle‐ear mucosa has heretofore been collected. The hypotheses motivating the current study were: 1) biofilms of nontypable Hemophilus influenzae will form on the middle‐ear mucosa of chinchillas in an experimental model of OME, 2) these biofilms will exhibit changes in density or structure over time, and 3) biofilms are also present on tympanostomy tubes in children with refractory post‐tympanostomy otorrhea. The objective of this study was to collect visual evidence of the formation of bacterial biofilms in these situations.

Characterization of biofilm matrix, degradation by DNase treatment and evidence of capsule downregulation in Streptococcus pneumoniae clinical isolates

BackgroundStreptococcus pneumoniae is a common respiratory pathogen and a major causative agent of respiratory infections, including otitis media (OM). Pneumococcal biofilms have been demonstrated on biopsies of the middle ear mucosa in children receiving tympanostomy tubes, supporting the hypothesis that chronic OM may involve biofilm development by pathogenic bacteria as part of the infectious process. To better understand pneumococcal biofilm formation six low-passage encapsulated nasopharyngeal isolates of S. pneumoniae were assessed over a six-eight day period in vitro.ResultsMultiparametric analysis divided the strains into two groups. Those with a high biofilm forming index (BFI) were structurally complex, exhibited greater lectin colocalization and were more resistant to azithromycin. Those with a low BFI developed less extensive biofilms and were more susceptible to azithromycin. dsDNA was present in the S. pneumoniae biofilm matrix in all strains and treatment with DNase I significantly reduced biofilm biomass. Since capsule expression has been hypothesized to be associated with decreased biofilm development, we also examined expression of cpsA, the first gene in the pneumococcal capsule operon. Interestingly, cpsA was downregulated in biofilms in both high and low BFI strains.ConclusionAll pneumococcal strains developed biofilms that exhibited extracellular dsDNA in the biofilm matrix, however strains with a high BFI correlated with greater carbohydrate-associated structural complexity and antibiotic resistance. Furthermore, all strains of S. pneumoniae showed downregulation of the cpsA gene during biofilm growth compared to planktonic culture, regardless of BFI ranking, suggesting downregulation of capsule expression occurs generally during adherent growth.

The role of biofilms in otolaryngologic infections: update 2007.

Purpose of reviewBiofilms have been shown to play a role in otitis media, sinusitis, cholesteatoma, tonsillitis, adenoiditis, and device infections. This article is written to review recent advances in the field. Recent findingsThe role of biofilms in the persistence of chronic, mucosal-based ENT-related infections was first recognized in otitis media. Definitive proof was lacking until the demonstration of bacterial biofilms on the middle-ear mucosa of children, not only with chronic otitis media with effusion, but also with recurrent otitis media. Strains of Pseudomonas aeruginosa isolated from cholesteatoma are avid biofilm formers. Biofilms have been reported in the adenoids of children with chronic rhinosinusitis, helping to explain the clinical observation that adenoidectomy can be beneficial to children with chronic otitis or chronic rhinosinusiti. Additional studies have confirmed the presence of biofilms in chronic tonsillitis. Biofilms have also been shown to be involved in infected cochlear implants and tracheotomy tubes. SummaryThe recognition that chronic otolaryngologic bacterial infections are biofilm related has been the impetus for the development of new technologies for the study of biofilms and their prevention and treatment. Understanding that chronic bacterial infections are biofilm related is fundamental to developing rationale strategies for treatment and prevention.

Phenotypic Characterization of Streptococcus pneumoniae Biofilm Development

Streptococcus pneumoniae is among the most common pathogens associated with chronic otitis media with effusion, which has been hypothesized to be a biofilm disease. S. pneumoniae has been shown to form biofilms, however, little is known about the developmental process, the architecture, and the changes that occur upon biofilm development. In the current study we made use of a continuous-culture biofilm system to characterize biofilm development of 14 different S. pneumoniae strains representing at least 10 unique serotypes. The biofilm development process was found to occur in three distinct stages, including initial attachment, cluster formation, and biofilm maturation. While all 14 pneumococcal strains displayed similar developmental stages, the mature biofilm architecture differed significantly among the serotypes tested. Overall, three biofilm architectural groups were detected based on biomass, biofilm thickness, and cluster size. The biofilm viable cell counts and total protein concentration increased steadily over the course of biofilm development, reaching approximately 8 x 10(8) cells and approximately 15 mg of protein per biofilm after 9 days of biofilm growth. Proteomic analysis confirmed the presence of distinct biofilm developmental stages by the detection of multiple phenotypes over the course of biofilm development. The biofilm development process was found to correlate not only with differential production of proteins but also with a dramatic increase in the number of detectable proteins, indicating that biofilm formation by S. pneumoniae may be a far more complex process than previously anticipated. Protein identification revealed that proteins involved in virulence, adhesion, and resistance were more abundant under biofilm growth conditions. A possible role of the identified proteins in biofilm formation is discussed.

PCR-based detection of bacterial DNA after antimicrobial treatment is indicative of persistent, viable bacteria in the chinchilla model of otitis media

PURPOSE Bacterial deoxyribonucleic acid (DNA) has been previously detected by polymerase chain reactions (PCR) in a significant percentage of culturally-sterile pediatric middle-ear effusions. The current study was designed to determine whether this represents the existence of viable bacteria or the persistence of residual DNA in the middle-ear cleft. MATERIALS AND METHODS The middle-ear cavities of two sets of chinchillas were inoculated with either: 1) 100 colony-forming units (CFU) of live Haemophilus influenzae, 2.2 x 10(6) CFU of pasteurized Moraxella catarrhalis, and 1000 ng of DNA (>10(8) genomic equivalents) from Streptococcus pneumoniae; or 2) 100 CFU of live S pneumoniae, 2.2 x 10(6) CFU of pasteurized M catarrhalis and 1000 ng of purified DNA from H influenzae. Animals were treated with ampicillin for 5 days beginning on day 3. A single-point longitudinal study design was used for sampling to eliminate the possibility of contamination. RESULTS No DNA was detectable from the heat-killed bacteria or the purified DNA after day 3. However, DNA from the live bacteria persisted through day 21, even though all specimens were culture-negative following the initiation of antimicrobial therapy. CONCLUSION These findings indicate that purified DNA and DNA from intact but nonviable bacteria do not persist in the middle-ear cleft in the presence of an effusion, even following high copy inoculation. In contrast, antibiotic-treated bacteria persist in some viable state for weeks as evidenced by the differential ability of the PCR-based assay systems to detect the live bacteria, but not detect the heat-killed organisms.

Bacterial Plurality as a General Mechanism Driving Persistence in Chronic Infections

Classical methods for the study of bacterial pathogens have proven to be inadequate to inform with respect to chronic infections including those associated with arthroplasties. Modern methods of analysis have demonstrated that bacterial growth patterns, ecology, and intra-species heterogeneity are more complex than were envisioned by early microbiologists. Cultural methods were developed to study acute, epidemic infections, but it is now recognized that the phenotype associated with these diseases represents only a minor aspect of the bacterial life cycle, which consists of planktonic, attachment, biofilm, and dispersal phases. Over 99% of bacteria in natural populations are found in biofilms which contain multiple ecological niches and numerous phenotypes. Unfortunately, the effort to develop antibiotics has been directed solely at the planktonic minority (associated with systemic illness) which explains our inability to eradicate chronic infections. In this study we establish a new rubric, bacterial plurality, for the understanding of bacterial ecology and evolution with respect to chronic infection. The fundamental tenets of bacterial plurality are that the bacteria within an infecting population display multiple phenotypes and possess multiple genotypes. Phenotypic plurality is embodied in the biofilm paradigm and genotypic plurality is embodied in the concepts of the supra-genome and the distributed genome hypothesis. It is now clear that bacterial diversity provides bacterial populations, as a whole, the ability to persist in the face of a multi-faceted host response.

Generation of Genic Diversity among Streptococcus pneumoniae Strains via Horizontal Gene Transfer during a Chronic Polyclonal Pediatric Infection

Although there is tremendous interest in understanding the evolutionary roles of horizontal gene transfer (HGT) processes that occur during chronic polyclonal infections, to date there have been few studies that directly address this topic. We have characterized multiple HGT events that most likely occurred during polyclonal infection among nasopharyngeal strains of Streptococcus pneumoniae recovered from a child suffering from chronic upper respiratory and middle-ear infections. Whole genome sequencing and comparative genomics were performed on six isolates collected during symptomatic episodes over a period of seven months. From these comparisons we determined that five of the isolates were genetically highly similar and likely represented a dominant lineage. We analyzed all genic and allelic differences among all six isolates and found that all differences tended to occur within contiguous genomic blocks, suggestive of strain evolution by homologous recombination. From these analyses we identified three strains (two of which were recovered on two different occasions) that appear to have been derived sequentially, one from the next, each by multiple recombination events. We also identified a fourth strain that contains many of the genomic segments that differentiate the three highly related strains from one another, and have hypothesized that this fourth strain may have served as a donor multiple times in the evolution of the dominant strain line. The variations among the parent, daughter, and grand-daughter recombinant strains collectively cover greater than seven percent of the genome and are grouped into 23 chromosomal clusters. While capturing in vivo HGT, these data support the distributed genome hypothesis and suggest that a single competence event in pneumococci can result in the replacement of DNA at multiple non-adjacent loci.