The diversity of the proline-rich domain of pneumococcal surface protein A (PspA): Potential relevance to a broad-spectrum vaccine.

Pneumococcal surface protein A (PspA) is a surface exposed, highly immunogenic protein of Streptococcus pneumoniae. Its N-terminal α-helical domain (αHD) elicits protective antibody in humans and animals that can protect mice from fatal infections with pneumococci and can be detected in vitro with opsonophagocytosis assays. The proline-rich domain (PRD) in the center of the PspA sequence can also elicit protection. This study revealed that although the sequence of PRD was diverse, PRD from different pneumococcal isolates contained many shared elements. The inferred amino acid sequences of 123 such PRDs, which were analyzed by assembly and alignment-free (AAF) approaches, formed three PRD groups. Of these sequences, 45 were classified as Group 1, 19 were classified as Group 2, and 59 were classified as Group 3. All Group 3 sequences contained a highly conserved 22-amino acid non-proline block (NPB). A significant polymorphism was observed, however, at a single amino acid position within NPB. Each of the three PRD groups had characteristic patterns of short amino acid repeats, with most of the repeats being found in more than one PRD group. One of these repeats, PKPEQP as well as the NPB were previously shown to elicit protective antibodies in mice. In this study, we found that sera from 12 healthy human adult volunteers contained antibodies to all three PRD groups. This suggested that a PspA-containing vaccine containing carefully selected PRDs and αHDs could redundantly cover the known diversity of PspA. Such an approach might reduce the chances of PspA variants escaping a PspA vaccine's immunity.

Prevalence and genetic diversity of nontypeable haemophilus influenzae in the respiratory tract of infants and primary caregivers.

BACKGROUND: Nontypeable Haemophilus influenzae (NTHi) causes otitis media, sinusitis, and likely lower respiratory tract infections in children. Colonization, strain diversity, transmission, and antimicrobial susceptibility have implications for both children and their caregivers. METHODS: For 13 months, we conducted a cross-sectional study of NTHi colonization. Upper respiratory tract cultures were performed in 273 infants and children 2 to 26 months of age and their primary caregivers. NTHi isolates were characterized by multilocus sequence typing (MLST), and antibiotic resistance was examined. RESULTS: Of the 273 infants, 44 (16.1%) were colonized with NTHi. Prevalence of NTHi varied from 14% in infants less than 6 months of age to 32% in infants between 19 and 26 months of age (P = 0.003). NTHi-colonized infants were more likely to attend day care (30% vs. 12%), have a recent respiratory infection (68% vs. 38%), have recently taken an antibiotic (27% vs. 9%), and have a primary caregiver who reported asthma (11% vs. 1%), compared with other infants (P < 0.01). In the 44 infants colonized with NTHi, we identified 33 different MLSTs. Of the 44 infant-primary caregiver dyads, 9 (20.5%) were colonized with NTHi, and 7 of these 9 shared identical NTHi strains. We also found beta-lactamase-negative NTHi with minimum inhibitory concentrations >2 µg/mL for amoxicillin and beta-lactamase-positive NTHi with minimum inhibitory concentrations >2 µg/mL for amoxicillin clavulanate. CONCLUSIONS: We found substantial diversity by MLST analysis among NTHi isolates from this community. Infant-primary caregiver dyads usually carried the same strain of NTHi, suggesting that infant-primary caregiver transmission is occurring.

Molecular genetic basis of ribotyping.

Nearly 2,000 ribotyping-based studies exist, ranging from epidemiology to phylogeny and taxonomy. None precisely reveals the molecular genetic basis, with many incorrectly attributing detected polymorphisms to rRNA gene sequences. Based on in silico genomics, we demonstrate that ribotype polymorphisms result from sequence variability in neutral housekeeping genes flanking rRNA operons, with rRNA gene sequences serving solely as conserved, flank-linked tags. We also reveal that from such an informatics perspective, it is readily feasible a priori to design an interpretable ribotyping scheme for a genomically sequenced microbial species, and we discuss limitations to the basic restriction fragment length polymorphism-based method as well as alternate PCR ribotyping-based schemes.

Strain characteristics of Streptococcus pneumoniae carriage and invasive disease isolates during a cluster-randomized clinical trial of the 7-valent pneumococcal conjugate vaccine.

Widespread use of 7-valent pneumococcal conjugate vaccine (PCV7) has led to significant reductions in disease while changing pneumococcal population dynamics via herd immunity and serotype replacement. We performed multilocus sequence typing (MLST) on 590 pneumococcal isolates obtained during the American Indian clinical trial of PCV7, in which communities were randomized for eligible children to receive either PCV7 or a meningococcal conjugate vaccine (MCV). Sequence types (STs) were analyzed to determine the impact of the vaccine on pneumococcal population structure and to assess the possible impact of pneumococcal genetic background on vaccine effects. One hundred forty-three STs were obtained, the most frequent being ST199, the only one that included vaccine serotypes (VTs), non-vaccine-associated nonvaccine serotypes (NVA/NVTs), and vaccine-associated serotypes (VATs). Serotype replacement observed in the PCV communities was due to a diverse population of STs, most of which also existed in the MCV communities. Possible capsular switching to create novel ST associations with NVA/NVTs was detected only once. Reductions in VTs and changes in VATs in PCV communities did not show evidence of variation by ST, after accounting for lower vaccine effectiveness against serotype 19F. These observations suggest the hypothesis that the vaccine acts as a "serotype filter": its effect on a particular strain can be predicted on the basis of the serotype of the strain, with little effect of genetic background (as assessed by MLST) over and above capsule. If sustained, such patterns provide some cause for optimism that rapid evolution of PCV escape strains with drug resistance or high virulence is unlikely.

Multiple consecutive lavage samplings reveal greater burden of disease and provide direct access to the nontypeable Haemophilus influenzae biofilm in experimental otitis media.

The typically recovered quantity of nontypeable Haemophilus influenzae (NTHi) bacteria in an ex vivo middle ear (ME) aspirate from the chinchilla model of experimental otitis media is insufficient for direct analysis of gene expression by microarray or of lipopolysaccharide glycoforms by mass spectrometry. This prompted us to investigate a strategy of multiple consecutive lavage samplings to increase ex vivo bacterial recovery. As multiple consecutive lavage samples significantly increased the total number of bacterial CFU collected during nasopharyngeal colonization or ME infection, this led us to evaluate whether bacteria sequentially acquired from consecutive lavages were similar. Comparative observation of complete ex vivo sample series by microscopy initially revealed ME inflammatory fluid consisting solely of planktonic-phase NTHi. In contrast, subsequent lavage samplings of the same infected ear revealed the existence of bacteria in two additional growth states, filamentous and biofilm encased. Gene expression analysis of such ex vivo samples was in accord with different bacterial growth phases in sequential lavage specimens. The existence of morphologically distinct NTHi subpopulations with varying levels of gene expression indicates that the pooling of specimens requires caution until methods for their separation are developed. This study based on multiple consecutive lavages is consistent with prior reports that NTHi forms a biofilm in vivo, describes the means to directly acquire ex vivo biofilm samples without sacrificing the animal, and has broad applicability for a study of mucosal infections. Moreover, this approach revealed that the actual burden of bacteria in experimental otitis media is significantly greater than was previously reported. Such findings may have direct implications for antibiotic treatment and vaccine development against NTHi.

Host-derived sialic acid is incorporated into Haemophilus influenzae lipopolysaccharide and is a major virulence factor in experimental otitis media.

Otitis media, a common and often recurrent bacterial infection of childhood, is a major reason for physician visits and the prescription of antimicrobials. Haemophilus influenzae is the cause of approximately 20% of episodes of bacterial otitis media, but most strains lack the capsule, a factor known to play a critical role in the virulence of strains causing invasive H. influenzae disease. Here we show that in capsule-deficient (nontypeable) strains, sialic acid, a terminal residue of the core sugars of H. influenzae lipopolysaccharide (LPS), is a critical virulence factor in the pathogenesis of experimental otitis media in chinchillas. We used five epidemiologically distinct H. influenzae isolates, representative of the genetic diversity of strains causing otitis media, to inoculate the middle ear of chinchillas. All animals developed acute bacterial otitis media that persisted for up to 3 wk, whereas isogenic sialic acid-deficient mutants (disrupted sialyltransferase or CMP-acetylneuraminic acid synthetase genes) were profoundly attenuated. MS analysis indicated that WT bacteria used to inoculate animals lacked any sialylated LPS glycoforms. In contrast, LPS of ex vivo organisms recovered from chinchilla middle ear exudates was sialylated. We conclude that sialylated LPS glycoforms play a key role in pathogenicity of nontypeable H. influenzae and depend on scavenging the essential precursors from the host during the infection.

High rates of recombination in otitis media isolates of non-typeable Haemophilus influenzae.

Non-typeable (NT) or capsule-deficient, Haemophilus influenzae (Hi) is a common commensal of the upper respiratory tract of humans and can be pathogenic resulting in diseases such as otitis media, sinusitis and pneumonia. The lipopolysaccharide (LPS) of NTHi is a major virulence factor that displays substantial intra-strain and inter-strain variation of its oligosaccharide structures. To investigate the genetic basis of LPS variation we sequenced internal regions of each of seven genes required for the biosynthesis of either the inner or the outer core oligosaccharide structures. These sequences were obtained from 25 representative NTHi isolates from episodes of otitis media. We found abundant evidence of recombination among LPS genes of NTHi, a finding in marked contrast to previous analyses of biosynthetic genes for capsular polysaccharide, a well-documented virulence factor of Hi. We found mosaic sequences, linkage equilibrium between loci and a lack of congruence between gene trees. These high rates were not confined to LPS genes since evidence for similar amounts of recombination was also found in eight housekeeping genes in a subset of the same 25 isolates. These findings provide a population based foundation for a better understanding of the role of NTHi LPS as a virulence factor and its potential as a candidate vaccine.