Impact of Experimental Human Pneumococcal Carriage on Nasopharyngeal Bacterial Densities in Healthy Adults

Colonization of the nasopharynx by Streptococcus pneumoniae is a necessary precursor to pneumococcal diseases that result in morbidity and mortality worldwide. The nasopharynx is also host to other bacterial species, including the common pathogens Staphylococcus aureus, Haemophilus influenzae, and Moraxella catarrhalis. To better understand how these bacteria change in relation to pneumococcal colonization, we used species-specific quantitative PCR to examine bacterial densities in 52 subjects 7 days before, and 2, 7, and 14 days after controlled inoculation of healthy human adults with S. pneumoniae serotype 6B. Overall, 33 (63%) of subjects carried S. pneumoniae post-inoculation. The baseline presence and density of S. aureus, H. influenzae, and M. catarrhalis were not statistically associated with likelihood of successful pneumococcal colonization at this study’s sample size, although a lower rate of pneumococcal colonization in the presence of S. aureus (7/14) was seen compared to that in the presence of H. influenzae (12/16). Among subjects colonized with pneumococci, the number also carrying either H. influenzae or S. aureus fell during the study and at 14 days post-inoculation, the proportion carrying S. aureus was significantly lower among those who were colonized with S. pneumoniae (p = 0.008) compared to non-colonized subjects. These data on bacterial associations are the first to be reported surrounding experimental human pneumococcal colonization and show that co-colonizing effects are likely subtle rather than absolute

Repeat-Associated Plasticity in the Helicobacter pylori RD Gene Family▿ †

The bacterium Helicobacter pylori is remarkable for its ability to persist in the human stomach for decades without provoking sterilizing immunity. Since repetitive DNA can facilitate adaptive genomic flexibility via increased recombination, insertion, and deletion, we searched the genomes of two H. pylori strains for nucleotide repeats. We discovered a family of genes with extensive repetitive DNA that we have termed the H. pylori RD gene family. Each gene of this family is composed of a conserved 3′ region, a variable mid-region encoding 7 and 11 amino acid repeats, and a 5′ region containing one of two possible alleles. Analysis of five complete genome sequences and PCR genotyping of 42 H. pylori strains revealed extensive variation between strains in the number, location, and arrangement of RD genes. Furthermore, examination of multiple strains isolated from a single subject's stomach revealed intrahost variation in repeat number and composition. Despite prior evidence that the protein products of this gene family are expressed at the bacterial cell surface, enzyme-linked immunosorbent assay and immunoblot studies revealed no consistent seroreactivity to a recombinant RD protein by H. pylori-positive hosts. The pattern of repeats uncovered in the RD gene family appears to reflect slipped-strand mispairing or domain duplication, allowing for redundancy and subsequent diversity in genotype and phenotype. This novel family of hypervariable genes with conserved, repetitive, and allelic domains may represent an important locus for understanding H. pylori persistence in its natural host

The adult nasopharyngeal microbiome as a determinant of pneumococcal acquisition

BACKGROUND: Several cohort studies have indicated associations between S. pneumoniae and other microbes in the nasopharynx. To study causal relationships between the nasopharyngeal microbiome and pneumococcal carriage, we employed an experimental human pneumococcal carriage model. Healthy adult volunteers were assessed for pneumococcal carriage by culture of nasal wash samples (NWS). Those without natural pneumococcal carriage received an intranasal pneumococcal inoculation with serotype 6B or 23F. The composition of the nasopharyngeal microbiome was longitudinally studied by 16S rDNA pyrosequencing on NWS collected before and after challenge. RESULTS: Among 40 selected volunteers, 10 were natural carriers and 30 were experimentally challenged. At baseline, five distinct nasopharyngeal microbiome profiles were identified. The phylogenetic distance between microbiomes of natural pneumococcal carriers was particularly large compared to non-carriers. A more diverse microbiome prior to inoculation was associated with the establishment of pneumococcal carriage. Perturbation of microbiome diversity upon pneumococcal challenge was strain specific. Shifts in microbiome profile occurred after pneumococcal exposure, and those volunteers who acquired carriage more often diverted from their original profile. S. pneumoniae was little prominent in the microbiome of pneumococcal carriers. CONCLUSION: Pneumococcal acquisition in healthy adults is more likely to occur in a diverse microbiome and appears to promote microbial heterogeneity

Percent carrying Hi (A), Sa (B), and Mc (C) at 4 time points and density of Sa colonization at baseline and 14 days (D) segregated by success of pneumococcal colonization.

<p>Numbers of subjects carrying each bacteria ranged from 11<b>–</b>17 for <i>H. influenzae</i>, 5<b>–</b>10 for <i>M. catarrhalis</i>, and 13<b>–</b>14 for <i>S. aureus</i>. Asterisk indicates <i>p</i> = 0.008.</p

Colonization rates and maximum post-inoculation densities at four inoculation doses.

<p>Subjects were administered intra-nasal doses of 60 k, 80 k, 160 k, or 320 k CFU of <i>S. pneumoniae</i> 6B. Comparison of Sp colonization rates and maximum post-inoculation densities rates among subjects in the four dose groups revealed no statistically significant differences. Boxplots indicate median, interquartile range, and range with circles indicating outlier values.</p

Density of Sp (A), Hi (B), Sa (C), and Mc (D) in nasal wash samples at four time points.

<p>Boxplots of log bacterial density (in genomes/ml) of all samples with >100 genomes/ml at each time point. Boxplots indicate median, interquartile range, and range with circles indicating outlier values. Asterisk indicates <i>p</i> = 0.003.</p

Primers and probes used for qPCR assays.

a<p>All probes were labeled with Hex at 5′-end and Black Hole Quencher (BHQ) at the 3′-end with the exception for hpdPr762, which was labeled with BHQ at an internal “T” and SpC6 at the 3′-end.</p

Percent of subjects carrying Sp, Hi, Sa, and Mc at baseline and at 2, 7, and 14 days post-inoculation.

<p>Nasopharyngeal samples from 52 subjects were examined at four time points for bacterial DNA and carriage was defined as >100 genomes/ml.</p