Nontypeable pneumococcal isolates among navajo and white mountain apache communities: are these really a cause of invasive disease?

BACKGROUND: Pneumococci could evade pneumococcal conjugate vaccines (PCV) by modifying, mutating, or deleting vaccine-serotype capsule genes or by downregulating capsule production. We sought to assess whether pneumococci that are nontypeable (NT) by the Quellung reaction truly lack capsule genes or are failing to produce capsule in vitro. METHODS: We applied multilocus sequence typing and a microarray for detection of pneumococcal polysaccharide capsule biosynthesis genes to NT carriage (children aged <5 years; years 1997-2000, 2006-2008) and NT invasive disease (IPD) (all ages; years 1994-2007) isolates from Native American communities. RESULTS: Twenty-seven of 28 (96.4%) NT IPD isolates had sequence types (STs) typically found among typeable IPD isolates and contained whole or fragments of capsule genes that matched known serotypes; 1 NT-IPD isolate had a profile resembling NT carriage isolates. Forty-nine of 76 (64.5%) NT carriage isolates had STs that typically lack capsule genes and were similar to NT carriage isolates found globally. CONCLUSIONS: This is the first documentation of IPD from an NT strain confirmed to lack all known capsule genes. Most NT IPD isolates have or had the capacity to produce capsule, whereas a majority of NT carriage isolates lack this capacity. We found no evidence of pneumococcal adaptation to PCV7 via downregulation or deletion of vaccine-serotype capsule genes.

Impact of more than a decade of pneumococcal conjugate vaccine use on carriage and invasive potential in Native American communities.

BACKGROUND: We assessed the impact of 12 years of pneumococcal conjugate vaccine (PCV7) use on pneumococcal nasopharyngeal carriage and serotype-specific invasive disease potential among Native Americans. METHODS: Families were enrolled in a carriage study from 2006 to 2008; nasopharyngeal specimens and risk factor information were collected monthly for 7 visits. Pneumococcal carriage prevalence was compared with that before (1998-2000) and during (2001-2002) PCV7 introduction. We compared invasive disease incidence and carriage prevalence before and after PCV7 introduction to estimate changes in serotype-specific invasive potential. RESULTS: We enrolled 1077 subjects from 302 households. There was an absolute reduction in carriage prevalence of 8.0% (95% confidence interval [CI], 4.5%-11.4%) in children aged <5 years and 3.1% (95% CI, 1.1%-5.1%) in adults. In children aged <5 years, vaccine-serotype carriage prevalence decreased by 22.8% (95% CI, 20.1%-25.3%), and nonvaccine serotype (NVT) increased by 15.9% (95% CI, 12.4%-19.3%). No significant change was detected in serotype-specific invasive potential after PCV7 introduction. CONCLUSIONS: Pneumococcal carriage prevalence decreased in all ages since PCV7 introduction; vaccine-serotype carriage has been nearly eliminated, whereas the prevalence of NVT carriage has increased. The increase in the NVT invasive disease rate seems to be proportional to the increase in colonization prevalence.

Identification and characterization of novel catalytic bioscavengers of organophosphorus nerve agents.

In an effort to discover novel catalytic bioscavengers of organophosphorus (OP) nerve agents, cell lysates from a diverse set of bacterial strains were screened for their capacity to hydrolyze the OP nerve agents VX, VR, and soman (GD). The library of bacterial strains was identified using both random and rational approaches. Specifically, two representative strains from eight categories of extremophiles were chosen at random. For the rational approach, the protein sequence of organophosphorus hydrolase (OPH) from Brevundimonas diminuta was searched against a non-redundant protein database using the Basic Local Alignment Search Tool to find regions of local similarity between sequences. Over 15 protein sequences with significant sequence similarity to OPH were identified from a variety of bacterial strains. Some of these matches were based on predicted protein structures derived from bacterial genome sequences rather than from bona fide proteins isolated from bacteria. Of the 25 strains selected for nerve agent testing, three bacterial strains had measurable levels of OP hydrolase activity. These strains are Ammoniphilus oxalaticus, Haloarcula sp., and Micromonospora aurantiaca. Lysates from A. oxalaticus had detectable hydrolysis of VR; Haloarcula sp. had appreciable hydrolysis of VX and VR, whereas lysates from M. aurantiaca had detectable hydrolysis of VR and GD.

Pneumococcal sequence type replacement among American Indian children: a comparison of pre- and routine-PCV7 eras.

BACKGROUND: Multi-locus sequence typing (MLST) of pneumococcal isolates collected during an efficacy trial of the 7-valent pneumococcal conjugate vaccine (PCV7) among Navajo and White Mountain Apache children from 1998 to 2000 showed a non-differential expansion of pre-existing sequence types (STs) and only one capsule-switching event in the PCV7-randomized communities. PCV7 was introduced as a routine infant vaccine in October 2000. We assessed variability in PCV7 effectiveness and mechanisms of ST replacement after prolonged routine PCV7 use. METHODS: We applied MLST to 267 non-vaccine type pneumococcal carriage and invasive disease isolates from Navajo and White Mountain Apache children from 2006 to 2008, and compared them to those from 1998 to 2000. Microarray was used to confirm capsule switching events. RESULTS: The primary mechanism of ST replacement among Navajo and White Mountain Apache children was expansion of existing STs, although introduction of new STs was an important secondary mechanism. ST199, a majority being serotype 19A, was the most common ST in both eras. Only ST193 (serotype 21) was preferentially expanding in the PCV7 era. Three examples of capsule switching were identified. No variability in vaccine effectiveness by ST was observed. CONCLUSION: We did not observe an influence of ST on PCV7 serotype-specific effectiveness, although some STs may be favored in replacement.

Marked increase in biofilm-derived rough pneumococcal variants and rifampin-resistant strains not due to hex gene mutations.

Otitis, pneumonia, and meningitis are tissue-based pneumococcal infections that can be associated with biofilms. The emergence of phenotypic rough variants, also known as acapsular small-colony variants, is essential for pneumococcal biofilm formation. These rough variants can increase nearly 100-fold in biofilms over time and can arise through single nucleotide polymorphisms (SNPs), deletions, or tandem duplications in the first gene of the capsular operon, cps3D. We detected a 100-fold increase in rifampin-resistant (Rif(r)) mutants in biofilms compared to planktonic cultures using a nonvaccine serotype 3 strain, which is causing an increasing number of cases of otitis in the 7-valent pneumococcal conjugate vaccine era. Since both rough variants and Rif(r) strains can arise through SNPs, they could emerge due to alteration of the mismatch repair (MMR) system. The Hex system, a pneumococcal MMR system, repairs mismatches during replication and transformation. In this study, no mutations were detected in the hexAB gene sequences among several rough variants with unique mutations in the cps3D gene. Within a hexA null mutant grown in broth, we detected only a 17.5-fold increase in rough variants compared to the wild-type parental strain. Taken together, these data suggest that mutations in the hex genes and modulation of hexA activity are unlikely to account for the generation of biofilm-derived rough variants.