Novel Type of Streptococcus pneumoniae Causing Multidrug-Resistant Acute Otitis Media in Children

A new multidrug-resistant strain of serotype 19A has been characterized in upstate New York

Dual Orientation of the Outer Membrane Lipoprotein P6 of Nontypeable Haemophilus influenzae

The majority of outer membrane (OM) lipoproteins in Gram-negative bacteria are tethered to the membrane via an attached lipid moiety and oriented facing in toward the periplasmic space; a few lipoproteins have been shown to be surface exposed. The outer membrane lipoprotein P6 from the Gram-negative pathogenic bacterium nontypeable Haemophilus influenzae (NTHi) is surface exposed and a leading vaccine candidate for prevention of NTHi infections. However, we recently found that P6 is not a transmembrane protein as previously thought (L. V. Michel, B. Kalmeta, M. McCreary, J. Snyder, P. Craig, M. E. Pichichero, Vaccine 29:1624–1627, 2011). Here we pursued studies to show that P6 has a dual orientation, existing infrequently as surface exposed and predominantly as internally oriented toward the periplasmic space. Flow cytometry using three monoclonal antibodies with specificity for P6 showed surface staining of whole NTHi cells. Confocal microscopy imaging confirmed that antibodies targeted surface-exposed P6 of intact NTHi cells and not internal P6 in membrane-compromised or dead cells. Western blots of two wild-type NTHi strains and a mutant NTHi strain that does not express P6 showed that P6 antibodies do not detect a promiscuous epitope on NTHi. Depletion of targets to nonlipidated P6 significantly decreased bactericidal activity of human serum. Protease digestion of surface-exposed P6 demonstrated that P6 is predominantly internally localized in a manner similar to its homologue Pal in Escherichia coli. We conclude that P6 of NTHi is likely inserted into the OM in two distinct orientations, with the predominant orientation facing in toward the periplasm

Safety and Efficacy of Gatifloxacin Therapy for Children with Recurrent Acute Otitis Media (AOM) and/or AOM Treatment Failure

Background. Because of concerns about arthrotoxicity, fluoroquinolones are restricted for use in children. This study describes the safety and efficacy of gatifloxacin when used for treatment of children with recurrent acute otitis media (ROM) or acute otitis media (AOM) treatment failure (AOMTF). Methods. We performed an analysis of 867 children included in 4 clinical trials who had ROM and/or AOMTF and were treated with gatifloxacin (10 mg/kg once daily for 10 days). Results. Gatifloxacin had adverse event rates that were similar overall to those of a comparator antibiotic (amoxicillin-clavulanate), except for increased diarrhea in children !2 years old receiving amoxicillin-clavulanate. There was no evidence of arthrotoxicity, hepatotoxicity, alteration of glucose homeostasis, or central nervous system toxicity acutely or during 1 year follow-up in any child. Regarding efficacy, in 2 noncomparative trials, the gatifloxacin cure rate of AOM was 89% (95% confidence interval [CI], 83%-95%) at the test of cure (TOC) visit, 3-10 days after completion of therapy. In 2 comparative trials of gatifloxacin versus amoxicillin-clavulanate, the efficacy of gatifloxacin was 88% (95% CI, 82%-94%). Gatifloxacin led to better clinical outcomes than amoxicillinclavulanate for AOMTF (91% vs. 81%; ), for AOMTF and age !2 years old (89% vs. 69%; ), P p .029 P p .009 and for severe AOM in children !2 years old (90% vs. 75%; ). Among children with AOMTF previously P p .012 treated with amoxicillin-clavulanate or ceftriaxone injections, gatifloxacin cure rates were high (88% and 75%, respectively). Conclusions. Gatifloxacin appears to be safe for children, with no evidence of producing arthrotoxicity in 867 children exposed to the antibiotic when used as treatment for ROM and AOMTF

Molecular Surveillance of True Nontypeable Haemophilus influenzae: An Evaluation of PCR Screening Assays

BackgroundUnambiguous identification of nontypeable Haemophilus influenzae (NTHi) is not possible by conventional microbiology. Molecular characterisation of phenotypically defined NTHi isolates suggests that up to 40% are Haemophilus haemolyticus (Hh); however, the genetic similarity of NTHi and Hh limits the power of simple molecular techniques such as PCR for species discrimination.Methodology/Principal FindingsHere we assess the ability of previously published and novel PCR-based assays to identify true NTHi. Sixty phenotypic NTHi isolates, classified by a dual 16S rRNA gene PCR algorithm as NTHi (n = 22), Hh (n = 27) or equivocal (n = 11), were further characterised by sequencing of the 16S rRNA and recA genes then interrogated by PCR-based assays targeting the omp P2, omp P6, lgtC, hpd, 16S rRNA, fucK and iga genes. The sequencing data and PCR results were used to define NTHi for this study. Two hpd real time PCR assays (hpd#1 and hpd#3) and the conventional iga PCR assay were equally efficient at differentiating study-defined NTHi from Hh, each with a receiver operator characteristic curve area of 0.90 [0.83; 0.98]. The hpd#1 and hpd#3 assays were completely specific against a panel of common respiratory bacteria, unlike the iga PCR, and the hpd#3 assay was able to detect below 10 copies per reaction.Conclusions/SignificanceOur data suggest an evolutionary continuum between NTHi and Hh and therefore no single gene target could completely differentiate NTHi from Hh. The hpd#3 real time PCR assay proved to be the superior method for discrimination of NTHi from closely related Haemophilus species with the added potential for quantification of H. influenzae directly from specimens. We suggest the hpd#3 assay would be suitable for routine NTHi surveillance and to assess the impact of antibiotics and vaccines, on H. influenzae carriage rates, carriage density, and disease

Streptococcus pneumoniae Clonal Complex 199: Genetic Diversity and Tissue-Specific Virulence

Streptococcus pneumoniae is an important cause of otitis media and invasive disease. Since introduction of the heptavalent pneumococcal conjugate vaccine, there has been an increase in replacement disease due to serotype 19A clonal complex (CC)199 isolates. The goals of this study were to 1) describe genetic diversity among nineteen CC199 isolates from carriage, middle ear, blood, and cerebrospinal fluid, 2) compare CC199 19A (n = 3) and 15B/C (n = 2) isolates in the chinchilla model for pneumococcal disease, and 3) identify accessory genes associated with tissue-specific disease among a larger collection of S. pneumoniae isolates. CC199 isolates were analyzed by comparative genome hybridization. One hundred and twenty-seven genes were variably present. The CC199 phylogeny split into two main clades, one comprised predominantly of carriage isolates and another of disease isolates. Ability to colonize and cause disease did not differ by serotype in the chinchilla model. However, isolates from the disease clade were associated with faster time to bacteremia compared to carriage clade isolates. One 19A isolate exhibited hypervirulence. Twelve tissue-specific genes/regions were identified by correspondence analysis. After screening a diverse collection of 326 isolates, spr0282 was associated with carriage. Four genes/regions, SP0163, SP0463, SPN05002 and RD8a were associated with middle ear isolates. SPN05002 also associated with blood and CSF, while RD8a associated with blood isolates. The hypervirulent isolate's genome was sequenced using the Solexa paired-end sequencing platform and compared to that of a reference serotype 19A isolate, revealing the presence of a novel 20 kb region with sequence similarity to bacteriophage genes. Genetic factors other than serotype may modulate virulence potential in CC199. These studies have implications for the long-term effectiveness of conjugate vaccines. Ideally, future vaccines would target common proteins to effectively reduce carriage and disease in the vaccinated population

Pneumococcal whole-cell and protein-based vaccines: changing the paradigm

Epidemiologic evaluations of Streptococcus pneumoniae nasopharyngeal (NP) colonization and pneumococcal disease suggest that newer serotypes in future formulations of pneumococcal conjugate vaccines (PCVs) are needed and there may need to be continued reformulations because there are many new emerging serotypes expressed by pneumococci. Areas covered: Mechanisms of protection by next-generation whole-cell vaccine (WCV) and/or multi-component pneumococcal purified protein vaccines (PPVs) in development for prevention of pneumococcal infections. Expert commentary: A long-term strategy for prevention of pneumococcal disease will likely include WCV and PPVs. However these vaccines will impact disease pathogenesis in a different manner than PCVs. Prevention of pneumococcal NP colonization should not be expected, nor is it desirable because risks for NP colonization by other replacement organisms into the ecological niche vacated by all pneumococci may have consequences. The expression biology of capsule and surface protein antigens are phase dependent. Therefore, the immune response will be different and the mechanism of protection divergent. WCVs and PPVs may be alternative strategies in low income developing countries to protect against invasive disease and reduce NP carriage load