Karen A. Barbadora

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.

Treatment of Acute Otitis Media in Children under 2 Years of Age

BACKGROUND Recommendations vary regarding immediate antimicrobial treatment versus watchful waiting for children younger than 2 years of age with acute otitis media. METHODS We randomly assigned 291 children 6 to 23 months of age, with acute otitis media diagnosed with the use of stringent criteria, to receive amoxicillin-clavulanate or placebo for 10 days. We measured symptomatic response and rates of clinical failure. RESULTS Among the children who received amoxicillin-clavulanate, 35% had initial resolution of symptoms by day 2, 61% by day 4, and 80% by day 7; among children who received placebo, 28% had initial resolution of symptoms by day 2, 54% by day 4, and 74% by day 7 (P=0.14 for the overall comparison). For sustained resolution of symptoms, the corresponding values were 20%, 41%, and 67% with amoxicillin-clavulanate, as compared with 14%, 36%, and 53% with placebo (P=0.04 for the overall comparison). Mean symptom scores over the first 7 days were lower for the children treated with amoxicillin-clavulanate than for those who received placebo (P=0.02). The rate of clinical failure--defined as the persistence of signs of acute infection on otoscopic examination--was also lower among the children treated with amoxicillin-clavulanate than among those who received placebo: 4% versus 23% at or before the visit on day 4 or 5 (P<0.001) and 16% versus 51% at or before the visit on day 10 to 12 (P<0.001). Mastoiditis developed in one child who received placebo. Diarrhea and diaper-area dermatitis were more common among children who received amoxicillin-clavulanate. There were no significant changes in either group in the rates of nasopharyngeal colonization with nonsusceptible Streptococcus pneumoniae. CONCLUSIONS Among children 6 to 23 months of age with acute otitis media, treatment with amoxicillin-clavulanate for 10 days tended to reduce the time to resolution of symptoms and reduced the overall symptom burden and the rate of persistent signs of acute infection on otoscopic examination. (Funded by the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT00377260.).

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.

Group A Streptococci Among School-Aged Children: Clinical Characteristics and the Carrier State

Objective. A 4-year longitudinal study of school-aged children was conducted to describe the clinical characteristics and epidemiologic features of infections with group A streptococci (GAS). Methods. Between 1998 and 2002, surveillance throat cultures were performed twice per month (October to May) for a cohort of elementary school children in Pittsburgh, Pennsylvania. In addition, throat cultures were obtained during any respiratory illness. Erythromycin and clindamycin susceptibility testing was performed for all isolates. Molecular typing was performed with field-inversion gel electrophoresis. Representative isolates from each field-inversion gel electrophoresis group were emm typed. Strict definitions were used to characterize each GAS infection. Children were classified into 4 categories each year, ie, single episode, recurrent episodes, carriers of GAS, and no infections. Results. A total of 48 to 100 children per year were studied for 4 years; 61 (49%) were male. The mean age was 9.6 years (range: 5–15 years). A total of 5658 throat cultures were performed; 878 (15.5%) were positive for GAS. Antimicrobial agents were used to treat 209 episodes of infection. Thirteen emm types were observed during the 4-year period. GAS were isolated most often from children who were carriers; isolates from single episodes were next most common. Children carried a single emm type for a mean of 10.8 weeks (range: 3–34 weeks). Carriers were likely to be classified again as carriers in subsequent years and frequently switched emm types. Sixty-two percent of the children had ≥1 year with no infections. Conclusions. GAS infections are common among school-aged children. The majority of positive throat cultures observed in this longitudinal study were obtained from children who were carriers of GAS. Carriers switched emm types but tended to become carriers repeatedly during the study. Practitioners should consider treating children known to be GAS carriers when they develop a new illness that is consistent with streptococcal pharyngitis, because they may acquire new emm types and be at risk for rheumatic heart disease.

A streptococcal score card revisited.

Objective To evaluate the utility of a simple scoring system as a predictor of obtaining a positive throat culture for group A streptococci (GAS). Design Prospective descriptive study. Scores were assigned prior to the availability of the results of throat cultures. Setting Emergency department and walk-in clinic of the Childrens Hospital of Pittsburgh. Patients Patients were 365 children between the ages of two and 16 years with acute onset of sore throat and a history of or documentation of fever within the preceding 24 hours. Interventions A streptococcal score was assigned on the basis of a 6-point schema in which the features were 1) age; 2) season; 3) temperature of at least 38.3°C; 4) adenopathy; 5) pharyngeal erythema, edema, or exudate; and 6) no symptoms of a viral upper respiratory infection (conjunctivitis, rhinorrhea, or cough). A throat culture was performed for the isolation of GAS. Main outcome measure Positive predictive value of the streptococcal score in identifying children with a positive throat culture for GAS. Results A score of 5 or 6 predicted a positive culture for GAS in 59 and 75% of children, respectively. In patients with evidence of acute pharyngitis, the combination of age between five and 15 years, fever and absence of upper respiratory symptoms predicted a positive culture for GAS in 72% of patients. Conclusions The score can be used to predict the likelihood that a throat culture will be positive for GAS.

Reemergence of Macrolide Resistance in Pharyngeal Isolates of Group A Streptococci in Southwestern Pennsylvania

ABSTRACT We previously reported on the emergence of macrolide-resistant pharyngeal isolates of group A streptococci (GAS) in our community. The purpose of the present study was to track longitudinal trends in macrolide resistance in these isolates in southwestern Pennsylvania. Testing for susceptibility to erythromycin and clindamycin was performed for all pharyngeal GAS isolates recovered at the Childrens Hospital of Pittsburgh and a local pediatric practice between September 2001 and May 2002. Macrolide resistance phenotypes and genotypes were determined by double-disk diffusion and PCR, respectively. Strain relatedness was determined by field inversion gel electrophoresis and emm gene sequence typing. A total of 708 isolates of GAS were recovered during the study period; 68 (9.6%) were macrolide resistant, while all isolates were sensitive to clindamycin. The monthly prevalence of macrolide resistance ranged from 0 to 41%. Only 21 of 573 (3.7%) strains recovered from September 2001 through March 2002 were macrolide resistant. A sudden increase in the rate of macrolide resistance (47 of 135 isolates [35%]) was seen in April and May 2002. Sixty-two isolates demonstrated the M phenotype (resistance to macrolide antibiotics), and six isolates demonstrated the MLSB phenotype (resistance to most macrolide, lincosamide, and streptogramin B antibiotics); these isolates were confirmed to be mef(A) and erm(A), respectively. Three unique mef(A) clones and four unique erm(A) clones were identified among the resistant isolates. The MIC at which 50% of isolates are inhibited (MIC50) for the mef(A) strains was 16 μg/ml, while the MIC50 for erm(A) strains was 8 μg/ml. The finding of high levels of macrolide resistance among pharyngeal isolates of GAS for a second successive year in our community raises the concern that this problem may be more common in the United States than was previously appreciated. Longitudinal surveillance of isolates from multiple centers is needed to define the prevalence of antimicrobial agent-resistant GAS in the United States.

Characterization, Distribution, and Expression of Novel Genes among Eight Clinical Isolates of Streptococcus pneumoniae

ABSTRACT Eight low-passage-number Streptococcus pneumoniae clinical isolates, each of a different serotype and a different multilocus sequence type, were obtained from pediatric participants in a pneumococcal vaccine trial. Comparative genomic analyses were performed with these strains and two S. pneumoniae reference strains. Individual genomic libraries were constructed for each of the eight clinical isolates, with an average insert size of ∼1 kb. A total of 73,728 clones were picked for arraying, providing more than four times genomic coverage per strain. A subset of 4,793 clones were sequenced, for which homology searches revealed that 750 (15.6%) of the sequences were unique with respect to the TIGR4 reference genome and 263 (5.5%) clones were unrelated to any available streptococcal sequence. Hypothetical translations of the open reading frames identified within these novel sequences showed homologies to a variety of proteins, including bacterial virulence factors not previously identified in S. pneumoniae. The distribution and expression patterns of 58 of these novel sequences among the eight clinical isolates were analyzed by PCR- and reverse transcriptase PCR-based analyses, respectively. These unique sequences were nonuniformly distributed among the eight isolates, and transcription of these genes in planktonic cultures was detected in 81% (172/212) of their genic occurrences. All 58 novel sequences were transcribed in one or more of the clinical strains, suggesting that they all correspond to functional genes. Sixty-five percent (38/58) of these sequences were found in 50% or less of the clinical strains, indicating a significant degree of genomic plasticity among natural isolates.

Strain-specific virulence phenotypes of Streptococcus pneumoniae assessed using the Chinchilla laniger model of otitis media.

Background Streptococcus pneumoniae [Sp] infection is associated with local and systemic disease. Our current understanding of the differential contributions of genetic strain variation, serotype, and host response to disease phenotype is incomplete. Using the chinchilla model of otitis media [OM] we investigated the disease phenotype generated by the laboratory strain TIGR4 and each of thirteen clinical strains (BS68-75, BS290, BS291, BS293, BS436 and BS437); eleven of the thirteen strains have been genomically sequenced. Methodology/Principal Findings For each strain 100 colony forming units were injected bilaterally into the tympanic bullae of 6 young adult chinchillas under general anesthesia. All animals were examined daily for local and systemic disease by a blinded observer. Pneumatic otoscopy was used to evaluate local disease, and behavioral assessments served as the measure of systemic disease. Virulence scoring was performed using a 4-point scale to assess four clinical parameters [severity and rapidity of local disease onset; and severity and rapidity of systemic disease onset] during a 10-day evaluation period. Highly significant variation was observed among the strains in their ability to cause disease and moribundity. Conclusions/Significance As expected, there was a significant correlation between the rapidity of systemic disease onset and severity of systemic disease; however, there was little correlation between the severity of otoscopic changes and severity of systemic disease. Importantly, it was observed that different strains of the same serotype produced as broad an array of disease phenotypes as did strains of different serotypes. We attribute these phenotypic differences among the strains to the high degree of genomic plasticity that we have previously documented.

Emergence of streptococcus pneumoniae Serogroups 15 and 35 in Nasopharyngeal Cultures From Young Children With Acute Otitis Media

Background: Surveillance of children with acute otitis media (AOM) for nasopharyngeal colonization with Streptococcus pneumoniae before, during and after the introduction of 7-valent pneumococcal conjugate vaccine (PCV7) indicated the near-complete elimination of PCV7 strains and the emergence of pneumococcal serotype 19A. Methods: To determine effects of the introduction of 13-valent pneumococcal conjugate vaccine (PCV13) on pneumococcal nasopharyngeal colonization, we obtained nasopharyngeal cultures from 228 children 6 through 23 of age months presenting with a new episode of AOM during 2012 and 2013 and enrolled in an ongoing clinical trial of antimicrobial efficacy. All children had received at least 2 doses of PCV13. The S. pneumoniae isolates were subjected to serotyping and testing for antimicrobial susceptibility. We compared the findings with results obtained in 3 earlier studies. Results: We found nasopharyngeal colonization with S. pneumoniae in 113 (50%) of the children with AOM. PCV7 and PCV13 serotypes accounted for 2% and 12%, respectively, of the pneumococcal isolates. Of the 14 PCV13 isolates, 8 were serotype 19A. Nonvaccine serotypes accounted for 69% of the isolates. Most frequently occurring were subtypes of serotype 15 (23%) and serotype 35B (9%). Overall, 33% of the isolates were penicillin nonsusceptible, a proportion not significantly different from proportions found in our 3 earlier studies (26%, 36% and 37%, respectively). Serotypes 15 and 35B accounted for 51% of penicillin-nonsusceptible isolates. Conclusions: Expansion of contents of pneumococcal vaccine administered to children is followed by not-fully-predictable changes in nasopharyngeal pneumococcal colonization. Continued surveillance is required to help inform future vaccine development.

Pneumococcal Resistance and Serotype 19A in Pittsburgh-Area Children With Acute Otitis Media Before and After Introduction of 7-Valent Pneumococcal Polysaccharide Vaccine

Methods: Before and after introduction of pneumococcal conjugate vaccine (PCV7), the authors obtained nasopharyngeal (NP) specimens from 3 groups of children aged 6 to 23 months with acute otitis media (AOM): group 1 (pre-PCV7), group 2 (early post-PCV7), and group 3 (late post-PCV7). Results: Of the Streptococcus pneumoniae isolates, the proportion that were vaccine serotypes (VTs) declined progressively (60.4% vs 48.6% vs 5.2% in groups 1, 2, and 3, respectively; P < .001). Concurrently, increases occurred in the proportion of penicillin-nonsusceptible isolates (minimum inhibitory concentration >0.1 µg/mL; 26.7% vs 37.8% vs. 38.5%; P = .12); the proportion of isolates that were serotype 19A (4.0% vs 0% vs 25.9%; P < .001); and the proportion of 19A isolates that were penicillin-nonsusceptible (0% in group 1, 68.6% in group 3; P = .004). Conclusion: Shifts in pneumococcal serotype distribution and increases in penicillin nonsusceptibility among pneumococcal isolates from children with AOM underscore the need for continuing bacteriological surveillance for future vaccine development.