Melinda M. Pettigrew

Microbial interactions during upper respiratory tract infections.

Competitive interactions between bacteria differ by number and species present; thus, vaccination and treatment strategies may alter nasopharyngeal flora and disease susceptibility.

Variation in the Presence of Neuraminidase Genes among Streptococcus pneumoniae Isolates with Identical Sequence Types

ABSTRACT Streptococcus pneumoniae frequently colonizes the upper respiratory tract of young children and is an important cause of otitis media and invasive disease. Carriage is more common than disease, yet the genetic factors that predispose a given clone for disease are not known. The relationship between capsule type, genetic background, and virulence is complex, and important questions remain regarding how pneumococcal clones differ in their ability to cause disease. Pneumococcal neuraminidase cleaves sialic acid-containing substrates and is thought to be important for pneumococcal virulence. We describe the distribution of multilocus sequence types (ST), capsule type, and neuraminidase genes among 342 carriage, middle ear, blood, and cerebrospinal fluid (CSF) pneumococcal strains from young children. We found 149 STs among our S. pneumoniae isolates. nanA was present in all strains, while nanB and nanC were present in 96% and 51% of isolates, respectively. The distribution of nanC varied among the strain collections from different tissue sources (P = 0.03). The prevalence of nanC was 1.41 (95% confidence interval, 1.11, 1.79) times higher among CSF isolates than among carriage isolates. We identified isolates of the same ST that differed in the presence of nanB and nanC. These studies demonstrate that virulence determinants, other than capsule loci, vary among strains of identical ST. Our studies suggest that the presence of nanC may be important for tissue-specific virulence. Studies that both incorporate MLST and take into account additional virulence determinants will provide a greater understanding of the pneumococcal virulence potential.

Microbial Communities of the Upper Respiratory Tract and Otitis Media in Children

ABSTRACT Streptococcus pneumoniae asymptomatically colonizes the upper respiratory tract of children and is a frequent cause of otitis media. Patterns of microbial colonization likely influence S. pneumoniae colonization and otitis media susceptibility. This study compared microbial communities in children with and without otitis media. Nasal swabs and clinical and demographic data were collected in a cross-sectional study of Philadelphia, PA, children (6 to 78 months) (n = 108) during the 2008-2009 winter respiratory virus season. Swabs were cultured for S. pneumoniae. DNA was extracted from the swabs; 16S rRNA gene hypervariable regions (V1 and V2) were PCR amplified and sequenced by Roche/454 Life Sciences pyrosequencing. Microbial communities were described using the Shannon diversity and evenness indices. Principal component analysis (PCA) was used to group microbial community taxa into four factors representing correlated taxa. Of 108 children, 47 (44%) were colonized by S. pneumoniae, and 25 (23%) were diagnosed with otitis media. Microbial communities with S. pneumoniae were significantly less diverse and less even. Two PCA factors were associated with a decreased risk of pneumococcal colonization and otitis media, as follows: one factor included potentially protective flora (Corynebacterium and Dolosigranulum), and the other factor included Propionibacterium, Lactococcus, and Staphylococcus. The remaining two PCA factors were associated with an increased risk of otitis media. One factor included Haemophilus, and the final factor included Actinomyces, Rothia, Neisseria, and Veillonella. Generally, these taxa are not considered otitis media pathogens but may be important in the causal pathway. Increased understanding of upper respiratory tract microbial communities will contribute to the development of otitis media treatment and prevention strategies. IMPORTANCE Otitis media (middle ear infection) is the most common reason for pediatric sick visits in the United States. Streptococcus pneumoniae is a leading otitis media pathogen. S. pneumoniae must colonize the upper respiratory tract and compete with a complex community of nonpathogenic bacteria before infecting the middle ear. We compared microbial communities in the upper respiratory tract of children who had otitis media and those who did not. Members of the normal flora, i.e., Corynebacterium and Dolosigranulum, were protective for S. pneumoniae colonization and otitis media. As expected, the genera Haemophilus was associated with otitis media. Surprisingly, Actinomyces, Rothia, Neisseria, and Veillonella were associated with an increased risk of otitis media. These bacteria are not otitis media pathogens but may be associated with antibiotic use or involved in the causal pathway to disease. Increased understanding of upper respiratory tract microbial communities will lead to new ways to prevent middle ear infections, including probiotics. Otitis media (middle ear infection) is the most common reason for pediatric sick visits in the United States. Streptococcus pneumoniae is a leading otitis media pathogen. S. pneumoniae must colonize the upper respiratory tract and compete with a complex community of nonpathogenic bacteria before infecting the middle ear. We compared microbial communities in the upper respiratory tract of children who had otitis media and those who did not. Members of the normal flora, i.e., Corynebacterium and Dolosigranulum, were protective for S. pneumoniae colonization and otitis media. As expected, the genera Haemophilus was associated with otitis media. Surprisingly, Actinomyces, Rothia, Neisseria, and Veillonella were associated with an increased risk of otitis media. These bacteria are not otitis media pathogens but may be associated with antibiotic use or involved in the causal pathway to disease. Increased understanding of upper respiratory tract microbial communities will lead to new ways to prevent middle ear infections, including probiotics.

Viral-Bacterial Interactions and Risk of Acute Otitis Media Complicating Upper Respiratory Tract Infection

ABSTRACT Acute otitis media (AOM) is a common complication of upper respiratory tract infection whose pathogenesis involves both viruses and bacteria. We examined risks of acute otitis media associated with specific combinations of respiratory viruses and acute otitis media bacterial pathogens. Data were from a prospective study of children ages 6 to 36 months and included viral and bacterial culture and quantitative PCR for respiratory syncytial virus (RSV), human bocavirus, and human metapneumovirus. Repeated-measure logistic regression was used to assess the relationship between specific viruses, bacteria, and the risk of acute otitis media complicating upper respiratory tract infection. In unadjusted analyses of data from 194 children, adenovirus, bocavirus, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis were significantly associated with AOM (P < 0.05 by χ2 test). Children with high respiratory syncytial virus loads (≥3.16 × 107 copies/ml) experienced increased acute otitis media risk. Higher viral loads of bocavirus and metapneumovirus were not significantly associated with acute otitis media. In adjusted models controlling for the presence of key viruses, bacteria, and acute otitis media risk factors, acute otitis media risk was independently associated with high RSV viral load with Streptococcus pneumoniae (odds ratio [OR], 4.40; 95% confidence interval [CI], 1.90 and 10.19) and Haemophilus influenzae (OR, 2.04; 95% CI, 1.38 and 3.02). The risk was higher for the presence of bocavirus and H. influenzae together (OR, 3.61; 95% CI, 1.90 and 6.86). Acute otitis media risk differs by the specific viruses and bacteria involved. Acute otitis media prevention efforts should consider methods for reducing infections caused by respiratory syncytial virus, bocavirus, and adenovirus in addition to acute otitis media bacterial pathogens.

Molecular Epidemiology of Pediatric Pneumococcal Empyema from 2001 to 2007 in Utah

ABSTRACT Utah had a high rate of pediatric pneumococcal empyema (PPE) prior to licensure of the pneumococcal conjugate vaccine (PCV-7) in 2000. The majority (62%) of PPE cases was due to nonvaccine serotypes, primarily Streptococcus pneumoniae serotype 1, multilocus sequence type (MLST) 227. PPE in Utah children has increased over the last decade. It is unclear whether the increase was due to serotype replacement or switch. In this study, we describe the incidence and molecular epidemiology of PPE by MLST in Utah children after the licensure of PCV-7. Empyema rates increased from 8.5/100,000 children in the state of Utah in 2001 to 12.5/100,000 children in 2007 (P = 0.006). Ninety-eight percent was due to nonvaccine serotypes (P < 0.001 when compared to the pre-PCV-7 period). PPE was primarily due to serotypes 1, 3, 19A, and 7F, with MLST demonstrating sequence types (ST) that were commonly present in the United States prior to licensure of PCV-7. Serotype switch was not documented. Replacement disease with common ST of serotypes 1,3, 7F, and 19A rather than serotype switch was responsible for the increase in PPE in Utah children.

Diversity and sharing of Haemophilus influenzae strains colonizing healthy children attending day-care centers

Background. Children attending day-care centers (DCCs) are at risk for Haemophilus influenzae nasopharyngeal colonization and acute otitis media. The degree to which a given strain circulates within a day-care center and the heterogeneity of strains among DCCs in a geographic area are not well-characterized. This study describes the prevalence rates of H. influenzae colonization in a large number of children attending day-care centers and examines the genetic diversity of colonizing strains and the degree of sharing among children. Methods. Throat cultures were collected from 198 healthy children <3 years old attending 16 day-care centers in Michigan. All H. influenzae isolates were genetically typed by enterobacterial repetitive intergenic consensus PCR as the initial screening technique to identify unique strains within each child. Pulsed field gel electrophoresis was used subsequently to examine the genetic diversity of strains between children. Results. There were 127 (64%) children colonized with H. influenzae. Wide variation in rates of colonization was identified among day-care centers (0 to 95%). A total of 179 genetically unique H. influenzae strains were isolated, and 47 children (37%) were colonized with 2 or more genetically distinct H. influenzae organisms. Evidence of sharing of the same strain in different children was found in 13 of 15 colonized DCCs and 23% of genotypically unique strains were shared. Conclusion. The degree of sharing of H. influenzae among children in this study suggests transmission of these potentially pathogenic microorganisms in day-care centers.

Upper respiratory tract microbial communities, acute otitis media pathogens and antibiotic use in healthy and sick children

ABSTRACT The composition of the upper respiratory tract microbial community may influence the risk for colonization by the acute otitis media (AOM) pathogens Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. We used culture-independent methods to describe upper respiratory tract microbial communities in healthy children and children with upper respiratory tract infection with and without concurrent AOM. Nasal swabs and data were collected in a cross-sectional study of 240 children between 6 months and 3 years of age. Swabs were cultured for S. pneumoniae, and real-time PCR was used to identify S. pneumoniae, H. influenzae, and M. catarrhalis. The V1-V2 16S rRNA gene regions were sequenced using 454 pyrosequencing. Microbial communities were described using a taxon-based approach. Colonization by S. pneumoniae, H. influenzae, and M. catarrhalis was associated with lower levels of diversity in upper respiratory tract flora. We identified commensal taxa that were negatively associated with colonization by each AOM bacterial pathogen and with AOM. The balance of these relationships differed according to the colonizing AOM pathogen and history of antibiotic use. Children with antibiotic use in the past 6 months and a greater abundance of taxa, including Lactococcus and Propionibacterium, were less likely to have AOM than healthy children (odds ratio [OR], 0.46; 95% confidence interval [CI], 0.25 to 0.85). Children with no antibiotic use in the past 6 months, a low abundance of Streptococcus and Haemophilus, and a high abundance of Corynebacterium and Dolosigranulum were less likely to have AOM (OR, 0.51; 95% CI, 0.31 to 0.83). An increased understanding of polymicrobial interactions will facilitate the development of effective AOM prevention strategies.

Streptococcus pneumoniae biofilm formation and dispersion during colonization and disease

Streptococcus pneumoniae (the pneumococcus) is a common colonizer of the human nasopharynx. Despite a low rate of invasive disease, the high prevalence of colonization results in millions of infections and over one million deaths per year, mostly in individuals under the age of 5 and the elderly. Colonizing pneumococci form well-organized biofilm communities in the nasopharyngeal environment, but the specific role of biofilms and their interaction with the host during colonization and disease is not yet clear. Pneumococci in biofilms are highly resistant to antimicrobial agents and this phenotype can be recapitulated when pneumococci are grown on respiratory epithelial cells under conditions found in the nasopharyngeal environment. Pneumococcal biofilms display lower levels of virulence in vivo and provide an optimal environment for increased genetic exchange both in vitro and in vivo, with increased natural transformation seen during co-colonization with multiple strains. Biofilms have also been detected on mucosal surfaces during pneumonia and middle ear infection, although the role of these biofilms in the disease process is debated. Recent studies have shown that changes in the nasopharyngeal environment caused by concomitant virus infection, changes in the microflora, inflammation, or other host assaults trigger active release of pneumococci from biofilms. These dispersed bacteria have distinct phenotypic properties and transcriptional profiles different from both biofilm and broth-grown, planktonic bacteria, resulting in a significantly increased virulence in vivo. In this review we discuss the properties of pneumococcal biofilms, the role of biofilm formation during pneumococcal colonization, including their propensity for increased ability to exchange genetic material, as well as mechanisms involved in transition from asymptomatic biofilm colonization to dissemination and disease of otherwise sterile sites. Greater understanding of pneumococcal biofilm formation and dispersion will elucidate novel avenues to interfere with the spread of antibiotic resistance and vaccine escape, as well as novel strategies to target the mechanisms involved in induction of pneumococcal disease.

Prevalence of the hifBC, hmw1A, hmw2A, hmwC, and hia Genes in Haemophilus influenzae Isolates

ABSTRACT Adherence of Haemophilus influenzae to respiratory epithelial cells is the first step in the pathogenesis of H. influenzae infection and is facilitated by the action of several adhesins located on the surface of the bacteria. In this study, prevalences of hifBC, which represent the pilus gene cluster; hmw1A, hmw2A, and hmwC, which represent high-molecular-weight (HMW) adhesin genes; and hia, which represents H. influenzae adhesin (Hia) genes were determined among clinical isolates of encapsulated type b (Hib) and nonencapsulated (NTHi) H. influenzae. hifBC genes were detected in 109 of 170 (64%) Hib strains and in 46 of 162 (28%) NTHi isolates (P = 0.0001) and were more prevalent among the invasive type b strains than invasive NTHi strains (P = 0.00003). Furthermore, hifBC genes were significantly more prevalent (P = 0.0398) among NTHi throat isolates than NTHi middle ear isolates. hmw1A, hmw2A, hmwC, and hia genes were not detected in Hib strains. Among NTHi isolates, the prevalence of hmw1A was 51%, the prevalence of hmw2A was 23%, the prevalence of hmwC was 48%, and the prevalence of hia was 33%. The hmw genes were significantly more prevalent among middle ear than throat isolates, while hia did not segregate with a respiratory tract site. These results show the variability of the presence of adhesin genes among clinical H. influenzae isolates and suggest that hemagglutinating pili may play a larger role in H. influenzae nasopharyngeal colonization than in acute otitis media whereas the HMW adhesins may be virulence factors for acute otitis media.

Dynamic Changes in the Streptococcus pneumoniae Transcriptome during Transition from Biofilm Formation to Invasive Disease upon Influenza A Virus Infection

ABSTRACT Streptococcus pneumoniae is a leading cause of infectious disease globally. Nasopharyngeal colonization occurs in biofilms and precedes infection. Prior studies have indicated that biofilm-derived pneumococci are avirulent. However, influenza A virus (IAV) infection releases virulent pneumococci from biofilms in vitro and in vivo. Triggers of dispersal include IAV-induced changes in the nasopharynx, such as increased temperature (fever) and extracellular ATP (tissue damage). We used whole-transcriptome shotgun sequencing (RNA-seq) to compare the S. pneumoniae transcriptome in biofilms, bacteria dispersed from biofilms after exposure to IAV, febrile-range temperature, or ATP, and planktonic cells grown at 37°C. Compared with biofilm bacteria, actively dispersed S. pneumoniae, which were more virulent in invasive disease, upregulated genes involved in carbohydrate metabolism. Enzymatic assays for ATP and lactate production confirmed that dispersed pneumococci exhibited increased metabolism compared to those in biofilms. Dispersed pneumococci also upregulated genes associated with production of bacteriocins and downregulated colonization-associated genes related to competence, fratricide, and the transparent colony phenotype. IAV had the largest impact on the pneumococcal transcriptome. Similar transcriptional differences were also observed when actively dispersed bacteria were compared with avirulent planktonic bacteria. Our data demonstrate complex changes in the pneumococcal transcriptome in response to IAV-induced changes in the environment. Our data suggest that disease is caused by pneumococci that are primed to move to tissue sites with altered nutrient availability and to protect themselves from the nasopharyngeal microflora and host immune response. These data help explain pneumococcal virulence after IAV infection and have important implications for studies of S. pneumoniae pathogenesis.