Heidi C. Smith-Vaughan

Respiratory Bacterial Pathogens in the Nasopharynx and Lower Airways of Australian Indigenous Children with Bronchiectasis

OBJECTIVE To test the hypothesis that bacterial density, strain diversity, and concordance of pathogens between upper and lower airways are higher in children with bronchiectasis than in those with non-bronchiectatic conditions. STUDY DESIGN Nasopharyngeal (NP) swabs and bronchoalveolar lavage (BAL) fluid were cultured from 45 Indigenous children with bronchiectasis and 30 non-Indigenous children with non-bronchiectatic respiratory symptoms. Lower airway infection was defined as >10(4) colony-forming units of respiratory bacteria/mL of BAL fluid. Concordance was determined by phenotype or genotype. RESULTS NP carriage of Streptococcus pneumoniae, nontypable Haemophilus influenzae (NTHi), and Moraxella catarrhalis, and lower airway infection by NTHi (47% vs 3%), were detected significantly more often in the children with bronchiectasis than in those without this condition. BAL specimens from the infected Indigenous children also showed greater strain diversity (71% vs 0%). Strain concordance in NP and BAL cultures was high in both infected subgroups. CONCLUSIONS The high density and diversity of respiratory bacteria, along with strain concordance between upper and lower airways, found in Indigenous children with bronchiectasis suggest a possible pathogenic role of recurrent aspiration of NP secretions.

Molecular surveillance of true nontypeable Haemophilus influenzae: An evaluation of PCR screening assays

Background Unambiguous 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 Findings Here 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/Significance Our 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.

Pseudomonas pseudomallei isolates collected over 25 years from a non-tropical endemic focus show clonality on the basis of ribotyping

Between 1966 and 1991, melioidosis, a disease caused by Pseudomonas pseudomallei that is mostly confined to tropical regions, occurred in farm animals and a farmer in temperate south-west Western Australia. Using an Escherichia coli probe containing a ribosomal RNA operon, P. pseudomallei DNA from isolates from 8 animals, a soil sample and the human case showed an identical ribotype on Southern blotting. The ribotype was different from the 3 commonest ribotypes seen in tropical Australia. This molecular typing supports the theory of clonal introduction of P. pseudomallei into a non-endemic region, with environmental contamination, local dissemination and persistence over 25 years. As melioidosis is often fatal in humans, such persistence in a temperate region is cause for concern.

Deriving accurate microbiota profiles from human samples with low bacterial content through post-sequencing processing of Illumina MiSeq data

BackgroundThe rapid expansion of 16S rRNA gene sequencing in challenging clinical contexts has resulted in a growing body of literature of variable quality. To a large extent, this is due to a failure to address spurious signal that is characteristic of samples with low levels of bacteria and high levels of non-bacterial DNA. We have developed a workflow based on the paired-end read Illumina MiSeq-based approach, which enables significant improvement in data quality, post-sequencing. We demonstrate the efficacy of this methodology through its application to paediatric upper-respiratory samples from several anatomical sites.ResultsA workflow for processing sequence data was developed based on commonly available tools. Data generated from different sample types showed a marked variation in levels of non-bacterial signal and ‘contaminant’ bacterial reads. Significant differences in the ability of reference databases to accurately assign identity to operational taxonomic units (OTU) were observed. Three OTU-picking strategies were trialled as follows: de novo, open-reference and closed-reference, with open-reference performing substantially better. Relative abundance of OTUs identified as potential reagent contamination showed a strong inverse correlation with amplicon concentration allowing their objective removal. The removal of the spurious signal showed the greatest improvement in sample types typically containing low levels of bacteria and high levels of human DNA. A substantial impact of pre-filtering data and spurious signal removal was demonstrated by principal coordinate and co-occurrence analysis. For example, analysis of taxon co-occurrence in adenoid swab and middle ear fluid samples indicated that failure to remove the spurious signal resulted in the inclusion of six out of eleven bacterial genera that accounted for 80% of similarity between the sample types.ConclusionsThe application of the presented workflow to a set of challenging clinical samples demonstrates its utility in removing the spurious signal from the dataset, allowing clinical insight to be derived from what would otherwise be highly misleading output. While other approaches could potentially achieve similar improvements, the methodology employed here represents an accessible means to exclude the signal from contamination and other artefacts.

Carriage of multiple ribotypes of non-encapsulated Haemophilus influenzae in Aboriginal infants with otitis media

Ribotyping with the restriction enzyme XbaI was used to study the dynamics of Carriage of non-encapsulated Haemophilus influenzae (NCHi) in Aboriginal infants at risk of otitis media. Carriage rates of NCHi in the infants in the community were very high; the median age for detection was 50 days and colonization was virtually 100% by 120 days of age and persisted at a high level throughout the first year of life [1]. Eighteen different ribotypes of NCHi were identified from 34 positive swabs taken from 3 infants over a period of 9 months. The same ribotypes were recovered for up to 3 months from consecutive swabs of individual infants, and 12 of 27 swabs (44.4%) yielded two ribotypes from four colonies typed. Statistical analysis suggested that most swabs would have been positive for two ribotypes if enough colonies had been typed although the second most frequent ribotype was detected on average in only 13% of strains. Early colonization and carriage of multiple ribotypes of NCHi may help to explain the chronicity of carriage and thus the persistence of otitis media in Aboriginal infants.

Longitudinal Nasopharyngeal Carriage and Antibiotic Resistance of Respiratory Bacteria in Indigenous Australian and Alaska Native Children with Bronchiectasis

Background Indigenous children in Australia and Alaska have very high rates of chronic suppurative lung disease (CSLD)/bronchiectasis. Antibiotics, including frequent or long-term azithromycin in Australia and short-term beta-lactam therapy in both countries, are often prescribed to treat these patients. In the Bronchiectasis Observational Study we examined over several years the nasopharyngeal carriage and antibiotic resistance of respiratory bacteria in these two PCV7-vaccinated populations. Methods Indigenous children aged 0.5–8.9 years with CSLD/bronchiectasis from remote Australia (n = 79) and Alaska (n = 41) were enrolled in a prospective cohort study during 2004–8. At scheduled study visits until 2010 antibiotic use in the preceding 2-weeks was recorded and nasopharyngeal swabs collected for culture and antimicrobial susceptibility testing. Analysis of respiratory bacterial carriage and antibiotic resistance was by baseline and final swabs, and total swabs by year. Results Streptococcus pneumoniae carriage changed little over time. In contrast, carriage of Haemophilus influenzae declined and Staphylococcus aureus increased (from 0% in 2005–6 to 23% in 2010 in Alaskan children); these changes were associated with increasing age. Moraxella catarrhalis carriage declined significantly in Australian, but not Alaskan, children (from 64% in 2004–6 to 11% in 2010). While beta-lactam antibiotic use was similar in the two cohorts, Australian children received more azithromycin. Macrolide resistance was significantly higher in Australian compared to Alaskan children, while H. influenzae beta-lactam resistance was higher in Alaskan children. Azithromycin use coincided significantly with reduced carriage of S. pneumoniae, H. influenzae and M. catarrhalis, but increased carriage of S. aureus and macrolide-resistant strains of S. pneumoniae and S. aureus (proportion of carriers and all swabs), in a ‘cumulative dose-response’ relationship. Conclusions Over time, similar (possibly age-related) changes in nasopharyngeal bacterial carriage were observed in Australian and Alaskan children with CSLD/bronchiectasis. However, there were also significant frequency-dependent differences in carriage and antibiotic resistance that coincided with azithromycin use.

A variable region within the genome of Streptococcus pneumoniae contributes to strain-strain variation in virulence.

The bacterial factors responsible for the variation in invasive potential between different clones and serotypes of Streptococcus pneumoniae are largely unknown. Therefore, the isolation of rare serotype 1 carriage strains in Indigenous Australian communities provided a unique opportunity to compare the genomes of non-invasive and invasive isolates of the same serotype in order to identify such factors. The human virulence status of non-invasive, intermediately virulent and highly virulent serotype 1 isolates was reflected in mice and showed that whilst both human non-invasive and highly virulent isolates were able to colonize the murine nasopharynx equally, only the human highly virulent isolates were able to invade and survive in the murine lungs and blood. Genomic sequencing comparisons between these isolates identified 8 regions >1 kb in size that were specific to only the highly virulent isolates, and included a version of the pneumococcal pathogenicity island 1 variable region (PPI-1v), phage-associated adherence factors, transporters and metabolic enzymes. In particular, a phage-associated endolysin, a putative iron/lead permease and an operon within PPI-1v exhibited niche-specific changes in expression that suggest important roles for these genes in the lungs and blood. Moreover, in vivo competition between pneumococci carrying PPI-1v derivatives representing the two identified versions of the region showed that the version of PPI-1v in the highly virulent isolates was more competitive than the version from the less virulent isolates in the nasopharyngeal tissue, blood and lungs. This study is the first to perform genomic comparisons between serotype 1 isolates with distinct virulence profiles that correlate between mice and humans, and has highlighted the important role that hypervariable genomic loci, such as PPI-1v, play in pneumococcal disease. The findings of this study have important implications for understanding the processes that drive progression from colonization to invasive disease and will help direct the development of novel therapeutic strategies.

Nasopharyngeal microbial interactions in the era of pneumococcal conjugate vaccination

The nasopharynx of children is often colonised by microorganisms such as Streptococcus pneumoniae (the pneumococcus) that can cause infections including pneumonia and otitis media. In this complex environment, bacteria and viruses may impact each other through antagonistic as well as synergistic interactions. Vaccination may alter colonisation dynamics, evidenced by the rise in non-vaccine serotypes following pneumococcal conjugate vaccination. Discovery of an inverse relationship between S. pneumoniae and Staphylococcus aureus carriage generated concern that pneumococcal vaccination could increase S. aureus carriage and disease. Here we review data on co-colonisation of pathogens in the nasopharynx, focusing on S. pneumoniae and the impact of pneumococcal vaccination. Thus far, pneumococcal vaccination has not had a sustained impact on S. aureus carriage but it is associated with an increase in non-typeable Haemophilus influenzae in acute otitis media aetiology. Advances in bacterial and viral detection methodologies have facilitated research in nasopharyngeal microbiology and will aid investigation of potential vaccine-induced changes, particularly when baseline studies can be conducted prior to pneumococcal vaccine introduction.

Random colony selection versus colony morphology for detection of multiple pneumococcal serotypes in nasopharyngeal swabs

Detection of multiple serotype carriage is important in understanding pneumococcal epidemiology. We compared random selection of 4 pneumococcal colonies per swab with selection by colony morphology. Multiple serotypes were detected in 20% of 98 swabs; 14% by morphology and 17% by random selection. Selection by morphology was more efficient per colony, but underestimated the true rate of multiple carriage.

Viral-bacterial co-infection in Australian Indigenous children with acute otitis media

BackgroundAcute otitis media with perforation (AOMwiP) affects 40% of remote Indigenous children during the first 18 months of life. Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are the primary bacterial pathogens of otitis media and their loads predict clinical ear state. Our hypothesis is that antecedent respiratory viral infection increases bacterial density and progression to perforation.MethodsA total of 366 nasopharyngeal swabs from 114 Indigenous children were retrospectively examined. A panel of 17 respiratory viruses was screened by PCR, and densities of S. pneumoniae, H. influenzae and M. catarrhalis were estimated by quantitative real time PCR. Data are reported by clinical ear state.ResultsM. catarrhalis (96%), H. influenzae (91%), S. pneumoniae (89%) and respiratory viruses (59%) were common; including rhinovirus (HRV) (38%), polyomavirus (HPyV) (14%), adenovirus (HAdV) (13%), bocavirus (HBoV) (8%) and coronavirus (HCoV) (4%). Geometric mean bacterial loads were significantly higher in children with acute otitis media (AOM) compared to children without evidence of otitis media. Children infected with HAdV were 3 times more likely (p < 0.001) to have AOM with or without perforation.ConclusionThis study confirms a positive association between nasopharyngeal bacterial load and clinical ear state, exacerbated by respiratory viruses, in Indigenous children. HAdV was independently associated with acute ear states.