Effects of Vaccination with 10-Valent Pneumococcal Non-Typeable Haemophilus influenza Protein D Conjugate Vaccine (PHiD-CV) on the Nasopharyngeal Microbiome of Kenyan Toddlers.

OBJECTIVE: Pneumococcal conjugate vaccines reduce the prevalence of vaccine serotypes carried in the nasopharynx. Because this could alter carriage of other potential pathogens, we assessed the nasopharyngeal microbiome of children who had been vaccinated with 10-valent pneumococcal non-typeable Haemophilus influenzae protein-D conjugate vaccine (PHiD-CV). METHODS: Profiles of the nasopharyngeal microbiota of 60 children aged 12-59 months, who had been randomized to receive 2 doses of PHiD-CV (n=30) or Hepatitis A vaccine (n=30) 60 days apart, were constructed by 16S rRNA gene pyrosequencing of swab specimens collected before vaccination and 180 days after dose 1. RESULTS: Prior to vaccination, Moraxella catarrhalis (median of 12.3% of sequences/subject), Streptococcus pneumoniae (4.4%) and Corynebacterium spp. (5.6%) were the most abundant nasopharyngeal bacterial species. Vaccination with PHiD-CV did not significantly alter the species composition, abundance, or prevalence of known pathogens. Distinct microbiomes were identified based on the abundances of Streptococcus, Moraxella, and Haemophilus species. These microbiomes shifted in composition over the study period and were independent of age, sex, school attendance, antibiotic exposure, and vaccination. CONCLUSIONS: Vaccination of children with two doses of PHiD-CV did not significantly alter the nasopharyngeal microbiome. This suggests limited replacement carriage with pathogens other than non-vaccine strains of S. pneumoniae. TRIAL REGISTRATION: clinicaltrials.gov NCT01028326.

Sinus microbiota varies among chronic rhinosinusitis phenotypes and predicts surgical outcome.

BACKGROUND: Chronic rhinosinusitis (CRS) is a prevalent multifactorial disease process in which bacteria are believed to play a role in the propagation of inflammation. Multiple subtypes of CRS have been described based on clinical and pathologic features, but a detailed examination of the sinus microbiota in patients with CRS and its clinical subtypes has yet to be performed. OBJECTIVE: We sought to examine the resident microbiota of CRS subtypes and determine whether bacterial diversity is a predictor of disease outcomes. METHODS: Sinus swabs from patients with CRS and healthy subjects collected during endoscopic sinus surgery were analyzed by means of molecular phylogenetic analysis of 16S rDNA pyrosequences. RESULTS: Fifty-six patients with CRS and 26 control subjects were studied. Biodiversity was similar between the CRS and control groups. Among the CRS subtypes examined, only 2 conditions (presence of purulence and comorbid condition of asthma) were associated with significant alterations in microbial community composition. In 27 patients with CRS who were followed postoperatively, those with better outcomes had more diverse bacterial communities present at the time of surgery, along with higher relative abundances of Actinobacteria. CONCLUSION: Analysis of microbiota in a large cohort reveals that particular CRS phenotypes (asthma and purulence) are characterized by distinct compositions of resident bacterial communities. We found that bacterial diversity and composition are predictors of surgical outcome, promoting the concept of community ecology in patients with CRS.

Sinus culture poorly predicts resident microbiota.

BACKGROUND: Chronic rhinosinusitis (CRS) is an inflammatory disorder of the paranasal sinuses in which bacteria are implicated. Culture-based assays are commonly used in clinical and research practice; however, culture conditions may not accurately detect the full range of microorganisms present in a sample. The objective of this study was to determine the accuracy of clinical culture of CRS specimens compared with DNA-based molecular techniques. METHODS: Ethmoid samples from 54 CRS patients collected during endoscopic sinus surgery were analyzed by both clinical culture and 16S ribosomal RNA (rRNA) gene sequencing. The association between 16S relative abundance and detection by culture was determined using logistic regression. RESULTS: Each subject had an average of 3 isolates identified by bacterial culture and 21.5 ± 12.5 species identified by 16S sequencing. On average, 1.6 dominant taxa (>10% abundance) per subject were identified using molecular techniques, but only 47.7% of these taxa were identified by culture. Low abundance taxa (abundance <1%) were detected in only 4.5% of cultures. The odds that any organism would be detected by culture were 2.3 times higher with each 10% increase in relative abundance (p < 0.01). Conversely, only 29.5% of isolates identified by culture represented the dominant species, whereas 40% accounted for species with 1% to 10% abundance. Interestingly, 12% of isolates detected by culture were not identified by 16S pyrosequencing. CONCLUSION: Standard clinical culture is a poor representation of resident microbiota. The incorporation of modern culture-independent techniques into clinical and research practices provides additional information that may be relevant for CRS.

Effect of antibiotic stewardship programmes on Clostridium difficile incidence: a systematic review and meta-analysis.

OBJECTIVES: Despite vigorous infection control measures, Clostridium difficile continues to cause significant disease burden. Antibiotic stewardship programmes (ASPs) may prevent C. difficile infections by limiting exposure to certain antibiotics. Our objective was to perform a meta-analysis of published studies to assess the effect of ASPs on the risk of C. difficile infection in hospitalized adult patients. METHODS: Searches of PubMed, Web of Science, Cumulative Index to Nursing and Allied Health Literature and two Cochrane databases were conducted to find all published studies on interventions related to antibiotic stewardship and C. difficile. Two investigators independently assessed study eligibility and extracted data. Risk of bias was assessed using the Downs and Black tool. Risk ratios were pooled using random effects models. Heterogeneity was evaluated using the I(2) statistic. RESULTS: The final search yielded 891 articles; 78 full articles were reviewed and 16 articles were identified for inclusion. Included articles used quasi-experimental (interrupted time series or before-after) or observational (case-control) study designs. When the results of all studies were pooled in a random effects model, a significant protective effect (pooled risk ratio 0.48; 95% CI: 0.38, 0.62) was observed between ASPs and C. difficile incidence. When stratified by intervention type, a significant effect was found for restrictive ASPs (complete removal of drug or prior approval requirement). Furthermore, ASPs were particularly effective in geriatric settings. CONCLUSIONS: Restrictive ASPs can be used to reduce the risk of C. difficile infection.

Explicet: graphical user interface software for metadata-driven management, analysis and visualization of microbiome data.

Studies of the human microbiome, and microbial community ecology in general, have blossomed of late and are now a burgeoning source of exciting research findings. Along with the advent of next-generation sequencing platforms, which have dramatically increased the scope of microbiome-related projects, several high-performance sequence analysis pipelines (e.g. QIIME, MOTHUR, VAMPS) are now available to investigators for microbiome analysis. The subject of our manuscript, the graphical user interface-based Explicet software package, fills a previously unmet need for a robust, yet intuitive means of integrating the outputs of the software pipelines with user-specified metadata and then visualizing the combined data.

Effects of different complementary feeding regimens on iron status and enteric microbiota in breastfed infants.

OBJECTIVE: To compare iron status in breastfed infants randomized to groups receiving complementary feeding regimens that provided iron from fortified infant cereals or meats, and to examine the development of the enteric microbiota in these groups. STUDY DESIGN: Forty-five exclusively breastfed 5-month-old infants were randomized to 1 of 3 feeding groups (FGs)-commercially available pureed meats, iron- and zinc-fortified infant cereals, or iron-only fortified infant cereals-as the first and primary complementary food through 9-10 months of age. Dietary iron was determined by monthly 3-day diet records. Iron status was assessed at the end of the study by measurements of hemoglobin, serum ferritin, and soluble transferrin receptor levels. In a subsample of 14 infants, enteric microbiota were profiled in monthly stool samples (5-9 months) by 16S ribosomal RNA gene pyrosequencing. RESULTS: Infants in the 2 cereal FGs had 2- to 3-fold greater daily iron intakes versus the meat FG (P < .0001). More than one-quarter (27%) of the infants had a low serum ferritin level, and 36% were mildly anemic, with no significant differences by FG; more infants in the meat FG had a high soluble transferrin receptor value (P = .03). Sequence analysis identified differences by time and FG in the abundances of several bacterial groups, including significantly more abundant butyrate-producing Clostridium group XIVa in the meat FG (P = .01) CONCLUSION: A high percentage of healthy infants who were breastfed-only were iron-deficient, and complementary feeding, including iron exposure, influenced the development of the enteric microbiota. If these findings are confirmed, then reconsideration of strategies to both meet infants' iron requirements and optimize the developing microbiome may be warranted.

Sex differences in the gut microbiome drive hormone-dependent regulation of autoimmunity.

Microbial exposures and sex hormones exert potent effects on autoimmune diseases, many of which are more prevalent in women. We demonstrate that early-life microbial exposures determine sex hormone levels and modify progression to autoimmunity in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D). Colonization by commensal microbes elevated serum testosterone and protected NOD males from T1D. Transfer of gut microbiota from adult males to immature females altered the recipient's microbiota, resulting in elevated testosterone and metabolomic changes, reduced islet inflammation and autoantibody production, and robust T1D protection. These effects were dependent on androgen receptor activity. Thus, the commensal microbial community alters sex hormone levels and regulates autoimmune disease fate in individuals with high genetic risk.

Prevalence and abundance of Staphylococcus aureus in the middle meatus of patients with chronic rhinosinusitis, nasal polyps, and asthma.

BACKGROUND: Chronic rhinosinusitis (CRS) is an idiosyncratic and multifactorial disease process. Bacteria play a role in some patients, by infection or stimulation of inflammation. Staphylococcus aureus (SA) appears to be implicated in a number of infectious and inflammatory mechanisms, and may be particularly relevant in CRS patients with nasal polyps and asthma. METHODS: Middle meatus swabs from control and CRS patients collected during endoscopic sinus surgery were analyzed by quantitative polymerase chain reaction (QPCR). Total bacterial count, SA prevalence, and SA abundance were examined with respect to patient demographics and disease characteristics. RESULTS: Total bacteria, as measured by QPCR, was not statistically different between controls, CRS without nasal polyps (CRSsNP), CRS with nasal polyps (CRSwNP), or CRS with asthma groups (p < 0.09). Total bacterial counts did not correlate with disease severity as measured by Lund-Mackay computed tomography (CT) scores (p = 0.65). The prevalence of SA was similar between groups (15-25%); however, the abundance increased in CRS patients with allergic rhinitis, nasal polyps, and asthma. CONCLUSION: The paranasal sinuses are not sterile. SA is implicated in a subset of CRS patients with nasal polyps and/or asthma. Further study is required to predict this subset of patients, and to define the mechanisms of SA pathogenesis.

The microbiome of the middle meatus in healthy adults.

Rhinitis and rhinosinusitis are multifactorial disease processes in which bacteria may play a role either in infection or stimulation of the inflammatory process. Rhinosinusitis has been historically studied with culture-based techniques, which have implicated several common pathogens in disease states. More recently, the NIH Human Microbiome Project has examined the microbiome at a number of accessible body sites, and demonstrated differences among healthy and diseased patients. Recent DNA-based sinus studies have suggested that healthy sinuses are not sterile, as was previously believed, but the normal sinonasal microbiome has yet to be thoroughly examined. Middle meatus swab specimens were collected from 28 consecutive patients presenting with no signs or symptoms of rhinosinusitis. Bacterial colonization was assessed in these specimens using quantitative PCR and 16S rRNA pyrosequencing. All subjects were positive for bacterial colonization of the middle meatus. Staphylococcus aureus, Staphylococcus epidermidis and Propionibacterium acnes were the most prevalent and abundant microorganisms detected. Rich and diverse bacterial assemblages are present in the sinonasal cavity in the normal state, including opportunistic pathogens typically found in the nasopharynx. This work helps establish a baseline for understanding how the sinonasal microbiome may impact diseases of the upper airways.

Phototrophic phylotypes dominate mesothermal microbial mats associated with hot springs in Yellowstone National Park.

The mesothermal outflow zones (50-65°C) of geothermal springs often support an extensive zone of green and orange laminated microbial mats. In order to identify and compare the microbial inhabitants of morphologically similar green-orange mats from chemically and geographically distinct springs, we generated and analyzed small-subunit ribosomal RNA (rRNA) gene amplicons from six mesothermal mats (four previously unexamined) in Yellowstone National Park. Between three and six bacterial phyla dominated each mat. While many sequences bear the highest identity to previously isolated phototrophic genera belonging to the Cyanobacteria, Chloroflexi, and Chlorobi phyla, there is also frequent representation of uncultured, unclassified members of these groups. Some genus-level representatives of these dominant phyla were found in all mats, while others were unique to a single mat. Other groups detected at high frequencies include candidate divisions (such as the OP candidate clades) with no cultured representatives or complete genomes available. In addition, rRNA genes related to the recently isolated and characterized photosynthetic acidobacterium "Candidatus Chloracidobacterium thermophilum" were detected in most mats. In contrast to microbial mats from well-studied hypersaline environments, the mesothermal mats in this study accrue less biomass and are substantially less diverse, but have a higher proportion of known phototrophic organisms. This study provides sequences appropriate for accurate phylogenetic classification and expands the molecular phylogenetic survey of Yellowstone microbial mats.

Microbiome complexity and Staphylococcus aureus in chronic rhinosinusitis.

OBJECTIVES/HYPOTHESIS: The aim of this study was to compare microbiological culture-based and culture-independent (16S rRNA gene sequencing) methodologies for pathogen identification in chronic rhinosinusitis (CRS) patients. We hypothesized that bacterial culture and DNA sequencing would yield largely concurrent results, although sequencing would detect greater bacterial diversity, and the sinonasal microbiomes of CRS patients would differ in composition and diversity compared with non-CRS controls. STUDY DESIGN: Cross-sectional observational study. METHODS: Middle meatus swabs from CRS patients collected during endoscopic sinus surgery were analyzed by both clinical culture and broad-range analysis of 16S rRNA gene pyrosequences. RESULTS: A total of 21 swab samples from 15 CRS patients and five non-CRS controls were analyzed. One CRS patient was also swabbed 3 weeks postoperatively due to evidence of purulence during a clinical visit. All subjects had positive bacterial cultures, with a mean of 2.8 isolates per subject. The most prevalent cultivars were coagulase-negative staphylococci (15/20 specimens, 75%), Staphylococcus aureus (10/20, 50%), and Propionibacterium acnes (6/20, 30%). Among 57,407 pyrosequences generated, the most prevalent were from coagulase-negative staphylococci (21/21 specimens, 100%), Corynebacterium spp (18/21, 85.7%), P acnes (16/21, 76.2%), and S aureus (14/21, 66.7%). Bacterial diversity correlated with recent antibiotic use, asthma, prior sinus surgery, and relative abundance of S aureus. CONCLUSIONS: DNA pyrosequencing revealed greater biodiversity than culture, although in most cases culture results represented a subset of the abundant DNA sequence types. CRS patients were characterized by altered microbial composition (P = .02) and greater abundance of S aureus (P = .03).

Microbiological Water Quality Monitoring in a Resource-Limited Urban Area: a Study in Cameroon; Africa

In resource-limited developing nations; such as Cameroon; the expense of modern water-quality monitoring techniques is prohibitive to frequent water testing; as is done in the developed world. Inexpensive; shelf-stable 3MT PetrifilmT Escherichia coli/Coliform Count Plates potentially can provide significant opportunity for routine water-quality monitoring in the absence of infrastructure for state-of-the-art testing. We used shelf-stable E. coli/coliform culture plates to assess the water quality at twenty sampling sites in Kumbo; Cameroon. Culture results from treated and untreated sources were compared to modern bacterial DNA pyrosequencing methods using established bioinformatics and statistical tools. Petrifilms were reproducible between replicates and sampling dates. Additionally; cultivation on Petrifilms suggests that treatment by the Kumbo Water Authority (KWA) greatly improves water quality as compared with untreated river and rainwater. The majority of sequences detected were representative of common water and soil microbes; with a minority of sequences (40) identified as belonging to genera common in fecal matter and/or causes of human disease. Water sources had variable DNA sequence counts that correlated significantly with the culture count data and may therefore be a proxy for bacterial load. Although the KWA does not meet Western standards for water quality (less than one coliform per 100 mL); KWA piped water is safer than locally available alternative water sources such as river and rainwater. The culture-based technology described is easily transferrable to resource-limited areas and provides local water authorities with valuable microbiological safety information with potential to protect public health in developing nations

Update on bacterial detection methods in chronic rhinosinusitis: implications for clinicians and research scientists.

BACKGROUND: The exact etiologic role of microbes in chronic rhinosinusitis (CRS) remains unclear, due in part to inconsistencies and difficulties of microbiological detection. However, methodological advances now permit comprehensive analysis of microbial communities of the sinonasal region. This review summarizes recent developments in microbial detection technologies as exemplified by their application in the study of CRS. RECENT FINDINGS: A variety of novel methods for specimen collection, microbial culture, cultivar identification, and microscopic analysis of CRS specimens have been used in recent studies. Moreover, the advent of culture-independent methodologies based on the detection of microbial nucleic acids has greatly expanded the range of microorganisms, including fastidious species, that can be assayed. Many techniques use biochemical interactions to identify nucleic acids as markers for specific species. SUMMARY: Technological innovations in microbiology have radically improved the sensitivity and accuracy of microbial detection and identification in CRS specimens. Further application of these microbiological tools to CRS research should provide greater insight into the roles of microbial pathogens and commensals in sinus health and disease. Ultimately, translation of research results into clinical diagnostic technology will improve patient outcomes in this chronic disease.

The human nasal microbiota and Staphylococcus aureus carriage.

BACKGROUND: Colonization of humans with Staphylococcus aureus is a critical prerequisite of subsequent clinical infection of the skin, blood, lung, heart and other deep tissues. S. aureus persistently or intermittently colonizes the nares of approximately 50% of healthy adults, whereas approximately 50% of the general population is rarely or never colonized by this pathogen. Because microbial consortia within the nasal cavity may be an important determinant of S. aureus colonization we determined the composition and dynamics of the nasal microbiota and correlated specific microorganisms with S. aureus colonization. METHODOLOGY/PRINCIPAL FINDINGS: Nasal specimens were collected longitudinally from five healthy adults and a cross-section of hospitalized patients (26 S. aureus carriers and 16 non-carriers). Culture-independent analysis of 16S rRNA sequences revealed that the nasal microbiota of healthy subjects consists primarily of members of the phylum Actinobacteria (e.g., Propionibacterium spp. and Corynebacterium spp.), with proportionally less representation of other phyla, including Firmicutes (e.g., Staphylococcus spp.) and Proteobacteria (e.g. Enterobacter spp). In contrast, inpatient nasal microbiotas were enriched in S. aureus or Staphylococcus epidermidis and diminished in several actinobacterial groups, most notably Propionibacterium acnes. Moreover, within the inpatient population S. aureus colonization was negatively correlated with the abundances of several microbial groups, including S. epidermidis (p = 0.004). CONCLUSIONS/SIGNIFICANCE: The nares environment is colonized by a temporally stable microbiota that is distinct from other regions of the integument. Negative association between S. aureus, S. epidermidis, and other groups suggests microbial competition during colonization of the nares, a finding that could be exploited to limit S. aureus colonization.

Opportunistic pathogens enriched in showerhead biofilms.

The environments we humans encounter daily are sources of exposure to diverse microbial communities, some of potential concern to human health. In this study, we used culture-independent technology to investigate the microbial composition of biofilms inside showerheads as ecological assemblages in the human indoor environment. Showers are an important interface for human interaction with microbes through inhalation of aerosols, and showerhead waters have been implicated in disease. Although opportunistic pathogens commonly are cultured from shower facilities, there is little knowledge of either their prevalence or the nature of other microorganisms that may be delivered during shower usage. To determine the composition of showerhead biofilms and waters, we analyzed rRNA gene sequences from 45 showerhead sites around the United States. We find that variable and complex, but specific, microbial assemblages occur inside showerheads. Particularly striking was the finding that sequences representative of non-tuberculous mycobacteria (NTM) and other opportunistic human pathogens are enriched to high levels in many showerhead biofilms, >100-fold above background water contents. We conclude that showerheads may present a significant potential exposure to aerosolized microbes, including documented opportunistic pathogens. The health risk associated with showerhead microbiota needs investigation in persons with compromised immune or pulmonary systems.

Eucaryotic diversity in a hypersaline microbial mat.

To determine the eucaryotic diversity of the hypersaline Guerrero Negro microbial mat, we amplified 18S rRNA genes from DNA extracted from this mat and constructed and analyzed clone libraries. The extent of eucaryotic diversity detected was remarkably low, only 15 species among 890 clones analyzed. Six eucaryotic kingdoms were represented, as well as a novel cluster of sequences. Nematode sequences dominated the clone libraries.