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1.  Oral and Airway Microbiota in HIV-Infected Pneumonia Patients 
Journal of Clinical Microbiology  2012;50(9):2995-3002.
Despite the increased frequency of recurrent pneumonia in HIV-infected patients and recent studies linking the airway bacterial community (microbiota) to acute and chronic respiratory infection, little is known of the oral and airway microbiota that exist in these individuals and their propensity to harbor pathogens despite antimicrobial treatment for acute pneumonia. This pilot study compared paired samples of the oral and airway microbiota from 15 hospitalized HIV-infected patients receiving antimicrobial treatment for acute pneumonia. Total DNA was extracted, bacterial burden was assessed by quantitative PCR, and amplified 16S rRNA was profiled for microbiome composition using a phylogenetic microarray (16S rRNA PhyloChip). Though the bacterial burden of the airway was significantly lower than that of the oral cavity, microbiota in both niches were comparably diverse. However, oral and airway microbiota exhibited niche specificity. Oral microbiota were characterized by significantly increased relative abundance of multiple species associated with the mouth, including members of the Bacteroides, Firmicutes, and TM7 phyla, while airway microbiota were primarily characterized by a relative expansion of the Proteobacteria. Twenty-two taxa were detected in both niches, including Streptococcus bovis and Chryseobacterium species, pathogens associated with HIV-infected populations. In addition, we compared the airway microbiota of five of these patients to those of five non-HIV-infected pneumonia patients from a previous study. Compared to the control population, HIV-infected patients exhibited relative increased abundance of a large number of phylogenetically distinct taxa, which included several known or suspected pathogenic organisms, suggesting that recurrent pneumonia in HIV-infected populations may be related to the presence of these species.
doi:10.1128/JCM.00278-12
PMCID: PMC3421777  PMID: 22760045
2.  A Persistent and Diverse Airway Microbiota Present during Chronic Obstructive Pulmonary Disease Exacerbations 
Abstract
Acute exacerbations of chronic obstructive pulmonary disease (COPD) are a major source of morbidity and contribute significantly to healthcare costs. Although bacterial infections are implicated in nearly 50% of exacerbations, only a handful of pathogens have been consistently identified in COPD airways, primarily by culture-based methods, and the bacterial microbiota in acute exacerbations remains largely uncharacterized. The aim of this study was to comprehensively profile airway bacterial communities using a culture-independent microarray, the 16S rRNA PhyloChip, of a cohort of COPD patients requiring ventilatory support and antibiotic therapy for exacerbation-related respiratory failure. PhyloChip analysis revealed the presence of over 1,200 bacterial taxa representing 140 distinct families, many previously undetected in airway diseases; bacterial community composition was strongly influenced by the duration of intubation. A core community of 75 taxa was detected in all patients, many of which are known pathogens. Bacterial community diversity in COPD airways is substantially greater than previously recognized and includes a number of potential pathogens detected in the setting of antibiotic exposure. Comprehensive assessment of the COPD airway microbiota using high-throughput, culture-independent methods may prove key to understanding the relationships between airway bacterial colonization, acute exacerbation, and clinical outcomes in this and other chronic inflammatory airway diseases.
doi:10.1089/omi.2009.0100
PMCID: PMC3116451  PMID: 20141328
3.  Comprehensive Census of Bacteria in Clean Rooms by Using DNA Microarray and Cloning Methods▿ †  
Applied and Environmental Microbiology  2009;75(20):6559-6567.
A census of clean room surface-associated bacterial populations was derived from the results of both the cloning and sequencing of 16S rRNA genes and DNA microarray (PhyloChip) analyses. Samples from the Lockheed Martin Aeronautics Multiple Testing Facility (LMA-MTF), the Kennedy Space Center Payload Hazard and Servicing Facility (KSC-PHSF), and the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) clean rooms were collected during the various assembly phases of the Phoenix and Mars Science Laboratory (MSL) spacecraft. Clone library-derived analyses detected a larger bacterial diversity prior to the arrival of spacecraft hardware in these clean room facilities. PhyloChip results were in agreement with this trend but also unveiled the presence of anywhere from 9- to 70-fold more bacterial taxa than cloning approaches. Among the facilities sampled, the JPL-SAF (MSL mission) housed a significantly less diverse bacterial population than either the LMA-MTF or KSC-PHSF (Phoenix mission). Bacterial taxa known to thrive in arid conditions were frequently detected in MSL-associated JPL-SAF samples, whereas proteobacterial lineages dominated Phoenix-associated KSC-PHSF samples. Comprehensive bacterial censuses, such as that reported here, will help space-faring nations preemptively identify contaminant biomatter that may compromise extraterrestrial life detection experiments. The robust nature and high sensitivity of DNA microarray technologies should prove beneficial to a wide range of scientific, electronic, homeland security, medical, and pharmaceutical applications and to any other ventures with a vested interest in monitoring and controlling contamination in exceptionally clean environments.
doi:10.1128/AEM.01073-09
PMCID: PMC2765134  PMID: 19700540
4.  Application of Molecular Techniques To Elucidate the Influence of Cellulosic Waste on the Bacterial Community Structure at a Simulated Low-Level-Radioactive-Waste Site▿ † 
Applied and Environmental Microbiology  2010;76(10):3106-3115.
Low-level-radioactive-waste (low-level-waste) sites, including those at various U.S. Department of Energy sites, frequently contain cellulosic waste in the form of paper towels, cardboard boxes, or wood contaminated with heavy metals and radionuclides such as chromium and uranium. To understand how the soil microbial community is influenced by the presence of cellulosic waste products, multiple soil samples were obtained from a nonradioactive model low-level-waste test pit at the Idaho National Laboratory. Samples were analyzed using 16S rRNA gene clone libraries and 16S rRNA gene microarray (PhyloChip) analyses. Both methods revealed changes in the bacterial community structure with depth. In all samples, the PhyloChip detected significantly more operational taxonomic units, and therefore relative diversity, than the clone libraries. Diversity indices suggest that diversity is lowest in the fill and fill-waste interface (FW) layers and greater in the wood waste and waste-clay interface layers. Principal-coordinate analysis and lineage-specific analysis determined that the Bacteroidetes and Actinobacteria phyla account for most of the significant differences observed between the layers. The decreased diversity in the FW layer and increased members of families containing known cellulose-degrading microorganisms suggest that the FW layer is an enrichment environment for these organisms. These results suggest that the presence of the cellulosic material significantly influences the bacterial community structure in a stratified soil system.
doi:10.1128/AEM.01688-09
PMCID: PMC2869122  PMID: 20305022
5.  Assessment of Microbial Diversity in Biofilms Recovered from Endotracheal Tubes Using Culture Dependent and Independent Approaches 
PLoS ONE  2012;7(6):e38401.
Ventilator-associated pneumonia (VAP) is a common nosocomial infection in mechanically ventilated patients. Biofilm formation is one of the mechanisms through which the endotracheal tube (ET) facilitates bacterial contamination of the lower airways. In the present study, we analyzed the composition of the ET biofilm flora by means of culture dependent and culture independent (16 S rRNA gene clone libraries and pyrosequencing) approaches. Overall, the microbial diversity was high and members of different phylogenetic lineages were detected (Actinobacteria, beta-Proteobacteria, Candida spp., Clostridia, epsilon-Proteobacteria, Firmicutes, Fusobacteria and gamma-Proteobacteria). Culture dependent analysis, based on the use of selective growth media and conventional microbiological tests, resulted in the identification of typical aerobic nosocomial pathogens which are known to play a role in the development of VAP, e.g. Staphylococcus aureus and Pseudomonas aeruginosa. Other opportunistic pathogens were also identified, including Staphylococcus epidermidis and Kocuria varians. In general, there was little correlation between the results obtained by sequencing 16 S rRNA gene clone libraries and by cultivation. Pyrosequencing of PCR amplified 16 S rRNA genes of four selected samples resulted in the identification of a much wider variety of bacteria. The results from the pyrosequencing analysis suggest that these four samples were dominated by members of the normal oral flora such as Prevotella spp., Peptostreptococcus spp. and lactic acid bacteria. A combination of methods is recommended to obtain a complete picture of the microbial diversity of the ET biofilm.
doi:10.1371/journal.pone.0038401
PMCID: PMC3367921  PMID: 22693635
6.  Multidrug-Resistant Pseudomonas aeruginosa Ventilator-Associated Pneumonia: The Role of Endotracheal Aspirate Surveillance Cultures 
The Annals of pharmacotherapy  2008;43(1):28-35.
BACKGROUND
Inappropriate antibacterial treatment of ventilator-associated pneumonia (VAP) due to multidrug-resistant (MDR) pathogens is associated with increased mortality. Endotracheal aspirate (ETA) surveillance cultures potentially identify MDR pathogens, particularly MDR Pseudomonas aeruginosa, resulting in improved selection of therapy in patients who subsequently develop VAP.
OBJECTIVE
To investigate the role of ETA surveillance cultures in the identification of MDR P. aeruginosas newly intubated adults who subsequently develop VAP.
METHODS
Daily ETA surveillance cultures for P. aeruginosa were collected in all adults newly intubated for 48 hours or more. Patients with preexisting lung disease or colonization or infection with P. aeruginosa were excluded. Risk factors and outcomes of patients newly colonized with MDR P. aeruginosa were assessed.
RESULTS
Seventy-five patients newly colonized with P. aeruginosa were identified. Twenty (27%) of these patients were colonized with a P. aeruginosa isolate that was MDR (resistant to ≥3 classes of antibiotics). Six patients were colonized by an isolate resistant to all tested classes of antibiotics. Forty-five percent of patients colonized with MDR P. aeruginosa subsequently developed VAP. Prior receipt of fluoroquinolones was an independent predictor of colonization with MDR P. aeruginosa (OR 11.82; 95% CI 2.10 to 66.46; p = 0.005).
CONCLUSIONS
Performance of routine surveillance cultures may aid in the early detection of MDR P. aeruginosa, improving the initiation of early and appropriate antibiotic therapy for patients who subsequently develop VAP.
doi:10.1345/aph.1L210
PMCID: PMC2711849  PMID: 19033484
antibiotics; multidrug resistance; Pseudomonas aeruginosa; ventilator-associated pneumonia
7.  Multidrug-Resistant Pseudomonas aeruginosa Ventilator-Associated Pneumonia: The Role of Endotracheal Aspirate Surveillance Cultures 
The Annals of pharmacotherapy  2007;41(9):1427-1435.
BACKGROUND
Inappropriate antibacterial treatment of ventilator-associated pneumonia (VAP) due to multidrug-resistant (MDR) pathogens is associated with increased mortality. Endotracheal aspirate (ETA) surveillance cultures potentially identify MDR pathogens, particularly MDR Pseudomonas aeruginosa, resulting in improved selection of therapy in patients who subsequently develop VAP.
OBJECTIVE
To investigate the role of ETA surveillance cultures in the identification of MDR P. aeruginosa in newly intubated adults who subsequently develop VAP.
METHODS
Daily ETA surveillance cultures for P. aeruginosa were collected in all adults newly intubated for 48 hours or more. Patients with preexisting lung disease or colonization or infection with P. aeruginosa were excluded. Risk factors and outcomes of patients newly colonized with MDR P. aeruginosa were assessed.
RESULTS
Seventy-five patients newly colonized with P. aeruginosa were identified. Twenty (27%) of these patients were colonized with a P. aeruginosa isolate that was MDR (resistant to ≥3 classes of antibiotics). Six patients were colonized by an isolate resistant to all tested classes of antibiotics. Forty-five percent of patients colonized with MDR P. aeruginosa subsequently developed VAP. Prior receipt of fluoroquinolones was an independent predictor of colonization with MDR P. aeruginosa (OR 11.82; 95% CI 2.10 to 66.46; p = 0.005).
CONCLUSIONS
Performance of routine surveillance cultures may aid in the early detection of MDR P. aeruginosa, improving the initiation of early and appropriate antibiotic therapy for patients who subsequently develop VAP.
doi:10.1345/aph.1K213
PMCID: PMC3194036  PMID: 17666573
antibiotics; multidrug resistance; Pseudomonas aeruginosa; ventilator-associated pneumonia
8.  Bacterial Diversity Analysis of Huanglongbing Pathogen-Infected Citrus, Using PhyloChip Arrays and 16S rRNA Gene Clone Library Sequencing▿ †  
The bacterial diversity associated with citrus leaf midribs was characterized for citrus groves that contained the Huanglongbing (HLB) pathogen, which has yet to be cultivated in vitro. We employed a combination of high-density phylogenetic 16S rRNA gene microarrays and 16S rRNA gene clone library sequencing to determine the microbial community composition for symptomatic and asymptomatic citrus midribs. Our results revealed that citrus leaf midribs can support a diversity of microbes. PhyloChip analysis indicated that 47 orders of bacteria in 15 phyla were present in the citrus leaf midribs, while 20 orders in 8 phyla were observed with the cloning and sequencing method. PhyloChip arrays indicated that nine taxa were significantly more abundant in symptomatic midribs than in asymptomatic midribs. “Candidatus Liberibacter asiaticus” was detected at a very low level in asymptomatic plants but was over 200 times more abundant in symptomatic plants. The PhyloChip analysis results were further verified by sequencing 16S rRNA gene clone libraries, which indicated the dominance of “Candidatus Liberibacter asiaticus” in symptomatic leaves. These data implicate “Candidatus Liberibacter asiaticus” as the pathogen responsible for HLB disease.
doi:10.1128/AEM.02404-08
PMCID: PMC2655442  PMID: 19151177
9.  Diversity of Anaerobic Microbes in Spacecraft Assembly Clean Rooms ▿ †  
Although the cultivable and noncultivable microbial diversity of spacecraft assembly clean rooms has been previously documented using conventional and state-of-the-art molecular techniques, the occurrence of obligate anaerobes within these clean rooms is still uncertain. Therefore, anaerobic bacterial communities of three clean-room facilities were analyzed during assembly of the Mars Science Laboratory rover. Anaerobic bacteria were cultured on several media, and DNA was extracted from suitable anaerobic enrichments and examined with conventional 16S rRNA gene clone library, as well as high-density phylogenetic 16S rRNA gene microarray (PhyloChip) technologies. The culture-dependent analyses predominantly showed the presence of clostridial and propionibacterial strains. The 16S rRNA gene sequences retrieved from clone libraries revealed distinct microbial populations associated with each clean-room facility, clustered exclusively within gram-positive organisms. PhyloChip analysis detected a greater microbial diversity, spanning many phyla of bacteria, and provided a deeper insight into the microbial community structure of the clean-room facilities. This study presents an integrated approach for assessing the anaerobic microbial population within clean-room facilities, using both molecular and cultivation-based analyses. The results reveal that highly diverse anaerobic bacterial populations persist in the clean rooms even after the imposition of rigorous maintenance programs and will pose a challenge to planetary protection implementation activities.
doi:10.1128/AEM.02167-09
PMCID: PMC2863428  PMID: 20228115
10.  Comparative Analyses of the Bacterial Microbiota of the Human Nostril and Oropharynx 
mBio  2010;1(3):e00129-10.
The nose and throat are important sites of pathogen colonization, yet the microbiota of both is relatively unexplored by culture-independent approaches. We examined the bacterial microbiota of the nostril and posterior wall of the oropharynx from seven healthy adults using two culture-independent methods, a 16S rRNA gene microarray (PhyloChip) and 16S rRNA gene clone libraries. While the bacterial microbiota of the oropharynx was richer than that of the nostril, the oropharyngeal microbiota varied less among participants than did nostril microbiota. A few phyla accounted for the majority of the bacteria detected at each site: Firmicutes and Actinobacteria in the nostril and Firmicutes, Proteobacteria, and Bacteroidetes in the oropharynx. Compared to culture-independent surveys of microbiota from other body sites, the microbiota of the nostril and oropharynx show distinct phylum-level distribution patterns, supporting niche-specific colonization at discrete anatomical sites. In the nostril, the distribution of Actinobacteria and Firmicutes was reminiscent of that of skin, though Proteobacteria were much less prevalent. The distribution of Firmicutes, Proteobacteria, and Bacteroidetes in the oropharynx was most similar to that in saliva, with more Proteobacteria than in the distal esophagus or mouth. While Firmicutes were prevalent at both sites, distinct families within this phylum dominated numerically in each. At both sites there was an inverse correlation between the prevalences of Firmicutes and another phylum: in the oropharynx, Firmicutes and Proteobacteria, and in the nostril, Firmicutes and Actinobacteria. In the nostril, this inverse correlation existed between the Firmicutes family Staphylococcaceae and Actinobacteria families, suggesting potential antagonism between these groups.
IMPORTANCE
The human nose and throat, though connected, contain distinct niches that are important sites of colonization by pathogenic bacteria. For many of these pathogens, colonization increases the risk of infection. Most research on the microbiota of nose and throat habitats has focused on carriage of one or a few pathogens. We hypothesized that increased knowledge of the composition of the complex bacterial communities in which these pathogens reside would provide new insights into why some individuals become colonized with pathogens, while others do not. Indeed, in the nostril microbiota of participants, there was an inverse correlation between the prevalences of the Staphylococcaceae family (Firmicutes), whose members include important pathogens, and the Corynebacteriaceae and Propionibacteriaceae families (both Actinobacteria), whose members are more commonly benign commensals. An improved understanding of competitive bacterial colonization will increase our ability to define predispositions to pathogen carriage at these sites and the subsequent risk of infection.
doi:10.1128/mBio.00129-10
PMCID: PMC2925076  PMID: 20802827
11.  Phylogenetic Microarray Analysis of a Microbial Community Performing Reductive Dechlorination at a TCE-contaminated Site 
Environmental science & technology  2011;46(2):1044-1054.
A high-density phylogenetic microarray (PhyloChip) was applied to track bacterial and archaeal populations through different phases of remediation at Ft. Lewis, WA, a trichloroethene (TCE)-contaminated groundwater site. Biostimulation with whey, and bioaugmentation with a Dehalococcoides-containing enrichment culture were strategies implemented to enhance dechlorination. As a measure of species richness, over 1300 operational taxonomic units (OTUs) were detected in DNA from groundwater samples extracted during different stages of treatment and in the bioaugmentation culture. In order to determine active members within the community, 16S rRNA from samples were analyzed by microarray and ~600 OTUs identified. A cDNA clone library of the expressed 16S rRNA corroborated the observed diversity and activity of some of the phyla. Principle component analysis of the treatment plot samples revealed that the microbial populations were constantly changing during the course of the study. Dynamic analysis of the archaeal population showed significant increases in methanogens at the later stages of treatment that correlated with increases in methane concentrations of over two orders of magnitude. Overall, the PhyloChip analyses in this study have provided insights into the microbial ecology and population dynamics at the TCE-contaminated field site useful for understanding the in situ reductive dechlorination processes.
doi:10.1021/es203005k
PMCID: PMC3461955  PMID: 22091783
16S rRNA; PhyloChip; ecology; microbial community; bioremediation; chlorinated solvents
12.  Oligonucleotide Microarray for 16S rRNA Gene-Based Detection of All Recognized Lineages of Sulfate-Reducing Prokaryotes in the Environment 
Applied and Environmental Microbiology  2002;68(10):5064-5081.
For cultivation-independent detection of sulfate-reducing prokaryotes (SRPs) an oligonucleotide microarray consisting of 132 16S rRNA gene-targeted oligonucleotide probes (18-mers) having hierarchical and parallel (identical) specificity for the detection of all known lineages of sulfate-reducing prokaryotes (SRP-PhyloChip) was designed and subsequently evaluated with 41 suitable pure cultures of SRPs. The applicability of SRP-PhyloChip for diversity screening of SRPs in environmental and clinical samples was tested by using samples from periodontal tooth pockets and from the chemocline of a hypersaline cyanobacterial mat from Solar Lake (Sinai, Egypt). Consistent with previous studies, SRP-PhyloChip indicated the occurrence of Desulfomicrobium spp. in the tooth pockets and the presence of Desulfonema- and Desulfomonile-like SRPs (together with other SRPs) in the chemocline of the mat. The SRP-PhyloChip results were confirmed by several DNA microarray-independent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase (dsrAB).
doi:10.1128/AEM.68.10.5064-5081.2002
PMCID: PMC126405  PMID: 12324358
13.  Phylogenetic and metabolic diversity of bacteria associated with cystic fibrosis 
The ISME journal  2010;5(1):20-29.
In patients afflicted with cystic fibrosis (CF), morbidity and mortality are primarily associated with the adverse consequences of chronic microbial bronchial infections, which are thought to be caused by a few opportunistic pathogens. However, recent evidence suggests the presence of other microorganisms, which may significantly affect the course and outcome of the infection. Using a combination of 16S rRNA gene clone libraries, bacterial culturing and pyrosequencing of barcoded 16S rRNA amplicons, the microbial communities present in CF patient sputum samples were examined. In addition to previously recognized CF pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus, >60 phylogenetically diverse bacterial genera that are not typically associated with CF pathogenesis were also detected. A surprisingly large number of fermenting facultative and obligate anaerobes from multiple bacterial phyla was present in each sample. Many of the bacteria and sequences found were normal residents of the oropharyngeal microflora and with many containing opportunistic pathogens. Our data suggest that these undersampled organisms within the CF lung are part of a much more complex microbial ecosystem than is normally presumed. Characterization of these communities is the first step in elucidating potential roles of diverse bacteria in disease progression and to ultimately facilitate advances in CF therapy.
doi:10.1038/ismej.2010.88
PMCID: PMC3105664  PMID: 20631810
CF; lung; microbial diversity; polymicrobial disease; Pseudomonas aeruginosa; pyrosequencing
14.  Structure of the human gastric bacterial community in relation to Helicobacter pylori status 
The ISME Journal  2010;5(4):574-579.
The human stomach is naturally colonized by Helicobacter pylori, which, when present, dominates the gastric bacterial community. In this study, we aimed to characterize the structure of the bacterial community in the stomach of patients of differing H. pylori status. We used a high-density 16S rRNA gene microarray (PhyloChip, Affymetrix, Inc.) to hybridize 16S rRNA gene amplicons from gastric biopsy DNA of 10 rural Amerindian patients from Amazonas, Venezuela, and of two immigrants to the United States (from South Asia and Africa, respectively). H. pylori status was determined by PCR amplification of H. pylori glmM from gastric biopsy samples. Of the 12 patients, 8 (6 of the 10 Amerindians and the 2 non-Amerindians) were H. pylori glmM positive. Regardless of H. pylori status, the PhyloChip detected Helicobacteriaceae DNA in all patients, although with lower relative abundance in patients who were glmM negative. The G2-chip taxonomy analysis of PhyloChip data indicated the presence of 44 bacterial phyla (of which 16 are unclassified by the Taxonomic Outline of the Bacteria and Archaea taxonomy) in a highly uneven community dominated by only four phyla: Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. Positive H. pylori status was associated with increased relative abundance of non-Helicobacter bacteria from the Proteobacteria, Spirochetes and Acidobacteria, and with decreased abundance of Actinobacteria, Bacteroidetes and Firmicutes. The PhyloChip detected richness of low abundance phyla, and showed marked differences in the structure of the gastric bacterial community according to H. pylori status.
doi:10.1038/ismej.2010.149
PMCID: PMC3105737  PMID: 20927139
gastric; microbiota; H. pylori; microarray; Amerindians
15.  Autoinducer production and quorum-sensing dependent phenotypes of Pseudomonas aeruginosa vary according to isolation site during colonization of intubated patients 
BMC Microbiology  2007;7:33.
Background
Pseudomonas aeruginosa frequently colonizes and is responsible for severe ventilator-associated pneumonia in intubated patients. A quorum-sensing (QS) circuit, depending on the production of the two QS-signaling molecules (autoinducers, AIs) 3-oxo-C12-HSL and C4-HSL, regulates the production by P. aeruginosa of several virulence factors and is required for biofilm formation. Therefore QS-inhibition has been suggested as a new target for preventive and/or therapeutic strategies. However the precise role of QS during colonization and subsequent infections of intubated patients remains unclear.
Results
We wondered whether QS is active during colonization of intubated patients, and whether P. aeruginosa isolates growing inside the biofilm covering the intubation devices and those resident in the lungs of colonized patients differ in their QS-dependent phenotypes. We collected the intubation devices of eight patients colonized by P. aeruginosa. We detected 3-oxo-C12-HSL on eight, and C4-HSL on six of these devices. In three of these patients we also obtained P. aeruginosa isolates from tracheal aspirates at the time of extubation (n = 18), as well as isolates from the intubation devices (n = 25). We genotyped these isolates, quantified their AIs production, and determined three QS-dependent phenotypes (adherence capacity, biofilm and elastase production). The production of 3-oxo-C12-HSL was consistently increased for isolates from the intubation devices, whereas the production of C4-HSL was significantly higher for isolates from tracheal aspirates. Isolates from tracheal aspirates produced significantly higher amounts of elastase but less biofilm, and had a marginally reduced adhesion capacity than isolates from the intubation devices. Levels of 3-oxo-C12-HSL and elastase production correlated statistically for tracheal intubation isolates, whereas levels of 3-oxo-C12-HSL production and adhesion ability, as well as biofilm production, correlated weakly amongst intubation device isolates.
Conclusion
Our findings demonstrate that autoinducers are produced during the colonization of intubated patients by P. aeruginosa. The microenvironment, in which P. aeruginosa grows, may select for bacteria with different capacities to produce autoinducers and certain QS-dependent phenotypes. QS-inhibition might therefore affect differently isolates growing inside the biofilm covering intubation devices and those resident in the lungs.
doi:10.1186/1471-2180-7-33
PMCID: PMC1868030  PMID: 17442101
16.  16S rRNA Gene-Based Oligonucleotide Microarray for Environmental Monitoring of the Betaproteobacterial Order “Rhodocyclales” 
For simultaneous identification of members of the betaproteobacterial order “Rhodocyclales” in environmental samples, a 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes was developed. Probe design was based on phylogenetic analysis of available 16S rRNA sequences from all cultured and as yet uncultured members of the “Rhodocyclales.” The multiple nested probe set was evaluated for microarray hybridization with 16S rRNA gene PCR amplicons from 29 reference organisms. Subsequently, the RHC-PhyloChip was successfully used for cultivation-independent “Rhodocyclales” diversity analysis in activated sludge from an industrial wastewater treatment plant. The implementation of a newly designed “Rhodocyclales”-selective PCR amplification system prior to microarray hybridization greatly enhanced the sensitivity of the RHC-PhyloChip and thus enabled the detection of “Rhodocyclales” populations with relative abundances of less than 1% of all bacteria (as determined by fluorescence in situ hybridization) in the activated sludge. The presence of as yet uncultured Zoogloea-, Ferribacterium/Dechloromonas-, and Sterolibacterium-related bacteria in the industrial activated sludge, as indicated by the RHC-PhyloChip analysis, was confirmed by retrieval of their 16S rRNA gene sequences and subsequent phylogenetic analysis, demonstrating the suitability of the RHC-PhyloChip as a novel monitoring tool for environmental microbiology.
doi:10.1128/AEM.71.3.1373-1386.2005
PMCID: PMC1065177  PMID: 15746340
17.  Differences in biofilm formation and antimicrobial resistance of Pseudomonas aeruginosa isolated from airways of mechanically ventilated patients and cystic fibrosis patients 
Pseudomonas aeruginosa biofilms exhibit increased antimicrobial resistance compared with planktonic isolates and are implicated in the pathogenesis of both acute and chronic lung infections. Whilst antibiotic choices for both infections are based on planktonic antibiotic susceptibility results, differences in biofilm-forming ability between the two diseases have not previously been explored. The aim of this study was to compare differences in biofilm formation and antibiotic resistance of P. aeruginosa isolated from intubated patients and from patients with chronic pulmonary disease associated with cystic fibrosis (CF). The temporal evolution of antibiotic resistance in clonal P. aeruginosa strains isolated from CF patients during periods of chronic infection and acute pulmonary exacerbation was also evaluated. Biofilm formation and biofilm antibiotic susceptibilities were determined using a modified microtitre plate assay and were compared with antibiotic susceptibility results obtained using traditional planktonic culture. Clonality was confirmed using random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) analysis. Pseudomonas aeruginosa isolates collected from intubated patients produced substantially more biofilms compared with CF isolates. There was considerable heterogeneity in biofilm-forming ability among the CF isolates and this was unrelated to pulmonary status. Biofilm antibiotic resistance developed rapidly among clonal CF isolates over time, whilst traditional antibiotic resistance determined using planktonic cultures remained stable. There was a significant positive correlation between imipenem/cilastatin and ceftazidime resistance and biofilm-forming ability. The variability in biofilm-forming ability in P. aeruginosa and the rapid evolution of biofilm resistance may require consideration when choosing antibiotic therapy for newly intubated patients and CF patients.
doi:10.1016/j.ijantimicag.2010.12.017
PMCID: PMC3176759  PMID: 21382698
Pseudomonas aeruginosa; Bacterial biofilm; Antimicrobial resistance; Mechanical ventilation; Cystic fibrosis
18.  Lactobacillus casei Abundance Is Associated with Profound Shifts in the Infant Gut Microbiome 
PLoS ONE  2010;5(1):e8745.
Colonization of the infant gut by microorganisms over the first year of life is crucial for development of a balanced immune response. Early alterations in the gastrointestinal microbiota of neonates has been linked with subsequent development of asthma and atopy in older children. Here we describe high-resolution culture-independent analysis of stool samples from 6-month old infants fed daily supplements of Lactobacillus casei subsp. Rhamnosus (LGG) or placebo in a double-blind, randomized Trial of Infant Probiotic Supplementation (TIPS). Bacterial community composition was examined using a high-density microarray, the 16S rRNA PhyloChip, and the microbial assemblages of infants with either high or low LGG abundance were compared. Communities with high abundance of LGG exhibited promotion of phylogenetically clustered taxa including a number of other known probiotic species, and were significantly more even in their distribution of community members. Ecologically, these aspects are characteristic of communities that are more resistant to perturbation and outgrowth of pathogens. PhyloChip analysis also permitted identification of taxa negatively correlated with LGG abundance that have previously been associated with atopy, as well as those positively correlated that may prove useful alternative targets for investigation as alternative probiotic species. From these findings we hypothesize that a key mechanism for the protective effect of LGG supplementation on subsequent development of allergic disease is through promotion of a stable, even, and functionally redundant infant gastrointestinal community.
doi:10.1371/journal.pone.0008745
PMCID: PMC2807455  PMID: 20090909
19.  Pre-Hospital Intubation Factors and Pneumonia in Trauma Patients 
Surgical Infections  2011;12(5):339-344.
Abstract
Background
We reported similar rates of ventilator-associated pneumonia (VAP) previously in trauma patients intubated either in a pre-hospital (PH) venue or the emergency department. A subset of PH intubations with continuous quality assessment was re-examined to identify the intubation factors associated with VAP.
Methods
The subgroup was derived from an existing data set of consecutive adult trauma patients intubated prior to Level I trauma center admission July 2007–July 2008. Intubation details recorded included bag-valve mask ventilation (BVM) and the presence of material in the airway. The diagnosis of VAP was made preferentially by quantitative bronchoalveolar lavage (BAL) cultures (≥104 colony-forming units indicating infection). Baseline data, injury characteristics, and circumstances of intubation of patients with and without VAP were compared by univariable analysis.
Results
Detailed data were available for 197 patients; 32 (16.2%) developed VAP, on average 6.0±0.7 days after admission. Baseline characteristics were similar in the groups, but diabetes mellitus was more common in the VAP group (4 [12.5%] vs. 5 [3.0%]; p=0.02). There was a higher rate of blunt injury in the VAP patients (28 [87.5%] vs. 106 [64.2%]; p=0.01) and higher injury severity scores (33.1±2.8 vs. 23.0±1.0; p=0.0002) and chest Abbreviated Injury Scores (2.6±0.3 vs. 1.5±0.1; p=0.002). Lower Glasgow Coma Scale scores (7.9±0.9 vs. 9.9±0.4; p=0.04) and greater use of BVM (18 [56.3%] vs. 56 [34.0%]; p=0.02) were observed in patients who developed VAP. Among aspirations, 10 (31.3%) of patients with emesis developed VAP compared with only 4 (12.5%) with blood in the airway (p=0.003).
Conclusion
Aspiration, along with depressed consciousness and greater injury severity, may predispose trauma patients to VAP. Prospective studies should focus on the quality and timing of aspiration relative to intubation to determine if novel interventions can prevent aspiration or decrease the risk of VAP after aspiration.
doi:10.1089/sur.2010.074
PMCID: PMC3607967  PMID: 21933010
20.  Prevalence of Streptococci and Increased Polymicrobial Diversity Associated with Cystic Fibrosis Patient Stability 
Journal of Bacteriology  2012;194(17):4709-4717.
Diverse microbial communities chronically colonize the lungs of cystic fibrosis patients. Pyrosequencing of amplicons for hypervariable regions in the 16S rRNA gene generated taxonomic profiles of bacterial communities for sputum genomic DNA samples from 22 patients during a state of clinical stability (outpatients) and 13 patients during acute exacerbation (inpatients). We employed quantitative PCR (qPCR) to confirm the detection of Pseudomonas aeruginosa and Streptococcus by the pyrosequencing data and human oral microbe identification microarray (HOMIM) analysis to determine the species of the streptococci identified by pyrosequencing. We show that outpatient sputum samples have significantly higher bacterial diversity than inpatients, but maintenance treatment with tobramycin did not impact overall diversity. Contrary to the current dogma in the field that Pseudomonas aeruginosa is the dominant organism in the majority of cystic fibrosis patients, Pseudomonas constituted the predominant genera in only half the patient samples analyzed and reported here. The increased fractional representation of Streptococcus in the outpatient cohort relative to the inpatient cohort was the strongest predictor of clinically stable lung disease. The most prevalent streptococci included species typically associated with the oral cavity (Streptococcus salivarius and Streptococcus parasanguis) and the Streptococcus milleri group species. These species of Streptococcus may play an important role in increasing the diversity of the cystic fibrosis lung environment and promoting patient stability.
doi:10.1128/JB.00566-12
PMCID: PMC3415522  PMID: 22753064
21.  Airway Microbiota and Bronchial Hyperresponsiveness in Patients with Sub-optimally Controlled Asthma 
Background
Improvement in lung function following macrolide antibiotic therapy has been attributed to reduction in bronchial infection due to specific bacteria. However, the airway may be populated by a more diverse microbiota, and clinical features of asthma may be associated with characteristics of the airway microbiota present.
Objective
To determine if relationships exist between the composition of the airway bacterial microbiota and clinical features of asthma, using culture-independent tools capable of detecting the presence and relative abundance of most known bacteria.
Methods
In this pilot study, bronchial epithelial brushings were collected from sixty-five adults with sub-optimally controlled asthma participating in a multicenter study of the effects of clarithromycin on asthma control, and ten healthy subjects. A combination of high-density 16S rRNA microarray and parallel clone library-sequencing analysis was used to profile the microbiota and examine relationships with clinical measurements.
Results
Compared to controls, 16S rRNA amplicon concentrations (a proxy for bacterial burden) and bacterial diversity were significantly higher among asthmatic patients. In multivariate analyses, airway microbiota composition and diversity were significantly correlated with bronchial hyperresponsiveness. Specifically, the relative abundance of particular phylotypes, including members of the Comamonadaceae, Sphingomonadaceae, Oxalobacteraceae and other bacterial families, were highly correlated with the degree of bronchial hyperresponsiveness.
Conclusion
The composition of bronchial airway microbiota is associated with the degree of bronchial hyperresponsiveness among patients with sub-optimally controlled asthma. These findings support the need for further functional studies to examine the potential contribution of members of the airway microbiota in asthma pathogenesis.
doi:10.1016/j.jaci.2010.10.048
PMCID: PMC3037020  PMID: 21194740
microbiome; bacteria; asthma; 16S rRNA; PhyloChip
22.  A feasibility study on bedside upper airway ultrasonography compared to waveform capnography for verifying endotracheal tube location after intubation 
Background
In emergency settings, verification of endotracheal tube (ETT) location is important for critically ill patients. Ignorance of oesophageal intubation can be disastrous. Many methods are used for verification of the endotracheal tube location; none are ideal. Quantitative waveform capnography is considered the standard of care for this purpose but is not always available and is expensive. Therefore, this feasibility study is conducted to compare a cheaper alternative, bedside upper airway ultrasonography to waveform capnography, for verification of endotracheal tube location after intubation.
Methods
This was a prospective, single-centre, observational study, conducted at the HRPB, Ipoh. It included patients who were intubated in the emergency department from 28 March 2012 to 17 August 2012. A waiver of consent had been obtained from the Medical Research Ethics Committee. Bedside upper airway ultrasonography was performed after intubation and compared to waveform capnography. Specificity, sensitivity, positive and negative predictive value and likelihood ratio are calculated.
Results
A sample of 107 patients were analysed, and 6 (5.6%) had oesophageal intubations. The overall accuracy of bedside upper airway ultrasonography was 98.1% (95% confidence interval (CI) 93.0% to 100.0%). The kappa value (Κ) was 0.85, indicating a very good agreement between the bedside upper airway ultrasonography and waveform capnography. Thus, bedside upper airway ultrasonography is in concordance with waveform capnography. The sensitivity, specificity, positive predictive value and negative predictive value of bedside upper airway ultrasonography were 98.0% (95% CI 93.0% to 99.8%), 100% (95% CI 54.1% to 100.0%), 100% (95% CI 96.3% to 100.0%) and 75.0% (95% CI 34.9% to 96.8%). The likelihood ratio of a positive test is infinite and the likelihood ratio of a negative test is 0.0198 (95% CI 0.005 to 0.0781). The mean confirmation time by ultrasound is 16.4 s. No adverse effects were recorded.
Conclusions
Our study shows that ultrasonography can replace waveform capnography in confirming ETT placement in centres without capnography. This can reduce incidence of unrecognised oesophageal intubation and prevent morbidity and mortality.
Trial registration
National Medical Research Register NMRR11100810230.
doi:10.1186/2036-7902-5-7
PMCID: PMC3772703  PMID: 23826756
Bedside upper airway ultrasonography; Endotracheal intubation; Verification; Waveform capnography
23.  Comparison of the TruView infant EVO2 PCD™ and C-MAC video laryngoscopes with direct Macintosh laryngoscopy for routine tracheal intubation in infants with normal airways 
Clinics  2014;69(1):23-27.
OBJECTIVE:
Videolaryngoscopy has mainly been developed to facilitate difficult airway intubation. However, there is a lack of studies demonstrating this method's efficacy in pediatric patients. The aim of the present study was to compare the TruView infant EVO2 and the C-MAC videolaryngoscope with conventional direct Macintosh laryngoscopy in children with a bodyweight ≤10 kg in terms of intubation conditions and the time to intubation.
METHODS:
In total, 65 children with a bodyweight ≤10 kg (0-22 months) who had undergone elective surgery requiring endotracheal intubation were retrospectively analyzed. Our database was screened for intubations with the TruView infant EVO2, the C-MAC videolaryngoscope, and conventional direct Macintosh laryngoscopy. The intubation conditions, the time to intubation, and the oxygen saturation before and after intubation were monitored, and demographic data were recorded. Only children with a bodyweight ≤10 kg were included in the analysis.
RESULTS:
A total of 23 children were intubated using the C-MAC videolaryngoscope, and 22 children were intubated using the TruView EVO2. Additionally, 20 children were intubated using a standard Macintosh blade. The time required for tracheal intubation was significantly longer using the TruView EVO2 (52 sec vs. 28 sec for C-MAC vs. 26 sec for direct LG). However, no significant difference in oxygen saturation was found after intubation.
CONCLUSION:
All devices allowed excellent visualization of the vocal cords, but the time to intubation was prolonged when the TruView EVO2 was used. The absence of a decline in oxygen saturation may be due to apneic oxygenation via the TruView scope and may provide a margin of safety. In sum, the use of the TruView by a well-trained anesthetist may be an alternative for difficult airway management in pediatric patients.
doi:10.6061/clinics/2014(01)04
PMCID: PMC3870305  PMID: 24473556
Videolaryngoscopy; Tracheal Intubation; Infants
24.  Molecular Epidemiology of Chronic Pseudomonas aeruginosa Airway Infections in Cystic Fibrosis 
PLoS ONE  2012;7(11):e50731.
Background/Methods
The molecular epidemiology of the chronic airway infections with Pseudomonas aeruginosa in individuals with cystic fibrosis (CF) was investigated by cross-sectional analysis of bacterial isolates from 51 CF centers and by longitudinal analysis of serial isolates which had been collected at the CF centers Hanover and Copenhagen since the onset of airway colonization over 30 years.
Results
Genotyping revealed that the P. aeruginosa population in CF is dominated by a few ubiquitous clones. The five most common clones retrieved from the CF host also belonged to the twenty most frequent clones in the environment and in other human disease habitats. Turnover of clones in CF airways was rare. At the Hanover clinic more than half of the patient cohort was still harbouring the initially acquired clone after twenty years of airway colonization. At the Copenhagen clinic, however, two rare clones replaced the initially acquired individual clones in all but one patient.
Conclusion
The divergent epidemiology at the two sites is explained by their differential management of hygiene and antipseudomonal chemotherapy. Hygienic measures to prohibit patient-to-patient transmission and the modalities of antipseudomonal chemotherapy modify the epidemiology of the chronic P. aeruginosa infections in CF.
doi:10.1371/journal.pone.0050731
PMCID: PMC3508996  PMID: 23209821
25.  Huanglongbing, a Systemic Disease, Restructures the Bacterial Community Associated with Citrus Roots▿  
Applied and Environmental Microbiology  2010;76(11):3427-3436.
To examine the effect of pathogens on the diversity and structure of plant-associated bacterial communities, we carried out a molecular analysis using citrus and huanglongbing as a host-disease model. 16S rRNA gene clone library analysis of citrus roots revealed shifts in microbial diversity in response to pathogen infection. The clone library of the uninfected root samples has a majority of phylotypes showing similarity to well-known plant growth-promoting bacteria, including Caulobacter, Burkholderia, Lysobacter, Pantoea, Pseudomonas, Stenotrophomonas, Bacillus, and Paenibacillus. Infection by “Candidatus Liberibacter asiaticus” restructured the native microbial community associated with citrus roots and led to the loss of detection of most phylotypes while promoting the growth of bacteria such as Methylobacterium and Sphingobacterium. In pairwise comparisons, the clone library from uninfected roots contained significantly higher 16S rRNA gene diversity, as reflected in the higher Chao 1 richness estimation (P ≤ 0.01) of 237.13 versus 42.14 for the uninfected and infected clone libraries, respectively. Similarly, the Shannon index of the uninfected clone library (4.46) was significantly higher than that of the infected clone library (2.61). Comparison of the uninfected clone library with the infected clone library using LIBSHUFF statistics showed a significant difference (P ≤ 0.05). Quantitative PCR analysis revealed that the bacterial community changes not only qualitatively but also quantitatively. The relative proportions of different groups of bacteria changed significantly after infection with the pathogen. These data indicate that infection of citrus by “Ca. Liberibacter asiaticus” has a profound effect on the structure and composition of the bacterial community associated with citrus roots.
doi:10.1128/AEM.02901-09
PMCID: PMC2876436  PMID: 20382817

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