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1.  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
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  Macroscopic Biofilms in Fracture-Dominated Sediment That Anaerobically Oxidize Methane ▿ †  
Applied and Environmental Microbiology  2011;77(19):6780-6787.
Methane release from seafloor sediments is moderated, in part, by the anaerobic oxidation of methane (AOM) performed by consortia of archaea and bacteria. These consortia occur as isolated cells and aggregates within the sulfate-methane transition (SMT) of diffusion and seep-dominant environments. Here we report on a new SMT setting where the AOM consortium occurs as macroscopic pink to orange biofilms within subseafloor fractures. Biofilm samples recovered from the Indian and northeast Pacific Oceans had a cellular abundance of 107 to 108 cells cm−3. This cell density is 2 to 3 orders of magnitude greater than that in the surrounding sediments. Sequencing of bacterial 16S rRNA genes indicated that the bacterial component is dominated by Deltaproteobacteria, candidate division WS3, and Chloroflexi, representing 46%, 15%, and 10% of clones, respectively. In addition, major archaeal taxa found in the biofilm were related to the ANME-1 clade, Thermoplasmatales, and Desulfurococcales, representing 73%, 11%, and 10% of archaeal clones, respectively. The sequences of all major taxa were similar to sequences previously reported from cold seep environments. PhyloChip microarray analysis detected all bacterial phyla identified by the clone library plus an additional 44 phyla. However, sequencing detected more archaea than the PhyloChip within the phyla of Methanosarcinales and Desulfurococcales. The stable carbon isotope composition of the biofilm from the SMT (−35 to −43‰) suggests that the production of the biofilm is associated with AOM. These biofilms are a novel, but apparently widespread, aggregation of cells represented by the ANME-1 clade that occur in methane-rich marine sediments.
doi:10.1128/AEM.00288-11
PMCID: PMC3187087  PMID: 21821755
9.  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
10.  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
11.  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
12.  Deciphering the Bacterial Microbiome of Citrus Plants in Response to ‘Candidatus Liberibacter asiaticus’-Infection and Antibiotic Treatments 
PLoS ONE  2013;8(11):e76331.
The bacterial microbiomes of citrus plants were characterized in response to ‘Candidatus Liberibacter asiaticus’ (Las)-infection and treatments with ampicillin (Amp) and gentamicin (Gm) by Phylochip-based metagenomics. The results revealed that 7,407 of over 50,000 known Operational Taxonomic Units (OTUs) in 53 phyla were detected in citrus leaf midribs using the PhyloChip™ G3 array, of which five phyla were dominant, Proteobacteria (38.7%), Firmicutes (29.0%), Actinobacteria (16.1%), Bacteroidetes (6.2%) and Cyanobacteria (2.3%). The OTU62806, representing ‘Candidatus Liberibacter’, was present with a high titer in the plants graft-inoculated with Las-infected scions treated with Gm at 100 mg/L and in the water-treated control (CK1). However, the Las bacterium was not detected in the plants graft-inoculated with Las-infected scions treated with Amp at 1.0 g/L or in plants graft-inoculated with Las-free scions (CK2). The PhyloChip array demonstrated that more OTUs, at a higher abundance, were detected in the Gm-treated plants than in the other treatment and the controls. Pairwise comparisons indicated that 23 OTUs from the Achromobacter spp. and 12 OTUs from the Methylobacterium spp. were more abundant in CK2 and CK1, respectively. Ten abundant OTUs from the Stenotrophomonas spp. were detected only in the Amp-treatment. These results provide new insights into microbial communities that may be associated with the progression of citrus huanglongbing (HLB) and the potential effects of antibiotics on the disease and microbial ecology.
doi:10.1371/journal.pone.0076331
PMCID: PMC3826729  PMID: 24250784
13.  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
14.  Changes of Soil Bacterial Diversity as a Consequence of Agricultural Land Use in a Semi-Arid Ecosystem 
PLoS ONE  2013;8(3):e59497.
Natural scrublands in semi-arid deserts are increasingly being converted into fields. This results in losses of characteristic flora and fauna, and may also affect microbial diversity. In the present study, the long-term effect (50 years) of such a transition on soil bacterial communities was explored at two sites typical of semi-arid deserts. Comparisons were made between soil samples from alfalfa fields and the adjacent scrublands by two complementary methods based on 16S rRNA gene fragments amplified from total community DNA. Denaturing gradient gel electrophoresis (DGGE) analyses revealed significant effects of the transition on community composition of Bacteria, Actinobacteria, Alpha- and Betaproteobacteria at both sites. PhyloChip hybridization analysis uncovered that the transition negatively affected taxa such as Acidobacteria, Chloroflexi, Acidimicrobiales, Rubrobacterales, Deltaproteobacteria and Clostridia, while Alpha-, Beta- and Gammaproteobacteria, Bacteroidetes and Actinobacteria increased in abundance. Redundancy analysis suggested that the community composition of phyla responding to agricultural use (except for Spirochaetes) correlated with soil parameters that were significantly different between the agricultural and scrubland soil. The arable soils were lower in organic matter and phosphate concentration, and higher in salinity. The variation in the bacterial community composition was higher in soils from scrubland than from agriculture, as revealed by DGGE and PhyloChip analyses, suggesting reduced beta diversity due to agricultural practices. The long-term use for agriculture resulted in profound changes in the bacterial community and physicochemical characteristics of former scrublands, which may irreversibly affect the natural soil ecosystem.
doi:10.1371/journal.pone.0059497
PMCID: PMC3603937  PMID: 23527207
15.  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
16.  GASTROINTESTINAL MICROBIOME SIGNATURES OF PEDIATRIC PATIENTS WITH IRRITABLE BOWEL SYNDROME 
Gastroenterology  2011;141(5):1782-1791.
BACKGROUND AND AIMS
The intestinal microbiomes of healthy children and pediatric patients with irritable bowel syndrome (IBS) are not well defined. Studies in adults have indicated that the gastrointestinal microbiota could be involved in IBS.
METHODS
We analyzed 71 samples from 22 children with IBS (pediatric Rome III criteria) and 22 healthy children, ages 7–12 years, by 16S rRNA gene sequencing, with an average of 54,287 reads/stool sample (average 454 read length = 503 bases). Data were analyzed using phylogenetic-based clustering (Unifrac), or an operational taxonomic unit (OTU) approach using a supervised machine learning tool (randomForest). Most samples were also hybridized to a microarray that can detect 8,741 bacterial taxa (16S rRNA PhyloChip).
RESULTS
Microbiomes associated with pediatric IBS were characterized by a significantly greater percentage of the class Gammaproteobacteria (0.07% vs 0.89% of total bacteria; P <.05); one prominent component of this group was Haemophilus parainfluenzae. Differences highlighted by 454 sequencing were confirmed by high-resolution PhyloChip analysis. Using supervised learning techniques, we were able to classify different subtypes of IBS with a success rate of 98.5%, using limited sets of discriminant bacterial species. A novel Ruminococcus-like microbe was associated with IBS, indicating the potential utility of microbe discovery for gastrointestinal disorders. A greater frequency of pain correlated with an increased abundance of several bacterial taxa from the genus Alistipes.
CONCLUSIONS
Using16S metagenomics by Phylochip DNA hybridization and deep 454 pyrosequencing, we associated specific microbiome signatures with pediatric IBS. These findings indicate the important association between gastrointestinal microbes and IBS in children; these approaches might be used in diagnosis of functional bowel disorders in pediatric patients.
doi:10.1053/j.gastro.2011.06.072
PMCID: PMC3417828  PMID: 21741921
17.  Tackling the minority: sulfate-reducing bacteria in an archaea-dominated subsurface biofilm 
The ISME Journal  2012;7(3):635-651.
Archaea are usually minor components of a microbial community and dominated by a large and diverse bacterial population. In contrast, the SM1 Euryarchaeon dominates a sulfidic aquifer by forming subsurface biofilms that contain a very minor bacterial fraction (5%). These unique biofilms are delivered in high biomass to the spring outflow that provides an outstanding window to the subsurface. Despite previous attempts to understand its natural role, the metabolic capacities of the SM1 Euryarchaeon remain mysterious to date. In this study, we focused on the minor bacterial fraction in order to obtain insights into the ecological function of the biofilm. We link phylogenetic diversity information with the spatial distribution of chemical and metabolic compounds by combining three different state-of-the-art methods: PhyloChip G3 DNA microarray technology, fluorescence in situ hybridization (FISH) and synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectromicroscopy. The results of PhyloChip and FISH technologies provide evidence for selective enrichment of sulfate-reducing bacteria, which was confirmed by the detection of bacterial dissimilatory sulfite reductase subunit B (dsrB) genes via quantitative PCR and sequence-based analyses. We further established a differentiation of archaeal and bacterial cells by SR-FTIR based on typical lipid and carbohydrate signatures, which demonstrated a co-localization of organic sulfate, carbonated mineral and bacterial signatures in the biofilm. All these results strongly indicate an involvement of the SM1 euryarchaeal biofilm in the global cycles of sulfur and carbon and support the hypothesis that sulfidic springs are important habitats for Earth's energy cycles. Moreover, these investigations of a bacterial minority in an Archaea-dominated environment are a remarkable example of the great power of combining highly sensitive microarrays with label-free infrared imaging.
doi:10.1038/ismej.2012.133
PMCID: PMC3578563  PMID: 23178669
Archaea; microbial ecology; PhyloChip; SR-FTIR; SRB; CTC
18.  Comparing Bacterial Community Composition between Healthy and White Plague-Like Disease States in Orbicella annularis Using PhyloChip™ G3 Microarrays 
PLoS ONE  2013;8(11):e79801.
Coral disease is a global problem. Diseases are typically named or described based on macroscopic changes, but broad signs of coral distress such as tissue loss or discoloration are unlikely to be specific to a particular pathogen. For example, there appear to be multiple diseases that manifest the rapid tissue loss that characterizes ‘white plague.’ PhyloChip™ G3 microarrays were used to compare the bacterial community composition of both healthy and white plague-like diseased corals. Samples of lobed star coral (Orbicella annularis, formerly of the genus Montastraea [1]) were collected from two geographically distinct areas, Dry Tortugas National Park and Virgin Islands National Park, to determine if there were biogeographic differences between the diseases. In fact, all diseased samples clustered together, however there was no consistent link to Aurantimonas coralicida, which has been described as the causative agent of white plague type II. The microarrays revealed a large amount of bacterial heterogeneity within the healthy corals and less diversity in the diseased corals. Gram-positive bacterial groups (Actinobacteria, Firmicutes) comprised a greater proportion of the operational taxonomic units (OTUs) unique to healthy samples. Diseased samples were enriched in OTUs from the families Corynebacteriaceae, Lachnospiraceae, Rhodobacteraceae, and Streptococcaceae. Much previous coral disease work has used clone libraries, which seem to be methodologically biased toward recovery of Gram-negative bacterial sequences and may therefore have missed the importance of Gram-positive groups. The PhyloChip™data presented here provide a broader characterization of the bacterial community changes that occur within Orbicella annularis during the shift from a healthy to diseased state.
doi:10.1371/journal.pone.0079801
PMCID: PMC3835879  PMID: 24278181
19.  Characterization of the microbial community structure in Candidatus Liberibacter asiaticus-infected citrus plants treated with antibiotics in the field 
BMC Microbiology  2013;13:112.
Background
Huanglongbing (HLB) is a worldwide devastating disease of citrus. There are no effective control measures for this newly emerging but century-old disease. Previously, we reported a combination of Penicillin G and Streptomycin was effective in eliminating or suppressing the associated bacterium, ‘Candidatus Liberibacter asiaticus’ (Las).
Results
Here we report the bacterial composition and community structure in HLB-affected citrus plants during a growing season and while being treated with antibiotic combinations PS (Penicillin G and Streptomycin) and KO (Kasugamycin and Oxytetracycline) using the Phylochip™ G3 array. Both antibiotic treatments resulted in significantly lower Las bacterial titers (Pr<0.05) and hybridization scores. Of the 50,000+ available operational taxonomic units (OTUs) on PhyloChip™ G3, 7,028 known OTUs were present in citrus leaf midribs. These OTUs were from 58 phyla, of which five contained 100 or more OTUs, Proteobacteria (44.1%), Firmicutes (23.5%), Actinobacteria (12.4%), Bacteroidetes (6.6%) and Cyanobacteria (3.2%). In the antibiotic treated samples, the number of OTUs decreased to a total of 5,599. The over-all bacterial diversity decreased with the antibiotic treatments, as did the abundance of 11 OTUs within Proteobacteria, Firmicutes, Bacteroidetes and Planctomycetes. Within the Proteobacteria, ten OTUs representing the class γ-proteobacteria increased in abundance after four months of treatment, when the Las bacterium was at its lowest level in the HLB-affected citrus field plants.
Conclusions
Our data revealed that Proteobacteria was constantly the dominant bacterial phylum recovered from citrus leaf midribs, with the α-proteobacterial and the γ-proteobacterial classes vying for prevalence. In addition, the level of bacterial diversity found in the leaf midribs of field citrus was greater than previously described. Bacterial cells in close proximity may be able to modify their microenvironment, making the composition of the microbial community an important factor in the ability of Las to cause HLB progression. A low Las level was seen as an annual fluctuation, part of the bacterial population dynamics, and as a response to the antibiotic treatments.
doi:10.1186/1471-2180-13-112
PMCID: PMC3672075  PMID: 23701743
20.  New perspectives on viable microbial communities in low-biomass cleanroom environments 
The ISME Journal  2012;7(2):312-324.
The advent of phylogenetic DNA microarrays and high-throughput pyrosequencing technologies has dramatically increased the resolution and accuracy of detection of distinct microbial lineages in mixed microbial assemblages. Despite an expanding array of approaches for detecting microbes in a given sample, rapid and robust means of assessing the differential viability of these cells, as a function of phylogenetic lineage, remain elusive. In this study, pre-PCR propidium monoazide (PMA) treatment was coupled with downstream pyrosequencing and PhyloChip DNA microarray analyses to better understand the frequency, diversity and distribution of viable bacteria in spacecraft assembly cleanrooms. Sample fractions not treated with PMA, which were indicative of the presence of both live and dead cells, yielded a great abundance of highly diverse bacterial pyrosequences. In contrast, only 1% to 10% of all of the pyrosequencing reads, arising from a few robust bacterial lineages, originated from sample fractions that had been pre-treated with PMA. The results of PhyloChip analyses of PMA-treated and -untreated sample fractions were in agreement with those of pyrosequencing. The viable bacterial population detected in cleanrooms devoid of spacecraft hardware was far more diverse than that observed in cleanrooms that housed mission-critical spacecraft hardware. The latter was dominated by hardy, robust organisms previously reported to survive in oligotrophic cleanroom environments. Presented here are the findings of the first ever comprehensive effort to assess the viability of cells in low-biomass environmental samples, and correlate differential viability with phylogenetic affiliation.
doi:10.1038/ismej.2012.114
PMCID: PMC3554398  PMID: 23051695
viability; microarray; 454 pyrosequencing; PMA; PhyloChip; 16S rRNA gene
21.  Differential Growth Responses of Soil Bacterial Taxa to Carbon Substrates of Varying Chemical Recalcitrance 
Soils are immensely diverse microbial habitats with thousands of co-existing bacterial, archaeal, and fungal species. Across broad spatial scales, factors such as pH and soil moisture appear to determine the diversity and structure of soil bacterial communities. Within any one site however, bacterial taxon diversity is high and factors maintaining this diversity are poorly resolved. Candidate factors include organic substrate availability and chemical recalcitrance, and given that they appear to structure bacterial communities at the phylum level, we examine whether these factors might structure bacterial communities at finer levels of taxonomic resolution. Analyzing 16S rRNA gene composition of nucleotide analog-labeled DNA by PhyloChip microarrays, we compare relative growth rates on organic substrates of increasing chemical recalcitrance of >2,200 bacterial taxa across 43 divisions/phyla. Taxa that increase in relative abundance with labile organic substrates (i.e., glycine, sucrose) are numerous (>500), phylogenetically clustered, and occur predominantly in two phyla (Proteobacteria and Actinobacteria) including orders Actinomycetales, Enterobacteriales, Burkholderiales, Rhodocyclales, Alteromonadales, and Pseudomonadales. Taxa increasing in relative abundance with more chemically recalcitrant substrates (i.e., cellulose, lignin, or tannin–protein) are fewer (168) but more phylogenetically dispersed, occurring across eight phyla and including Clostridiales, Sphingomonadalaes, Desulfovibrionales. Just over 6% of detected taxa, including many Burkholderiales increase in relative abundance with both labile and chemically recalcitrant substrates. Estimates of median rRNA copy number per genome of responding taxa demonstrate that these patterns are broadly consistent with bacterial growth strategies. Taken together, these data suggest that changes in availability of intrinsically labile substrates may result in predictable shifts in soil bacterial composition.
doi:10.3389/fmicb.2011.00094
PMCID: PMC3153052  PMID: 21833332
soil; bacteria; carbon; substrate quality; bromo-deoxyuridine; microarray; rRNA copy number
22.  Fast UniFrac: Facilitating high-throughput phylogenetic analyses of microbial communities including analysis of pyrosequencing and PhyloChip data 
The ISME journal  2009;4(1):17-27.
Next-generation sequencing techniques, and PhyloChip, have made simultaneous phylogenetic analyses of hundreds of microbial communities possible. Insight into community structure has been limited by the inability to integrate and visualize such vast datasets. Fast UniFrac overcomes these issues, allowing integration of larger numbers of sequences and samples into a single analysis. Its new array-based implementation offers orders of magnitude improvements over the original version. New 3D visualization of principal coordinates analysis (PCoA) results, with the option to view multiple coordinate axes simultaneously, provides a powerful way to quickly identify patterns that relate vast numbers of microbial communities. We demonstrate the potential of Fast UniFrac using examples from three data types: Sanger-sequencing studies of diverse free-living and animal-associated bacterial assemblages and from the gut of obese humans as they diet, pyrosequencing data integrated from studies of the human hand and gut, and PhyloChip data from a study of citrus pathogens. We show that a Fast UniFrac analysis using a reference tree recaptures patterns that could not be detected without considering phylogenetic relationships and that Fast UniFrac, coupled with BLAST-based sequence assignment, can be used to quickly analyze pyrosequencing runs containing hundreds of thousands of sequences, revealing patterns relating human and gut samples. Finally, we show that the application of Fast UniFrac to PhyloChip data could identify well-defined subcategories associated with infection. Together, these case studies point the way towards a broad range of applications and demonstrate some of the new features of Fast UniFrac.
doi:10.1038/ismej.2009.97
PMCID: PMC2797552  PMID: 19710709
beta diversity; community ecology; multiplex pyrosequencing of 16S rDNA; PhyloChips
23.  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
24.  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
25.  Quorum Sensing Inhibition Selects for Virulence and Cooperation in Pseudomonas aeruginosa 
PLoS Pathogens  2010;6(5):e1000883.
With the rising development of bacterial resistance the search for new medical treatments beyond conventional antimicrobials has become a key aim of public health research. Possible innovative strategies include the inhibition of bacterial virulence. However, consideration must be given to the evolutionary and environmental consequences of such new interventions. Virulence and cooperative social behaviour of the bacterium Pseudomonas aeruginosa rely on the quorum-sensing (QS) controlled production of extracellular products (public goods). Hence QS is an attractive target for anti-virulence interventions. During colonization, non-cooperating (and hence less virulent) P. aeruginosa QS-mutants, benefiting from public goods provided by wild type isolates, naturally increase in frequency providing a relative protection from invasive infection. We hypothesized that inhibition of QS-mediated gene expression removes this growth advantage and selection of less virulent QS-mutants, and maintains the predominance of more virulent QS-wild type bacteria. We addressed this possibility in a placebo-controlled trial investigating the anti-QS properties of azithromycin, a macrolide antibiotic devoid of bactericidal activity on P. aeruginosa, but interfering with QS, in intubated patients colonized by P. aeruginosa. In the absence of azithromycin, non-cooperating (and hence less virulent) lasR (QS)-mutants increased in frequency over time. Azithromycin significantly reduced QS-gene expression measured directly in tracheal aspirates. Concomitantly the advantage of lasR-mutants was lost and virulent wild-type isolates predominated during azithromycin treatment. We confirmed these results in vitro with fitness and invasion experiments. Azithromycin reduced growth rate of the wild-type, but not of the lasR-mutant. Furthermore, the lasR-mutant efficiently invaded wild-type populations in the absence, but not in the presence of azithromycin. These in vivo and in vitro results demonstrate that anti-virulence interventions based on QS-blockade diminish natural selection towards reduced virulence and therefore may increase the prevalence of more virulent genotypes in the Hospital environment. More generally, the impact of intervention on the evolution of virulence of pathogenic bacteria should be assessed.
Trial Registration: ClinicalTrials.gov NCT00610623
Author Summary
With the rising development of antibiotic resistance and rapid spread of nosocomial pathogens, the search for new treatments beyond conventional antibiotics becomes a key aim of public health research. As such, anti-virulence therapies might be alternative antimicrobial strategies. However, consideration must be given to the potential evolutionary and environmental consequences of such interventions. Here we demonstrate a significant evolutionary impact of an anti-virulence intervention. Virulence and cooperative social behaviour of Pseudomonas aeruginosa rely on the quorum-sensing (QS) controlled production of extracellular products. In the absence of a specific intervention, non-cooperating (and hence less virulent) QS-mutants exploit and benefit from products provided by wild type isolates. As a consequence these less virulent mutants increase in frequency and provide a relative protection against infection to a colonized patient. In contrast, when QS-gene expression is reduced by the QS-inhibiting drug azithromycin, this advantage of QS-mutants is lost and virulent isolates predominate both in colonized patients and during in vitro experiments. These results suggest that QS-blockade may increase the prevalence of more virulent QS-responders among colonizing isolates in the hospital environment. More generally, the impact of anti-virulence interventions on the ecology and evolution of virulence of pathogenic bacteria needs to be assessed.
doi:10.1371/journal.ppat.1000883
PMCID: PMC2865528  PMID: 20463812

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