Loss of Bacterial Diversity during Antibiotic Treatment of Intubated Patients Colonized with Pseudomonas aeruginosa

ABSTRACT Management of airway infections caused by Pseudomonas aeruginosa is a serious clinical challenge, but little is known about the microbial ecology of airway infections in intubated patients. We analyzed bacterial diversity in endotracheal aspirates obtained from intubated patients colonized by P. aeruginosa by using 16S rRNA clone libraries and microarrays (PhyloChip) to determine changes in bacterial community compositions during antibiotic treatment. Bacterial 16S rRNA genes were absent from aspirates obtained from patients briefly intubated for elective surgery but were detected by PCR in samples from all patients intubated for longer periods. Sequencing of 16S rRNA clone libraries demonstrated the presence of many orally, nasally, and gastrointestinally associated bacteria, including known pathogens, in the lungs of patients colonized with P. aeruginosa. PhyloChip analysis detected the same organisms and many additional bacterial groups present at low abundance that were not detected in clone libraries. For each patient, both culture-independent methods showed that bacterial diversity decreased following the administration of antibiotics, and communities became dominated by a pulmonary pathogen. P. aeruginosa became the dominant species in six of seven patients studied, despite treatment of five of these six with antibiotics to which it was sensitive in vitro. Our data demonstrate that the loss of bacterial diversity under antibiotic selection is highly associated with the development of pneumonia in ventilated patients colonized with P. aeruginosa. Interestingly, PhyloChip analysis demonstrated reciprocal changes in abundance between P. aeruginosa and the class Bacilli, suggesting that these groups may compete for a similar ecological niche and suggesting possible mechanisms through which the loss of microbial diversity may directly contribute to pathogen selection and persistence.

[1]  P. Hugenholtz,et al.  Design and evaluation of 16S rRNA-targeted oligonucleotide probes for fluorescence in situ hybridization. , 2002, Methods in molecular biology.

[2]  D. Lane.  16S/23S rRNA sequencing , 1991 .

[3]  J. Banfield,et al.  Phylogeny of Microorganisms Populating a Thick, Subaerial, Predominantly Lithotrophic Biofilm at an Extreme Acid Mine Drainage Site , 2000, Applied and Environmental Microbiology.

[4]  B. Cunha,et al.  Activated protein C resistance and Sneddon's syndrome. , 1999 .

[5]  Eoin L. Brodie,et al.  Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB , 2006, Applied and Environmental Microbiology.

[6]  B. Bassler,et al.  Quorum sensing: cell-to-cell communication in bacteria. , 2005, Annual review of cell and developmental biology.

[7]  Steven A. Frank,et al.  Models of Parasite Virulence , 1996, The Quarterly Review of Biology.

[8]  Michel Wolff,et al.  Comparison of 8 vs 15 days of antibiotic therapy for ventilator-associated pneumonia in adults: a randomized trial. , 2003, JAMA.

[9]  R. Kent,et al.  The microbiota of young children from tooth and tongue samples. , 2002, Journal of dental research.

[10]  J. Karlowsky,et al.  Factors Associated with Relative Rates of Antibiotic Resistance in Pseudomonas aeruginosa Isolates Tested in Clinical Laboratories in the United States from 1999 to 2002 , 2004, Antimicrobial Agents and Chemotherapy.

[11]  G. Ray,et al.  Hospital-level rates of fluoroquinolone use and the risk of hospital-acquired infection with ciprofloxacin-nonsusceptible Pseudomonas aeruginosa. , 2005, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[12]  M. Salaspuro,et al.  Acetaldehyde production and other ADH-related characteristics of aerobic bacteria isolated from hypochlorhydric human stomach. , 2001, Alcoholism, clinical and experimental research.

[13]  D. Prough,et al.  Nosocomial pulmonary infection: Possible etiologic significance of bacterial adhesion to endotracheal tubes , 1985, Critical care medicine.

[14]  K. Ward,et al.  Duration of action of a single, early oral application of chlorhexidine on oral microbial flora in mechanically ventilated patients: a pilot study. , 2004, Heart & lung : the journal of critical care.

[15]  A. Torres,et al.  The role of the gastric reservoir in ventilator-associated pneumonia. , 1995, Clinical intensive care : international journal of critical & coronary care medicine.

[16]  Thomas Huber,et al.  Bellerophon: a program to detect chimeric sequences in multiple sequence alignments , 2004, Bioinform..

[17]  E. Zoetendal,et al.  A microbial world within us , 2006, Molecular microbiology.

[18]  I. Thompson,et al.  Characterisation of the culturable heterotrophic bacterial community in a small eutrophic lake (Priest Pot). , 2001, FEMS microbiology ecology.

[19]  E. Delong,et al.  Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing , 1991, Journal of bacteriology.

[20]  A. Torres,et al.  Stomach as a source of colonization of the respiratory tract during mechanical ventilation: association with ventilator-associated pneumonia. , 1996, The European respiratory journal.

[21]  K. Yuen,et al.  Identification by 16S ribosomal RNA gene sequencing of Arcobacter butzleri bacteraemia in a patient with acute gangrenous appendicitis , 2002, Molecular pathology : MP.

[22]  J. Doré,et al.  Direct Analysis of Genes Encoding 16S rRNA from Complex Communities Reveals Many Novel Molecular Species within the Human Gut , 1999, Applied and Environmental Microbiology.

[23]  A. Read,et al.  The Ecology of Genetically Diverse Infections , 2001, Science.

[24]  L. T. Angenent,et al.  Molecular identification of potential pathogens in water and air of a hospital therapy pool. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Philip Hugenholtz,et al.  NAST: a multiple sequence alignment server for comparative analysis of 16S rRNA genes , 2006, Nucleic Acids Res..

[26]  M. Nadkarni,et al.  Molecular Analysis of Microbial Diversity in Advanced Caries , 2005, Journal of Clinical Microbiology.

[27]  J. Handelsman,et al.  Status of the Microbial Census , 2004, Microbiology and Molecular Biology Reviews.

[28]  W. Wade,et al.  Molecular and Cultural Analysis of the Microflora Associated with Endodontic Infections , 2002 .

[29]  J. Handelsman,et al.  Introducing DOTUR, a Computer Program for Defining Operational Taxonomic Units and Estimating Species Richness , 2005, Applied and Environmental Microbiology.

[30]  D. M. Ward,et al.  16S rRNA sequences reveal numerous uncultured microorganisms in a natural community , 1990, Nature.

[31]  Sam P. Brown,et al.  Does multiple infection select for raised virulence? , 2002, Trends in microbiology.

[32]  A. Buckling,et al.  Interference competition and parasite virulence , 2004, Proceedings of the Royal Society of London. Series B: Biological Sciences.

[33]  Gary L. Andersen,et al.  High-Density Universal 16S rRNA Microarray Analysis Reveals Broader Diversity than Typical Clone Library When Sampling the Environment , 2007, Microbial Ecology.

[34]  S. Giovannoni,et al.  Genetic diversity in Sargasso Sea bacterioplankton , 1990, Nature.

[35]  L. Aarons,et al.  Molecular Analysis of the Subgingival Microbiota in Health and Disease , 2006, Applied and Environmental Microbiology.

[36]  Gary L. Andersen,et al.  High-Density Microarray of Small-Subunit Ribosomal DNA Probes , 2002, Applied and Environmental Microbiology.

[37]  E. Purdom,et al.  Diversity of the Human Intestinal Microbial Flora , 2005, Science.

[38]  P Green,et al.  Base-calling of automated sequencer traces using phred. II. Error probabilities. , 1998, Genome research.

[39]  Eoin L Brodie,et al.  Application of a High-Density Oligonucleotide Microarray Approach To Study Bacterial Population Dynamics during Uranium Reduction and Reoxidation , 2006, Applied and Environmental Microbiology.

[40]  J. Sanderson,et al.  Molecular characterization of the bacteria adherent to human colorectal mucosa , 2006, Journal of applied microbiology.

[41]  N. Pace,et al.  Characterization of a Yellowstone hot spring microbial community by 5S rRNA sequences , 1985, Applied and environmental microbiology.

[42]  F. Dewhirst,et al.  Bacterial Diversity in Human Subgingival Plaque , 2001, Journal of bacteriology.

[43]  S. Ott,et al.  Reduced microbial diversity in inflammatory bowel diseases. , 2006, Gut.

[44]  J. Cantrell,et al.  The presence and sequence of endotracheal tube colonization in patients undergoing mechanical ventilation. , 1999, The European respiratory journal.

[45]  Gary L Andersen,et al.  Rapid quantification and taxonomic classification of environmental DNA from both prokaryotic and eukaryotic origins using a microarray. , 2005, FEMS microbiology letters.

[46]  P. Green,et al.  Base-calling of automated sequencer traces using phred. I. Accuracy assessment. , 1998, Genome research.

[47]  J. Bernstein,et al.  Molecular analysis of bacterial flora associated with chronically inflamed maxillary sinuses. , 2003, Journal of medical microbiology.

[48]  S. Lewenza,et al.  Interspecies communication between Burkholderia cepacia and Pseudomonas aeruginosa. , 2002, Canadian journal of microbiology.

[49]  F. Dewhirst,et al.  Bacterial diversity in necrotizing ulcerative periodontitis in HIV-positive subjects. , 2002, Annals of Periodontology.

[50]  N. Saitou,et al.  The neighbor-joining method: a new method for reconstructing phylogenetic trees. , 1987, Molecular biology and evolution.

[51]  Zhang Sb,et al.  Analysis of the c- kit gene in patients with slow transit constipation , 2005 .

[52]  S. Levy.  Antibiotic resistance: an ecological imbalance. , 2007, Ciba Foundation symposium.

[53]  J. Kreft,et al.  The evolution of groups of cooperating bacteria and the growth rate versus yield trade-off. , 2005, Microbiology.

[54]  G. Roberts,et al.  Statistical Analyses of Complex Denaturing Gradient Gel Electrophoresis Profiles , 2005, Journal of Clinical Microbiology.