CenterCenter forfor EnvironmentalEnvironmental BiotechnologyBiotechnology Environmental Influences on Emerging Pathogens, Development of a High- Density Microarray to Measure Microbial Community Dynamics Gary L. Andersen Lawrence Berkeley National Lab [email protected] Culture enrichment has been used for over 100 years for bacterial pathogen discovery Bacillus Bordetella Clostridium Escherichia Spirulina Staphylococcus Streptococcus Salmonella http://www.pbrc.hawaii.edu/kunkel/gallery/ Stable Biomarkers: the16S rRNA gene 1. Along with 23S 16S rDNArRNA, dnaK, differences used to infer phylogeny. 2. Less prone to homologous rRNA (functionalrecombination. molecule) 3. Conserved regions ncbi.nlm.nih.gov of sequence allow 200,000 180,000 for PCR 160,000 140,000 amplification in 120,000 100,000 most bacteria. 80,000 60,000 4. Rapidly expanding 40,000 20,000 gene database. Cumulative NCBI 16S records 0 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 Year Novel Pathogens Found with 16SrRNA •Bartonella henselae - the causative agent for bacillary angiomatosis (cannot be cultured) •Ehrlichia chaffeensis- a new species associated with tick bites, similar to Rocky Mt. Spotted fever •Tropheryma whipplei- causative agent of Whipple’s disease in duodenum. Disease is chronic--diarrhea, weight loss, lymphadenopathies and polyarthritis. Whipple noted bacilli in lymph node in a patient with weight loss, joint pain and abdominal pain in 1907 but not identified until application of sequence analysis in 1992 A PhyloChip to study microbial communities • Massive parallelism - 500,000 probes in a 1.28 cm2 array • Identification of multiple species in a mixed population • Organism specific region of amplified DNA products are used for targets • Mismatch control oligo for each probe used to minimize effect of cross-hybridization cctagcatgCattctgcata cctagcatgGattctgcata MATCH MISMATCH Probe Design is Critical for Distinguishing Bacterial Groups (http://greengenes.lbl.gov) • Cluster similar 16S sequences: – Remove chimeras – High quality • Create probe sets – Identify multiple unique regions – Test for cross- hybridization Each probe-set is placed in a phylogenetic context • Hierarchical assignment of probe-sets is based on variation in 16S sequence • Minimum of 11 probe-pairs • Detection of over 90% of the assigned probes required for positive identification • Each probe-set assigned to one of 455 families 2 domains Å 63 phyla Å136 classes Å 262 orders Å 455 families 842 sub-families (~94% identity) 8,989 Probe-Sets (~99% identity) Example of the Location of Probes Used for the Desulfovibrio vulgaris Probe Set Desulfovibrio sp. str. DMB. Desulfovibrio sp. 'Bendigo A' Desulfovibrio vulgaris DSM 644 Regions not unique to taxon Regions unique to taxon A A C C G A G G A C G T C G T C A G G C G C G T G A G T C A G C G T G A C A G Overview of SampleA Preparation18 µ 18 µ Extract Genomic DNA PCR Amplify DNA Hybridize Fractionate DNA End-label with biotin Affymetrix CHIP Computer Screen display The CHIP Impact of microbial community dynamics on human health What are the “typical” pathogens ¥ in the air that we breath and do they change over time? ¥ How does antibiotic use affect microbial communities in lung? ¥ What is the diversity of bacteria in the GI tract? Climate change will alter microbial composition of aerosols Microbial Diversity in Aerosols: A Pathogen Surveillance Perspective •Major US programs, >$100M/Y to monitor urban locations for pathogens •A number of documented “false-positive” detections each year •Background levels of microbes, including pathogens is unknown •Sequential 17 weekly samples for 2 cities to determine scope of variability San Antonio AustinGulf of Mexico Assess diversity by time (week) and location (city). Comparing the phylogenetic diversity detected using DNA Microarrays or Cloning & Sequencing •Dominant Types •Bacillus megaterium 48 •Bacillus 81 taxa pseudomegaterium 59 170 taxa •Terrabacter sp. YK10 8 detected by array detected by library 8 taxa 97 taxa 73 taxa detected by detected detected by clones only by array both methods only 9 taxa confirmed with specific PCR primers Is Nitrospira Present? Chip says “YES” Clone library says “NO” Bacteria;Nitrospira;Nitrospira; Nitrospirales;Nitrospiraceae; Confirmation sf_1; OTU 864 prokMSA_id:1456 X82558.1 Nitrospira moscoviensis PCR with OTU Specific prokMSA_id:1468 Y14644.1 nitrifying sludge clone GC86 prokMSA_id:1490 AF035813.1 nitrifying sludge clone Hovanec Primers says “YES” prokMSA_id:1491 AF155152.1 nitrifying sludge clone SBR1015 prokMSA_id:38830 AF293010.1 ferromanganous micronodule clone MNC2 prokMSA_id:85357 AY221079.2 Mammoth cave clone CCU23 prokMSA_id:88224 AY328760.1 drinking water system simulator clone DSSD62 prokMSA_id:102267 AY532585.1 uranium-contaminated aquifer clone 1013-28-CG50 prokMSA_id:110431 AJ617919.1 oxic-anoxic interphase flooded paddy soil clone D14423 800 bp 200 bp 27 1492 Sequencing of PCR products says “YES” Nitrospira moscoviensis X82558 (98.3%) Does Mycobacterium detection increase with temperature? 6.5 r = 0.64, p=0.026527 6 (adjusted for multiple testing) Amount of DNA 5.5 M. tuberculosis detected subgroup'Myb.tuberculosis_subgrou on chip 5 p Mycobacterium PENDANT-38. 4.5 4 75 80 85 90 degrees F Multivariate regression tree analysis: Interaction of environmental factors with aerosol bacterial dynamics Actinomycetes Environmental factors Firmicutes 1.) Week Bacteroides 2.) Temp Cyanobacteria 3.) PM 2.5 Alpha-proteobacteria Beta-proteobacteria 4.) Mean SLP Gamma-proteobacteria n Austin City Week n San Antonio City Week 22 24 27 31 32 29 35 33 24 34 30 34 25 26 23 28 19 20 21 19 20 21 23 26 28 29 32 35 25 22 27 30 31 33 Microbial ecology of Ventilator-associated pneumonia Microarray analysis Many anaerobes, Alcaligenes Mycoplasma Pseudomonas aeruginosa Staphylococcus Alcanivorax Prevotella Campylobacter Coriobacter Helicobacter Streptococcus Bifidobacterium Many anaerobes including sulfate reducers 10/25/0410/14/04 10/29/04 98 151 139 Diversity (number of phylotypes detected) declines and rebounds High-density array analysis of gut microflora Previous studies Ley et al. 2005. PNAS 31:11070 Eckburg et al. 2005. Science 308:1635 Obesity alters gut microbial ecology Diversity of the Human Intestinal Microbial Flora 5,088 clones - mouse 11,831 clones - human Additional phyla/divisions detected in human gut samples by high-density array From: Ley et al. 2005. PNAS 31:11070 Microbial ecology of the human gut 214-324 subfamilies detected • Variation between individuals highest • No significant change in diversity from healthy to sick • Gut ecosystem is dynamic – large temporal abundance changes • May be changes in activity – not composition Direct rRNA analysis Aliquot treated with DNase Intact 16S rRNA RNA fragmented using RNase III purified from dephosphorylated agarose gel using SAP DNA/RNA extracted from environmental sample RNA terminally labeled with biotin In house analysis scripts using novel donor molecule and T4 RNA ligase 16S GeneChip Biotin labeled RNA labeled hybridized to washed 16S GeneChip and scanned Cole et al NAR 32:e86 2004 Direct rRNA analysis Archaea More active in 100 mM OC Acidobacteria δ-proteobacteria Azoarcus spp. More active in 0 mM OC Conclusions • Microarray data complements clone and sequence libraries in the assessment of bacterial diversity. • Confirmation of array data by specific PCR and qPCR. • Clone library consistently underestimates microbial richness. • Organisms identified at the subgroup or probe-set level may be followed by more precise sampling techniques.. • Candidate organisms may be identified without prior selection by monitoring population shifts Acknowledgements Todd DeSantis Sonya Murray Jordan Moberg Ingrid Zubieta Eoin Brodie Terry Hazen Phil Hugenholtz Kyle Cole Chuan Chen.