presented by seungdae oh
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Metagenomic Investigation of Microorganisms exposed to Benzalkonium Chlorides: Induction of Antibiotic Resistance. Presented by Seungdae Oh. School of Civil and Environmental Engineering Georgia Institute of Technology April, 26, 2012. Antibiotic resistance. S. aureus. Affect anyone - PowerPoint PPT PresentationTRANSCRIPT
Metagenomic Investigation of Microorganismsexposed to Benzalkonium Chlorides:
Induction of Antibiotic Resistance
Presented by
Seungdae Oh
School of Civil and Environmental EngineeringGeorgia Institute of Technology
April, 26, 2012
Antibiotic resistance
2
- Affect anyone
- Mortality
- Hamper health care systems
- Spread rapidly
- New antibiotics are drying up.
(IDSA, 2004)
No action today, no cure tomorrow.
S. aureus
P. aeruginosa
Entercocci
Benzalkonium chlorides (BAC)
-Disinfectant, cationic surfactant, phase transfer agents
- Cell membrane-active agents: Membrane perturbation: Inhibition of respiratory functions: Osmotic/oxidative stress
- BAC resistance mechanisms: Cell envelope modification: Efflux pumps: Oxidative stress defense systems
3
BAC resistance mechanisms also may work against antibiotics.
N
Cl−
R
BAC (R=C8H17 - C18H37)
B:BAC
DPB:Dextrin/Peptone + BAC
Inoculum
Aerobic fed-batch reactor - 14 days retention time at RT- >2 years operation
Substrates: Dextrin/Peptone (2,200 mg/L COD): BAC (140 mg/L COD)
http://www.csert.com/emergency.asp
Calcasieu River Sediment, LA
Development of microbial communities
DP: Dextrin/Peptone
DPB:Dextrin/Peptone + BAC
4
Minimum Inhibitory Concentrations (MICs, mg/L)
5
(Tandukar et al., unpublished)
BAC exposure induces antibiotic resistance.
Antimicrobials DP DPB B
BAC 100 250 460
Tetracycline <0.5 250 95
Ciprofloxacin <0.5 16 18
Metagenomics for the entire microbes
6
Whole genomic DNA
AssemblyGene prediction
Bioinformatics
ATGCATCCAATCCATGCA
Metabolism
Phylogeny
Evolution
Microbial communitySamples
Data preparation
7
Assembly Gene predictionFunctional characterization
Function DP-1 … DP-100
F-1 0.1 … 0.08
F-2 0.08 . 0.07
. . . .
F-11,912 0.05 … 0.07
Function DPB-1 … DPB-100
F-1 0.09 … 0.07
F-2 0.3 . 0.33
. . . .
F-11,912 0.04 … 0.08
Culture DP DPB B
Gene # 32,053 85,942
62,365
100 times of bootstrap to sample 5000 genes
100 subsets
5000 sampled genes normalized by the size and categorized into 11,912 functional categories
Before hypothesis testing, what the distributions in each function look like should be checked (normal or not normal?).
equal
Significantly different?
equal
Normality test
8
Jarque-Bera tests for distributions in each function
-Null hypothesis: Data come from a normal distribution with unknown mean and variance.
~7% of distributions are not normally distributed.
Not allowed to use Student’s T-test or F-test
DP
DPB
B
8%
6%
6%
Non-parametric tests
9
Distribution free tests, which do not rely on assumptions that the data are drawn from a given probability distribution (e.g., normal distribution).
-Ansari-Bradley test
-Mann-Whitney test
-Kolmogorov-Smirnov (KS) test
Null hypothesis: The samples are drawn from the same distributions.
Quantifies a distance between the empirical distributions of two samples.
KS test is not sensitive to the underlying distribution and adequate for metagenomic community comparison (Wang et al., 2011).
Gene functions that reject the null hypothesis
10
There are ~1000 functions where there is a statistical evidence that two distributions (control vs. DPB or B) are not identical (P < 10-4).
Some of the functions may relate to antimicrobial resistance mechanisms.
# functions
Log2 (DPB/DP or B/DP)
Gene functions enriched in DPB and B communities
11BAC exposure enriches antimicrobial resistance capabilities.
Oxidative stress defense
Membrane stability
Efflux pumps
Drug inactivation
Questions?
12
Biocides induce antibiotic resistance.(American Academy of Microbiology report, 2009; Karatzas et al., 2008; Loughlin et al., 2002; Mc Cay et al., 2010; Romanova et al., 2006; Tattawasart et al., 1999)
Biocide-resistant bacteria are not necessarily more resistant to antibiotics than biocide-sensitive bacteria.
(Anderson et al., 1997; Cole et al., 2003; Kucken et al., 2000; Lear et al., 2006; Sidhu et al., 2001a; Stecchini et al., 1992)
QAC: agents of spreading antibiotic resistance?
13
Conclusive evidence is lacking.
vs.
Research questions
14
1. Do QAC exposure induce antibiotic resistance? 2. What mechanisms enable the biocide-induced antibiotic resistance?