Metagenomic Investigation of Microorganisms exposed to Benzalkonium Chlorides: Induction of...
-
Upload
rhoda-holmes -
Category
Documents
-
view
228 -
download
0
description
Transcript of Metagenomic Investigation of Microorganisms exposed to Benzalkonium Chlorides: Induction of...
![Page 1: Metagenomic Investigation of Microorganisms exposed to Benzalkonium Chlorides: Induction of Antibiotic Resistance Presented by Seungdae Oh School of Civil.](https://reader034.fdocuments.in/reader034/viewer/2022051008/5a4d1b427f8b9ab0599a1792/html5/thumbnails/1.jpg)
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
![Page 2: Metagenomic Investigation of Microorganisms exposed to Benzalkonium Chlorides: Induction of Antibiotic Resistance Presented by Seungdae Oh School of Civil.](https://reader034.fdocuments.in/reader034/viewer/2022051008/5a4d1b427f8b9ab0599a1792/html5/thumbnails/2.jpg)
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
![Page 3: Metagenomic Investigation of Microorganisms exposed to Benzalkonium Chlorides: Induction of Antibiotic Resistance Presented by Seungdae Oh School of Civil.](https://reader034.fdocuments.in/reader034/viewer/2022051008/5a4d1b427f8b9ab0599a1792/html5/thumbnails/3.jpg)
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.
NCl−
R
BAC (R=C8H17 - C18H37)
![Page 4: Metagenomic Investigation of Microorganisms exposed to Benzalkonium Chlorides: Induction of Antibiotic Resistance Presented by Seungdae Oh School of Civil.](https://reader034.fdocuments.in/reader034/viewer/2022051008/5a4d1b427f8b9ab0599a1792/html5/thumbnails/4.jpg)
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
![Page 5: Metagenomic Investigation of Microorganisms exposed to Benzalkonium Chlorides: Induction of Antibiotic Resistance Presented by Seungdae Oh School of Civil.](https://reader034.fdocuments.in/reader034/viewer/2022051008/5a4d1b427f8b9ab0599a1792/html5/thumbnails/5.jpg)
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
![Page 6: Metagenomic Investigation of Microorganisms exposed to Benzalkonium Chlorides: Induction of Antibiotic Resistance Presented by Seungdae Oh School of Civil.](https://reader034.fdocuments.in/reader034/viewer/2022051008/5a4d1b427f8b9ab0599a1792/html5/thumbnails/6.jpg)
Metagenomics for the entire microbes
6
Whole genomic DNA
AssemblyGene prediction
Bioinformatics
ATGCATCCAATCCATGCA
Metabolism
Phylogeny
Evolution
Microbial communitySamples
![Page 7: Metagenomic Investigation of Microorganisms exposed to Benzalkonium Chlorides: Induction of Antibiotic Resistance Presented by Seungdae Oh School of Civil.](https://reader034.fdocuments.in/reader034/viewer/2022051008/5a4d1b427f8b9ab0599a1792/html5/thumbnails/7.jpg)
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
![Page 8: Metagenomic Investigation of Microorganisms exposed to Benzalkonium Chlorides: Induction of Antibiotic Resistance Presented by Seungdae Oh School of Civil.](https://reader034.fdocuments.in/reader034/viewer/2022051008/5a4d1b427f8b9ab0599a1792/html5/thumbnails/8.jpg)
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%
![Page 9: Metagenomic Investigation of Microorganisms exposed to Benzalkonium Chlorides: Induction of Antibiotic Resistance Presented by Seungdae Oh School of Civil.](https://reader034.fdocuments.in/reader034/viewer/2022051008/5a4d1b427f8b9ab0599a1792/html5/thumbnails/9.jpg)
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).
![Page 10: Metagenomic Investigation of Microorganisms exposed to Benzalkonium Chlorides: Induction of Antibiotic Resistance Presented by Seungdae Oh School of Civil.](https://reader034.fdocuments.in/reader034/viewer/2022051008/5a4d1b427f8b9ab0599a1792/html5/thumbnails/10.jpg)
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
![Page 11: Metagenomic Investigation of Microorganisms exposed to Benzalkonium Chlorides: Induction of Antibiotic Resistance Presented by Seungdae Oh School of Civil.](https://reader034.fdocuments.in/reader034/viewer/2022051008/5a4d1b427f8b9ab0599a1792/html5/thumbnails/11.jpg)
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
![Page 12: Metagenomic Investigation of Microorganisms exposed to Benzalkonium Chlorides: Induction of Antibiotic Resistance Presented by Seungdae Oh School of Civil.](https://reader034.fdocuments.in/reader034/viewer/2022051008/5a4d1b427f8b9ab0599a1792/html5/thumbnails/12.jpg)
Questions?
12
![Page 13: Metagenomic Investigation of Microorganisms exposed to Benzalkonium Chlorides: Induction of Antibiotic Resistance Presented by Seungdae Oh School of Civil.](https://reader034.fdocuments.in/reader034/viewer/2022051008/5a4d1b427f8b9ab0599a1792/html5/thumbnails/13.jpg)
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.
![Page 14: Metagenomic Investigation of Microorganisms exposed to Benzalkonium Chlorides: Induction of Antibiotic Resistance Presented by Seungdae Oh School of Civil.](https://reader034.fdocuments.in/reader034/viewer/2022051008/5a4d1b427f8b9ab0599a1792/html5/thumbnails/14.jpg)
Research questions
14
1. Do QAC exposure induce antibiotic resistance? 2. What mechanisms enable the biocide-induced antibiotic resistance?