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
P. aeruginosa
Entercocci
- Mortality
- Hamper health care systems
- Spread rapidly
- New antibiotics are drying up.
No action today,
no cure tomorrow.
(IDSA, 2004)
2
Benzalkonium chlorides (BAC)
-Disinfectant, cationic surfactant,
phase transfer agents
Cl−
R
N
- Cell membrane-active agents
: Membrane perturbation
: Inhibition of respiratory functions
: Osmotic/oxidative stress
BAC (R=C8H17 - C18H37)
- BAC resistance mechanisms
: Cell envelope modification
: Efflux pumps
: Oxidative stress defense systems
BAC resistance mechanisms also may work against antibiotics.
3
Development of microbial communities
Aerobic fed-batch reactor
Calcasieu River Sediment, LA
- 14 days retention time at RT
- >2 years operation
Substrates
http://www.csert.com/emer
gency.asp
: Dextrin/Peptone (2,200 mg/L COD)
: BAC (140 mg/L COD)
Inoculum
DPB
:Dextrin/Peptone + BAC
DP: Dextrin/Peptone
DPB
:Dextrin/Peptone + BAC
B
:BAC
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Minimum Inhibitory Concentrations (MICs, mg/L)
Antimicrobials DP
DPB
B
BAC
100
250
460
Tetracycline
<0.5
250
95
Ciprofloxacin
<0.5
16
18
(Tandukar et al., unpublished)
BAC exposure induces antibiotic resistance.
5
Metagenomics for the entire microbes
Samples
Microbial community
Whole genomic DNA
Metabolism
Phylogeny
Bioinformatics
Evolution
ATGCATCCA
ATCCATGCA
Assembly
Gene prediction
6
Data preparation
Assembly
Gene prediction
Functional characterization
Culture
DP
DPB
B
Gene #
32,053
85,94
2
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
Function
DP-1
…
DP-100
F-1
0.1
…
0.08
F-2
0.08
.
0.07
.
.
.
.
F-11,912
0.05
…
0.07
equal
Function
DPB-1
…
DPB-100
F-1
0.09
…
0.07
0.3
.
0.33
.
.
.
0.04
…
0.08
F-2
Significantly
different? .
F-11,912
equal
Before hypothesis testing, what the distributions in each
function look like should be checked (normal or not normal?).
7
Normality test
Normal
Not normal
8%
D
P
Jarque-Bera tests for distributions in each function
-Null hypothesis: Data come from a normal
distribution with unknown mean and variance.
6%
 ~7% of distributions are not normally distributed.
DPB
 Not allowed to use Student’s T-test or F-test
6%
B
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Non-parametric tests
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).
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Gene functions that reject the null hypothesis
# functions
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.
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Gene functions enriched in DPB and B communities
Drug
inactivation
Efflux pumps
Membrane stability
Oxidative stress
defense
Log2 (DPB/DP or B/DP)
BAC exposure enriches antimicrobial resistance capabilities.11
Questions?
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QAC: agents of spreading antibiotic resistance?
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)
vs.
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)
Conclusive evidence is lacking.
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Research questions
1. Do QAC exposure induce antibiotic resistance?
2. What mechanisms enable the biocide-induced antibiotic
resistance?
14