Metagenomic Investigation of Microorganisms exposed to Benzalkonium Chlorides: Induction of Antibiotic Resistance

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Metagenomic Investigation of Microorganismsexpose
d to Benzalkonium ChloridesInduction of
Antibiotic Resistance

• Presented by
• Seungdae Oh

School of Civil and Environmental
Engineering Georgia Institute of
Technology April, 26, 2012
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Antibiotic resistance

• Affect anyone
• Mortality
• Hamper health care systems
• Spread rapidly
• New antibiotics are drying up.

S. aureus
P. aeruginosa
Entercocci
No action today, no cure tomorrow.
(IDSA, 2004)
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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

Cl-
R
N
BAC (RC8H17 - C18H37)
BAC resistance mechanisms also may work against
antibiotics.
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Development of microbial communities

• Aerobic fed-batch reactor
• 14 days retention time at RT
• gt2 years operation
• Substrates
• Dextrin/Peptone (2,200 mg/L COD)
• BAC (140 mg/L COD)

Calcasieu River Sediment, LA
http//www.csert.com/emergency.asp
Inoculum
DPB Dextrin/Peptone BAC
DP Dextrin/Peptone
B BAC
DPB Dextrin/Peptone BAC
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Minimum Inhibitory Concentrations (MICs, mg/L)
Antimicrobials DP DPB B
BAC 100 250 460
Tetracycline lt0.5 250 95
Ciprofloxacin lt0.5 16 18
(Tandukar et al., unpublished)
BAC exposure induces antibiotic resistance.
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Metagenomics for the entire microbes
Samples
Microbial community
Whole genomic DNA
Metabolism
Phylogeny
Assembly Gene prediction
Bioinformatics
ATGCATCCA ATCCATGCA
Evolution
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Data preparation
Assembly Gene prediction Functional
characterization
Culture DP DPB B
Gene 32,053 85,942 62,365
100 times of bootstrap to sample 5000 genes
100 subsets
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
5000 sampled genes normalized by the size and
categorized into 11,912 functional categories
equal
Significantly different?
equal
Before hypothesis testing, what the distributions
in each function look like should be checked
(normal or not normal?).
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Normality test
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• 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 Students T-test or F-test

DP
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DPB
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 lt
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.
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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) 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)
vs.
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?