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

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- 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

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BAC resistance mechanisms also may work against antibiotics.

NCl−

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

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Minimum Inhibitory Concentrations (MICs, mg/L)

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(Tandukar et al., unpublished)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

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Whole genomic DNA

AssemblyGene prediction

Bioinformatics

ATGCATCCAATCCATGCA

Metabolism

Phylogeny

Evolution

Microbial communitySamples

Data preparation

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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

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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 Student’s T-test or F-test

DP

DPB

B

8%

6%

6%

Non-parametric tests

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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

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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?

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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?

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Conclusive evidence is lacking.

vs.

Research questions

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1. Do QAC exposure induce antibiotic resistance? 2. What mechanisms enable the biocide-induced antibiotic resistance?