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Presented by Seungdae Oh

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

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Presented by Seungdae Oh

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  1. Metagenomic Investigation of Microorganismsexposed to Benzalkonium Chlorides:Induction of Antibiotic Resistance Presented by Seungdae Oh School of Civil and Environmental Engineering Georgia Institute of Technology April, 26, 2012

  2. Antibiotic resistance S. aureus • Affect anyone • Mortality • Hamper health care systems • Spread rapidly • New antibiotics are drying up. P. aeruginosa Entercocci No action today, no cure tomorrow. (IDSA, 2004)

  3. 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 (R=C8H17 - C18H37) BAC resistance mechanisms also may work against antibiotics.

  4. Development of microbial communities • 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) Calcasieu River Sediment, LA http://www.csert.com/emergency.asp Inoculum DPB :Dextrin/Peptone + BAC DP: Dextrin/Peptone B :BAC DPB :Dextrin/Peptone + BAC

  5. Minimum Inhibitory Concentrations (MICs, mg/L) (Tandukar et al., unpublished) BAC exposure induces antibiotic resistance.

  6. Metagenomicsfor the entire microbes Samples Microbial community Whole genomic DNA Metabolism Phylogeny Assembly Gene prediction Bioinformatics ATGCATCCA ATCCATGCA Evolution

  7. Data preparation Assembly Gene prediction Functional characterization 100 times of bootstrap to sample 5000 genes 100 subsets 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?).

  8. 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 6% DPB 6% B

  9. 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).

  10. 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.

  11. 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.

  12. Questions?

  13. 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.

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

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