1. 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

2. 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

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 3 BAC resistance mechanisms also may work against antibiotics. N Cl − R BAC (R=C 8 H 17 - C 18 H 37 )

4. 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/emer gency.asp Calcasieu River Sediment, LA Development of microbial communities DP: Dextrin/Peptone DPB :Dextrin/Peptone + BAC 4

5. Minimum Inhibitory Concentrations (MICs, mg/L) 5 (Tandukar et al., unpublished) BAC exposure induces antibiotic resistance. AntimicrobialsDPDPBB BAC100250460 Tetracycline<0.525095 Ciprofloxacin<0.51618

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

7. Data preparation 7 Assembly Gene prediction Functional characterization FunctionDP-1…DP-100 F-10.1…0.08 F-20.08.0.07.... F-11,9120.05…0.07 FunctionDPB-1…DPB-100 F-10.09…0.07 F-20.3.0.33.... F-11,9120.04…0.08 CultureDPDPBB Gene #32,05385,94 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 Before hypothesis testing, what the distributions in each function look like should be checked (normal or not normal?). equal Significantly different? equal

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 DPDP DPB B 8% 6%

9. 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).

10. 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

11. Log 2 (DPB/DP or B/DP) Gene functions enriched in DPB and B communities 11 BAC exposure enriches antimicrobial resistance capabilities. Oxidative stress defense Membrane stability Efflux pumps Drug inactivation

12. Questions? 12

13. 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.

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