Andrew Y. Z. ZHANG MSc. Candidate Supervisor: Dr. T. ZHANG Department of Civil Engineering

1. Antibiotic Resistant Bacteria and Antibiotic Resistance Genes in Hong Kong Potable Water Andrew Y. Z. ZHANG MSc. Candidate Supervisor: Dr. T. ZHANG Department of Civil Engineering

2. Contents • Introduction • Objective • Materials & Methods • Results and Discussion • Conclusion

3. Introduction • Background of antibiotics • Usage of antibiotics • Introduction of antibiotic residues to aquatic environments • Occurrence of resistant bacteria due to antibiotic discharge • Current situation in south China

4. Background of Antibiotics Antibiotics are among the most frequently prescribed medications in modern medicine. Antibiotics cure disease by killing or injuring bacteria. The first antibiotic was penicillin, discovered accidentally from a mold culture in 1929. Today, over hundreds of different antibiotics are available to doctors to cure minor discomforts as well as life-threatening infections.

5. Background of Antibiotics • Different antibiotics destroy bacteria in different ways. • Short-circuit the processes by which bacteria receive energy. • Disturb the structure of the bacterial cell wall. • Interfere the production of essential proteins.

6. Major Classes of Antibiotics • Ketolides • Lincosamides • Macrolides • Oxazolidinones • Streptogramins • Sulphonamides • Tetracyclines • Aminoglycosides • β-lactams - Penicillins - Cephalosporins - Carbapenems - Monobactams • Fluoroquinolones

7. Usage of Antibiotics Annual Consumption of Antibiotics Kummerer, K (2003) Significance of antibiotics in the environment. Journal of Antimicrobial Chemotherapy 52 (1): 5-7

8. Usage of Antibiotics Journal of Antimicrobial Chemotherapy

9. Usage of Antibiotics • Penicillins are the most frequently used group of antibiotics. (accounted for 50%-70% of total antibiotic use in most countries) • Half of annual use of antibiotics in China was utilized for agricultural purpose. • 50% of antibiotic consumption in Australia was used as veterinary medicines or growth promoters while 36% was used in human. • The proportion of human use and animal use antibiotics is approximately 50/50 ratio in many western countries. • Most of the veterinary antibiotics were applied for growth promotion than disease treatment. Reference: Kummerer , K (2009a) Antibiotics in the aquatic environment- a review – Part I Chemosphere 75: 417-434 Richardson, BJ, Lam, PKS, Martin, M (2005) Emerging chemicals of concern: Pharmaceuticals and personal care products (PPCPs) in Asia, with particular reference to Southern China. Marine Pollution Bulletin 50 : 913-920

10. Introduction of antibiotic residues to aquatic environments

11. Introduction pathways of antibiotics to aquatic environments Antibiotic Substances Human medical antibiotics Veterinary antibiotics Direct entry in waste water Excretion of drugs (feces) Aquatic culture ( fish farms) Growth promoters, coccidiostatica Treatment of livestock Municipal Treatment Plant ----------------------------------------- Conditioned Wastewater Aquatic Environment (Obst et al., 2006)

12. Occurrence of resistant bacteria due to antibiotic discharge • Apart from antibiotic residues, antibiotic resistant bacteria in humans and animals could also be excreted in urine or faeces and discharged to the aquatic environment. (Koksal et al., 2007; Ram et al., 2008) • Antibiotic abuse and discharge to the environment would be risky as it would assist the occurrence/maintenance/spread of antibiotic resistant bacteria and those bacteria could be pathogenic. (Baquero et al., 2008)

13. Functional mechanisms of environmental ARGs dfrA1, A5, A7, A12, A15, A17 and 18; sulI, II, III and A cmlA1 and A5; floR; otrB; tetA, A(41), B, C, D, E, G, H, J, Y, Z, 33 and 39 aacC1, C2, C3 andC4; aadA1, A2, A5, A13 and B; ampC; aphA1, D and (3")-Ic; blaOXA-1, blaOXA-2, blaOXA-10, blaOXA-30and blaPSE-1; catI, II, III, IV, B2 and B3;mphA;nptII; sat1and 2; strAand B ermA, B, C, E, F, T, V and X; mecA; otrA; penA; tetB(P), M, O, Q, S, T and W; vanA and B

14. Horizontal Transfer of Antibiotic Resistance Genes

15. Current situation in Hong Kong ◆ Pearl Region Delta (PRD) region and Hong Kong: More than 15,000 tons of antibiotics were consumed in the region alone for human and veterinary use in 2004. ◆ Different types of antibiotics have been detected in the final effluent of STPs in Hong Kong at the concentration reaching thousands ng L-1. Antibiotic resistant genes have been identified in activated sludge of STPs in Hong Kong (T. ZHANG, et al., 2009) ◆ Pathogenic strains of antibiotic resistant bacteria have been isolated from aquatic environments in Hong Kong. Reference: Xu-Xiang Zhang, Tong Zhang, Herbert. H.H.P. Fang (2009) Antibiotic resistance gnens in water environments 82: 397-414 Xu-Xiang Zhang, Tong Zhang, (2011) Occurrence, Abundance, and Diversity of Tetracycline Resistance Genes in 15 Sewage Treatment Plants across China and other Global Locations 45: 2598-2604 Tong. ZHANG, Ming. ZHANG, Xu-Xiang ZHANG, (2009) Tetracycline Resistance Genes and Tetracycline Resistant Lactose --Fermenting Enterobacteriacae in Activated Sludge in Sewage Treatment Plants 43: 3455-3460

16. BBC NWES: Hong Kong is threatened by SuperbugFebruary 22, 1999

17. Objective of this Study • To investigate the species of bacteria isolated from Hong Kong potable water. • To identify the antibiotic resistance genes carried by the bacteria. • To characterize the antibiotic resistance phenotypes of ARB in tap water.

18. Materials and Methods Isolation and cultivation of bacteria from Hong Kong potable water Bacteria species identification by 16S rDNA-sequencing Antibiotic susceptibility test and multi-antibiotic resistance survey Antibiotic resistance genes identification by PCR and sequencing

19. Isolation and Cultivation of Bacteria Filtration Ultrasonic Vibration Suction Filtration Cultivation Tap Water Pre-concentration of Bacteria ⊕ Filter Cores: different diameter; for the purpose of distilled water preparation for laboratory use. ⊕ Ultrasonic Vibration: each filter in 1.5 L distilled water; BRANSON 8200 ultrasonic vibration. ⊕ Suction Filtration: 750 ml of solution for each membrane. ⊕ Cultivation: R2A agar media; 37°C for 24 hrs.

20. Filtration Ultrasonic Vibration Suction Filtration Cultivation Tap Water Pre-concentration of Bacteria Filter Cores: to prepare distilled water from tap water for laboratory use. 5 micron Carbon Filter 1 micron

21. Filtration Ultrasonic Vibration Suction Filtration Cultivation Tap Water Pre-concentration of Bacteria The filter cores were bathed in 1.5 L distilled water in a 5 L beaker and then placed in the BRANSON 8200 ultrasonic vibrator for one hour. Each end vibrated for 30 minutes. Bacteria trapped on the filter core were then detached in the 1.5 L distilled water.

22. Filtration Ultrasonic Vibration Suction Filtration Cultivation Tap Water Pre-concentration of Bacteria Suction filtration was adopted to concentrate the re-suspended bacteria again. The 1.5 L bacteria containing solution was filtered by two 47 mm diameter and 0.45 um pore size filters (Millipore) (Koksalet al., 2007) Each filter membrane was used to filter 750 ml of solution.

23. Filtration Ultrasonic Vibration Suction Filtration Cultivation Tap Water Pre-concentration of Bacteria Step 2 Cut into pieces using sterile scissors Step 1 Get the membrane Step 3 Saturated in 6 ml distilled water

24. R2A Media Preparing & Petri Dish Culturing R2A medium is considered suitable to culture oligotrophic bacteria, including the ones in the environment of potable water. These bacteria grow slowly under oligotrophic conditions and would be competed by faster growing bacteria if cultured on other common media. R2A culturing preparation : ¤18.12 g per 1 L distilled water ¤autoclaved at 121 ◦C for 20 minutes for disinfection ¤solidification inside the safety cabinet ¤bacteria saturated water coating on the plate ¤incubated at 37◦C for 24 hours ¤ colonies were transferred to new R2A medium to make single colonies 200 Isolates

25. Bacteria species identification by 16S rDNA sequencing DNA preparation PCR Amplification Electrophoresis screening DNA sequencing & analysis Principle of 16S rDNA sequencing 16S rDNA gene sequences contain hypervariable regions that can provide species-specific signature sequences useful for bacterial identification. As a result, 16S rRNA gene sequencing has become prevalent method of bacterial identification.

26. DNA preparation • DNA extracted from bacterial strains by direct cell lysis. • Bacteria cells transferred from R2A plate to PCR reaction tubes(200 tubes). • Tubes heated at 99◦Cfor 10 minutes in PCR machine. • Cellular membrane broken and bacteria DNA released to the distilled water----template.

27. PCR amplification The Polymerase Chain Reaction (PCR) is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. In this study, PCR primer: 8F (sequence: 5’-AGAGTTGATCCTGGCTCAG 3’ -) 1392R(sequence: 5’-GGTTACCTTGTTAC-GACTT 3’-) Reaction Solution: ※ 3 μl of 10× PCR buffer ※ 1.2 μl of dNTP ※ 0.75 U Taqpolymerase ※ 0.8 μl of 10 μ M 8F primer ※ 0.8 μl of 10 μ M 1392R primer ※ 2 μlof DNA template ※ 20 μl of distilled water

28. PCR amplification After the preparation of PCR reaction solution, all 200 reaction tubes were placed in PCR machine and set for amplification. ●Initialization Step: 95 ◦Cfor 7 minutes for 1cycle ●Denaturation Step: 95 ◦Cfor 1 minute ●Annealing Step: 55 ◦Cfor 1 minute ●Elongation Step: 72 ◦Cfor 1.5 minutes ●Final Extention: 72 ◦Cfor 10 minutes 35 cycles The whole reaction cycle was completed in three hours.

29. Electrophoresis screening PCR results verified by gel electrophoresis PCR reaction products: 5 μl for each strain 6 × loading buffer : 1 μl for each strain ● Loaded on the 1% agarose gel and separated by electrophoresis at 120V for 20 minutes ● DL2000 DNA marker was used in electrophoresis. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 2000 bp 1000 bp 750 bp 500 bp 250 bp Successful PCR process 100 bp

30. DNA sequencing and analysis 150 successful PCR results were selected for further analysis by DNA sequencing. Amplified DNA sequences were purified with PCR quick-spinTMPCR Product Purification Kit (iNtRON Biotechnology, INC) DNA sequences had been send to the following sectors for sequencing: NDM-1 DNA Database DNA sequences BLAST DNA hormology search software

31. DNA Sequencing Result Part 1 Part 2 Part 3

32. Archive of Bacteria Isolated from H.K. Potable Water Pathogenic Pathogenic

33. Phylogenetic tree Herbaspirillum Aquabacterium Pseudomonas Acidovorax Acinetobacter Bacillus Microbacterium

34. Antibiotic Susceptibility Test • Screening: Tetracycline and Ampicillin • Based on the presence of growth colonies on the antibiotics pre-mixed agars. • Conc. For Tetracycline and Ampicilline: 20mg/L in the agar solution. • The result suggested existence of antibiotic resistant bacteria in H.K. potable water.

35. Antibiotic Resistant Genes Investigation • PCR technology was used to amplify the targeted resistance gene DNA fractions. • Various antibiotic resistant genes were investigated. • Gel Documentation System used for preliminary identification. • DNA sequencing of selected DNA samples for confirmation. tet(A) tet(C) TEM-1 tet(D) oxa-1 Tetracycline Ampicillin tet(E) amp(C) tet(O) tet(M) tet(G) Reference: ZHANG T, ZHANG M, ZHANG, XX (2009) Tetracycline resistance genes and tetracycline resistant lactose--fermenting Enterobacteriacaein activated sludge in sewage treatment plants 43: 3455-3460 Richardson, BJ, Lam, PKS, Martin, M(2005) Emerging chemicals of concern: Pharmaceuticals and personal care products (PPCPs) in Asia, with particular reference to Southern China. Marine Pollution Bulletin 50 : 913-920

36. Gel Documentation of Antibiotic Resistance Genes 250 bp tet(C) tet (A) 275 bp 250 bp 100 bp amp (C) tet(G) 189 bp

37. Tetracycline Resistance Genes Investigation

38. Tetracycline Resistance Profile 54 strains out of 150 isolates showed tetracycline resistance phenotypes.

39. Multi-Antibiotic Resistance Investigation Acinetobacer Chryseobacterium Herbaspirillum Aquabacter Bacillus Acidovorax Comamonas Microbacterium Pseudomonas

40. Preliminary Conclusions • More than 13 species of bacteria could be culturable. Two genera of them are pathogenic(Bacillus and Pseudomonas). • Over 35% of the bacteria isolated are resistant to tetracycline and 64% are resistant to ampicilin. Bacteria are multi-resistant to various antibiotics. • Seven tetracycline resistance genes had been identified. Among them,tet(A), tet(C) and tet(G) are most frequently carried by tetracycline resistant bacteria. • Hong Kong potable water is polluted by antibiotic resistant bacteria.

41. Acknowledgements • Dr. T. Zhang • Dr. Xu-xiang Zhang • Lin Ye, Bing Li, KeYu, Ying Yang and other lab-mates • Enviromental Biotechnology Laboratory

42. Thank You ! Andrew Y. Z. ZHANG Department of Civil Engineering University of Hong Kong June 20, 2011