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

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Andrew Y. Z. ZHANGMSc. Candidate

Supervisor: Dr. T. ZHANGDepartment of Civil Engineering

Antibiotic Resistant Bacteria and Antibiotic Resistance Genes in Hong Kong Potable Water

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Contents

• Introduction

• Objective

• Materials & Methods

• Results and Discussion

• Conclusion

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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

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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.

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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.

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Major Classes of Antibiotics

• Aminoglycosides• β-lactams - Penicillins - Cephalosporins - Carbapenems - Monobactams• Fluoroquinolones

• Ketolides• Lincosamides• Macrolides• Oxazolidinones• Streptogramins• Sulphonamides• Tetracyclines

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Usage of AntibioticsAnnual Consumption of Antibiotics

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

Country or Region Comsuption Persentage

All Over World 100,000-200,000 tons N/A

China 25,000 tons 12.5%-25%

European Union 10,200 tons 5.1%-10.2%

United States 22,700 tons 11.35%-22.7%

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Usage of AntibioticsJournal of Antimicrobial Chemotherapy

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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

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Introduction of antibiotic residues to aquatic environments

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Introduction pathways of antibiotics to aquatic environments

Antibiotic Substances

Human medical antibiotics

Direct entry in waste water

Municipal Treatment Plant -----------------------------------------

Conditioned Wastewater

Veterinary antibiotics

Excretion of drugs (feces)

Aquatic culture

( fish farms)

Growth promoters,

coccidiostatica

Treatment of livestock

Aquatic Environment (Obst et al., 2006)

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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)

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aacC1, C2, C3 and C4; aadA1, A2, A5, A13 and B; ampC; aphA1, D and (3")-Ic; blaOXA-1, blaOXA-2, blaOXA-

10, blaOXA-30 and blaPSE-1; catI, II, III, IV, B2 and B3; mphA; nptII; sat1 and 2; strA and 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

Functional mechanisms of environmental ARGs

cmlA1 and A5; floR; otrB; tetA, A(41), B, C, D, E, G, H, J, Y, Z, 33 and 39

dfrA1, A5, A7, A12, A15, A17 and 18; sulI, II, III and A

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Horizontal Transfer of Antibiotic Resistance Genes

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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

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

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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.

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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

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Isolation and Cultivation of Bacteria

Tap Water

Filtration Ultrasonic Vibration Suction Filtration Cultivation

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.

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Tap Water



Filter Cores: to prepare distilled water from tap water for laboratory use.

5 micron Carbon Filter 1 micron

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Tap Water



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.

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Tap Water



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) (Koksal et al., 2007)

Each filter membrane was used to filter 750 ml of solution.

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Tap Water



Step 1 Get the membrane

Step 2 Cut into pieces using sterile scissors

Step 3 Saturated in 6 ml distilled water

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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

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Bacteria species identification by 16S rDNA sequencing

DNA preparation

PCR Amplification

Electrophoresis screening

DNA sequencing &

analysis

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.

Principle of 16S rDNA sequencing

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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 ◦C for 10 minutes in PCR machine. • Cellular membrane broken and bacteria DNA released to the distilled water----template.

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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 Taq polymerase ※ 0.8 μl of 10 μ M 8F primer ※ 0.8 μl of 10 μ M 1392R primer ※ 2 μl of DNA template ※ 20 μl of distilled water

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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 ◦C for 7 minutes for 1 cycle

● Denaturation Step: 95 ◦C for 1 minute

● Annealing Step: 55 ◦C for 1 minute

● Elongation Step: 72 ◦C for 1.5 minutes

● Final Extention : 72 ◦C for 10 minutes

The whole reaction cycle was completed in three hours.

35 cycles

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Electrophoresis screening

PCR results verified by gel electrophoresis

PCR reaction products: 5 μl for each strain6 × 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 1112 13141516

2000 bp

1000 bp

750 bp

500 bp

250 bp

100 bpSuccessful PCR process

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DNA sequencing and analysis

150 successful PCR results were selected for further analysis by DNA sequencing.

Amplified DNA sequences were purified with PCR quick-spinTM PCR 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

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Code Species Coverage SimilarityA02_19 Herbaspirillum frisingense 草螺菌属 100% 99%A03_12 Uncultured bacterium clone N/A 100% 100%A04_30 Chryseobacterium sp. 金黄杆菌属 100% 100%A06_4-2 Acinetobacter sp. 不动杆菌属 100% 100%A07_14-2 Duganella sp. 杜檊氏属 99% 99%A08_7-2 Uncultured bacterium clone N/A 100% 99%B02_27 Comamonas sp. 毛单胞菌属 100% 99%B04_46 Uncultured Acidovorax sp. 嗜酸菌属 100% 99%B05_62 Acidovorax sp 嗜酸菌属 100% 99%B06_9 Bacillus thuringiensis strain 苏云金芽孢杆菌 100% 100%C01_56 Acidovorax sp. 嗜酸菌属 100% 99%C02_36 Acidovorax sp. 嗜酸菌属 100% 100%C03_10-2 Uncultured bacterium clone N/A 100% 100%C04_57 Acinetobacter sp. 不动杆菌属 100% 96%C06_17 Uncultured bacterium clone N/A 100% 99%C07_39 Acinetobacter sp. 2A18N1 不动杆菌属 100% 100%C08_13 Uncultured bacterium clone N/A 100% 99%D01_3-2 Uncultured bacterium clone N/A 100% 100%D02_52 Uncultured Acidovorax sp. 嗜酸菌属 100% 100%D03_6 Uncultured bacterium clone N/A 100% 99%D04_35 Acidovorax sp. 嗜酸菌属 100% 99%D05_37 Acidovorax sp. 嗜酸菌属 100% 99%D06_48 Uncultured Acidovorax sp. 嗜酸菌属 100% 100%D07_17 Acinetobacter sp. 不动杆菌属 100% 96%D08_65 Comamonas sp. 毛单胞菌属 100% 99%E01_32 Acinetobacter sp. 不动杆菌属 100% 100%E03_31 Comamonas sp. 毛单胞菌属 100% 99%E04_47 Uncultured Acidovorax sp. 嗜酸菌属 100% 99%E05_41 Acinetobacter sp. 不动杆菌属 100% 99%E06_60 Uncultured Acidovorax sp. 嗜酸菌属 100% 97%E07_8 Uncultured bacterium clone N/A 100% 99%E08_58 Comamonas sp. 毛单胞菌属 100% 99%F01_28 Naxibacter haematophilus strain 草酸杆菌属 100% 98%F02_29 Uncultured Comamonas sp. 毛单胞菌属 100% 97%F03_3 Uncultured bacterium clone N/A 100% 99%F04_22 Herbaspirillum seropedicae 草螺菌属 100% 89%F05_26 Uncultured bacterium clone N/A 99% 99%F06_66 Uncultured bacterium clone N/A 100% 99%F08_5 Acinetobacter sp. 不动杆菌属 100% 100%

DNA Sequencing Result

Part 1

G02_15 Duganella sp. / Massilia sp.

杜檊氏属/ 100%

99%

G03_40 Acinetobacter sp. 不动杆菌属 100%

97%

G04_4 Acinetobacter sp.不动杆菌属 100%

99%

G05_33 Chryseobacterium sp.

金黄杆菌属

100%100%

G07_50_ Uncultured Acidovorax sp. 嗜酸菌属 100%

99%

H02_23 Acinetobacter sp.不动杆菌属 100%

99%

H03_38 Acinetobacter sp. 不动杆菌属 99%

99%

H05_21Comamonas sp. CONC14

毛单胞菌属 100%

99%

H06_49Uncultured Acidovorax sp. 嗜酸菌属 100%

99%

H07_1 Acinetobacter sp. 不动杆菌属 100%

99%

A02_99 Herbaspirillum sp. 草螺菌属 100% 98%A03_103 Uncultured bacterium N/A 100% 100%A04_67 Microbacterium sp. 细杆菌属 100% 99%A07_137 Acidovorax sp. 嗜酸菌属 100% 99%A08_123 Acidovorax sp. 嗜酸菌属 100% 99%A09_129 Acidovorax sp. 嗜酸菌属 100% 99%

B02_81 Microbacterium sp. 141-3 细杆菌属 100% 99%

B03_105 Delftia sp. N/A 100% 99%B05_107 Herbaspirillum sp. 草螺菌属 100% 99%B06_134 Acidovorax sp. 嗜酸菌属 100% 99%B08_141 Acidovorax sp. 嗜酸菌属 100% 99%B10_160 Acidovorax sp. 嗜酸菌属 100% 99%C01_90 Aquabacterium sp. N/A 99% 97%C02_114 Aquabacterium sp. N/A 100% 99%C03_108 Aquabacterium sp. N/A 99% 99%C04_86 Microbacterium sp. 细杆菌属 100% 98%C06_131 Bacillus cereus strain 芽孢杆菌 99% 99%C07_139 Acidovorax sp. 嗜酸菌属 100% 100%C08_142 Acidovorax sp. 嗜酸菌属 100% 99%C09_126 Bacillus cereus strain 芽孢杆菌 100% 99%C10_156 Bacillus cereus strain 芽孢杆菌 100% 99%D01_83 Microbacterium sp. 细杆菌属 100% 99%

D03_69 Aquabacterium sp. Aqua2 N/A 99% 99%

D04_116 Herbaspirillum putei strain 草螺菌属 100% 100%

D05_164 Acidovorax sp. 嗜酸菌属 100% 98%D08_124 Acidovorax sp. 嗜酸菌属 100% 99%D09_147 Delftia sp. 100% 98%E01_84 Microbacterium sp. 细杆菌属 100% 99%E02_76 Microbacterium sp. 细杆菌属 100% 99%

E03_75Microbacterium ginsengisoli 细杆菌属 100%

96%

E04_111 Herbaspirillum huttiense strain 草螺菌属 100% 100%

E05_161 Bacillus sp. 芽孢杆菌 100% 96%

E06_132 Acidovorax temperans 嗜酸菌属 100% 99%

E09_151 Delftia sp. N/A 100% 99%

E10_154 Delftia sp. N/A 100% 100%

F01_78 Microbacterium sp. 细杆菌属 100% 99%

F03_88 Microbacterium sp. 细杆菌属 100% 99%

F06_121 Acidovorax sp. 嗜酸菌属 100% 97%



G01_96 Aquabacterium sp. N/A 100% 99%

G02_71 Microbacterium sp. 细杆菌属 100% 99%

G03_85 Microbacterium sp. 细杆菌属 99% 99%

G04_115 Herbaspirillum sp. 草螺菌属 100% 100%

G07_127 Bacillus cereus strain R13 芽孢杆菌 100% 98%

G08_125 Acidovorax sp. 嗜酸菌属 100% 99%



H01_100 Aquabacterium sp. N/A 100% 99%

H02_106 Aquabacterium sp. N/A 100% 99%

H03_72 Microbacterium sp. 细杆菌属 100% 98%

H04_73 Microbacterium sp. 细杆菌属 100% 99%

H05_135 Acidovorax sp. 嗜酸菌属 100% 99%

H07_130 Bacillus thuringiensis strain 芽孢杆菌 100% 99%

H08_145 Delftia tsuruhatensis partial N/A 100% 99%

H09_150 Uncultured Acidovorax sp. N/A 100% 99%

A 3 Delftia sp. R-41392 代尔夫特菌 100% 99%

A 12 Bacillus sp. 芽孢杆菌 100% 99%

A 22 Pseudomonas fluorescens 荧光假单胞菌 100% 99%

A 29 Pseudomonas sp 假单胞菌 100% 99%

B 5 Acidovorax sp 嗜酸菌 100% 99%

A 14 Pseudomonas poae 假单胞镰刀菌 100% 99%

A 30 Acidovorax sp 嗜酸菌 100% 99%

B 6 Acidovorax facilis 敏捷食酸菌 100% 99%

A 6 Bacillus sp 芽孢杆菌 100% 99%

A 15 Pseudomonas brenneri 假单胞菌 * 100% 99%

A 23 Pseudomonas poae 假单胞镰刀菌 100% 99%

B 7 Acinetobacter 不动杆菌 100% 99%

A 7 Janthinobacterium sp 紫色杆菌 100% 99%

A 24 Sphingomonas subterranean 单胞菌 100% 99%

A 32 Acinetobacter sp 不动杆菌 99% 99%

A 1 Pseudomonas poae 假单胞菌 100% 99%


A 17 Sphingomonas 单胞菌 100% 99%





B 11 Acidovorax delafieldii 德式嗜酸菌 100% 99%





A 4 Bacillus cereus 蜡样芽胞杆菌 100% 99%

A 11 Bacillus cereus 蜡样芽胞杆菌 100% 99%



B 13 Acinetobacter sp 不动杆菌 100% 99%

Part 2 Part 3

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Archive of Bacteria Isolated from H.K. Potable Water

Bacteria genera 菌种 Isolate amount Percentage Type of species

Acidovorax 嗜酸菌 38 28.8% 5

Acinetobacter 不动杆菌 15 11.3% 1

Aquabacterium N/A 7 5.3% 1

Bacillus 芽孢杆菌 15 11.3% 3

Comamonas 毛单胞菌属 6 4.5% 1

Chryseobacterium 金黄杆菌 2 1.5% 1

Delftia 代尔夫特菌 7 5.3% 2

Duganella 杜檊氏属 2 1.5% 1

Herbaspirillum 草螺菌属 7 5.3% 5

Janthinobacterium 紫色杆菌 1 0.75% 1

Microbacterium 细杆菌属 13 9.8% 2

Pseudomonas 假单胞菌 18 13.6% 4

Naxibacter haematophilus N/A 1 0.75% 1

Pathogenic

Pathogenic

Phylogenetic tree

Aquabacterium

Acinetobacter

Herbaspirillum

AcidovoraxMicrobacteriumBacillus

Pseudomonas

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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.

Bacteria Isolates Percentage

Total Bacteria Strains

150 N/A

Tetracycline Resistant

54 35%

Ampicillin Resistant

96 64%

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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.

Tetracycline

tet(A)tet(C)tet(D)tet(E)tet(O)tet(M)tet(G)

Reference: ZHANG T, ZHANG M, ZHANG, XX (2009) Tetracycline resistance genes and tetracycline resistant lactose--fermenting Enterobacteriacae in 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

AmpicillinTEM-1oxa-1

amp(C)

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Gel Documentation of Antibiotic Resistance Genes

tet(C) tet (A)

tet(G) amp (C)

275 bp

250 bp

100 bp

189 bp

250 bp

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Code Species tet(A) tet(C) tet(D) tet(E) tet(O) tet(M) tet(G)16 Uncultured bacterium + - - + - + +17 Acinetobacter sp. + + - - - - +20 Uncultured bacterium + - - + - - +21 Comamonas sp. - + - - - - -27 Comamonas sp. + + - - - - -29 Comamonas sp. - + - - - - -30 Chryseobacterium sp. + + - - - - +31 Comamonas sp. + + - - - - +32 Acinetobacter sp. - + + - - + -33 Chryseobacterium sp. - + - - - - +34 Uncultured bacterium + - - - - - +37 Acidovorax sp. + + - - - - -42 Uncultured bacterium - + - + - - +44 Uncultured bacterium + - - - - - +45 Uncultured bacterium + - - - - - +46 Acidovorax sp. + + - - - - +47 Acidovorax sp. + + - - - - +48 Acidovorax sp. + + - - - - +49 Acidovorax sp. + + - - - - +50 Acidovorax sp. + + - - - - +53 Uncultured bacterium + - - + - + +60 Acidovorax sp. + + - + - - +61 Uncultured bacterium + + - - + - +65 Uncultured bacterium - + - - - - +70 Uncultured bacterium + - - + - - +79 Uncultured bacterium + - - - + - +87 Uncultured bacterium + - - - - - -90 Aquabacterium sp. - + - - - - -91 Uncultured bacterium + - - + - - -93 Uncultured bacterium - - - - - + -94 Uncultured bacterium - - - - - - -95 Uncultured bacterium - - - - - - -96 Aquabacterium sp. - + - - - - -99 Herbaspirillum sp + + - + - - -

100 Aquabacterium sp. + + - - - - +103 Uncultured bacterium + + + - - - +106 Aquabacterium sp. - + - - - - +108 Aquabacterium sp. - + - - - - -110 Uncultured bacterium + - - - - - -114 Aquabacterium sp. - + - - - - -115 Herbaspirillum sp - + - + - - -116 Herbaspirillum sp - + - + - - -137 Acidovorax sp. + + - - - - -159 Uncultured bacterium + + - - - + -161 Uncultured bacterium - + - - - + -A8 Bacillus sp - + - + - - -

A22 Pseudomonas fluorescens + - - - - - -A15 Pseudomonas brenneri + - - - - - -A14 Pseudomonas poae + - - - - - -A17 Sphingomonas - - - + - - -A18 Bacillus sp - + - - - - -A20 Pseudomonas brenneri - - - - - - -A28 Acidovorax sp - + - - + - -A29 Pseudomonas sp - - - + - - -

Tetracycline Resistance Genes Investigation

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Tetracycline Resistance Profile54 strains out of 150 isolates showed tetracycline resistance phenotypes.

Resistant Bacteria Strains Amount percentage

Acidovorax 10 18.5%Acinetobacter 3 5.5%

Aquabacter 7 12.9%Bacillus 2 3.7%

Chryseobacterium 2 3.7%Comamonos 5 9.3%

Herbaspirillum 3 5.5%Pseudomonas 7 12.9%

Unknown 15 28%

tet(A) tet(C) tet(D) tet(E) tet(O) tet(M) tet(G)strains 31 34 2 13 3 6 24

**% 57.4% 62.9% 3.7% 24.1% 5.5% 11.1% 44.4%

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Multi-Antibiotic Resistance Investigation

56 A27 17 96 69 108 A6 A8 130 A2 30 65 22 115 84 71 88 A15 A21 A29 Pt

AMP + + + + + + + + + + + + + + - + + + + + 95%

CEF - + + + + + + + + + - + - + - + + + + + 80%

GEN - - - - + + - - - - - - - - - - + - - - 15%

COT - + - + - + + + + - - - - + + + + + - + 60%

LEV - - - - - - - - - - - - - - + + - - - - 10%

CEF - - - + - + + + + + - - - + + + + + - - 55%

AUG - + + + - + + + + - - - - + - - + + + + 60%

TET - + + + + + + + + - - - - + - - + + + + 65%

NEO - - - - + - - - - - - - - - - - + - - - 10%

TAZ - - - - - - - - - - - + - - - - - + + - 15%

STR - - - - + - - - - - - + - - - - + + - - 20%

CHL - + + - + - - - - - - - - - - - + - - + 25%

AcidovoraxAcinetobacer

Aquabacter BacillusChryseobacterium

ComamonasHerbaspirillum

Microbacterium Pseudomonas

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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.

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Acknowledgements

• Dr. T. Zhang

• Dr. Xu-xiang Zhang

• Lin Ye, Bing Li, Ke Yu, Ying Yang and other lab-mates

• Enviromental Biotechnology Laboratory

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Thank You !

Andrew Y. Z. ZHANGDepartment of Civil Engineering

University of Hong KongJune 20, 2011

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

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