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

aquaaniseΒιοτεχνολογία

6 Δεκ 2012 (πριν από 4 χρόνια και 8 μήνες)

234 εμφανίσεις

Andrew Y. Z. ZHANG

MSc. Candidate

Supervisor: Dr. T. ZHANG

Department of Civil Engineering

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

1

Contents


Introduction



Objective



Materials & Methods



Results and Discussion



Conclusion

2

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

3

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
.


4

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.



5

Major Classes of Antibiotics


Aminoglycosides


β
-
lactams


-

Penicillins



-

Cephalosporins


-

Carbapenems


-

Monobactams


Fluoroquinolones







Ketolides


Lincosamides


Macrolides


Oxazolidinones


Streptogramins


Sulphonamides


Tetracyclines








6

Usage of Antibiotics

Annual

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%

7

Usage of Antibiotics

Journal

of

Antimicrobial

Chemotherapy






















8

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

promoter
s

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

(
2009
a)

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





9

Introduction of antibiotic residues to aquatic environments



10

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)

11

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
)



12

aac
C1, C2, C3 and

C4;

aad
A1
,
A2, A5
,
A13

and
B
;
amp
C
; aph
A1, D

and
(3")
-
Ic
; bla
OXA
-
1
, bla
OXA
-
2
,
bla
OXA
-
10
, bla
OXA
-
30

and
bla
PSE
-
1
;
catI
, II, III, IV, B2
and B3;

mph
A
;

npt
II
;
sat
1

and 2;
str
A

and B


erm
A
, B, C, E, F, T, V and X;
mec
A
;
otr
A
;
pen
A
;
tet
B
(P), M,
O, Q, S, T and W;
van
A

and B



Functional mechanisms of environmental ARGs

cml
A1 and A5;
flo
R
;
otr
B;
tet
A, A(41), B, C,
D, E, G, H, J, Y, Z, 33
and 39

dfr
A1, A5, A7,
A12, A15, A17
and 18;
sul
I
, II,
III and A

13

Horizontal

Transfer

of

Antibiotic

Resistance

Genes


14

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
.

Ant i bi ot i c

r e s i s t a nt

ge ne s

ha ve

be e n

i de nt i f i e d

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










15

BBC NWES: Hong Kong is threatened by
Superbug

February 22, 1999

16

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
.





17

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

18

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.

19

Tap Water

Filtration

Ultrasonic Vibration

Suction Filtration

Cultivation

Pre
-
concentration of Bacteria

Filter

Cores
:

to

prepare

distilled

water

from

tap

water

for

laboratory

use
.


5 micron

Carbon Filter

1 micron

20

Tap Water

Filtration

Ultrasonic Vibration

Suction Filtration

Cultivation

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
.

21

Tap Water

Filtration

Ultrasonic Vibration

Suction Filtration

Cultivation

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)

(
Koksal

et

al
.
,

2007
)



Each

filter

membrane

was

used

to

filter

750

ml

of

solution
.



22

Tap Water

Filtration

Ultrasonic Vibration

Suction Filtration

Cultivation

Pre
-
concentration of Bacteria


Step 1

Get the membrane


Step 2

Cut into pieces
using sterile scissors


Step 3

Saturated in 6 ml
distilled water

23

R2
A Media Preparing & Petri Dish Culturing




R
2
A

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

24

Bacteria species identification by 16S
rDNA

sequencing

DNA
preparation

PCR
Amplification

Electrophoresis
screening

DNA
sequencing &
analysis

16
S

rDNA

gene

sequences

contain

hypervariable

regions

that

can

provide

species
-
specific

signature

sequences

useful

for

bacterial

identification
.

As

a

result,

16
S

rRNA

gene

sequencing

has

become

prevalent

method

of

bacterial

identification
.

Principle of 16S
rDNA

sequencing

25

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.


26

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
μ
l of 10
×


PCR buffer



1.2
μ
l of
dNTP



0.75 U
Taq

polymerase



0.8
μ
l of 10
μ
M 8F primer



0.8
μ
l of 10
μ
M 1392R primer



2
μ
l of DNA template



20
μ
l of distilled water










27

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
.

28

35 cycles

Electrophoresis screening

PCR

results

verified

by

gel

electrophoresis


PCR

reaction

products
:

5

μ
l

for

each

strain

6

×

loading

buffer

:

1

μ
l

for

each

strain




Loaded

on

the

1
%

agarose

gel

and

separated

by

electrophoresis

at

120
V

for

20

minutes



DL
2000

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

100
bp

Successful PCR process

29

DNA sequencing and analysis

150

successful

PCR

results

were

selected

for

further

analysis

by

DNA

sequencing
.



Amplified

DNA

sequences

were

purified

with

PCR

quick
-
spin
TM

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

30

Code

Species

Coverage

Similarity

A02_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


100%

97%

G04_4

Acinetobacter sp.

不动杆菌


100%

99%

G05_33

Chryseobacterium sp.

金黄杆菌




100%

100%

G07_50_

Uncultured Acidovorax
sp.

嗜酸菌属

100%

99%

H02_23

Acinetobacter sp.

不动杆菌


100%

99%

H03_38

Acinetobacter sp.

不动杆菌


99%

99%

H05_21

Comamonas sp.
CONC14

毛单胞菌



100%

99%

H06_49

Uncultured Acidovorax
sp.

嗜酸菌属

100%

99%

H07_1

Acinetobacter sp.

不动杆菌


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_75

Microbacterium
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%

F09_152

Acidovorax sp.

嗜酸菌属

100%

99%

F10_155

Acidovorax sp.

嗜酸菌属

100%

99%

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%

G09_149

Acidovorax sp.

嗜酸菌属

99%

99%

G10_153

Acidovorax sp.

嗜酸菌属

100%

100%

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 8

Bacillus sp

芽孢杆菌

100%

99%

A 17

Sphingomonas

单胞菌


100%

99%

A 25

Pseudomonas brenneri

假单胞菌
*

100%

99%

A 2

Bacillus sp

芽孢杆菌

100%

99%

A 18

Bacillus sp

芽孢杆菌

100%

99%

A 26

Pseudomonas poae

假单胞菌

100%

99%

B 11

Acidovorax delafieldii

德式嗜酸菌

100%

99%

A 20

Pseudomonas brenneri

假单胞菌
*

100%

99%

A 27

Acidovorax sp

嗜酸菌

100%

99%

B 3

Acidovorax sp

嗜酸菌

100%

99%

B 12

Acidovorax sp

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%

A 21

Pseudomonas poae

假单胞菌


100%

99%

A 28

Acidovorax sp

嗜酸菌

100%

99%

B 13

Acinetobacter

sp

不动杆菌

100%

99%

Part 2

Part 3

31

Archive of Bacteria Isolated from H.K. Potable Water

Bacteria
genera





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

32

Phylogenetic tree

Aquabacterium

Acinetobacter

Herbaspirillum

Acidovorax

Microbacterium

Bacillus

Pseudomonas

33

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
:

20
mg/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%

34

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





Ampicillin

TEM
-
1

oxa
-
1

amp(C)


35

Gel Documentation of Antibiotic Resistance Genes

tet
(C)

tet

(A)

tet
(G)

amp (C)

36

275
bp

250
bp

100
bp

189
bp

250
bp

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

37

Tetracycline Resistance Profile

38

54

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%

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%

Acidovorax

Acinetobacer

Aquabacter

Bacillus

Chryseobacterium

Comamonas

Herbaspirillum

Microbacterium

Pseudomonas

39

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
.


40

Acknowledgements


Dr. T. Zhang


Dr.
Xu
-
xiang

Zhang


Lin Ye, Bing Li,
Ke

Yu, Ying Yang and other lab
-
mates


Enviromental

Biotechnology Laboratory

41

Thank You !

Andrew Y. Z. ZHANG

Department
of Civil
Engineering

University of Hong Kong

June 20, 2011

42