DSpace at VNU: Antibiotic contamination and occurrence of antibiotic-resistant bacteria in aquatic environments of northern Vietnam

Science of the Total Environment 409 (2011) 2894–2901

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Science of the Total Environment
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s c i t o t e n v

Antibiotic contamination and occurrence of antibiotic-resistant bacteria in aquatic
environments of northern Vietnam
Phan Thi Phuong Hoa a, Satoshi Managaki b, Norihide Nakada b, Hideshige Takada b, Akiko Shimizu b,
Duong Hong Anh c, Pham Hung Viet c, Satoru Suzuki a,⁎
a
b
c

Center for Marine Environmental Studies (CMES), Ehime University, Matsuyama 790-8577, Japan
Institute of Symbiotic Science and Technology, Tokyo University of Agriculture and Technology, Fuchu 183-8509, Japan
Research Center for Environmental Technology and Sustainable Development (CETASD), Hanoi University of Science, Hanoi, Vietnam

a r t i c l e

i n f o

Article history:
Received 2 September 2010
Received in revised form 8 April 2011
Accepted 13 April 2011
Keywords:
Sulfonamide
Resistant bacteria
Vietnam

Acinetobacter
Sul
Animal farm

a b s t r a c t
The ubiquitous application and release of antibiotics to the environment can result in bacterial antibiotic
resistance, which in turn can be a serious risk to humans and other animals. Southeast Asian countries
commonly apply an integrated recycling farm system called VAC (Vegetable, Aquaculture and Caged animal).
In the VAC environment, antibiotics are released from animal and human origins, which would cause
antibiotic-resistant bacteria (ARB). This study evaluated occurrence of ARB in the VAC environment in
northern Vietnam, with quantitative analysis of antibiotic pollution. We found that sulfonamides were
commonly detected at all sites. In dry season, while sulfamethazine was a major contaminant in pig farm pond
(475–6662 ng/l) and less common in city canal and aquaculture sites, sulfamethoxazole was a major one in
city canal (612–4330 ng/l). Erythromycin (154–2246 ng/l) and clarithromycin (2.8–778 ng/ml) were the
common macrolides in city canal, but very low concentrations in pig farm pond and aquaculture sites. High
frequencies of sulfamethoxazole-resistant bacteria (2.14–94.44%) were found whereas the occurrence rates of
erythromycin-resistant bacteria were lower (b 0.01–38.8%). A positive correlation was found between
sulfamethoxazole concentration and occurrence of sulfamethoxazole-resistant bacteria in dry season. The
sulfamethoxazole-resistant isolates were found to belong to 25 genera. Acinetobacter and Aeromonas were the
major genera. Twenty three of 25 genera contained sul genes. This study showed specific contamination
patterns in city and VAC environments and concluded that ARB occurred not only within contaminated sites
but also those less contaminated. Various species can obtain resistance in VAC environment, which would be
reservoir of drug resistance genes. Occurrence of ARB is suggested to relate with rainfall condition and
horizontal gene transfer in diverse microbial community.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction
It is known that antibiotics cause antibiotic-resistant bacteria
(ARB) in hospital-inquired infection. In recent years, antibiotics
contamination is recognized as an emerging environmental pollution

in aquatic environments, because of their potential adverse effects on
the ecosystem and human health (Huang et al., 2001; Kümmerer,
2009). Majority of antibiotics used for human, plants and animals are
excreted into the environment as intact or decomposed form via
various pathways, including wastewater effluent discharge, runoff
from land to which agricultural or human waste has been applied, and
leaching (Zhang et al., 2009). Antibiotic residues in the environment
impose selective pressure on bacterial populations, which results
prevalence of resistant bacteria even at sub-inhibitory low concen-

⁎ Corresponding author. Tel./fax: + 81 89 927 8552.
E-mail address: (S. Suzuki).
0048-9697/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.scitotenv.2011.04.030

trations. Other pollutants are also known as selective agents
(Stepanauskas et al., 2005, 2006). Additionally, the raw wastewater
contaminated by antibiotics released into aquatic environments often
carries human and animal pathogenic bacteria, in addition to
commensal bacteria, and many of these organisms harbor antibiotic-resistance genes. Therefore, water constitutes a way of dissemination of not only antibiotic-resistant bacteria, but also the resistance
genes, which genetically change in natural bacterial ecosystems
(Baquero et al., 2008; Rosenblatt-Farrell, 2009). The ARB has been
found in various aquatic environments (Kümmerer, 2004; Kim and
Aga, 2007; Schluter et al., 2007; Watkinson et al., 2007;Caplin et al.,
2008; Vanneste et al., 2008). In particular, our previous studies
showed that aquatic environment is potential reservoirs of ARB
(Nonaka et al., 2000, 2007; Kim et al., 2003, 2004; Hoa et al., 2008)
even in pristine conditions (Kobayashi et al., 2007; Rahman et al.,
2008). On the other hand, a variety of antibiotics have been detected
in the aquatic environments (Hirsch et al., 1999; Göbel et al., 2005;

Zhang et al., 2009), from ng/l to μg/l levels, which are lower than


P.T.P. Hoa et al. / Science of the Total Environment 409 (2011) 2894–2901

therapeutic levels (Göbel et al., 2005; Managaki et al., 2007, Zhang et
al., 2009). In some cases, high concentration with mg/l order was
found (Le and Munekage, 2004; Le et al., 2005). At the sub-therapeutic
concentrations of antibiotics detected in the aquatic environments,
the question is whether antibiotics could have an impact on bacterial
populations (Kümmerer, 2009). Very few studies have investigated
the relationship between antibiotic contamination and antibiotic
resistance in aquatic environments relating to human and agricultural
activities. Furthermore the diversity of ARB within integrated
recycling farm VAC (Vegetable, Aquaculture and Caged animal)
systems is virtually unknown.
From these background, we hypothesized that Asian integrated
agriculture environment should be polluted with various antibiotics
derived from animal and human origins, which selects ARB in the
environment. To clarify this, we conducted monitoring of concentrations of residual antibiotics and ARB in the VAC environments.
Antibiotic residues were often detected in the human-impacted
aquatic environments of Southeast Asian countries and China (Le and
Munekage, 2004; Richardson et al., 2005; Managaki et al., 2007). In
Vietnam, agriculture and aquaculture are the major economic
activities, and excessive and unregulated use of antibiotics was
commonly found in human medicine, management of livestock, and
aquaculture (Le et al., 2005; Managaki et al., 2007; Duong et al., 2008).
The Red River delta of northern Vietnam is an appropriate study site
because of following conditions. This area is one of the largest deltas in
Southeast Asia, including 9 provinces along with 2 municipalities, the

capital city of Hanoi, and the main seaport of Haiphong. The delta is an
agriculturally rich area, which is densely populated by 11,000,000
people (Berg et al., 2001). The heavy and unregulated use of
antibiotics along with the discharge of untreated wastewater into
the aquatic environments might cause significant contamination of
both antibiotic residues and ARB. On the basis of the information
obtained during our pre-study onsite interview-based survey, we
identified sulfonamides, trimethoprim, and macrolides as the target
antibiotics.
In the present study, we first characterized the pattern of
contamination by antibiotics and ABR in the rainy and dry seasons
in the Red River delta area. To better understand whether the
antibiotic residues in the aquatic environment is an important source
in selecting and creating an increasingly resistant bacteria in the
environments, the statistical correlation between the concentration of
antibiotic residues and ARB occurrence was analyzed. A part of
sulfonamide-resistant (SR) bacteria was isolated and classified in
January when the relationship between sulfonamide and occurrence
of SR bacteria was possibly observed. The possession of sul genes was
also monitored. The variation and distribution of sul genes in manure
were examined in Europe, and the molecular information for a
method of detection is available (Heuer et al., 2009). Therefore sul
genes were the priority to survey in this study.
2. Materials and methods
2.1. Sampling area and procedure
Specific integrated aquaculture-agriculture system is major in
Vietnam, at which freshwater fishponds directly receive excreta from
intensive pig farms (termed “pig farm/fish ponds”) (Hoa et al., 2008).
Sampling was performed at 10 sites in the Red River delta of northern
Vietnam, including 3 sites of a city canal in Hanoi (HNC-1–3), 3 sites at

3 pig farm/fish ponds in Hatay (HNP-1–3), and 4 sites at 4 coastal
shrimp ponds of Haiphong (HNAQ-1–4) (Fig. 1). The HNC sites were
upper-, middle- and down-stream of the main canal of Hanoi City, and
the HNP and HNAQ sites were representative farms of animal and
shrimp culture. The sampling sites were at least 3 km away from each
other. These 3 habitats were selected because they were representative of the aquatic environments exposed to antibiotics. The city canal

2895

in Hanoi directly receives various types of untreated municipal
wastewater, such as wastewater from households and hospital
(Duong et al., 2008). According to the onsite interviews conducted
during our field trips in both sampling periods, antimicrobials were
rarely used for the freshwater fishponds in Hatay province, but very
frequently used in the pig farms. The categories of the drugs and their
amounts varied widely among the individual pig farms. We chose
coastal shrimp ponds in Haiphong because this province is one of the
centers of fish and shrimp cultivation, which is an industry that
supplies seafood to domestic and foreign markets (Tran et al., 2006).
The use of antibiotics in the Vietnamese shrimp industry has been
reported by Le and Munekage (2004) and Le et al. (2005). The
investigated shrimp ponds were located near the estuary mouths, and
the water level of the ponds was somewhat influenced by tidal
movement; the water level in the ponds was adjusted by using
pumps. The collection of water samples was repeated twice, once in
January (dry season) and then in July (rainy season) in 2007. A
detailed description of the characteristics of water samples and the
climate conditions of the sampling locations is provided in Tables S1
and 2. The sampling procedure was identical to that described in our
previous studies (Managaki et al., 2007; Hoa et al., 2008). Then, the

samples were preserved on ice and analyzed in the laboratory within
3–6 h after sampling.
2.2. Analysis of antibiotics
The concentrations of sulfonamides, trimethoprim, and macrolides
were determined by tandem mass spectrometry equipped with highperformance liquid chromatography (LC–MS/MS), which was performed after solid-phase extraction. The detailed procedure has been
described by Managaki et al. (2007), and the outline of the procedure
is as follows. We extracted 50 ml (city canal samples), 20 ml (pig
farm/fish pond samples), and 250 ml (shrimp pond samples) on 6-ml
Oasis HLB sorbent cartridges (200 mg; Waters) (flow rate, b5 ml/min;
pH, 4). After extraction, the cartridges were stored at − 30 °C,
transported to the laboratory in Tokyo, and defrosted before elution
of the antibiotics. The cartridges were washed with 5 ml of water–
methanol (75:25) and dried in a nitrogen flow for 30 min. The
analytes were eluted with 2 × 1.5 ml of methanol–ethyl acetate (1:1)
and 2 × 1.5 ml of methanol containing 1% (v/v) ammonia. A fixed
amount 25 ng (500 pg/μl × 50 μl) of each antibiotic surrogate standard
(sulfamethoxazole-d4, clarithromycin-d3, and roxithromycin-d9)
was spiked to the sample extracts. The extracts were evaporated to
dryness using a rotary dryer and dissolved in 1 ml of water–methanol
(1:1), and the antibiotic contents were determined by separating the
extracts by LC–MS/MS.
HPLC analyses were performed on a Hewlett-Packard G1310. The
antibiotics were separated on a reverse-phase column (YMC Pro C18;
3 μm, 150 mm × 2 mm) that was operated at 30 °C at a flow rate of
0.15 ml/min. The mobile-phase solvents were water-acidified with 1%
(v/v) formic acid (eluent A) and methanol-acidified with 1% (v/v)
formic acid (eluent B) to pH 2.5 by using a gradient program. The
antibiotics were detected using a triple-quadrupole mass spectrometer (TSQ Quantum 7000; Thermo Finnigan, Japan) equipped with
electrospray ionization. The analyses were performed in the positiveion mode. The detection was performed in the selected-reactionmonitoring mode (SRM) using the 2 most intense and specific
fragment ions.

To compensate for matrix effects and experimental losses during
sample treatment, we corrected the concentrations of the target
antibiotics with the recovery values of the corresponding surrogates
spiked in the same extracts. The following compounds were used as
recovery surrogate standards: sulfamethoxazole-d4 for all sulfonamides and trimethoprim; clarithromycin-d3 for clarithromycin,
erythromycin-H2O, and azithromycin; and roxithromycin-d9 for
roxithromycin. The analytical precision was examined by performing


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P.T.P. Hoa et al. / Science of the Total Environment 409 (2011) 2894–2901

106E

Red River

Red River

China

107E

21N

Red River Delta

Hanoi

Vietnam

HNC-3
HNC-2
HNC-1

Hanoi

HNAQ-1
HNAQ-4
HNAQ-3
HNAQ-2

Laos
HNP 3
HNP-3

Haiphong

HNP-2
HNP-1

20N

Thailand

Hue

Hatay
0

25

50 km

Fig. 1. Study area and sampling sites.

4 replicate analyses of 250 ml of water from the Tamagawa River. The
relative standard deviations of the target antibiotics ranged from 2% to
11%. For the recovery studies, 25 ng of each antibiotic (500 pg/μl in
50 μl) and the recovery surrogate were spiked into 250 ml of water
from the Tamagawa River. Recoveries of the spiked standards ranged
from 72% to 93% (SPY: 87% ± 6%, STZ: 91% ± 3%, SMR: 86% ± 3%, ; SMT:
84% ± 3%; SMZ: 86% ± 3%; SMX: 85% ± 6%; SDX: 86% ±2%; TRI: 72% ±
11%; AZI: 90% ±4%; ERY: 90% ±2%; CLA: 93% ± 6%; ROX: 93% ± 3%).
Most of the target compounds showed recoveries of over 84%, with
the exception of trimethoprim with 72% recovery. If we consider such
a trace amount (e.g., low ng/L) of the target compound from a
complex environmental matrix, recovery of over 70% is normally
acceptable for this type of monitoring study. The limits of quantification were defined as 10 times the procedural blank value or 10
times the noise level of the baseline in the chromatograms if there
were no peaks in the procedural blank analysis. Limits of quantifications ranged from 0.1 ng/l to 1.2 ng/l. Travel contamination and
laboratory contamination were determined by analyzing the travel
blanks and procedural blanks after extraction. Calibration curves for
each compound are shown in Fig. S1.
2.3. Enumeration of antibiotic-resistant bacteria (ARB)
The colony forming unit (CFU) was measured by using the plate
spreading method described in our previous study (Hoa et al., 2008).
Briefly, 1 m1 of each water sample was suspended in 9 ml of phosphatebuffered saline (PBS), and serial 10-fold dilutions were prepared.
Nutrient broth (Difco, Detroit, MD, USA) with 1.5% agar was used for
fresh water samples collected from HNPs and HNCs, and marine broth
(Difco, Detroit, MD, USA) with 1.5% agar was used for the brackish water

samples collected from HNAQs. The total viable count was obtained
from the antibiotic-free media, and the ARB was counted on media
supplemented with 60 μg/ml of each antibiotic: sulfamethoxazole
(SMX) and erythromycin (ERY) (both from Sigma-Aldrich, St. Louis,
MO, USA). These two compounds are appropriate representatives of
sulfonamide and macrolide, which are commonly used drugs in
Indochina and are well studied (Managaki et al., 2007). In this study,
‘resistance’ was determined as growth after 5 days at 30 °C in the media

containing antibiotic at concentrations of 60 μg/ml and additionally our
previous study showed that 92%, 72% and 43% of SMX-resistant (SMXr,
for 60 μg/ml) isolates from HNPs, HNCs and HNAQs, respectively
contained the sul genes (Hoa et al., 2008). The criterion for indicating
resistance has been designated to be 32–60 μg/ml (Hoa et al., 2008;
Toleman et al., 2007), and this concentration is appropriate for
comparison to other drugs (Nonaka et al., 2007). The bacterial count
was determined by plating 0.1 ml of 10-fold dilution (10−3 and 10−4
dilution for most cases of total viable bacteria, and 10−1 and 10−2
dilution for the selection of ARB). CFUs were counted in double plates.
2.4. Classification of SMXr isolates
The bacterial isolates that were randomly picked from the
duplicate plates in January samples (Hoa et al., 2008) were classified
to the genus level by 16S rRNA gene sequencing. Addition to 43 strains
carrying sul genes reported in our previous study (Hoa et al., 2008), 78
SMXr strains were newly identified in this study. The primers (F984,
R1378) and polymerase chain reaction (PCR) conditions were the
same as described by Heuer et al. (1997). The 394-bp PCR product was
purified and sequenced using the Big Dye terminator version 3.1 cycle
reaction kit (Applied Biosystems, Foster City, CA, USA) on a 3100 ABI
Prism DNA sequencer (Applied Biosystems). The DNA sequences

obtained were analyzed by the Basic Local Alignment Search Tool
(BLAST) at the National Center for Biotechnology Information (NCBI,
website. When the sequence of
amplified PCR product showed similarity ≥ 95%, the isolate was
recognized as the closest genus.
2.5. Data analysis
The correlation between the antibiotic concentration and incidence of ARB was calculated by Spearman correlation coefficients.
Continuous variables were compared by the t-test. A p value b 0.05
was considered statistically significant. Beside that linear correlation
was used for calculation the correlation between SMX and trimethoprim concentrations detected.


P.T.P. Hoa et al. / Science of the Total Environment 409 (2011) 2894–2901

3. Results and discussion
3.1. Antibiotic contamination status
We quantified the contamination of the 3 antibiotic groups,
sulfonamides, trimethoprim, and macrolides at 10 sites in different
seasons (Table 1). Among sulfonamides, SMX and sulfamethazine
were the major compounds detected. Trimethoprim was also detected
with same manner of sulfonamide. Concentrations of SMX and
trimethoprim were positively correlated (R2 = 0.758, p b 0.001),
which is caused by combination use of sulfonamide with trimethoprim (Huovinen et al., 1986 Houvinen et al., 1995). Present study
showed that SMX is used with trimethoprim in this area especially in
human medicine, because of high concentration in city canal samples.
Positive correlation was not found between sulfamethazine and
trimethoprim, suggesting that the combination of the two compounds
in pig farming is not frequent (Table 1). Other four sulfonamides
(sulfathiazole, sulfamerazine, sulfamethizol, and sulfadimethoxine)
were not detected at any investigated sites, which have a similar

tendency to our previous study conducted at the Mekong River delta,
Vietnam and the Tamagawa River, Japan (Managaki et al., 2007).
When the contamination was compared between rainy and dry
seasons, clear difference was not observed (p = 0.96) despite that the
precipitation in July (rainy season: 286 mm of rainfall/month) should
dilute drugs much more than January (dry season: 2 mm of rainfall/
month). This may be due to the intensive use of drugs in the rainy
season, which could compensate for the large dilution effect by rain.
The concentrations of sulfonamides and trimethoprim residues in
the city canal and the pig farm/fish ponds (16.1–4330.0 ng/l and 16.8–
6662.0 ng/l, respectively) were, on average, approximately 7-fold
higher than those in the shrimp ponds (2.38–914 ng/l) (Table 1). The
high concentrations of SMX, sufamethazine, and trimethoprim
detected in the water samples of the city canal and the pig farm/fish
ponds could be due to the unregulated consumption of these
compounds by humans (Duong et al., 2008) and livestock (Managaki
et al., 2007). The maximum detected concentration of sulfamethoxazole in the city canal (4330 ng/l) was higher than that in municipal
wastewater in Switzerland (1900 ng/l) (Göbel et al., 2005), the
Mekong River delta (360 ng/l) of southern Vietnam, and the
Tamagawa River of Japan (132 ng/l) (Managaki et al., 2007), but
lower than reported in the German study (9000 ng/l) conducted at
10 years ago (Hartig et al., 1999). In the present study, while the
maximum concentration of SMX was detected in municipal raw
wastewater, the maximum concentration of sulfamethazine was
detected in the pig farm/fish ponds (Table 1). The contamination
profile showed that sulfamethazine was a major contaminant in the
pig farm/fish ponds, while SMX was a major sulfonamide in the city
canal and the coastal shrimp ponds; these findings were repeatedly
observed during the 2 sampling trials, i.e., during both rainy and dry
seasons (Table1). These findings suggested that sulfamethazine was

intensively used in pig farms, and SMX was mainly used in human
medicine. A previous study also detected high concentrations of
sulfamethazine (18,512–19,153 ng /l) in the Mekong River delta of
Vietnam and suggested that this sulfamethazine had a livestock origin
(Managaki et al., 2007). The studies by Le and Munekage (2004), Le
et al. (2005) and Managaki et al. (2007) also suggested that SMX was
primarily used for human medication and coastal aquaculture, but not
for livestock production in Vietnam. The data obtained in the present
study showed that contamination by sulfonamides and trimethoprim
did not occur frequently in the shrimp ponds. Low concentrations of
these drugs (0–914 ng/l) were detected in the shrimp ponds;
however, the profile of the relative composition of sulfonamides
suggested that the sulfonamides and trimethoprim detected in this
study were derived from humans and livestock.
In terms of the number of compounds and residue concentrations,
we found that the macrolide contamination in municipal raw

2897

wastewater was more severe than that of agricultural wastewater in
both pig farm/fish ponds and the coastal shrimp ponds. All the 4
investigated macrolides were detected at relatively high concentrations
in the city canal (azithromycin, 0–90.8 ng/l; ERY, 61.1–2246.0 ng/l;
clarithromycin, 1.60–778.0 ng/l; and roxithromycin, 0–125 ng/l), while
concentrations in the pig farm/fish ponds (ERY, 0–63.9 ng/l and
clarithromycin, 0–0.40 ng/l) and the shrimp ponds (ERY, 0–0.28 ng/l)
were very low (Table 1). Erythromycin and clarithromycin were
abundant in the city canal and the pig farm/fish ponds, where the
highest erythromycin concentration (2246 ng/l from city canal) was
approximately 55-fold higher than that from the Mekong River delta

(41 ng/l) (Managaki et al., 2007), and 3-fold higher than that from
wastewater in Hong Kong (810 ng/l) (Gulkowska et al., 2008). The
findings in this study suggested that human medication was the primary
source of macrolide contamination in this area.
As a conclusion, the contamination data showed specific contamination patterns in the city canal, pig farm and aquaculture sites.
3.2. Occurrence of ARB
We enumerated SMX-resistant (SMXr) and ERY-resistant (ERYr)
bacteria from the same samples that were evaluated for antibiotic
concentrations. Overall, the occurrence rates of SMXr bacteria were
generally higher than those of ERYr bacteria at any site across the 2
sampling times (Table 2), which was similar tendency to drug
contaminations, that is, SMX was detected at higher concentration
than ERY at all sites. The occurrence rates of SMXr and ERYr bacteria in
the city canal in January were higher than those in July, although other
sites did not show differences between January and July (Table 2). This
difference in city canal can be attributed to the high rainfall in July and
the frequent wide-range floods during Hanoi's rainy season (Table S2).
Antibiotic contamination was almost the same in both seasons as we
mentioned above; however, there is a possibility that high rainfall in July
disturbs microbial ecosystem in city canal, which could interfere the
occurrence of ARB. On the other hand, the water level was relatively
lower and water exchange should not be frequent in January. In
intensive agricultural systems like the pig farm/fish ponds and the
coastal shrimp ponds, the water level was less affected by rainfall
because these ponds function as water storage and are not subject to
frequent exchange. This condition would facilitate adequate time to
develop resistance by promoting cell-to-cell horizontal gene transfer.
The exposure time between bacteria and antibiotics in July might be as
equal as that in January; and therefore their occurrence rates were
nearly stable during the two seasons. It was found in this study that

occurrence of ARB is depending on rainfall condition in city canal, but
the ARB are reserved through the year in pig farm and aquaculture sites
without correlation to drug contamination. The relationship of
contamination and ARB will be discussed further below.
3.3. Relationship between antibiotic concentration and occurrence of
ARB
Relationship between the use of antibiotic and occurrence of ARB
is complicated. A number of studies have shown a positive correlation
between the use of antibiotics in humans and the development of
antibiotic resistance in pathogenic bacteria (e.g. β-lactam, aminoglycosides, fluoroquinolones, macrolides, reviewed in Cristino, 1999). In
contrast, a number of studies have shown that heavy use of antibiotics
did not necessarily accelerate the prevalence of resistance (Gaynes,
1997; Cristino, 1999; Kahlmeter et al., 2003; Le et al., 2005). In many
cases, the association between these 2 factors was not established
because of various contributing factors, including cross-transmission,
inter-hospital transfer of resistance, community contribution to
resistance, or a complex relationship between resistance and the
use of a variety of antibiotics (Gaynes, 1997; Cristino, 1999). On the
other hand, some studies have shown that the consumption of


2898

Table 1
Concentrations of sulfonamides, trimethoprim and macrolides detected from water samples.
Antibiotic concentrationa (ng/l)
Sulfapyridine

Sulfamethoxazole

Sulfamethazine

Sulfathiazol

Sulfamerazine

Sulfamethizol

Sulfadimethoxine

Trimethoprim

Azithromycin

Erythromycin

Clarithromycin

Roxithromycin

A. January
HNC-1
HNC-2
HNC-3
HNP-1
HNP-2
HNP-3
HNAQ-1
HNAQ-2
HNAQ-3

HNAQ-4
LOQ

n.d.
21.50
57.50
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
7.00

612.00
2982.00
4330.00
422.00
625.00
328.00
13.40
n.d.
2.38
n.d.
0.02

16.10
47.90
66.20

6662.00
475.00
2501.00
n.d.
n.d.
n.d.
n.d.
0.70

n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
16.00

n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.

n.d.
16.00

n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.40

n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
0.08

23.00
222.00
1808.00

16.80
34.60
22.30
n.d.
n.d.
n.d.
n.d.
0.10

n.d.
n.d.
90.80
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
7.00

154.00
2246.00
2191.00
58.90
63.90
62.80
n.d.
n.d.
n.d.

n.d.
1.00

2.80
778.00
674.00
0.20
0.12
0.40
n.d.
n.d.
n.d.
n.d.
0.20

0.72
103.00
125.00
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.07

B. July
HNC-1
HNC-2

HNC-3
HNP-1
HNP-2
HNP-3
HNAQ-1
HNAQ-2
HNAQ-3
HNAQ-4
LOQ

n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
3.00

818.00
3847.00
3170.00
326.00
68.20
69.90
10.80
n.d.

914.00
n.d.
2.00

n.d.
44.10
46.20
851.00
658.00
6.78
n.d.
n.d.
n.d.
n.d.
0.30

n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
7.00

n.d.
n.d.

n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
5.00

n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
8.00

n.a.
n.a.
n.a.
n.a.
n.a.
n.d.
n.a.
n.a.

n.a.
n.a.
0.3

91.40
730.00
726.00
26.40
26.00
n.a.
n.d.
n.d.
85.00
n.d.
0.02

n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.02

61.10
734.00

642.00
12.80
0.59
n.d.
0.28
n.d.
n.d.
n.d.
0.03

1.60
309.00
292.00
n.d.
0.01
n.d.
n.d.
n.d.
n.d.
n.d.
0.001

n.d.
77.90
726.00
n.d.
n.d.
n.d.
n.d.
n.d.

n.d.
n.d.
0.001

LOQ: limit of quantification; n.d., not detected; n.a., not available due to overlapped interfering peak.
a
t-test (p N 0.05) showed no significant difference between antibiotic concentration detected at the 2 campaigns, except for sulfamethazine concentration detected in HNPs.

P.T.P. Hoa et al. / Science of the Total Environment 409 (2011) 2894–2901

Site


P.T.P. Hoa et al. / Science of the Total Environment 409 (2011) 2894–2901

2899

Table 2
Antibiotic-resistant bacteria detected from water samples.
Site

HNC-1
HNC-2
HNC-3
HNP-1
HNP-2
HNP-3
HNAQ-1
HNAQ-2
HNAQ-3

HNAQ-4

Total viable count

Sulfamethoxazole-resistant bacteria

Erythromycin-resistant bacteria

CFU/ml

CFU/ml (%)

CFU/ml (%)

Jan

Jul

Jan

1.00 × 106
4.80 × 106
1.80 × 106
1.80 × 105
3.40 × 104
1.60 × 105
1.70 × 104
1.90 × 104
5.14 × 104
2.90 × 104

1.50 × 106
2.05 × 106
2.44 × 106
7.40 × 105
No data
9.00 × 105
3.40 × 105
3.65 × 105
7.60 × 105
4.65 × 105

5.00 × 105
2.50 × 106
1.70 × 106
1.00 × 104
3.88 × 103
5.60 × 103
2.30 × 103
1.04 × 104
1.10 × 103
2.40 × 103

Jul
(50.00)
(52.08)
(94.44)
(5.55)
(11.41)
(3.50)

(13.52)
(54.70)
(2.14)
(8.27)

Jan

9.14 × 104
1.31 × 105
8.92 × 104
7.10 × 104
9.62 × 103
2.83 × 104
6.73 × 104
5.16 × 104
6.59 × 104
7.97 × 104

(6.09)
(6.39)
(3.65)
(9.59)
(No data)
(3.14)
(19.79)
(14.1)
(8.7)
(17.1)

Jul

No data
5.00 × 105
7.00 × 105
7.00 × 103
2.98 × 103
5.51 × 102
1.00 × 102
6.00 × 102
6.00 × 101
2.00 × 101

(No data)
(10.41)
(38.80)
(3.88)
(8.76)
(0.34)
(0.58)
(0.31)
(0.11)
(0.06)

2.80 × 104
9.40 × 104
6.05 × 104
5.81 × 104
4.76 × 103
1.80 × 103
2.50 × 101

5.00 × 101
6.00 × 101
3.00 × 101

(1.86)
(4.58)
(2.47)
(7.85)
(No data)
(0.20)
(b0.01)
(b0.01)
(b0.01)
(b0.01)

No data is due to laboratory accident. Each count is an average of duplicate counts.
t-test for the number of antibiotic-resistant bacteria (January N July) in HNCs is significant (p b 0.05), but not in HNAQs and HNPs (p N 0.05).

that not only contamination of antibiotics but also other selective
pressures act as inducing factors of ARB in aquatic environments,
although further studies are needed to confirm these possibilities.
3.4. Diversity of SMXr isolates and possession of sul genes

SMXr bacteria (%)

We examined the species composition of the 121 isolates sampled
in January whose SMX resistance possibly correlated with SMX
contamination. These isolates were identified and classified into 25
different genera (Table 3). Among the identified SMXr bacteria,
Acinetobacter was the most abundant (24%), followed by Aeromonas

(19.8%), Bacillus (13.2%) and Pseudoalteromonas (10%); the other
bacterial genera occupied small fraction (less than 10%) of the total
isolates. Acinetobacter was major in pig farm/fish pond, whereas
Aeromonas was mainly detected in city canal. Shrimp pond showed

A (Jan)

100
90
80
70
60
50
40
30
20
10
0
1

Rs=0.803

10

100

1000

10000

1000

10000

SMX concentration (ng/L)

SMXr bacteria (%)

antibiotics in animal husbandry can also play a major role in the
selection and dissemination of ARB in the environment. For example,
Angulo et al. (2004) and Asai et al. (2005) observed strong positive
correlations between the usage of veterinary therapeutic antibiotics
and antibiotic resistance in Escherichia coli isolates obtained from the
feces of food-producing animals. The findings by these studies raise
great concerns regarding the long-term consequences of antibiotic
use in agricultural ecosystems. Furthermore, agricultural and aquacultural products are sometimes at risk from ARB through the food
chain and from handlers (Levy and Marshall, 2004).
The relationship between antibiotic contamination and ARB is not
always observed. Our statistical analysis revealed a significantly
positive correlation between the occurrence rate of SMXr bacteria and
the residual concentration of SMX (Rs = 0.803) in January, but this
finding was not observed in July. However, as shown in Fig. 2, SMXr
bacteria increased in January at only city canal cases. Thus it is
suggested that rainfall effect is appeared in city canal sites by the
reason mentioned above. There was no statistically significant
correlation between ERY concentration and the occurrence rate of
ERYr bacteria. Erythromycin resistance is mediated by various
mechanisms (Perichon and Courvalin, 2009), suggesting possibility
of cross-resistance occurred by other chemicals.
The coastal shrimp ponds, brackish water environments, are

habitats differed from the freshwater environments, the relationship
between antibiotic contamination and ARB was not observed in this
study. We found that at several sites in the coastal shrimp ponds
where antibiotic concentration was below or almost below the limit of
detection (Table 1), high incidence of ARB was still observed both in
rainy and dry seasons (Table 2); this was particularly the case with
SMXr bacteria. This result is in agreement with previous study in the
coastal shrimp ponds of Vietnam by Le et al. (2005); higher incidence
of bacteria resistant to antibiotics was found in the ponds where
antibiotic concentration was lower. There are many possible reasons
for this. For example, first, higher incidence of ARB could be due to
their persistence in the environments. Previous studies (Enne et al.,
2001 and Enne et al., 2002; Antunes et al., 2005; Bean et al., 2005)
have shown strong evidence that SR bacteria can persist for a long
time even after a great decrease in prescription of the antibiotic; this is
due to the genetic linkage of the SR to other resistance determinants
(Enne et al., 2001). Second, other factors could have impact on
incidence of ARB. Recent ecological studies have shown evidence for
the co-selection of ARB with various other resistance determinants in
aquatic environments (Stepanauskas et al., 2005 and Stepanauskas
et al., 2006). Hence, ARB may be abundant even when the
corresponding antibiotics are absent in the environment. Third,
horizontal gene transfer of SR genes might play an important role
on their dissemination in the environment in the presence of coselection by other antibiotics (Bean et al., 2005). This study suggested

100
90
80
70
60

50
40
30
20
10
0

B (Jul)

1

Rs=-0.610

10

100

SMX concentration (ng/L)
Fig. 2. Relationship between sulfamethoxazole (SMX) concentration and the occurrence
rate of sulfamethoxazole-resistant (SMXr) bacteria in January (A) and July (B) 2007. The
value of RS was calculated by the test for Spearman rank correlation. Symbol, rhombus
enclosed with oval indicates city canal (HNCs), circle indicates pig farm/fish pond
(NHPs) and triangle indicates shrimp pond (HNAQs).


2900

P.T.P. Hoa et al. / Science of the Total Environment 409 (2011) 2894–2901

Table 3

Sulfamethoxazole-resistant strains from three environments and their sul gene
possession.
Closest genus or
strain name (total #)

Isolate # in HNCs Isolate # in HNPs Isolate # in HNAQs
(sul positive #)
(sul positive #)
(sul positive #)

Acinetobacter (29)
Aeromonas (24)
Bacillus (16)
Pseudoalteromonas (12)
Halobacillus (8)
Shewanella (4)
Escherichia (4)
Pseudomonas (3)
Arthrobacter (2)
Brachybacterium (2)
Micobacterium (2)
Rheinheimera (2)
Agrococcus (1)
Cellulosimicrobium (1)
Citrobacter (1)
Sandaracinobacter (1)
Shigella (1)
Staphylococcus (1)
Tenacibaculum (1)
Uruburuella (1)

Vibrio (1)
Vitreosciella (1)
Wautersiella (1)
Enterobacter (1)
Marine bacterium
Tw-9 (1)
Total 25 genera
(121 strains)

2 (2)
15 (13)
0
0
0
0
2 (1)
0
0
0
0
0
1 (1)
0
1 (0)
1 (1)
0
0
0
0
0

0
0
0
0

17 (17)
8 (7)
1 (1)
1 (1)
0
2 (1)
2 (1)
3 (2)
2 (2)
2 (2)
2 (2)
2 (1)
0
1 (1)
0
0
1 (1)
1 (1)
0
1 (1)
0
1 (1)
1 (1)
1 (1)
0

10 (9)
1 (1)
15 (9)
11 (4)
8 (1)
2 (0)
0
0
0
0
0
0
0
0
0
0
0
0
1 (1)
0
1 (0)
0
0
0
1 (1)

22
(18 sul positive)

49
(44 sul positive)

50
(26 sul positive)

variety of bacterial genera, suggesting the shrimp pond was highly
diverse in microbial community. In addition, referring to the
distribution of the sulfonamide resistance genes (sul1, sul2 and
sul3), which detected by PCR and Southern hybridization as reported
in our previous study (Hoa et al., 2008), we detected a total of 23
genera conferring sul1, sul2 or sul3 genes; of these 23 genera, 13
genera, namely Pseudoalteromonas , Halobacillus, Arthrobacter, Brachybacterium, Microbacterium, Rheinheimera, Marine bacterium Tw-9,
Agrococcus, Cellulosimicrobium, Sandaracinobacter, Tenacibaculum,
Uruburuella, and Wautersiella were first reported in this study as
sul-containing bacteria. Interestingly, our results revealed a potential
reservoir of sul-containing bacterial genera from various habitats;
major genera were Acinetobacter (17/17 sul-positive) in the pig farm/
fish pond, Aeromonas (13/15 sul-positive) in the city canal and Bacillus
(9/15 sul-positive) and Acinetobacter (9/10 sul-positive) in the coastal
shrimp ponds. The findings are in agreement with recent studies that
Acinetobacter was a potential environmental reservoir for sulfonamide resistance genes in pig slurry, manured agricultural soils and
fish ponds that contaminated by sulfonamides and other antibiotics
(Petersen et al., 2002; Byrne-Beiley et al., 2009; Heuer et al., 2009).
Among the sites, pig farm/fish ponds and shrimp ponds showed
higher bacterial species diversity than from city canals (Table 3). A
previous study by Suzuki et al., 2008) suggested that the occurrence
rate of tetracycline-resistant bacteria positively correlated with
bacterial species diversity, accounting for the increase in tetracycline-resistant bacteria in the environment with higher microbial
diversity. The SMXr bacteria in pig farm/fish ponds and shrimp ponds

may also be a similar condition, and may be one of the reasons why
the occurrence rate of SMXr bacteria in July and January were nearly
equal.
Escherichia, Shigella, Staphylococcus and Enterobacter were found in
HNPs (Table 3), and these possessed sul genes. This suggests that pigs
and /or humans released ARB which was earlier selected in animal
and human bodies. In the VAC environment, animals and humans are
also candidates for the origins of ARB and resistance genes.

Bacillus was reported as the major bacteria possessing tet(M) in
marine sediments in Japan (Rahman et al., 2008), which was found to
be a potential reservoir sul genes in Vietnamese water in this study.
The three habitats investigated in this study showed a higher number
of sul-processing bacteria; 18 sul-positive genera /18 total genera in
the pig farm/fish pond, 5/6 in the city canal, and 7/9 in the shrimp
ponds. This finding suggests that the sul genes are widely distributed
in various bacterial groups in the bacterial isolates, and more diverse
than those genera reported in previous studies (Le et al., 2005;
Rahman et al., 2008;Byrne-Beiley et al., 2009. Our study concluded
that SMXr bacterial groups containing the sul genes in the environments were more diverse than known by previous studies. Various
species should be reservoirs of sul genes in VAC aquatic environments.
4. Conclusion
This study provides new results on the contamination status by
antibiotics in VAC environment in northern Vietnam. Sulfonamide
especially SMX and sulfamethazine were major drugs in city canal and
pig farm/fish pond, respectively. Sulfonamides were used intensively
in rainy season. Trimethoprim was used as combination drug with
SMX. Macrolides were detected in city canal, indicating human use
origin.
Occurrence of ARB and diversity of ARB were also evaluated. Result

of city canal site showed higher occurrence of SMXr in dry season than
rainy season; however, pig farm/fish pond and aquaculture sites
showed constant rate of SMXr in rainy and dry seasons. SMXr bacteria
were diverse, which included first recorded genera as SMXr.
Acinetobacter and Aeromonas were the major SMXr in aquatic
environment. Many of SMXr possessed sul genes, suggesting reservoir
of the sul genes.
This study first showed relationship of drug contamination and
ARB diversity in rainy and dry seasons.
Acknowledgements
This research was partly supported by the 21st Century and Global
COE programs (MEXT) and Grant-in-Aids from JSPS (19405004 and
19310039). We thank Dr. Nguyen Thi Mui, Hanoi University of
Science, Vietnam, for providing us with the data on rainfall and
temperature of Red River delta areas. Dr. A. Subramanian and Dr. T. W.
Miller are appreciated for their critical reading of this paper.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
doi:10.1016/j.scitotenv.2011.04.030.
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