Antimicrobial
Resistant
Escherichia coli from
Livestock
THE PHILIPPINE
AGRICULTURAL
SCIENTIST
Vol. 90 No. 1, 64-70
March 2007
S. C.
Jiao et al.
ISSN
0031-7454
Prevalence of Multiple Drug-Resistant Escherichia coli from Chicken, Pig
and Nile Tilapia (Oreochromis nilotica) Intestines Sold in Wet Markets in
Manila and the Conjugative Transferability of the Resistance
Stephanie C. Jiao, Rea Marie L. Fami, Vena Airene D. Pedernal and Esperanza C. Cabrera*
Biology Department, De La Salle University, 2401 Taft Ave., Manila, Philippines
*Author for correspondence; e-mail: [email protected]; telefax: (632) 536-0228
One hundred eighty (180) Escherichia coli isolates from chicken, pig and Nile tilapia intestines sold in
wet markets in Manila were studied for their antimicrobial susceptibility patterns using the disc diffusion method, and for conjugative transferability of the resistance. The highest prevalence of resistance in E. coli from all animals was to tetracycline and trimethoprim/sulfamethoxazole, the two most
widely used antimicrobials in feeds locally. Percentage resistance in isolates from pigs was 93.33%
for both antimicrobials, 58.33% for tetracycline and 41.67% for trimethoprim/sulfamethoxazole in isolates from chickens, and 38% and 12% to tetracycline and trimethoprim/sulfamethoxazole, respectively, in isolates from fish. A high 38.33% of E. coli from chickens was resistant to the quinolone
nalidixic acid, and 25% were resistant to the fluoroquinolone ciprofloxacin compared to those from
pigs, where only 10% of the isolates were resistant to nalidixic acid and 5% to ciprofloxacin, and those
from fish, with 5% isolates resistant to nalidixic acid and 0% to ciprofloxacin. Quinolones and
fluoroquinolones are mostly used in poultry.
The isolates from the different animals also showed varying resistance to ampicillin (2% of isolates from fish, 23.33% from pigs and 38.33% from chickens), chloramphenicol (0%, 6.67% and 20% of
isolates from fish, chickens and pigs, respectively), gentamicin (0% isolates from fish, 5% from pigs
and 13.33% from chickens), amikacin (0% isolates from fish and pigs, 3.33% from chickens), aztreonam
(1.67% of isolates from pigs and chickens, and 2% from fish), cefotaxime (0% resistant isolates from
fish and pigs, 1.67% from chickens), cefoxitin (0% resistant isolates from pigs, 3.33% and 5% from
chickens and fish, respectively) and ceftazidime (0% resistant isolates from fish and pigs, 3.33% from
chickens). All of the isolates were susceptible to ceftriaxone. Isolates that screened positive for
extended-spectrum β-lactamase (ESBL) production were negative for ESBL using the confirmatory
double-disc synergy method. All the tested E. coli isolates completely transferred their resistance
determinants to a susceptible E. coli through conjugation, converting it to a multiple resistant strain.
The study showed high prevalence of E. coli isolated from the intestines of the animals studied that
were resistant to multiple antimicrobials, especially to those that are most commonly incorporated in
feeds. It also demonstrated the conjugative transferability of all resistance determinants of the isolates.
Key words: agricultural animal, antibiotic-supplemented feeds, antimicrobial resistance, conjugation, Escherichia coli,
food animal, livestock
Abbreviations: BHIB – brain heart infusion broth, CLSI – Clinical Laboratory Standards Institute, EMB – eosin methylene
blue, ESBL – extended spectrum β-lactamases, NA – nalidixic acid, SXT – trimethroprim/sulfamethoxazole, TE – tetracycline
INTRODUCTION
Antimicrobial agents are commonly used in livestock to
prevent animal diseases and, at the same time, improve
animal productivity. However, studies suggest that the use
of antibiotics selects for resistant strains to survive and be
64
disseminated, contributing to the increase in antimicrobial
resistant bacteria (Aarestrup et al. 2001; Bryan et al. 2004;
Petersen et al. 2002; Sayah et al. 2005). Links between the
agricultural use of antibiotics and human infections caused
by antibiotic resistant bacteria have been suggested (Smith
et al. 1999; Witte 1998). In addition, antimicrobial resis-
The Philippine Agricultural Scientist Vol. 90 No. 1 (March 2007)
Antimicrobial Resistant Escherichia coli from Livestock
tance genes may be carried in resistance or R plasmids,
which are mobile extrachromosomal DNA that may be transmitted to different susceptible bacteria belonging to the
same species, or even to bacteria belonging to different
genera, by conjugation (Kruse and Sorum 1994; Poppe et
al. 2005). This further results in an increase in antimicrobial
resistant microorganisms that can be disseminated in the
environment, to humans, as well as to other animals that
can cause serious diseases.
Antimicrobial agents are still routinely used in the Philippines to promote the growth of food animals such as
pigs, chickens and fish. The antimicrobials administered
are those that are used, or are related to those used, in the
human population for treatment of infectious diseases.
Chicken and pig intestines, as well as Nile tilapia, are popular local delicacies. The animal intestines are directly sold
to consumers in wet markets, after these have been isolated from freshly butchered animals. Fish intestines are
usually removed during cleaning and are not eaten; however, the intestinal bacteria may contaminate the meat during further processing. When meats are not thoroughly
cooked, intestinal bacteria may be taken in with the food.
This study was conducted to determine the antimicrobial susceptibility patterns of non-clinical strains of E. coli
from the intestines of pigs, chickens and fish that were
obtained from different suppliers which utilized antibiotics
in animals for non-therapeutic purposes. It also determined
the production of extended-spectrum β-lactamases (ESBL)
by the isolates. ESBLs are coded by mutated β-lactamase
genes that are carried in R plasmids, which may be conjugative, and which frequently also carry genes for resistance to other drug classes (Paterson and Bonomo 2005).
The mutations extend the spectrum of β-lactams that is
inactivated by the enzymes. ESBLs render the bacterial
hosts resistant to the latter generation cephalosporins with
the oxyimino side chain and to the monobactams, which
are normally resistant to hydrolysis by the classical βlactamases. It has been recommended that diseases caused
by ESBL-producing isolates should not be treated with
S. C. Jiao et al.
any β-lactam antibiotic, even if the organisms were to test
susceptible to some of these agents in vitro (CLSI 2005).
This practice deprives the patients of the option for treatment with this potent group of antimicrobials. The study
also determined the conjugative transferability of the antimicrobial resistance determinants to the susceptible strain
E. coli J532. Literature search shows a paucity of local
studies in this area. The results of our study hope to sound
the call for the review and evaluation of the use of antimicrobials for non-therapeutic purposes in agricultural animals in the Philippines.
MATERIALS AND METHODS
Microbial Isolates from Animal Samples
The animal samples were purchased from three of the largest wet markets in Manila from July to August, 2005. These
are the major distributors of food products to small markets. Sixty samples each of fish and the intestines from
freshly slaughtered chickens and pigs were purchased.
Table 1 shows the sampling design. The suppliers of the
vendors varied each time. All the samples were collected
early in the morning to ensure their freshness. The samples
were individually placed in tightly sealed sterile plastic
bags, and immediately brought in an ice chest to the laboratory for analysis.
E. coli from the chicken, pig and fish intestines was
isolated and identified using conventional methods such
as colonial morphology on MacConkey and eosin methylene blue agar plates (EMB) and biochemical tests such as
reaction on triple sugar iron agar, indole, methyl red, Vogues
Proskauer, citrate utilization, urease and gelatin liquefaction tests. One isolate from each animal sample was included in the study.
Antimicrobial Susceptibility Testing
The antimicrobial susceptibility patterns of the E. coli isolates were determined following the standard disc diffu-
Table 1. Sampling design used in the procurement of test specimens.
Specimen
Market Source
No. of
Specimens
Duration of
Sampling
Chicken intestine
Quiapo Market
Trabajo Market
Divisoria Market
60
6 wk
2 samples from each of 5 vendors per wk
Pig intestine
Quiapo Market
Trabajo Market
Divisoria Market
60
6 wk
2 samples from each of 5 vendors per wk
Nile tilapia
Baclaran Market
60
3 wk
2 samples from each of 10 vendors per wk
The Philippine Agricultural Scientist Vol. 90 No. 1 (March 2007)
Sampling
Method
65
Antimicrobial Resistant Escherichia coli from Livestock
S. C. Jiao et al.
sion method of the Clinical and Laboratory Standard Institute (CLSI 2005). ATCC E. coli 25922 was used as the
reference strain that was tested together with the isolates.
The E. coli isolates were grown in brain heart infusion
broth (BHIB) at 37 oC for 16–18 h, and then subcultured in
BHIB at 37 oC for 4 h to bring the microorganisms to the log
phase. The turbidity of the cultures was adjusted to equal
that of 0.5 McFarland standard. This approximated 1.5 x
108 cells mL-1. The inocula were streaked on Mueller-Hinton
agar plates using sterile cotton swabs.
The antimicrobials and concentrations used were the
β-lactam antibiotics: ampicillin 10 µg (AP), ceftazidime 30
µg (CAZ), cefotaxime 30 µg (CTX), cefoxitin 30 µg (FOX),
aztreonam 30 µg (ATM), ceftriaxone 30 µg (CRO); the
aminoglycosides: gentamicin 10 µg (GM) and amikacin 30
µg (AN); the fluoroquinolone, ciprofloxacin 5 µg (CIP); the
quinolone, nalidixic acid 30 µg (NA); trimethoprim/
sulfamethoxazole 1.25/23.75 µg (SXT); chloramphenicol 30
µg (C); and tetracycline 30 µg (TE). The plates were incubated at 37 °C for 16-18 h and read.
Diameters of the zones of inhibition were measured to
the nearest millimeter (mm) and interpreted using the CLSI
(2005) breakpoints as follows: “Susceptible category implies that an infection due to the strain may be appropriately treated with the dosage of antimicrobial agent recommended for that type of infection and infecting species,
unless otherwise contraindicated. Intermediate category
includes isolates with antimicrobial agent MICs that approach usually attainable blood and tissue levels and for
which response rates may be lower than for susceptible
isolates. Resistant strains are not inhibited by the usually
achievable systemic concentrations of the agent with normal dosage schedules and/or fall in the range where specific microbial resistance mechanisms are likely and clinical
efficacy has not been reliable in treatment studies.”
Detection of Extended-Spectrum β-Lactamase (ESBL)
Production
E. coli isolates were screened for ESBL production using
the CLSI (2005) disc diffusion method as described above.
Isolates that had diameters of zones of inhibition that fell
within those set by CLSI for any of the extended-spectrum
β-lactam antibiotics were further studied to confirm their
ESBL production. The antibiotics used and their respective zones of inhibition were: ATM, <27 mm; CAZ, < 22 mm;
CTX, <27 mm; CRO, <25 mm (CLSI 2005).
The double-disc diffusion method, as standardized by
CLSI (2005), was used to confirm the ESBL production of
the isolates. Inoculum preparation was the same as earlier
described for the disc diffusion method. Amoxicillin/
clavulanate 20/10 µg (AmC) disc was placed in the center
of the inoculated Mueller-Hinton agar plate. ATM, CAZ,
66
CTX and CRO discs, with concentrations as used in the
disc diffusion method, were each placed at a distance of 14
mm side to side (or repeated at 20 mm distance for overlapping zones) from the AmC disc. The plate was incubated at
37 oC for 18 h, and the diameters of the zones of inhibition
produced by the extended-spectrum β-lactam antibiotics
on the side facing the AmC disc were compared with those
facing away from the AmC disc. A >5 mm enhancement by
AmC of the inhibition zone diameter produced by any of
the extended-spectrum β-lactam antibiotics was taken to
be a confirmed positive test for ESBL production. Each
test was done in triplicate.
Conjugation Studies
E. coli isolates from the animal intestines that were susceptible to NA, but resistant to some of the test antimicrobials, were tested for the conjugative transferability of their
resistance. E. coli J532, a strain that is resistant to NA and
susceptible to all the other antimicrobials, was used as the
recipient. E. coli J532 was courtesy of Dr. Jesus Silva
Sanchez of the Instituto Nacional de Salud Publica, Mexico.
The donor strain and the recipient strain were separately
grown in tryptic soy broth (TSB) at 37 oC for 16–18 h. A
loopful of each was separately transferred into BHIB and
grown for 4 h at 37 oC, allowing the bacteria to reach the log
phase of growth. The turbidity of the cultures was adjusted to that of 0.5 McFarland. Two hundred microliters
(µL) of the recipient and 20 µL of the donor were mixed and
spread plated on Mueller Hinton agar plate using a flamesterilized bent glass rod. The plates were then incubated at
37 °C for 6 h. The conjugation mixtures in the plates were
harvested by adding 2 mL of sterile distilled water, and
were scraped off from the plate using a bent glass rod.
Each conjugation mixture was inoculated into selective
plates of Mueller Hinton agar containing a combination of
NA (30 µg mL-1) and TE (30 µg mL-1), and another with NA
and SXT (25 µg mL-1). The suspected transconjugants
growing in the selective plates were further tested for their
antimicrobial susceptibility patterns using the disc diffusion method to determine the transfer of other resistance
determinants, and to confirm the identity of the recipient
as that of J532, which is resistant to nalidixic acid.
RESULTS AND DISCUSSION
One hundred eighty (180) E. coli were isolated from the
intestines of 60 samples each of tilapia, 60 pig intestines
and 60 chicken intestines sold in wet markets in Metro
Manila. The isolates produced pink colonies on
MacConkey agar plate, and dark-colored colonies with
greenish metallic sheen on EMB. They produced acid from
The Philippine Agricultural Scientist Vol. 90 No. 1 (March 2007)
Antimicrobial Resistant Escherichia coli from Livestock
lactose, gas from glucose, positive indole and methyl red
tests, negative Vogues Proskauer, citrate, urease and gelatin liquefaction tests.
Table 2 shows the number of antimicrobials to which
the isolates showed either a resistant or an intermediate
response. The E. coli from pigs were the most resistant to
the test antimicrobials compared to those from the chickens and the fish. Ninety-seven percent (97%) of the pig
isolates were resistant to at least one antimicrobial, with
93.33% showing multiple resistance. E. coli from chickens
(55%) also showed multiple resistance, while those from
fish showed resistance to only two antimicrobials at the
most. The results of Sayah et al. (2005) also showed that
the highest levels of multiple resistant E. coli were from
swine fecal samples compared to E. coli from cattle and
poultry. This may be explained by the longer exposure of
pig microbial flora to the antimicrobials due to the pig’s
longer stay before they are slaughtered for food. Moreover, multiplicity of resistance suggests the co-selection
of genes for resistance to other antimicrobials by agents to
which microorganisms were exposed as a consequence of
their use as feed additives.
The results also showed that several isolates shared
the same resistance phenotypes (Tables 3, 4 and 5). For
example, four isolates from chickens had the resistance
phenotype AP, SXT, TE, CIP, NA whereas three had the
resistance phenotype AP, GM, SXT, TE, CIP, NA (Table 3).
From pigs, most E. coli (34 of the isolates) had the resistance phenotype of TE, SXT (Table 4), whereas from fish,
six isolates exhibited the same phenotype TE, SXT (Table
5). Isolates with the same resistance phenotype may be of
the same strain (Guan et al. 2002), and suggests clonal
spread of the E. coli isolates in the environment to the
different animals. It may also be due to the conjugative
S. C. Jiao et al.
transfer of resistance genes in R plasmids to other bacterial hosts.
Figures 1, 2 and 3 show the responses of intestinal E.
coli from pigs, chickens and Nile tilapia to the test antimicrobials. The most frequently encountered antimicrobial
resistance in the E. coli isolates from all animal samples
tested was to tetracycline, followed by resistance to
trimethoprim/sulfamethoxazole. Escherichia coli from pigs
had the highest percentage of isolates that were resistant
to the two antimicrobials at 93.33% for both (Fig. 1). This
was followed by E. coli from chickens at 58.33% for tetracycline and 41.67% for trimethoprim/sulfamethoxazole (Fig.
2). The E. coli from fish had a much lower percentage of
isolates resistant to tetracycline and trimethoprim/
sulfamethoxazole at 38% and 12%, respectively (Fig. 3).
These two are the most common antimicrobials given to
chickens and pigs locally, while tetracycline is the most
common antimicrobial given to fishes (J. Victoria pers.
com.).
Tetracycline is a broad-spectrum antimicrobial agent
with a history of more than 50 yr of veterinary use. The
study of Bryan et al. in 2004 showed that more than 78%
Table 2. Number of antimicrobials to which intestinal Escherichia coli isolates from chickens, pigs and fish were
not susceptible (resistant and intermediate responses).
No. and Percentage of
Isolates from Intestines
No. of
Antimicrobials
Chicken
0
1
2
3
4
5
6
7
8
Total no. of
isolates
18
9
8
3
9
6
4
2
1
60
(30%)
(15%)
(13.33%)
(5%)
(15%)
(10%)
(6.67%)
(3.33%)
(1.67%)
(100%)
Pig
2
2
34
10
4
6
1
1
(3.33%)
(3.33%)
(56.66%)
(16.67%)
(6.67%)
(10%)
(1.67%)
(1.67%)
60 (100%)
Fish
29 (48.33%)
22 (36.67%)
9 (15%)
60 (100%)
The Philippine Agricultural Scientist Vol. 90 No. 1 (March 2007)
Table 3. Resistance phenotypes of Escherichia coli from
chicken intestines
Resistance Phenotype
AP, SXT, TE, CIP, NA
AP, CIP, NA
SXT, CIP, TE, NA
TE, NA
AP, CAZ TE, CIP, NA
AP, SXT. TE NA
AP, GM, SXT, C
AP, GM, TE, CIP, NA
AP, GM, SXT, TE, CIP, NA
AP, GM, SXT, C, TE, CIP, NA
AP, GM, FOX, SXT, C, TE, CIP, NA
AP, GM, FOX, SXT, TE, CIP, NA
AP, SXT, CIP, NA
AP, TE
TE
SXT, TE, NA
TE, AN
AP, SXT, C, TE, AN, NA
AP, C, CIP, NA
SXT, TE
CTX, AP, SXT
SXT, AP
CAZ, ATM
AN – amikacin
AP – ampicillin
ATM – aztreonam
C – chloramphenicol
CAZ – ceftazidime
CIP – ciprofloxacin
No. of Isolates with
R Phenotype
4
1
3
1
1
2
1
1
3
1
1
1
2
1
9
1
1
1
1
3
1
1
1
CTX – cefotaxime
FOX – cefoxitin
GM – gentamicin
NA – nalidixic acid
SXT – trimethoprim/sulfamethoxazole
TE – tetracycline
67
Antimicrobial Resistant Escherichia coli from Livestock
S. C. Jiao et al.
Table 4. Resistance phenotypes of Escherichia coli from
pig intestines.
Fig. 1. Responses of Escherichia coli isolates from
pig intestines to the test antimicrobials.
100%
GM – gentamicin
NA – nalidixic acid
SXT – trimethoprim/sulfamethoxazole
TE – tetracycline
80%
Intermediate
40%
Res istant
20%
No. of Isolates with
R Phenotype
Antimicrobials
17
2
1
1
6
1
1
1
1
NA – nalidixic acid
SXT – trimethoprim/sulfamethoxazole
TE – tetracycline
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
susceptibility
intermediate
resistance
tetracycline
trimethoprim/s
cefoxitin
ampicillin
aztreonam
nalidixic acid
amikacin
ciprofloxacin
chlorampheicol
ceftazidime
ceftriaxone
gentamicin
cefotaxime
% of isolates
Fig. 2. Responses of Escherichia coli isolates from
chicken intestines to the test antimicrobials.
and 47% of E. coli from pigs and chickens, respectively,
were resistant to tetracycline. The prevalence of resistance
is lower than that from the present study. Brian et al. (2004)
explained that this high occurrence of resistance might be
due to the continuous use of the drug in livestock at
subtherapeutic levels.
In addition, 38.33% and 25% of E. coli from chickens
were resistant to the quinolone nalidixic acid and the
fluoroquinolone ciprofloxacin, respectively (Fig. 2). In contrast to these observations, only 10% of the E. coli from
pigs was resistant to nalidixic acid and 5% to ciprofloxacin
68
Susceptible
60%
0%
Resistance Phenotype
AP – ampicillin
ATM – aztreonam
FOX – cefoxitin
Resistant
Antimicrobials
Table 5. Resistance phenotypes of Escherichia coli from
Nile tilapia (Oreochromys niloticus) intestines.
TE
FOX
NA
AP, NA
SXT, TE
SXT, NA
AP, FOX
ATM
AP
Intermediate
tetracycline
trimethopri
ampicillin
chloramph
nalidixic
ciprofloxaci
gentamicin
aztreonam
ceftriaxone
cefoxitine
ceftazidime
cefotaxime
amikacin
1
1
34
3
2
4
2
1
1
1
1
1
1
1
1
2
1
Susceptible
tetracycline
trimethoprim/sul
ampicillin
nalidixic acid
ciprofloxacin
gentamicin
chloramphenicol
ceftazidime
cefoxitine
aztreonam
amikacin
ceftriaxone
cefotaxime
AP – ampicillin
ATM – aztreonam
C – chloramphenicol
CIP – ciprofloxacin
FOX – cefoxitin
% of isolates
TE
SXT
SXT, TE
SXT, TE, C
SXT, TE, AP
SXT, TE, NA
SXT, TE, C, AP
SXT, TE, C, AP, NA
SXT, TE, AP, GM
SXT, TE, AP, NA, GM
SXT, TE, C, AP, GM
SXT, TE, AP, NA, CIP
SXT, TE, CIP
SXT, TE, C, AP, NA, CIP
SXT, TE, C, AP, NA, ATM, CIP
SXT, TE, C, AP, NA
TE, C, AP, NA
No. of Isolates with
R Phenotype
% of isolates
Resistance Phenotype
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
Antimicrobials
Fig. 3. Responses of Escherichia coli isolates from
tilapia (Oreochromys niloticus) intestines to the
test antimicrobials.
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Antimicrobial Resistant Escherichia coli from Livestock
(Fig. 1), whereas all of the isolates from fish were susceptible to ciprofloxacin, and only 5% were resistant to nalidixic acid (Fig. 3). The use of fluoroquinolones in poultry
locally may explain the higher prevalence of resistance to
these antimicrobials of E. coli from chickens compared to
those from the pigs and fish found in this study.
Luangtongkum et al. (2006) likewise showed that 46%
of Campylobacter isolates from broilers and 67% from turkeys that were conventionally raised with antimicrobials
were resistant to fluoroquinolones, in contrast to only less
than 2% resistant Campylobacter from poultry that were
organically raised, and were never exposed to antimicrobials. Unicomb et al. (2003) reported that fluoroquinolone
resistance in Campylobacter isolates from human infections acquired from meats consumed in Australia has not
emerged in the country. They attributed this to the fact
that fluoroquinolones have never been licensed for use in
livestock in Australia. In 2005, the U.S. Food and Drug
Administration (FDA) banned the use of enrofloxacin, a
fluoroquinolone, for treatment of bacterial infections in
chickens, citing a significant increase in infections by
fluoroquinolone-resistant Campylobacter since the use
of enrofloxacin in poultry was approved in the U.S. (FDA
2005). As early as 1999, the European Union banned the
use of many antibiotics for growth promotion purposes,
with the remaining antimicrobials to be phased out by 2006
(Collignon et al. 2005).
The results of the present study suggest that the popular local use of fluoroquinolones, as with the use of tetracycline and trimethoprim sulfamethoxazole in animals, positively selects for strains resistant to these antimicrobials,
allowing these to increase in the population and be disseminated, as was also suggested by other studies (Bryan
et al. 2004; Aarestrup et al. 2001; Sayah et al. 2005; Petersen
et al. 2002; Cabello 2006).
While only 1.67%, 2% and 1.67% of E. coli isolates
from pigs, fish and chickens, respectively, were resistant
to aztreonam, 3.33% and 1.67% of E. coli isolates from
chickens were resistant to ceftazidime and cefotaxime, respectively (Fig. 1, 2 and 3), 27% of the E. coli isolates from
chickens, 20% from Nile tilapia and 10% from pigs screened
positive for ESBL production based on the CLSI (2005)
guidelines. However, all of these 34 isolates were found to
be ESBL negative based on the double-disc synergy confirmatory test.
Other notable responses of the E. coli isolates were
their resistance to ampicillin as shown by 38.33% of the
isolates from chickens, 23.33% of those from pigs and 2%
of those from fish, and their resistance to chloramphenicol
(as shown by 20% of the isolates from pigs, 6.67% from
chickens, and none from Nile tilapia). Taken all together,
the isolates from all animals tested were still more susceptible to the aminoglycosides: gentamicin and amikacin, and
S. C. Jiao et al.
to the β-lactams: ceftazidime, cefoxitin, ceftriaxone,
cefotaxime and aztreonam (Fig. 1, 2 and 3).
Seventeen E. coli isolates that were susceptible to
nalidixic acid but resistant to at least two antimicrobial
agents were conjugated with E. coli J532, which was resistant to nalidixic acid but susceptible to the other antimicrobials. All donors completely transferred their resistance
determinants (Table 6), converting the susceptible recipient to a multiple resistant strain. The results suggest that
the antimicrobial resistance determinants in the isolates
tested are found in conjugative R plasmids. Thus, aside
from clonal spread, resistance may be transferred horizontally from one organism to another through conjugation.
The animal intestine is an ideal environment for the selection and transfer of antimicrobial resistance genes as shown
by the study of Lee et al. (2005). In addition, transfer of R
plasmids has also been shown between bacteria of diverse
origins in natural inanimate microenvironments (Kruse and
Sorum 1994).
The role played by the positive pressure from antimicrobials incorporated in animal feeds in selecting for resistant strains to survive and further be disseminated can not
be overemphasized. These strains may enter the food chain
due to undercooked livestock and other contaminated produce, which may, in turn, cross-contaminate other food
products because of common unsafe handling practices
(Smith et al. 2005). Once transmitted to humans, it is likely
that the resistance genes in these microorganisms can be
transmitted to more pathogenic bacteria, compromising the
treatment of serious infectious diseases.
Table 6. Resistance transferred from Escherichia coli
isolates from chicken, pig and Nile tilapia to Escherichia
coli J532.
Resistance Transferred
from E. coli
No. of Donor Isolates
From Chicken
SXT, AP
SXT, TE
2
3
From Pig
SXT, TE, GM, AP
SXT, TE, C
SXT, TE, AP
SXT, TE, AP, C
1
2
1
1
From Fish
SXT, TE
TE 3
AP – ampicilin
C – chloramphenicol
GM – gentamicin
The Philippine Agricultural Scientist Vol. 90 No. 1 (March 2007)
4
SXT – trimethoprim/sulfamethoxazole
TE – tetracycline
69
Antimicrobial Resistant Escherichia coli from Livestock
S. C. Jiao et al.
In summary, the study showed a high prevalence of E.
coli from the intestines of pigs, chickens and Nile tilapia
purchased from wet markets in Manila that were resistant
to multiple antimicrobials. These antimicrobials belong to
different classes. The highest resistance was to tetracycline and trimethoprim/sulfomethoxazole, the two antimicrobials that are most commonly incorporated in locallymanufactured animal feeds. In addition, a higher proportion of the isolates from chickens compared to those from
pigs and fish showed resistance to the quinolone, nalidixic
acid and the fluoroquinolone, ciprofloxacin.
Fluoroquinolones are more commonly utilized in chickens
than in the other animals.
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The Philippine Agricultural Scientist Vol. 90 No. 1 (March 2007)