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Research Article
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High Prevalence of Multi-drug Resistant Bacteria in Selected Poultry Farms in Selangor, Malaysia
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Yaqub Ahmed Geidam,
Zunita Zakaria,
Saleha Abdul Aziz,
Siti Khairani Bejo,
Jalila Abu
and
Sharina Omar
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ABSTRACT
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Prevalence of multidrug resistant bacteria in apparently healthy chickens from 3 selected poultry farms in Selangor area of Malaysia was investigated. Conventional isolation techniques such as growth on selective media, gram staining and biochemical tests were utilised for the identification of the different bacterial isolates. Antimicrobial sensitivity test was monitored with the disc diffusion assay against 12 antimicrobial agents. A total of 96 Staphylococcus aureus, 48 E. coli, 7 Pasteurella sp. and 6 Salmonella sp. were isolated. All E. coli and Salmonella spp. isolates were multidrug resistant while 77.2% of Staphylococcus aureus and 71.5% of Pasteurella sp. isolates were multidrug resistant. The study further revealed highest resistance to tetracycline while cephalothin as the best drug of choice for treatment of infections caused by the isolates in the study area. Since not only chickens are at risk, this study recommends urgent intervention by regulatory agencies to limit the emergence and spread of these bacteria as well as prudent use of antibacterial agents among farmers in Malaysia.
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Received: December 02, 2011;
Accepted: February 20, 2012;
Published: May 10, 2012
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INTRODUCTION
The introduction of antimicrobial agents in the early 20th Century is one of
the greatest achievements of scientific medicine. However, the use of these
antimicrobial agents for clinical purposes and growth promotion purposes (Gunal
et al., 2006) has increased the number of organisms that developed
resistance to these agents. Previous studies showed that for many antimicrobial
agents, resistant bacteria were found within three to five years from the introduction
of the antimicrobial agent into clinical use (Schwarz and
Chaslus-Dancla, 2001; Swartz, 2002). Antibiotic
resistance still remains a global problem today (Adeleke
and Omafuvbe, 2011).
Serious infections, notably in hospitals and other health care facilities are
associated with the emergence of antibiotic-resistant organisms (Schwartz
et al., 1997; Spellberg et al., 2008;
Taddele et al., 2012). The treatment of infections
caused by antibiotic-resistant organisms is difficult because of limited options
and the organisms appear to be biologically competent to cause serious threat
to life (Mulvey and Simor, 2009; Sibi
et al., 2011). The continued discovery of these large number of drug-resistant
organisms is occurring at a time of decreased discovery and development of new
anti-infective agents (Menghani et al., 2011;
Mamun-or-Rashid et al., 2012) and most of the
new agents are synthetic relatives of the older ones (Spellberg
et al., 2008). As a consequence, an increase in the number of infections
which cannot be treated is imminent in the near future.
Although most of the antimicrobial resistance problems in human medicine stem
from overuse, there is evidence that antimicrobial resistant enteric bacteria
can transfer from animals to humans and thereby establishing a reservoir of
resistant genes (Fey et al., 2000; Angulo
et al., 2004; Molbak, 2004; Maripandi
and Al-Salamah, 2010). The widespread use of antibiotics in animals has
also raised several concerns related to human and animal health. The principal
area of concern has been the increasing emergence of antibiotic resistance phenotypes
in both clinically relevant strains and normal commensal microbiota (Chikwendu
et al., 2008). The use of nontherapeutic levels of antibiotics in
poultry production can select for antibiotic resistance in commensal and pathogenic
bacteria in poultry (OBrien, 2002). Transfer of
resistant bacteria between animals and humans through food products has been
documented and can pose a threat to public health (OBrien,
2002; Angulo et al., 2004; Molbak,
2004; Sornplang et al., 2011; Akinjogunla
et al., 2011).
The World Health Organisation (WHO) has recognised that antimicrobial resistance
is a global problem that calls for a global response. Consequently, WHO issued
the global principles for the containment of antimicrobial resistance in animals
intended for food and the WHO global strategy for the containment of antimicrobial
resistance where some interventions were recommended that hopefully will enable
local authorities to slow down the emergence and reduce the spread of resistance
in diverse settings (WHO, 2000, 2001).
These guidelines recommend the establishment of surveillance programmes for
antimicrobial consumption and resistance, as well as guidelines for prudent
use of antimicrobials and further research.
Antimicrobial resistance rates of bacteria from animals and their products
are available for many countries (Centers for Disease Control
and Prevention, 2004; Bywater et al., 2004;
SWARM, 2007; Getachew et al.,
2010). However, continuous monitoring is important for the control of resistance
in animals and man (WHO, 2000, 2001).
Continuous monitoring and surveillance will efficiently evaluate the resistance
problem and detect trends and changes (WHO, 2000, 2001).
Monitoring will identify the prevalence of resistance and resistance trends
over time can determine the emergence of resistance, help to develop guidelines
for the prudent use of antimicrobials and limit the emergence and spread of
resistant organisms (WHO, 2000, 2001).
Multi-drug resistant bacteria have earlier been reported in other animal species
in Malaysia (Zunita et al., 2008; Ooi
et al., 2011). The present study is aimed at determining the prevalence
of multi-drug resistant bacteria from selected poultry farms in Selangor area,
Malaysia.
MATERIALS AND METHODSs
Sampling: Three commercial poultry farms were selected from Selangor area of Malaysia where a total of 80 samples from apparently healthy chickens were collected comprising of skin and feather swabs for Staphylococcus aureus, nasal and tracheal swabs for pasteurella and cloacal swabs for E. coli and Salmonella. All samples were collected in January 2011 using sterile swabs.
Isolation and identification of bacterial isolates: Conventional isolation
techniques such as growth on selective media, gram staining and biochemical
tests were utilised for the identification of the different bacterial isolates.
The Staphylococcus aureus isolates from both skin and feather were identified
and confirmed based on colony and cell morphology, gram positive staining, positive
catalase and coagulase tests and formation of yellow colonies on Mannitol salt
agar. E. coli isolates were identified and confirmed using colony and
cell morphology, pinkish colonies on MacConkey agar, gram negative staining,
indole and methyl red positive, Voges-Proskauer and citrate negative. Salmonella
isolates were identified and confirmed using colony and cell morphology,
pinkish colonies on brilliant green agar and positive for motility test. The
Pasteurella isolated were identified and confirmed based on colony and
cell morphology on blood agar, gram negative staining and oxidase positive test.
Antimicrobial sensitivity test: Antimicrobial sensitivity test was monitored
with the disc diffusion assay (Kirby-Bauer) recommended by the NCCLS
(2000) and CLSI (2010) on Muller Hinton agar (Oxoid,
Milan, Italy); with the following 12 antimicrobial agents : Ampicillin 10 μg
(AMP 10), Ciprofloxacin 5 μg (CIP 5), Sulphamethoxazole/Trimethoprim 25
μg (SXT 25), Streptomycin 10 μg (S 10), Tetracycline 10 μg (TE
10), Cephalothin 30 μg (KF 30), Erythromycin 15 μg (E 15), Chloramphenicol
30 μg (C 30), Penicillin G 10 units (P 10), Oxacilin 1 μg (OX 1),
Clindamycin 2 μg (DA 2) and Neomycin 30 μg (N 30) also obtained from
Oxiod (Milan, Italy). Antibiotic agents were selected based on the importance
in treatment of the bacterial isolates. The zone of inhibition was interpreted
according to National Laboratory Standard Institute (NLSI, 2010). Although multidrug
resistance is defined as resistance to ≥3 antibiotics tested (Oteo
et al., 2005), resistance to ≥4 antibiotics tested was considered
in this study.
RESULTS A total of 96 Staphylococcus aureus, 48 E. coli, 7 Pasteurella spp. and 6 Salmonella spp. were isolated from the 3 poultry farms (Table 1). Staphylococcus aureus and E. coli were isolated from all the skin and feather and cloacal swabs respectively from all the farms. However, out of the 7 Pasteurella spp. isolated 4 were from tracheal specimens of farm A and 3 from nasal specimens of farm B. Neither the tracheal nor the nasal specimens from farm C yielded positive colonies for Pasteurella. The only 6 Salmonella spp. isolated were from cloacal specimens of farm B, none was isolated from farm A and C (Table 1). The result of the antibiotic sensitivity tests for the bacterial isolates is presented in Table 2. More than 50% of the Staphylococcus aureus isolates showed resistance to ampicillin, tetracycline, erythromycin, penicillin and clindamycin, where as none of the isolates were resistance to cephalothin. Ninety four percent of E. coli isolates were resistant to tetracycline while on the hand only 33% of the isolates were resistant to cephalothin. All Salmonella isolated were found to be resistant to tetracycline and clindamycin while only 14% were resistant to cephalothin. Although no breakpoint for zone of inhibition was given for Pasteurella, isolates that have no zone of inhibition were considered as resistant. All the bacterial isolates in this study showed very high resistance (83-100%) to tetracycline while all isolates except Salmonella showed low resistance (0-33%) to cephalothin (Table 2).
Table 3 presents the resistance of the bacterial isolates
to the number of antibiotics used in the study. Highest numbers of Staphylococcus
aureus isolates (17.7%) were resistant to four antibiotic agents while on
the other hand only 3.1% of the isolates were resistant to 10 antibiotic agents.
A total of 77.2% of Staphylococcus aureus isolates were resistant to
4 or more antibiotic agents. For E. coli highest number of isolates (25%)
was resistant to 9 different antibiotic agents while on the other hand lowest
number of isolates (4.2%) was resistant to 5 and 12 antibiotic agents.
Table 1: |
Source of samples and number of bacteria isolated from selected
poultry farms in Selangor, Malaysia |
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*: Tracheal sample, **: Nasal sample |
Table 2: |
Antibiotic resistance of bacteria isolated from selected
poultry farms in Selangor, Malaysia |
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NA: Not applicable in the 2010 guidelines |
Table 3: |
Resistance of bacterial isolates to number of antibacterial
agents tested |
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All E. coli isolates (100%) were resistant to 5 or more antibiotic agents.
Pasteurella isolates presented 71.5% resistance to 8 and above antibiotic
agents where as 100% of Salmonella isolates were resistant to 6 or more
antibiotic agents.
DISCUSSION
The microbial isolates identified in this study include Staphylococcus aureus,
E. coli, Pasteurella sp. and Salmonella sp. Staphylococcus
aureus and E. coli appeared to be the most prevalent bacterial species
isolated. Staphylococcus aureus is known to be easily carried in the
nasopharynx, throat, skin, cuts, boils, nails and as such can easily contribute
to the normal microflora (Ekhaise et al., 2008).
Salmonella is the least bacteria isolated from the chickens and this trend
can be attributed to the good Salmonella control programme practiced
by most farms as examination of food to detect Salmonella is routinely
carried out for food safety and food-borne disease surveillance.
The result of this study revealed the presence multidrug resistant bacteria
from chickens. All isolates showed high resistance to tetracycline while on
the other hand all except Salmonella spp. showed very low resistance
to cephalothin. The result of this study clearly identified cephalothin as a
good choice antibiotic for treatment of infection in the study area. Also all
E. coli and Salmonella sp. isolated in this study were found to
be resistant to 5 or more antibacterial agents tested in this study, a finding
which is supported by earlier reports of Overdevest et
al. (2011) that drug resistance in Enterobacteriaceae has increased
dramatically during the past decade. The increase which was attributed mainly
to increased prevalence of extended-spectrum β-lactamase (ESBL)-producing
Enterobacteriaceae (Canton et al., 2008; Coque
et al., 2008; Hashim et al., 2011)
has caused increase in the use of last-resort antimicrobial drugs (i.e., carbapenems).
In addition, these results provide evidence that there is an increased emergence
of antibiotic resistance from commensal bacterial isolates, a finding which
is in agreement with the earlier reports of Chikwendu et
al. (2008) who found increasing emergence of antibiotic resistance phenotypes
in both clinically relevant strains and normal commensal microbiota. The presence
of multidrug-resistant bacteria significantly limits the treatment options available
for these life-threatening infections. This unfortunate trend is happening at
a time when the discovery and development of new antibacterial agents has slowed
down drastically as reported by Spellberg et al.
(2008) and Menghani et al. (2011).
Although the control of antibiotic usage in Malaysia is legislated, the findings
of this study indicated a probable indiscriminate use of antibiotics by the
poultry farms in Selangor. This is further supported by earlier reports of uncontrolled
used of antibiotics in feed and for treatment by some selected pig farms in
Malaysia by Ooi et al. (2011). Although the use
of antibiotics in human medicine has influenced the emergence of antibiotic-resistant
bacteria, the use of antibiotics in animal agriculture has markedly contributed
to this problem as indicated in this study. This has raised several concerns
related to human and animal health. The types of bacteria detected from the
poultry farms investigated in this study are associated with a variety of human
infections (Gehanno et al., 2009; Lim
et al., 2009; Fadel and Ismail, 2009). Especially
the fact that transfer of resistant bacteria between animals and humans through
food products has been documented and can pose a threat to public health ((OBrien,
2002; Angulo et al., 2004; Molbak,
2004). Although antimicrobial resistance rates of bacteria from animals
and their products are available for many countries (Bywater
et al., 2004; SWARM, 2007) including Malaysia
(Getachew et al., 2010), the guidelines issued
by WHO recommends continues surveillance and prudent use of antibacterial agents.
The findings in this investigation emphasize the importance of studying multiple
genera of bacteria from different animals as sources of human exposure to antibiotic
resistance strains. Therefore not only that the chickens are at risks, poultry
workers and consumers are equally exposed to serious hazards due to multidrug
resistance bacteria. This calls for urgent intervention by regulatory agencies
to limit the emergence and spread of these bacteria as well as prudent use of
antibacterial agents among farmers in Malaysia.
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