Antimicrobial Resistance of Staphylococcus aureus Isolated from Bovine, Sheep and Goat Raw Milk

Abstract

Objective of this study was to determine the prevalence rate of antimicrobial resistance of S. aureus isolated from dairy cow, sheep and goat milk in Iran. From September 2010 to September 2011, a total of 348 raw milk samples from cow, sheep and goat were collected from randomly selected herds in Fars, Chahar Mahal va Bakhtiari and Ghom, provinces, Iran. Overall, 46 raw milk samples (13.2%) were found to be contaminated with S. aureus. Susceptibilities of the isolates were determined for 11 antimicrobial drugs using the disk diffusion assay. Most of the isolates (82.6%) were resistant to one or more antimicrobial agent. Six isolates (13.0%) were resistant to single antibiotic and 16 isolates (34.8%) showed resistance to 2 antimicrobial agents. Multiresistance was found in 34.8% of S. aureus isolates. Resistance (resistance and intermediate resistance) to ampicillin was the most common finding (54.3%), followed by resistance to oxacillin (28.3%), tetracycline (26.1%), penicillin G (23.9%), erythromycin (23.9%), trimethoprim-sulfamethoxazole (17.4%) and cephalotin (2.2%). All isolates tested for antibiotic sensitivity were susceptible to methicillin, vancomycin, chloramphenicol and ciprofloxacin. Furthermore, impacts and dynamics of genetic antibiotic determinants should also be investigated using molecular methods.

Full text

Global Veterinaria 8 (2): 111-114, 2012
ISSN 1992-6197
© IDOSI Publications, 2012
Corresponding Author: Ebrahim Rahimi, Department of Food Hygiene, College of Veterinary Medicine,
Islamic Azad University, Shahrekord Branch, Shahrekord, Iran. Tel: +983813361060.
Fax: +983116259809. 111
Antimicrobial Resistance of Staphylococcus aureus
Isolated from Bovine, Sheep and Goat Raw Milk
Forough Alian, Ebrahim Rahimi, Amir Shakerian,
1 2 1
Hassan Momtaz, Majid Riahi and Manochehr Momeni
3 4 4
Young Researchers Club, Islamic Azad University, Shahrekord Branch, Shahrekord, Iran
1
Department of Food Hygiene, College of Veterinary Medicine,
2
Islamic Azad University, Shahrekord Branch, Shahrekord, Iran
Department of Microbiology, College of Veterinary Medicine,
3
Islamic Azad University, Shahrekord Branch, Shahrekord, Iran
Center of Biotechnology Researches, Islamic Azad University, Shahrekord Branch, Shahrekord, Iran
4
Abstract: Objective of this study was to determine the prevalence rate of antimicrobial resistance of S. aureus
isolated from dairy cow, sheep and goat milk in Iran. From September 2010 to September 2011, a total of 348 raw
milk samples from cow, sheep and goat were collected from randomly selected herds in Fars, Chahar Mahal va
Bakhtiari and Ghom, provinces, Iran. Overall, 46 raw milk samples (13.2%) were found to be contaminated with
S. aureus. Susceptibilities of the isolates were determined for 11 antimicrobial drugs using the disk diffusion
assay. Most of the isolates (82.6%) were resistant to one or more antimicrobial agent. Six isolates (13.0%) were
resistant to single antibiotic and 16 isolates (34.8%) showed resistance to 2 antimicrobial agents.
Multiresistance was found in 34.8% of S. aureus isolates. Resistance (resistance and intermediate resistance)
to ampicillin was the most common finding (54.3%), followed by resistance to oxacillin (28.3%), tetracycline
(26.1%), penicillin G (23.9%), erythromycin (23.9%), trimethoprim-sulfamethoxazole (17.4%) and cephalotin
(2.2%). All isolates tested for antibiotic sensitivity were susceptible to methicillin, vancomycin, chloramphenicol
and ciprofloxacin. Furthermore, impacts and dynamics of genetic antibiotic determinants should also be
investigated using molecular methods.
Key words: Antimicrobial Resistance % Cow % Sheep % Goat % Milk % Staphylococcus aureus
INTRODUCTION be other possible sources of bulk milk contamination. S.
Milk is an excellent bacteriological medium for a largeintoxications worldwide [2-4]. The ability of S. aureus to
number of microorganisms. When the milk is drawn from grow and produce staphylococcal enterotoxins (SEs)
the udder of a healthy animal, it contains organisms thatunder a wide range of conditions is evident from the
have entered the teat canal through its opening. They arevariety of foods implicated in staphylococcal food
mechanically flushed out during milking. The number ispoisoning (SFP) [2]. SFP is suspected when the symptoms
ranged during milking between several hundreds toincluding nausea, violent vomiting, abdominal cramps and
several thousand per milliliter [1]. Staphylococcus aureus diarrhea affect the patients between 1 and 8 h after food
isolates are normal inhabitants of skin and mucusconsumption [5].
membranes. The coagulase-positive staphylococciAntibiotics are used to treat diseases of cattle, sheep,
constitute the well known pathogenic species S. aureus.goat, water buffalo and other animals and as well as used
S. aureus mastitis is a serious problem in dairy production as preservatives for milk [6]. The indiscriminate use of
and infected animals may contaminate bulk milk.antibiotics has led to the development of multiple
Additionally, human handlers, milking equipment, theantibiotic resistances thereby rendering the antibiotic
environment and udder and teat skin of dairy animals may treatment ineffective. Resistant bacteria occur in soil,
aureus is still an important cause of foodborne

Global Veterinaria, 8 (2): 111-114, 2012
112
water, plants and animals. The resistant bacteria presentdisks (HiMedia Laboratories, Mumbai, India) were used:
in environments are in contact with human beings andpenicillin G (10 IU), cephalotin (30 µg), chloramphenicol
animals. It has been estimated that nearly equal tonnage(30 µg), ciprofloxacin (30 µg), erythromycin (15 µg),
of antimicrobial agents are used in man and in agriculture tetracycline (30 µg), oxacillin (15 µg), gentamycin (10 µg),
worldwide [1]. Antimicrobial resistance is a major public trimethoprim-sulfamethoxazole (25 µg), methicillin (5µg)
health concern in many countries due to the persistentand vancomycin (30 µg). After incubation at 37°C for
circulation of resistant strains of bacteria in the48 h, the susceptibility of the S. aureus isolates to each
environment and the possible contamination of water and antimicrobial agent was measured and the results were
food [7]. S. aureus has been reported to frequently showinterpreted in accordance with interpretive criteria
multiple antimicrobial resistance patterns [8]. provided by CLSI [10].
Determination of levels of S. aureus and an
evaluation of the antibiotic-resistant phenotypes of theRESULTS AND DISCUSSION
isolates could serve as a tool for determining the hygiene
standards implemented during milking. Data on antibiotic In this study we described the isolation and
resistance could also be used to characterize theseantibiotic susceptibility characterization of S. aureus from
opportunistic pathogens, which may further limit the risks cow, sheep and goat milk obtained from two provinces of
associated with the consumption of contaminated milkIran. Fourty six of 348 samples (13.2%) were positive for
and its products. The aims of this study were to determine S. aureus. Thirty four raw cow milk (17.9%), 7 raw sheep
the prevalence rate and antimicrobial resistance ofmilk (9.0%) and 5 raw goat milk (6.3%), samples were
S. aureus in cow, sheep and goat, raw milk in Fars, Chahar contaminated with S. aureus. This contamination rate is
Mahal va Bakhtyari and Ghom, Iran. similar that observed in the surveys previously conducted
MATERIALS AND METHODS The resistance pattern of S. aureus isolates to 11
Sample Collection: From September 2010 to SeptemberTable 1. Most of the isolates (82.6%; n=38) were resistant
2011 a total of 348 cow (190), sheep (78) and goat (80), rawto one or more antimicrobial agent. Six isolates (13.0%)
milk samples was collected from Fars, Chahar Mahal vawere resistant/ intermediate resistance to single antibiotic
Bakhtyari and Ghom provinces, Iran. The samples wereand 16 isolates (34.8%) showed resistance/ intermediate
immediately transported to the laboratory in a cooler with resistance to 2 antimicrobial agents. Multiresistance
ice packs and were processed within an hour of collection. which is defined as resistance to 3 or more antimicrobial
Detection of S. Aureus: The samples were processedResistance (resistance and intermediate resistance) to
immediately upon arrival using aseptic techniques. To
detect S. aureus, 1mL of each milk sample was inoculated
on Baird - Parker agar (Difco, Detroit, Michigan, USA).
After 24 - 48 h of incubation at 37°C, suspected colonies
were sub-cultured on blood agar plate (Difco, Detroit,
Michigan, USA) and incubated for 24 h at 37°C. To
identify S. aureus, Gram stain, catalase, coagulase and
Voges-Proskaver (VP) tests were conducted on suspected
colonies [4, 9].
Antimicrobial Susceptibility Testing: One strain from
each S. aureus-positive sample was selected for
susceptibility tests. Antimicrobial susceptibility testing
was performed by the Kirby-Bauer disc diffusion method
using Mueller-Hinton agar (HiMedia Laboratories,
Mumbai, India) supplemented with 5% defibrinated sheep
blood, according to the Clinical Laboratory Standards
Institute [10]. The following antimicrobial impregnated
in other countries on several kinds of raw milk [3, 7, 11-13].
antimicrobial agents tested in this study is shown in
agents was found in 34.8% of S. aureus isolates.
ampicillin was the most common finding (54.3%), followed
by oxacillin (28.3%), tetracycline (26.1%), penicillin G
(23.9%), erythromycin (23.9%), trimethoprim-
sulfamethoxazole (17.4%) and cephalotin (2.2%). All
isolates tested for antibiotic sensitivity were susceptible
to methicillin, vancomycin, chloramphenicol and
ciprofloxacin.
The antimicrobial resistance profile of the tested
S. aureus strains to different antibacterial agents revealed
that 82.6% (n=38) of the strains were resistant to at least
one antibiotic. As reported by other investigators, the
resistance of S. aureus isolates to â-lactams such as
ampicillin, penicillin, tetracycline and oxacillin was evident
[14-17]. The finding that a large number of S. aureus were
resistant to ampicillin, penicillin, tetracycline and oxacillin
are, however, a cause for concern and should be further
investigated. These drugs are used in veterinary medicine
in Iran.

Global Veterinaria, 8 (2): 111-114, 2012
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Table 1: Antimicrobial resistance profiles of Staphylococcus aureus isolated from dairy cow, sheep and goat raw milk samples in Iran
S. aureus (N = 46)
--------------------------------------------------------------------------------------------------------------------------------------------
Antimicrobial agent Resistance Intermediate resistance Susceptible
Ampicillin 18 (39.1%) 7 (15.2%) 21 (45.6%)
Cephalotin 1 (2.2%) 0 (0.0%) 45 (97.8%)
Chloramphenicol 0 (0.0%) 0 (0.0%) 0 (0.0%)
Ciprofloxacin 0 (0.0%) 0 (0.0%) 0 (0.0%)
Erythromycin 5(10.9%) 6 (13.0%) 35 (76.1%)
Methicillin 0 (0.0%) 0 (0.0%) 0 (0.0%)
Oxacillin 7 (15.2%) 6 (13.0%) 33 (71.7%)
Penicillin G 8 (17.4%) 3 (6.5%) 35 (76.1%)
Tetracycline 7 (15.2%) 5 (10.9%) 34 (73.9%)
Trimethoprim-sulfamethoxazole 4 (8.7%) 4 (8.7%) 38 (82.6%)
Vancomycin 0 (0.0%) 0 (0.0%) 0 (0.0%)
Erythromycin resistance was almost non-existent inbe the reason of infection in human beings if they take
the cow milk samples. Despite the fact that a large number raw milk. These can be treated by improving hygienic
of S. aureus isolated from both sheep and goat milkconditions and careful handling of cow during milking.
samples was resistant to erythromycin, it was evidentFurthermore, impacts and dynamics of genetic antibiotic
from our results that this antibiotic was not frequentlydeterminants should also be investigated using molecular
used in animals by large scale farmers. About 14% of themethods.
isolates from cow milk samples were resistant to
erythromycin. ACKNOWLEDGEMENTS
Trimethoprim-sulfamethoxazole was the drug to
which almost small proportion of the isolates wereThis study was supported by vice chancellor for
resistant (17.4%). Similarly, a previous study reportedresearch of Islamic Azad University, Shahrekord Branch,
that only a small percentage (15.7% to 23.8%) of S.Shahrekord, Iran.
aureus isolated from meat and dairy products was
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