1. Antibiotic Resistance of Milk Bacteria Isolated (Staphylococcus spp.
and Streptococcus spp.) from Clinically Normal Dairy Cows on the Basis
of Somatic Cell Count in Shiraz, Iran
Mohammad Amin Hanifpour1
; Abdolah Mirzaei2*
; Mohammad Reza Hatami1
; Razmik Beglarian3
;
Mehdi Roushan Zamir1
; Mohammad Hadi Eskandari4
1
Fars Pegah Pasteurized Milk Company, Iran; 2
Department of Clinical Science, School of
Veterinary Medicine, Shiraz University, Shiraz 71345, Iran; 3
Chief department of stock-
breeding's production Technology, Dean of Technology faculty, Armenian Agricultural
Academy, Yerevan, Armenia; 4
Department of Food Science and Technology,
College of Agriculture, Shiraz University, Shiraz, Iran
*Corresponding Author: e-mail: mirzaei@shirazu.ac.ir
Abstract
The aim of this study was to investigate the in vitro antibiotic resistance of
bacteria isolated from clinically normal lactating cows on the basis of somatic
cell count. Milk samples were taken aseptically from all quarters (n = 400) of
100 lactating cows in four farms just before morning milking. About 10 ml of
the foremilk were collected from each quarter of cows in two sterile tubes.
The milk samples were transported on ice to the laboratory for somatic cell
count and bacteriological culture of milk samples with somatic cell count of
greater than 100000 cells per milliliter. Culture plates were incubated at 37 °C
for 24–48 hours. Twelve genuses of bacteria were isolated from 133 (33.25%)
quarters and 48 (48%) cows. The most two isolated bacteria are
Staphylococcus Spp. and Streptococcus spp. which was 37.61 and 36.69
percent, respectively. Antibiotic resistance to Penicillin, Oxacillin,
Streptomycin, Ampicillin, Cephalotin, Cloxacillin, Erythromycin,
Gentamicin, Novobiocin, Tetracyclin and Chloramphenicol was performed.
The greatest antibiotic resistance of Staphylococcus spp. and Streptococcus
spp. to Penicillin and Cloxacillin were 39 and 22.5 percent, respectively.
Minimum inhibitory concentrations of the various antibiotics which are
conventionally used to treat mastitis for Staphylococcus Spp was < 8 and < 2
µg/ml based on the Percentage of the bacterial resistance. Control of milk
somatic cell count and the strategies of mastitis control can be responsible for
improving milk quality in dairy cows.
Keywords: Antibiotic resistance, Somatic cell count, Dairy cows.

2. 1. Introduction
Bovine mastitis is the inflammation of the mammary gland and a frequent
cause of economic loss in the dairy industry worldwide. Mastitis is a complex
and costly disease of dairy herds with different types and durations of infection.
The primary causes of mastitis are bacteria or other microorganisms such as
fungi and yeast (Wattiaux 1999; Ruegg 2001). The majority of clinical mastitis
is caused by Gram-positive pathogens such as Staphylococcus aureus,
Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis.
In contrast, coagulase- negative staphylococci (CNS) can cause mainly
subclinical mastitis, characterized by an elevated somatic cell count of milk
(Salmon et al. 1998; Gentilini et al. 2002). High incidence of SCC of milk
indicates the presence of infection (Bellamy 1999). A somatic cell count (SCC)
of greater than 200000/mL is a strong indicator of mastitis and many cows
maintain SCC values of less than 100000 cells per milliliter (Pamela 2003).
Coagulase negative staphylococci are the most frequent organisms isolated
from positive milk samples for California mastitis test in dairy cows in Tehran,
Iran (Atyabi et al. 2006). The polymerase chain reaction and bacterial culture
results showed that the most important streptococcal agents of bovine
subclinical mastitis are S. agalactiae, S. dysgalactiae and S. uberis in Ahvaz,
Iran (Moatamedi et al. 2007).
It has been a well-accepted fact that the antimicrobial agents are applied to
treat, control and prevent bacterial infections in lactating and dry cows.
Resistance of isolated microorganisms from mastitic quarters of cows to several
antimicrobial agents has been shown in several studies in some countries (Luthje
and Schwarz 2006; Roberts et al. 1999; Myllys et al. 1998; Gianneechini et al.
2002; Gentilini 2002; Moroni et al. 2006). The aim of this study was to
investigate the in vitro antibiotic resistance of bacteria isolated from clinically
normal lactating cows on the basis of somatic cell count in Shiraz, Iran. A
further objective was to identify isolated microorganisms and to determine the
bacterial resistance of Staphylococcus Spp to minimum inhibitory concentrations
(µg/ml) of the various antibiotics. Antibiogram tests for two most isolation
bacteria (Staphylococcus spp. and Streptococcus spp.) showed that the various
resistances to different antibiotic.
2. Materials and methods
This study was conducted on registered multiparous Holstein cows at the
farms in Shiraz, southern Iran. The present study was carried out during June
and July when the peak temperature reaches 40°C. Shiraz is located at a latitude
of 29° 38′ N and longitude 52° 36′ E. Its altitude is 1296 m above sea level.
The cows were housed in free-stall barns and the ration (total mixed ration)
included mainly alfalfa, corn silage, beet pulp, cotton seed, soybean, corn and

3. barley. The cows were machine-milked three times daily and subjected to post
milking teat dipping. All cows were dried off two months before expected
calving and dry cow therapy was performed following the last milking of
lactation. Milk samples were taken aseptically from all quarters (n = 400) of 100
lactating cows in four farms just before morning milking. Prior to sampling the
teats were washed and dried with a single-use disposable tissue. The first three
squirts of milk from each quarter were discarded and the teat end was
disinfected with cotton soaked in 70% ethanol and allowed to dry. About 10 ml
of the foremilk were collected from each quarter of cow in two sterile tubes. The
milk samples were transported on ice to the laboratory of the Shiraz University
for SCC and to the professor Alborzi Clinical Microbiology Research Center of
Shiraz University of Medical Sciences for bacteriological culture.
Quarter milk SCC was measured using the electronic cell counting method
(COMBIFOSS 5000, Fossomatic, Foss Electric, Denmark). Samples that had
somatic cell counts of greater than 100000 cells per milliliter were selected
for identification of bacteria. To detect the bacteria in the collected samples,
they were surface plated on 5% sheep blood agar supplemented with 10 mg L-
1
of amphotericin B. Culture plates were incubated at 37 °C for 24–48 hours,
the bacteria were isolated based on differentiation between the corresponding
colonies and each colony was purified afterwards. To identify the genius and
species of the isolated bacteria, gram stain, a proper differential biochemical
test and API test (120 series) were utilized. Following the identification of the
genius and the species of the bacteria, antibiogram test for each isolated
bacterium was done for the antibiotics including Penicillin, Oxacillin,
Streptomycin, Ampicillin, Cephalotin, Cloxacillin, Erythromycin,
Gentamicin, Novobiocin, Tetracyclin and Chloramphenicol, according to the
standard and up to date references. After 24 hours, the results were evaluated
based on the size of the zones and using the standards to determine the
resistance and sensitivity of the bacteria to the above-mentioned antibiotics.
Minimum inhibitory concentration (MIC) was recorded as the lowest
concentration of the antimicrobial agent that inhibited bacterial growth.
3. Results
Bacterial cultures were performed on the milk samples with SCC of greater
than 100000 cells per milliliter. Twelve genuses of bacteria were isolated
from 133 (33.25%) quarters and 48 (48%) cows (Table 1). The results of
bacterial cultures and identified bacteria by means of biochemical differential
and API tests are demonstrated in Table 1. Antibiogram tests for two most
isolated bacteria (Staphylococcus spp. and Streptococcus spp.) showed that the
various resistance to different antibiotics (Table 2). Minimum inhibitory
concentrations of the antibiotics including Tetracycline, Penicillin, Oxacilin,

4. Erythromycin and Streptomycin for milk Staphylococcus spp. are shown in
Table 3.
Minimum inhibitory concentrations test (agar dilution) was performed for
Staphylococcus spp. which showed greater resistance to the above antibiotics
which are conventionally used by the vets to treat mastitis.
Table 1 Proportion of isolated bacteria form milk samples with greater than 100000
cells per milliliter
Bacteria Percent
Staphylococcus spp. 37.61
Streptococcus spp. 36.69
Bacillus spp. 11
Acinetobacter spp. 4.58
E.coli 2.75
Pseudomonas spp. 1.83
Neisseria, Stenotrophomanos
maltiphilia, Pasteurella
pneumotropica,
Diphtheroides, Nonfermented
bacteria
5.54
Total 100
Table 2 Percentage for the resistance of Staphylococcus spp. and Streptococcus spp. to
different antibiotics
Antibiotic Staphylococcus spp. Streptococcus spp.
Ampicillin 14 15
Cephalotin 14 10
Cloxacillin 19 22.5
Erythromycin 12 0
Gentamicin 0 2.5
Novobiocin 2 0
Penicillin 39 20
Streptomycin 5 5
Tetracycline 29 7.5
Chloramphenicol 2 2.5
Table 3 Percentage of the bacterial resistance of Staphylococcus Spp to minimum
inhibitory concentrations (µg/ml) of the various antibiotics which are conventionally
used to treat mastitis
Antibiotic < 8 µg/ml < 2 µg/ml
Tetracycline 56.2 84.4
Penicillin 56.2 75
Oxacillin 56.2 65.6
Erythromycin 25 56.2
Streptomycin 93.7 96.8

5. 4. Discussion
The most important finding of the present study was that Staphylococcus spp.
and Streptococcus spp. are the two most commonly isolated bacteria form milk
samples with greater than 100000 cells per milliliter. Both of them are
widespread in the environment and on the skin of the teats. Mastitis can be
characterized by an increase in the somatic cell count in milk (Rodriguez-Zas et
al. 2000).
Most researchers consider composite cow milk with a cell count less than
200000/mL to be normal, while milk with a count above 200000 cells/mL is
considered abnormal (Nelson Philpot and Nickerson 1991). At quarter level, a
threshold value of < 100000 cell/mL has been proposed for a healthy quarter
(Ruegg and Reinemann, 2002; Schukken et al. 2003). So, in the present study all
samples with >100000 cell/mL were selected for culturing. The culture results
revealed that Staphylococcus spp. and Streptococcus spp. account for 37.61%
and 36.69 % of the isolated bacteria in this study, respectively.
Hawari and Al-Dabbas (2008) found that the major aetiological agents
responsible for subclinical mastitis were Staphylococcus aureus, Coliforms and
Streptococcus spp. in Jordan. The results of the culture on the clinical cases in
U.K. showed that E. coli was isolated from 32 % of samples, whereas it was
found that the presence of E. coli in only 2.75 % cases of healthy quarter cow
with SCC >200000 cell/mL. So, this bacterium can easily cause clinical mastitis
under specific conditions such as different stresses and reduction of innate udder
immunity. Atyabi et al., (2006) reported that cagulase negative staphylococci
(CNS) were the most frequent organisms causing contamination of quarters
around Tehran, Iran. They collected milk samples from mastitic cows and found
that S. epidermidis was isolated from the majority of samples. In the present
study, staphylococci species including S. aureus, S. lugdunensis, S. warneri, S.
simulans, S. saprophyticus, S. epidermidis, S. xylosus, S. cohnii, S. haemolyticus,
S. capitis were separated from milk samples of clinically healthy quarters. The
results in this study have similar patterns compared to the results of Atyabi et al.,
(2006); however, it is difficult to compare results of studies because of
differences in sample selection and cultivation techniques. Therefore, the
relative frequencies of these bacteria were high in healthy cows, and it is
important for producers and managers of farms to prevent mastitis by taking
care of hygiene such as disinfection of milking machine, teat and milker’s
hands.
In the Netherlands, the most common cause of subclinical mastitis was staph.
aureus when quarters had SCC > 250000 cell/mL (Poelarends et al. 2001).
Djarbi et al. (2002) reported that the mean SCC was > 300000 and 138000
cells/mL for staph. aureus and coagulase negative staphylococci, respectively.
In this study, mean SCC was 389347 cells/mL for all of staphylococci species,
while for CNS, the mean value was 408709 cells/mL. Staph. aureus was

6. separated from one quarter which had 130000 cell/mL. In Finland, the
percentages of bacterial resistance for Staph. aureus and CNS to ≥ 3
antimicrobials were 2 and 3.3 %, respectively. The MIC90 for Staph. aureus and
CNS to Penicillin were 16 and 2 µg/mL, respectively (Pitkala et al. 2004). In the
present study isolated Staphylococci and Streptococci species were more
resistant to Penicillin, Cloxacillin, Ampicillin, Cephalothin and Tetracycline
than the other antibiotics.
The overall resistance pattern of Staphyloccous spp. and Streptococcus spp. to
ten antimicrobials varied from 0 to 39 % in the present study. The results
showed that the MIC75 for Staphyloccous spp. to Penicillin was < 2 µg/mL.
Various staphylocci species were isolated from quarter samples but Staph.
aureus was isolated just from one quarter. This resistance pattern may be caused
by the wide use of broad spectrum antibiotics with low doses through the
intramammary and systemic routes and also insufficient periods of treatment. It
also might be due to the improper prescription of the antibiotics by the vets or
unwise use of them by the farmers.
In conclusion, the results underline the important role of hygiene in
controlling and preventing mastitis. This includes disinfection of teat, teat cup
liners and milker’s hand. It is worthwhile to use properly treatment procedures
and control strategies for mastitis to improve milk quality in dairy cows. Thus, it
seems to logical to keep in mind that in recent decades, the mastitis control and
prevention methods used have been effective.
References
Atyabi, N., M. Vodjgani, F. Gharagozloo and A. Bahonar, 2006. Prevalence of
bacterial mastitis in cattle from the farms around Tehran. Iranian Journal
of Veterinary Research 7 (3): 76-79.
Bellamy, K. 1999, Breeding cattle for mastitis resistance. MDC project.
Proceeding of the British Mastitis Conference 37-45.
Djarbi, B., N. Bareille, F. Beaudeau and H. Seegers, 2002. Quarter milk
somatic cell count in infected dairy cows: A meta-analysis. Vet. Res. 33:
335–357.
Gentilini, E., G. Denamiel, A. Betancor, M. Rebuelto, M. Rodriguez Fermepin
and R.A. De Torres, 2002. Antimicrobial susceptibility of coagulase-
negative staphylococci isolated from bovine mastitis in Argentina. J.
Dairy Sci. 85: 1913–1917.
Gianneechini, R.E., C. Concha and A. Franklin, 2002. Antimicrobial
susceptibility of udder pathogens isolated from dairy herds in the West
Littoral region of Uruguay. Acta. Vet. Scand. 43: 31–41.
Hawari, A.D. and F. Al-Dabbas, 2008. Prevalence and distribution of mastitis
pathogens and their resistance against antimicrobial agents in dairy cows

7. in Jordan. American Journal of Animal and Veterinary Sciences 3 (1):
36-39.
Luthje, P. and S. Schwarz, 2006. Antimicrobial resistance of coagulase-
negative staphylococci from bovine subclinical mastitis with particular
reference to macrolide–lincosamide resistance phenotypes and
genotypes. Journal of Antimicrobial Chemotherapy 57: 966–969.
Moatamedi, H., M. Seyfiabad Shapouri, M. Ghorbanpoor, M. Jamshidian and
S. Gooraninejad, 2007. A polymerase chain reaction based study on the
subclinical mastitis caused by Streptococcus agalactiae, S. dysgalactiae
and S. uberis in cattle in Ahvaz. Iranian Journal of Veterinary Research
8 (3): 260-265.
Moroni, P., G. Pisoni, M. Antonini, R. Villa, P. Boettcher and S. Carli, 2006.
Antimicrobial drug susceptibility of Staphylococcus aureus from
subclinical bovine mastitis in Italy. J. Dairy Sci. 89: 2973-2976.
Myllys, V., K. Asplund, E. Brofeldt, V. Hirvela-Koski, T. Honkanen-Buzalski,
J. Junttila, L. Kulkas, O. Myllykangas, M. Niskanen, H. Saloniemi, M.
Sandholm and T. Saranpaa, 1998. Bovine mastitis in Finland in 1988
and 1995-Changes in prevalence and antimicrobial resistance. Acta. Vet.
Scand. 39: 119–126.
Nelson Philpot, W., S.C. Nickerson, 1991. Mastitis: counter attack, a strategy to
combat mastitis. Babson Bros Co. Naperville, Illinois USA.
Pamela, L.R. 2003. Investigation of mastitis problems on farms. Vet. Clin.
Food Anim. 19: 47–73.
Pitkala, A., M. Haveri, S. Pyorala, V. Myllys and T. Honkanen-Buzalski, 2004.
Bovine Mastitis in Finland 2001—Prevalence, Distribution of Bacteria,
and Antimicrobial Resistance. J. Dairy Sci. 87: 2433–2441.
Poelarends, J.J., H. Hogeveen, O.C. Sampimon and J. Sol, 2001. Monitoring
subclinical mastitis in Dutch dairy herds. Pages 145–149 in Proc. 2nd
Int. Symp. Mastitis Milk Quality, Vancouver, BC, Canada. National
Mastitis Council, Madison, WI and American Association of Bovine
Practitioners, Rome, GA.
Roberts, M.C., J. Sutcliffe, P. Courvalin, L. Bogo Jensen, J. Rood and H.
Seppala, 1999. Nomenclature for macrolide and macrolide-lincosamide-
streptogramin B resistance determinants. Antimicrob. Agents
Chemother. 43: 2823–2830.
Rodriguez-Zas, S.L., D. Gianola and G.E. Shook, 2000. Evaluation of models
for somatic cell score lactation patterns in Holsteins. Livest. Prod. Sci.
67: 19-30.
Ruegg, P.L. 2001. Mastitis Control. In: Dairy Updates: Milking and Milk
Quality, The Babcock Institute, University of Wisconsin, 405: 10.
Ruegg, P.L. and D.J. Reinemann, 2002. Milk quality and mastitis tests. Bovine
Pract. 36: 41–54.

8. Salmon, S.A., J.L. Watts, F.M. Aarestrup, J.W. Pankey and R.J. Yancey Jr,
1998. Minimum inhibitory concentrations for selected antimicrobial
agents against organisms isolated from the mammary glands of dairy
heifers in New Zealand and Denmark. J. Dairy Sci. 81: 570–578.
Schukken, Y.H., D.J. Wilson, F. Welcome, L. Garrison-Tikofsky and R.N.
Gonzales, 2003. Monitoring udder health and milk quality using somatic
cell counts. Vet. Res. 34: 579–596.
Wattiaux, M.A. 1999. Mastitis: The Disease and its Transmission. In: Dairy
Essentials. Babcock Institute for International Dairy Research and
Development. University of Wisconsin-Madison, Chapter 23: 89-92.