Bull. Vet. Inst. Pulawy 46, 289-294, 2002
ANTIMICROBIAL SUSCEPTIBILITY OF STAPHYLOCOCCI
ISOLATED FROM AFFECTED WITH MASTITIS COWS
EDWARD MALINOWSKI, ANNA KŁOSSOWSKA,
MICHAŁ KACZMAROWSKI, HENRYKA LASSA
AND KRYSTYNA KUŹMA
Department of Pathophysiology of Reproduction and Mammary Gland,
National Veterinary Research Institute,
85-090 Bydgoszcz, Poland
e-mail: vetri@logonet.com.pl
The purpose of the study was to determine the in vitro susceptibility of Staphylococcus
aureus and coagulase-negative staphylococci (CNS) strains isolated from clinical and subclinical
cases of mastitis to antimicrobial drugs. Antimicrobial sensitivity was tested by the disk diffusion
method and performed according to NCCLS guidelines in the Mueller-Hinton agar. Strains
isolated from clinical mastitis did not differ in their sensitivity from that of strains isolated from
subclinical mastitis cases. However, the strains were more resistant to penicillin, ampicillin,
amoxicillin, cloxacillin, cefoperazone, tetracycline, lincomycin and neomycin than CNS strains,
but their sensitivity to erythromycin, bacitracin and novobiocin was almost the same.
Key words: dairy cows, mastitis, Staphylococcus, sensitivity to antibiotics.
Mastitis is continously the most frequent and most expensive disease of dairy
cows (1, 6, 9, 19). Staphylococcus aureus is a predominant etiological agent of both
subclinical and clinical forms of udder inflammations (13, 20, 23, 25). The role of
coagulase-negative staphylococci (CNS) has clearly increased during the last years.
These bacteria can cause mainly subclinical mastitis (13, 23, 25), but some authors
reported high percentage of clinical cases evoked by CNS (1, 8, 10, 22). Cure rates of
Staph. aureus infections are poor after antibiotic treatment (5, 16, 18). The efficacy of
bovine mastitis treatment depends on the cause, clinical manifestation, antibiotic
susceptibility of etiological agent and the efficiency of immunological system. Mastitis
therapy is commonly unsuccessful owing to pathological changes that occur in the
udder parenchyma as a result of the inflammatory reaction, mastitogenic bacteria
related factors, pharmacokinetic properties of antimicrobial drugs, poor animal
husbandry and inadequate veterinary service (5, 17, 20). The main reason of low
efficacy of antibiotic treatment of staphylococcal mastitis is among others the
resistance of bacteria. In addition, during the past decade, bacteria that cause human
diseases have developed resistance to many of the antibiotics commonly used for
treatment (7, 26).
The purpose of this work was to determine the in vitro activity of
antimicrobial drugs against Staph. aureus and CNS isolated from clinical and
subclinical cases of mastitis during 2001.
290
Material and Methods
Quarter milk (inflamed secretion) samples were collected aseptically by
scientific personnel of the Department of Pathophysiology of Reproduction and
Mammary Gland or (sometimes) by field veterinary surgeons. The teat ends were
cleaned with alcohol swabs and allowed to dry. The first few streams were discarded
and then 2 – 4 ml of secretion was collected in sterile tubes. Samples were cooled and
immediately transported to the laboratory. Bacteriological examinations were
performed according to the commonly accepted principles (11).
Antimicrobial sensitivity was tested by the disk diffusion method and
performed according to NCCLS guidelines (14) in the Mueller-Hinton agar. The
following antibacterial agents (Oxoid) were used: penicillin (10 i.u.), ampicillin (10
µg), amoxicillin (10 µg), cefoperazone (30 µg), tetracycline (30 µg), erythromycin (15
µg), bacitracin (10 µg), cloxacillin (5 µg), neomycin (30 µg), streptomycin (10 µg),
novobiocin (30 µg), lincomycin (15 µg ) and tylosin (150 µg - Rosco). Staphylococcus
aureus ATCC 25 923 and Escherichia coli ATCC 25 922 were the control strains.
Interpretation of the test results was based on NCCLS criteria (14).
Results
From quarter secretions of subclinically or clinically inflamed udders, 517
strains of Staphylococcus aureus and 514 CNS strains were isolated during the year
2001. Sensitivity of the strains and CNS to antibiotic agents are presented in Tables 1
and 2. Strains isolated from clinical mastitis did not differ in their sensitivity from
strains isolated from subclinical mastitis forms. It was noted that out of all sensitive (S)
strains isolated both from clinical and subclinical mastitis cases, Staph. aureus was
most sensitive to bacitracin (92.9%) and neomycin (90.2%), in average. The next in
vitro effective antibiotics were erythromycin (74.4%), tetracycline (74.0%), cloxacillin
(73.9%), cefoperazone (71.8%) and novobiocin (70.3%). Coagulase-negative
staphylococci were sensitive mostly to neomycin (96.5%), bacitracin (94.8%),
cefoperazone (86.5%), cloxacillin (84.2%), tetracycline (83.9%), tylosin (81.3%), and
amoxicillin (74.8%).
Strains of Staph. aureus were resistant mostly to penicillin, ampicillin,
amoxicillin, lincomycin and streptomycin versus CNS that were mainly resistant to
penicillin, ampicillin, streptomycin and lincomycin. Altogether Staph. aureus strains
were more resistant to penicillin, ampicillin, amoxicillin, cefoperazone, tetracycline,
cloxacillin, lincomycin and neomycin than CNS. The susceptibility to erythromycin,
bacitracin, novobiocin and streptomycin did not differ between staphylococcal strains
tested.
291
Table 1
Antibiotic sensitivity of Staphylococcus aureus strains isolated
from subclinical and clinical cases of mastitis
Antibiotic
Pe
Amp
Aml
Cfp
Te
E
B
Ob
N
S
Nov
My
n
141
103
144
130
131
110
132
101
129
84
109
109
Subclinical cases
S(%)
I(%)
R(%)
33.3
66.7
31.1
68.9
52.1
5.6
42.3
42.3
30.0
27.7
60.3
13.7
26.0
53.6
20.9
25.5
89.4
5.3
5.3
62.4
9.9
27.7
83.7
7.0
9.3
41.7
20.2
38.1
57.8
11.0
31.2
20.2
24.8
55.0
n
36
11
36
33
38
19
36
18
38
24
29
35
Clinical cases
S(%)
I(%)
36.1
45.5
50.0
5.6
42.4
24.2
57.9
15.8
31.6
42.1
72.2
13.9
66.7
16.7
87.9
37.5
25.0
62.1
13.8
22.9
22.9
R(%)
63.9
54.5
44.4
33.3
26.3
26.3
13.9
16.7
12.1
37.5
24.1
54.2
Explanation: S - sensitive, I – intermediate sensitive, R - resistance, Pe - penicillin,
Amp - ampicillin, Aml - amoxicillin, Cfp - cefoperazone, Te - tetracycline, E erythromycin, B - bacitracin, Ob - cloxacillin, N - neomycin, S - streptomycin,
Nov – novobiocin, My – lincomycin.
Table 2
Antibiotic sensitivity of CNS strains isolated from subclinical and clinical cases of mastitis
Antibiotic
Pe
Amp
Aml
Cfp
Te
E
B
Tyl
Ob
N
S
Nov
My
n
101
63
105
78
88
37
106
21
71
82
59
84
29
Subclinical cases
S(%)
I(%)
52.5
58.7
66.7
7.6
60.3
26.9
76.1
3.4
51.4
21.6
87.7
6.6
52.4
23.8
71.8
11.3
90.2
6.1
62.7
10.2
58.4
11.9
40.5
14.6
R(%)
42.5
41.3
25.7
12.8
20.5
27.0
5.7
23.8
16.9
3.7
27.1
29.7
44.9
n
49
29
50
33
45
22
48
11
30
32
22
36
39
Clinical cases
S(%)
I(%)
46.9
62.1
68.0
8.0
54.6
30.3
84.4
4.5
45.5
18.2
93.7
2.1
72.7
18.2
86.7
93.8
3.1
40.9
9.1
50.0
25.0
30.8
20.5
Explanation: S, I, R, Pe, Amp, Aml, Cfp, Te, E, B, Ob, N, S, Nov, My –
as in Table 1, Tyl – tylosin.
R(%)
53.1
37.9
24.0
15.1
11.1
36.3
4.2
9.1
13.3
3.1
50.0
25.0
48.7
292
Discussion
Staphylococcus aureus strains isolated from the secretion of inflamed
mammary glands were more resistant to antibiotics commonly used in treatment of
mastitis than coagulase-negative strains. Almost the same results were reported by
other authors (2, 4), especially on resistance to penicillin, erythromycin and neomycin.
Staphylococci examined in this work were more resistant to antibiotics than bacteria
isolated earlier (12). But the in vitro resistance to antibiotics of bacteria isolated in the
same farm can change from one year to the next one (3, 12).
The majority of authors have noted the increase in the resistance to antibiotics
of staphylococci isolated from mastitis (2, 3, 4, 13, 24). The proportion of strains
resistant to only one antimicrobial drug increased with regard to Staph. aureus from
36.9% in 1988, to 63.6% in 1995 and regarding CNS from 26.6% to 49.7%.
Multiresistance increased also (13). It seems that our results are in a close accordance
with them. The most significant factor affecting the cure rates from clinical Staph.
aureus mastitis was the ability of the strain to produce β-lactamase (18). Apart of this
Watts and Salmon (24) emphasize the need to identify MRSA accurately because these
strains are resistant to all compounds currently approved for treatment of bovine
mastitis. On the other hand, de Oliveira et al. (15) determined MIC concentration for
Staph. aureus strains from 11 countries and found that overall level of resistance was
generally low to all antimicrobial agents that are currently commercially available to
treat bovine mastitis, regardless a country. Independently of the last conclusion, the in
vitro testing of antibiotic sensitivity is considered to be a predictor of the results of
therapy of IMI caused by Staphylococcus sp. (2, 16). However, the effectiveness of
antibacterial therapy against udder pathogens depends not only on its exogenous
administration into intramammary gland tissues, but it is also related to the various
indigenous inhibitors in milk: Ig, complement, lactoferrin, lactoperoxidase, etc. Many
of these are activated by the inflammatory process. Interactions between antibiotics and
immunological factors could lead either to augmenting the antibacterial effect on the
target tissue, or diminishing it. Appropriate elimination of bacteria requires both the
effectiveness of an antimicrobial drug against bacteria and a well functioning defense
system of the animal (21).
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