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Short communication: antimicrobial drug susceptibility of staphylococcus aureus from subclinical bovine mastitis in italy
J. Dairy Sci. 89:2973–2976
American Dairy Science Association, 2006.
Short Communication: Antimicrobial Drug Susceptibility
of Staphylococcus aureus from Subclinical Bovine Mastitis in Italy
P. Moroni,*1 G. Pisoni,* M. Antonini,† R. Villa,‡ P. Boettcher,†2 and S. Carli‡
*Department of Veterinary Pathology, Hygiene and Public Health, University of Milan, via Celoria 10, 20133 Milan, Italy
†Institute of Agricultural Biology and Biotechnology (IBBA), Consiglio Nazionale delle Ricerche (CNR), Milan, Italy
‡Department of Veterinary Sciences and Technologies for Food Safety, University of Milan, via Celoria 10, 20133 Milan, Italy
nent of any mastitis control program, but the outcomefor treatment of mastitis caused by Staph. aureus
The antimicrobial susceptibility of 68 Staphylococcus
variable and the probability of curing the disease is not
isolates collected during 2004 from milk of cows
high, primarily because of poor distribution of the drug
affected by subclinical mastitis was examined. The anti-
in the inflamed udder and the occurrence of staphylo-
microbial agents tested were the β
cocci resistant to antimicrobial agents (Pyo¨ra¨la¨ and Py-
G, amoxicillin, ampicillin, cloxacillin, amoxicillin + cla-
o¨ra¨la¨, 1994). During lactation, the cure rate of subclini-
vulanate, cephalonium, and cefoperazone; and other
cal mastitis ranges widely, and this variability can be
drugs including lincomycin, oxytetracycline, doxycy-
due to the choice of antimicrobial agent as well as to
cline, and kanamycin. Minimum inhibitory concentra-
factors associated with the infected cow and the quar-
tions recorded show that only certain β
ter. Therefore, cure is likely not a random event (Schuk-
sistant penicillins (specifically cloxacillin) or penicillin
ken et al., 1994). Antimicrobial therapy is a primary
combinations (amoxicillin + clavulanate) were consis-
tool for controlling staphylococcal mastitis, and antimi-
tently effective against Staph. aureus,
crobial susceptibility tests can guide the veterinarian
-lactam derivatives and drugs from other phar-
in selecting the most appropriate antimicrobial agent
macological groups were either moderately effective or
for treatment of IMI by Staph. aureus.
ineffective. Thus, β
-lactamase–resistant penicillins are
a variety of available antimicrobial agents, success in
to be considered the antimicrobial agents of choice for
the treatment of Staph. aureus
treatment of bovine mastitis resulting from infection
during lactation, is still very low. In fact, Staph. aureus
pathogens have many characteristics that make them
Key words: Staphylococcus aureus,
difficult targets for antimicrobial therapy (Sol et al.,
2000). For example, they can penetrate the phagocyticcells and survive inside them. This process shields the
Bovine mastitis is the most costly disease to the dairy
pathogens from some of the activity of antimicrobial
industry worldwide, with losses estimated at 2 billion
agents, even with drugs that can penetrate the cells.
dollars per year in the United States alone. These rele-
The objective of the present study was to evaluate the
vant economic losses are attributable to rejected milk,
antimicrobial resistance patterns of Staph. aureus
reduced milk quality, early culling, drug costs, veteri-
lates collected from IMI in cows from 44 commercial
nary expenses, and increased labor costs (Hoblet et al.,
1991; Gruet et al., 2001). The disease is the most fre-
Sixty-eight isolates of Staph. aureus
taken from indi-
quent reason for the use of antimicrobial agents on
vidual quarters of 68 cows with subclinical mastitis
dairy farms (Erskine, 2000). Intramammary infection
were used. The isolates were obtained during 2004,
sustained by Staphylococcus aureus
may result in clini-
from milk samples collected from animals belonging to
cal or subclinical mastitis and is usually associated with
42 dairy herds from different regions of Italy. Animals
increased SCC. Appropriate treatment of mastitis dur-
were selected on the basis of having quarter milk SCC
ing the lactation or dry period is an important compo-
greater than 400,000 cells/mL but no sign of clinicalmastitis. Herds were selected to represent differentprevalences of Staph. aureus
IMI, ranging from 1 to100%. The number of isolates for each herd was deter-
Received October 17, 2005.
Accepted February 28, 2006.
mined according to the number of lactating animals in
1Corresponding author: email@example.com
each herd: One single isolate was collected from 26
2Current address: Animal Health and Production Section, Joint
IAEA/FAO Division, International Atomic Energy Agency, A-1400
small herds (≤50 lactating cows), 2 isolates were col-
lected from 11 medium-sized herds (≤100 lactating
cows), and more than 2 isolates were collected from
isolates collected from IMI of dairy cattle (Craven et
5 large herds (>100 lactating cows). All isolates were
al., 1986; Watts and Yancey, 1994; Owens et al., 1997;
identified on the basis of morphology, hemolysis pat-
Makovec and Ruegg, 2003; Pengov and Ceru, 2003; Ti-
tern, and gram staining. The gram-positive cocci were
kofsky et al., 2003). Nevertheless, obtaining continually
tested for catalase and coagulase production. The spe-
updated MIC values is important to prevent the use of
cies were identified by biochemical tests and by the API
ineffective antimicrobial drugs. Table 1 reports MIC50
Staph System (BioMe´rieux, Rome, Italy) and were then
and MIC90 values of the selected antibiotics against the
stored at −70°C in a nutrient broth enriched with
isolates examined in the present study.
All values obtained with the control strain were within
In the present study, the antibiotics were selected
the expected ranges for all antimicrobial agents tested.
by considering the approved and most frequently used
Of the 68 isolates evaluated, none were susceptible to
drugs for the treatment of bovine IMI in Italy, even
all antibiotics and 64 (94%) were resistant to 3 or more
if these drugs were not representative of a particular
antimicrobial agents. No isolates were resistant to CLX.
antibiotic class. The antimicrobial agents selected and
-lactams (penicillins and cephalosporins) are
provided by manufacturers as powders were penicillin
widely used for intramammary treatment of bovine
), ampicillin (AMP
), amoxicillin (AMX
mastitis, but in the present experiment we observed a
+ clavulanate, cloxacillin (CLX
), cephalonium (CFL
very poor activity of PEN (MIC50 = 0.5 and MIC90 =
and cefoperazone (CFP
), kanamycin, oxytetracycline,
2,000 g/mL). The MIC90 for PEN reported in previous
doxycycline, and lincomycin (LIN
). The antimicrobial
studies ranged from <0.06 to >100 g/mL (De Oliveira et
agents were dissolved in suitable solvents to make stock
al., 2000; Erskine et al., 2004). In our study, 47 isolates
solutions and then diluted in sterile distilled water ac-
(69%) of Staph. aureus
were PEN resistant. This pro-
cording to the methods recommended by the Clinical
portion was greater than those reported for comparable
Laboratory Standards Institute (2002). Minimum in-
studies in Argentina (40%; Gentilini et al., 2000), the
hibitory concentration tests were performed according
United States (38.4 to 60.9%; Erskine et al., 2002), and
to the microdilution broth method, as recommended by
Finland (50%; Myllys et al., 1998), but was lower than
the Clinical Laboratory Standards Institute (2002a),
that reported for strains isolated from mammary paren-
using U-bottomed 96-well microtiter plates. Serial 2-
chymas of slaughtered dairy cows in Brazil (75%; Costa
fold dilutions of the antimicrobial agents were prepared
et al., 2000). Similarly, in the penicillin group, AMP
starting from the stock solution of each drug. The dilu-
and AMX had very poor in vitro activity (MIC50 = 2 and
tion schemes differed according to the antimicrobial
4 g/mL, and MIC90 = 500 and 1,000 g/mL, respec-
agent. Inocula were prepared by diluting an overnight
tively), and 100 and 98.5% of the isolates were resistant
(16 to 18 h) Mueller–Hinton broth culture in buffered
to these respective antimicrobial agents. Results from
saline solution to a density of 0.5 on the McFarland
other studies for MIC90 for AMP differed remarkably
turbidity scale and finally diluting it again 40-fold be-
from our findings; they ranged from only 0.5 to 4 g/
fore testing. The MIC was defined as the lowest concen-
mL (Watts and Salmon, 1997; De Oliveira et al., 2000).
tration of the antimicrobial agent at which the bacterial
This high level of resistance was probably related to
growth was completely inhibited. A reference strain
the presence of strong β
-lactamase producers among
ATCC 29213) was inoculated as a con-
the tested staphylococcal isolates. The in vitro data
trol in each plate. The MIC data were summarized,
confirmed the influence of β
-lactamase production on
calculating the MIC values for which the isolates were
the microbial susceptibility to β
-lactams in general and
equal to or below 50 and 90% (MIC50 and MIC90, respec-
to PEN in particular. In fact, the difference between
tively), as well as the minimum and maximum MIC
MIC50 and MIC90 values, with reference to PEN, AMP,
values (range). Resistance and susceptibility, for most
and AMX, correlates very well with the identification
of the antimicrobial agents tested, were determined
of 28 β
-lactamase–producing isolates (58%). Recalcula-
according to Clinical Laboratory Standards Institute
tion of the MIC90 without these strains yielded values
(2002a) MIC breakpoints for veterinary pathogens. The
of 0.5 g/mL for PEN and 4 g/mL for both AMP and
interpretive criteria, however, were based on MIC data
AMX. On the other hand, the β
and drug pharmacokinetic data obtained in humans
CLX and amoxicillin + clavulanate (a widely used β
(taken from Clinical Laboratory Standards Institute,
lactamase inhibitor) were both highly effective, with
2002b). Staphylococcus aureus
was also tested for β
MIC50 of 0.25 and 1 to 0.5 g/mL and MIC90 of 0.5 and
lactamase production by the nitrocefin test (Cefinase,
Becton, Dickinson and Co., Sparks, MD).
The cephalosporins are usually classified into 3 differ-
Numerous data are available in the literature on the
ent generations on the basis of their respective antimi-
susceptibility to antimicrobial agents of Staph. aureus
crobial spectra. In the present study, CFL and CFP
Journal of Dairy Science Vol. 89 No. 8, 2006
SHORT COMMUNICATION: DRUG SUSCEPTIBILITY OF STAPH. AUREUS
Antimicrobial susceptibility of 68 isolates of Staphylococcus aureus
collected from different animals
throughout the lactation1
1MIC50 and MIC90 are the minimum concentrations of the various antimicrobial agents required to inhibit
growth of 50 and 90% of the isolates tested, respectively. PEN = benzylpenicillin; AMP = ampicillin; AMX =amoxicillin; AMC = AMX + clavulanate; CLX = cloxacillin; KAN = kanamycin; OXT = oxytetracycline; DOX =doxycycline; CFP = cephoperazone; CFL = cephalonium; and LIN = lincomycin; NA = not available.
2Interpretive criteria based on human data.
were included as first-generation (good to excellent ac-
broad-spectrum antibiotics widely used to treat respira-
tivity against gram-positive bacteria but strain-depen-
tory and other diseases in cattle. Because of this wide-
dent gram negative activity) and third-generation
spread use, tetracycline and aminoglycoside resistance,
drugs (good to moderate activity against gram-positive
coded by a wide variety of determinants, was demon-
bacteria and good to excellent gram negative activity),
strated by the high MIC90 observed in the present study.
respectively. Cefoperazone and CFL showed antistaph-
Several factors other than antimicrobial usage can
ylococcal activity greater than that of the β
influence the overall susceptibility patterns of mastitis
sensitive penicillins. Between these 2 drugs, CFL
pathogens. Scar tissue in the udders of cattle chroni-
showed greater efficacy than did CFP (MIC50 = 0.12 vs.
cally infected by Staph. aureus
often prevents the pene-
2 g/mL and MIC90 = 2 vs. 16 g/mL, respectively).
tration of antimicrobial agents (De Oliveira et al., 2000).
These results may indicate that these agents are resis-
Therefore, the general recommendation is to cull all
tant to β
-lactamase, which hydrolyzes penicillins. Lin-
animals with chronic Staph. aureus
IMI. The control
comycin, oxytetracycline, doxycycline, and kanamycin
of IMI sustained by Staph. aureus
should involve the
(selected as representative drugs of the lincosamide,
best management practices and selective antimicrobial
tetracycline, and aminoglycoside groups, respectively)
usage. Unfortunately, most antimicrobial agents used
had moderate to poor activity against the Staph. aureus
in veterinary medicine still rely on interpretive criteria
isolates tested in the present study, as demonstrated
developed for humans, and the validity of these inter-
by MIC values ranging from 1 to >500, 1 to >500, 0.5
pretive criteria for categorizing veterinary pathogens
to 250, and 2 to 250 g/mL, respectively.
as susceptible or resistant has not been established
The lincosamide antimicrobial agents (e.g., LIN and
(Watts and Yancey, 1994). Currently, only pirlimycin
clindamycin) act by inhibiting RNA-dependent bacte-
and a penicillin–novobiocin combination have had in-
rial protein synthesis (Yao and Moellering, 1995). Lin-
terpretive criteria developed using MIC data generated
comycin showed an MIC90 of 250 g/mL, and this value
with mastitis pathogens. Interpretation of antimicro-
was greater than those previously found in other coun-
bial susceptibility data for the remaining compounds
tries. For example, LIN MIC90 ranged from 16.0 to 64
relies on interpretive criteria developed with human
g/mL for isolates from the United States, Ireland, Ice- data. The interpretive criteria used for categorizing iso-land, and Germany and from 1.0 to 8.0 g/mL for iso-
lates as susceptible or resistant are based on human
lates from Denmark, England, Norway, Sweden, and
data for most of the drugs tested in this study. Thus,
Finland (De Oliveira et al., 2000). The LIN MIC90 value
the usefulness of susceptibility data is limited to moni-
obtained for the strains tested in this study may be
toring the percentage of Staph. aureus
with MIC above
linked with their carriage of the erm
gene, which en-
a threshold value, and these values may not be used to
codes resistance to lincosamides, macrolides, and strep-
predict clinical efficacy. The percentage of resistance
togramine B antimicrobial agents (Leclercq and Cour-
data presented in this study was used for comparative
valin, 1991). Tetracyclines and aminoglycosides are
purposes but not as an indicator of the actual resistance
Journal of Dairy Science Vol. 89 No. 8, 2006
level. The experimental tests performed showed im-
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