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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 is The antimicrobial susceptibility of 68 Staphylococcus variable and the probability of curing the disease is not aureus 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-lactams, penicillin 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-lactamase–re- 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, whereas the crobial susceptibility tests can guide the veterinarian other β-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. However, despite 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 mastitis, particularly 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, bovine, minimum
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 iso- 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: 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 Staph. aureus 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 The β-lactams (penicillins and cephalosporins) are provided by manufacturers as powders were penicillin widely used for intramammary treatment of bovine G (PEN), ampicillin (AMP), amoxicillin (AMX), 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 (Staph. aureus, 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-lactamase–resistant 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 Table 1. 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.
3Amoxicillin/clavulanate concentrations.
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-lactamase– 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- aureus isolated from bovine mastitis in Argentina. J. Dairy Sci.
portant in vitro activity against the Staph. aureus iso- Gruet, P., P. Maincent, X. Berthelot, and V. Kaltsatos. 2001. Bovine lates of the majority of antimicrobial agents currently mastitis and intramammary drug delivery: Review and perspec- used in Italy for control of IMI. However, we consider tives. Adv. Drug Deliv. Rev. 50:245–259.
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