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Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA), Faculdade de Medicina Veterinária, Av. Universidade Técnica, 1300-477 Lisbon, Portugal
1 Corresponding author: amsandronunes{at}fmv.utl.pt
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ABSTRACT |
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64, 8, 1, 32, 64, 64, and 0.06 µg/mL for Staph. epidermidis. All Staph. aureus isolates showed susceptibility to oxacillin, cefazolin, gentamicin, sulphamethoxazole/trimethoprim, and enrofloxacin. ß-Lactamase production was detected in 20 of these isolates (66.7%), all of which were resistant to penicillin. Of the 31 Staph. epidermidis tested, 24 (77.4%) were ß-lactamase positive. All isolates were susceptible to both cefazolin and enrofloxacin. Nine Staph. epidermidis isolates were resistant to oxacillin, with MIC values ranging from 4 to 8 µg/mL. The MIC values of 5 antimicrobial agents tested were higher than those reported in other countries. Enrofloxacin was the only exception, showing lower MIC values compared with other reports. Overall, the antimicrobial agents tested in our study, with the exception of penicillin, were active against the 61 isolates studied.
Key Words: bovine mastitis • antimicrobial susceptibility • Staphylococcus aureus • Staphylococcus epidermidis
Bovine mastitis remains the most costly disease affecting dairy cows and milk production worldwide. Microrganisms such as Staphylococcus aureus, Streptococcus agalactiae, and Streptococcus dysgalactiae are considered major pathogens, due to their prevalence in dairy herds, contagious nature of infection, and economic impact of treatment and control. Minor pathogens include CNS such as Staphylococcus epidermidis, Staphylococcus xylosus, and Staphylococcus chromogenes and are considered to infect the mammary gland from an environmental source. The CNS are the most frequently isolated bacteria from mastitic milk, more frequent in samples from first lactation and from un-bred dairy heifers. The CNS have been implicated in a large proportion of dry period and early lactation mastitis episodes (Rajala-Schultz et al., 2004). They may be responsible for both clinical and subclinical disease even though their role as subclinical pathogens is more often emphasized. Whenever preventive measures fail, antimicrobial therapy is a useful tool for the control of clinical, subclinical, and recurrent IMI in both lactating and dry dairy cows. Information on the prevalence and antimicrobial susceptibility of mastitis pathogens becomes crucial for a successful and rational mastitis control program. In the central region of Portugal, Staph. aureus and Staph. epidermidis have been identified as 2 of the most prevalent pathogens associated with subclinical mastitis in dairy cows (Bexiga et al., 2005). Until now, mastitis-specific susceptibility breakpoints were available only for pirlimycin and the penicillin/novobiocin combination. Updated information on MIC values for mastitis pathogens from different origins is continuously required to validate the clinical relevance of in vitro susceptibility testing results of these pathogens.
The aim of the present study was to determine the MIC of 7 antimicrobial drugs commonly used in mastitis control of subclinical mastitis isolates of Staph. aureus and Staph. epidermidis in Portugal. Thirty Staph. aureus and 31 Staph. epidermidis isolated from bovine intramammary subclinical infections, detected by the California Mastitis Test, were used in this study. Isolates were obtained from quarter milk samples from 7 commercial dairy farms of the Ribatejo-Oeste region of Portugal (Bexiga et al., 2005). Sample collection and bacterial isolation were performed according to the National Mastitis Council recommendations (NMC, 1987).
The MIC of penicillin, oxacillin, cefazolin, gentamicin, sulfamethoxazole/trimethoprim, oxytetracycline, and enrofloxacin were determined by broth microdilution, following the Clinical and Laboratory Standards Institute standards for veterinary susceptibility testing (CLSI, 2002). Antimicrobial agents were purchased as standard powders from Sigma, with exception of enrofloxacin, which was kindly supplied by Bayer Animal Health (Carbaxide, Portugal). Sulfamethoxazole and trimethoprim were tested in a 5:1 ratio (vol/vol) as available in commercial drugs for mastitis treatment.
Ten 2-fold dilutions of each drug were prepared in Mueller-Hinton broth (Oxoid, Basingstoke, UK) and distributed in 96-well microtitres plates (Nunc, Wiesbaden, Germany). Before testing, isolates were revived by plating twice on Columbia agar (bioMérieux, Marcy l’Etoile, France) and incubating at 37°C for 24 h. Isolated colonies were suspended in Mueller-Hinton broth, adjusted by optical density, distributed into wells to a final concentration of 5 x 105 cfu/mL, and incubated at 37°C for 22 ± 2 h under aerobic conditions. All isolates were tested at least twice in separate occasions. Reference strain Staph. aureus ATCC 29213 was used for quality control purposes. The dilution range and susceptibility breakpoints used, based on CLSI standards (document M31-A2; CLSI, 2002) are described in Table 1
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and 3. The MIC values for the quality control organism were within recommended ranges for all the antimicrobial agents tested; cephalothin ranges were used for cefazolin (CLSI, 2002)
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All of the Staph. aureus isolates tested were susceptible to oxacillin, cefazolin, gentamicin, sulphamethoxazole/trimethoprim, and enrofloxacin. Production of ß-lactamase was detected in 20 isolates (66.7%), all of which were resistant to penicillin. The isolate with the highest MIC value for penicillin (16 µg/mL) was also the only oxytetracycline-resistant strain. Of the 31 Staph. epidermidis isolates tested, 24 (77.4%) showed ß-lactamase production. All isolates were considered susceptible to cefazolin and enrofloxacin. High levels of resistance to penicillin, gentamicin, and oxytetracycline were found.
Penicillin susceptibility testing predicts the susceptibility to other ß-lactamase–sensitive antimicrobial drugs, such as ampicillin and amoxicillin. In the present study, levels of penicillin resistance were higher than in other countries. The MIC90 values of penicillin described for Staph. aureus isolated from bovine mastitis range from 
0.06 µg/mL in Norway to 4 µg/mL in New Zealand, the latter equal to the value found in our study. However, the most frequent MIC90 value reported in the literature is 0.5 µg/mL (Watts et al., 1995; Salmon et al., 1998; De Oliveira et al., 2000; Gentilini et al., 2000; Guerin-Faublee et al., 2003). The MIC values for penicillin for Staph. epidermidis are scarce, because CNS testing is seldom performed at the species level. Watts et al. (1995) reported a penicillin MIC90 of 0.5 µg/mL for 28 Staph. epidermidis isolates. For this species, our study showed a MIC90 64 µg/mL, a much higher value than that reported previously, even when all CNS are considered together (Owens et al., 1997; Salmon et al., 1998; Guerin-Faublee et al., 2003). The confirmation of ß-lactamase production in conjunction with susceptibility testing can provide useful information on penicillin resistance. The fact that none of the susceptible isolates to penicillin of both species were ß-lactamase producers indicate that the high levels of penicillin-resistance might be attributed to the production of these enzymes. In the present study, the percentage of isolates showing the ability to produce these enzymes is similar to that reported for isolates from Ireland and Iceland, respectively 63.6 and 76.2% (De Oliveira et al., 2000) and higher than the percentages found in Scandinavia, where antimicrobial drug use in humans and animals is carefully monitored and controlled (Myllys et al., 1998).
Although only cloxacillin is approved for intramammary administration in Portugal, oxacillin is the class representative recommended by CLSI for antimicrobial susceptibility testing and detection of methicillin resistance of staphylococcal strains. The MIC90 values obtained for Staph. aureus were similar to those found in other countries (Watts et al., 1995; De Oliveira et al., 2000; Gentilini et al., 2000, 2002; Guerin-Faublee et al., 2003). All 9 oxacillin-resistant isolates were also resistant to penicillin (MIC ranging from 2 to 64 µg/mL) and oxytetracycline (all with MIC 64 µg/mL), and they were ß-lactamase producers. Only one of these isolates showed susceptibility to gentamicin (MIC = 0.125 µg/mL). Subsequent testing revealed that all 9 isolates were mecA carriers (data not shown; Nunes et al., 2006). Methicillin resistance in mastitis pathogens is still uncommon. In a study carried out by Guerin-Faublee et al. (2003), only 1 out of 171 staphylococcal mastitis isolates was methicillin resistant. De Oliveira et al. (2000) reported that none of 12 isolates with MIC values corresponding to oxacillin resistance possessed the mecA gene and that the resistance might be associated with hyperproduction of ß-lactamase. Recently in Korea, higher levels of both methicillin resistance and mecA carriers were found in mastitis-related Staph. aureus and CNS (Moon et al., 2007). The presence of methicillin resistance in CNS isolates is of concern, because horizontal mecA gene transfer among different staphylococcal species has previously been reported (Archer et al., 1994).
In the present study, no resistance to cefazolin, a first-generation cephalosporin, was observed in any of the isolates of either species. The MIC90 was 1 µg/mL, higher than the value observed in studies in France and Japan (Yoshimura et al., 2002; Guerin-Faublee et al., 2003). However, according to CLSI recommendations, all methicillin-resistant strains, such as the 9 found in this study, should be considered also resistant to penicillins and cephalosporins (CLSI, 2002).
Gentamicin has limited use in farm animal antimicrobial therapy due to the long withdrawal period in milk and meat. In our study, gentamicin showed good activity against Staph. aureus, with a MIC90 of 1 µg/mL, higher than that reported in Japan (0.39 µg/mL) but lower than in Argentina with 1.5 µg/mL (Gentilini et al., 2000; Yoshimura et al., 2002).
Our results showed good activity for the association of sulfamethoxazole with trimethoprim against Staph. aureus with a MIC90 of 0.25 µg/mL. A greater number of resistant isolates was observed in Staph. epidermidis with MIC90 >64 µg/mL. However, the MIC distribution showed a wide range of susceptibility (0.06 to 8 µg/mL). This association was tested using a higher ratio than that recommended by the CLSI (19:1), which may be responsible for the high susceptibility levels observed in both species in this study.
In our study, no resistance to oxytetracycline was found in Staph. aureus, with a MIC90 of 0.25 µg/mL, which was lower than that reported by Yoshimura et al. (2002) with a value of 1.56 µg/mL. On the contrary, >80% of Staph. epidermidis isolates were resistant, with both MIC50 and MIC90 >64 µg/mL. The high levels of resistance of Staph. epidermidis to sulfamethoxazole/trimethoprim and oxytetracycline could be due to the extended use of these drugs in dairy cows. The CNS are found in the normal microbiota of human skin and the bovine udder and teat canal. It could be argued that the resistance levels observed may be related to a selective pressure whenever antimicrobial therapy is used, although human transmission of resistant isolates may also occur.
The enrofloxacin MIC90 values of Staph. aureus and Staph. epidermidis found in this study (both 0.06 µg/mL) are much lower than those reported in other regions (Watts et al., 1995; Owens et al., 1997; Salmon et al., 1998; De Oliveira et al., 2000; Yoshimura et al., 2002). These high susceptibility levels to enrofloxacin may be due to its infrequent use in cattle, in either intramammary or systemic administration, for treatment of mastitis in Portugal. Finally, when comparing our results on antimicrobial resistance with the published data, special attention should be given to the possible influence of previous exposure to drugs. Most of our isolates were obtained from mature cows, but some heifers presented subclinical mastitis and these isolates were also included in this study. Therefore, previous mastitis episodes or preventive treatments may have influenced the MIC values registered.
Overall, with the exception of penicillin, the antimicrobial agents tested in our study demonstrated good activity against the 61 isolates studied. The MIC values of the antimicrobial agents tested were, in general, higher than those reported in other countries, except for enrofloxacin, which showed the lowest MIC values. The major differences observed in the resistance traits of the 2 species tested emphasize the importance of the bacterial identification of the pathogens in mastitis control. Among CNS, a minor mastitis pathogen, special attention should be given to the Staph. epidermidis, due to the high incidence of resistance found in general and in particular to methicillin resistance. The high number of ß-lactamase–producing isolates found in our study suggests that the administration of ß-lactams, especially penicillin and related drugs, should be carefully considered for mastitis control in the Ribatejo-Oeste region.
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ACKNOWLEDGEMENTS |
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Received for publication November 7, 2006. Accepted for publication March 19, 2007.
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