Bull Vet Inst Pulawy 53, 401-405, 2009 USEFULNESS OF THE E-TEST FOR THE DETERMINATION OF THE SUSCEPTIBILITY OF STAPHYLOCOCCUS SP. ISOLATED FROM MILK OF SHEEP AND GOATS WITH SUBCLINICAL MASTITIS TO AMIKACIN AND AMOXICILLIN-CLAVULANIC ACID EKREM KIRECCI, YASAR ERGUN 1, GOKHAN DOGRUER 1, AND MUSTAFA KEMAL SARIBAY 1 Department of Microbiology, Faculty of Veterinary Medicine, Ataturk University, 25700, Erzurum, Turkey 1 Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, Mustafa Kemal University, 31040, Hatay, Turkey ekremkirecci@gmail.com Received for publication February 03, 2009 Abstract The aim of the study was to evaluate the Epsilometer-test (E-test) method to detect the susceptibility to amikacin and amoxicillin-clavulanic acid of 100 Staphylococcus species isolated from sheep and goats with subclinical mastitis. Of all the strains isolated, 24 were identified as Staphylococcus epidermidis, 17 as S. intermedius, 12 as S. xylosus, ten as S. warneri, nine as S. saprophyticus, nine as S. capitis, five as S. simulans, five as S. haemolyticus, three as S. sciuri, two as S. auricularis, two as S. hyicus, one as S. cohnii, and one as S. caprae. All isolates were sensitive to both antibiotics. While the MIC values of amoxicillin-clavulanic acid for the goat strains ranged from 0.125 to 1 µg/ml and of amikacin from 0.25 to 12 µg/ml, the MIC values for the sheep strains ranged from 0.047 to 2 µg/ml (amoxicillin-clavulanic acid) and 0.75 to 6 µg/ml (amikacin). The results of this study have shown that both antibiotics can be highly effective against Staphylococcus sp. that are often the reason for subclinical mastitis in small ruminants. Key words: sheep, goat, mastitis, milk, Staphylococcus, antibiotic resistance, E-test. Awassi sheep and domestic goats have been bred in Middle Eastern countries, including Jordan, Iraq, Syria, Lebanon, Palestine, and Israel, and, in particular, in the Hatay region of Southeast Anatolia, for centuries and are highly regarded for their milk, meat, wool, and skins. In these regions, sheep and goat milk is used extensively in the production of milk products, such as cheese and ice cream. Worldwide, mastitis is still one of the most important diseases in the dairy industry. Staphylococci are the most prevalent and widespread species isolated from milk samples from subclinical mastitis in goats and sheep (2, 6, 8). In subclinical mastitis caused by Staphylococcus sp., the udder and milk may show no macroscopic changes, although somatic cell increases and pathogenic microorganisms can be detected. Infection spreads readily between animals, threatening both the animals health and milk production. When optimum conditions are created, Staphylococcus first colonises the udder lactiterous ducts, and then builds up infection in the milk producing tissues. In subclinical mastitis, symptoms of disease remain hidden (7, 22). After determining the changes in the milk with special screening tests, such as the California Mastitis Test (CMT), identifying the microbial species and antimicrobial sensitivity and applying suitable antibiotics form the basis of mastitis diagnosis and treatment. In fighting back against mastitis, as well as anti-microbial therapy, executing mastitis control programs such as minimal stress, optimal nutrition, and herd management are mandatory. As a result of indiscriminate utilisation of antibiotics, resistant strains have evolved, and treatment has become difficult. Therefore, before starting mastitis treatment, sensitivity patterns should be specified by, at the very least, performing antimicrobial susceptibility. For this purpose, various susceptibility tests are used today. Of these tests, agar disk diffusion, agar and broth dilution (micro-or macro-dilution), E-test, and automated systems such as Vitek, MicroScan, and Sensititer have been widely applied (11, 12, 19, 24). The preference and utilisation of these tests in routine laboratories varies with the aim, technical capabilities, and economic conditions of the laboratory. Among these tests, the E- test (AB Biodisk, Solna, Sweden) was first introduced at an Antimicrobial Agents and Chemotherapy Conference in 1988 (11). The E-test is a method for obtaining a
402 minimal inhibitory concentration (MIC) result. The basis of the E-test is a strip impregnated with increasing concentrations of an antibiotic; therefore, each strip includes a concentration gradient of a specific antibiotic rather than a single concentration of that antibiotic. The MIC is determined directly from the scale of markings on the top surface of the strip. The E-test has been reported to be a simple and accurate alternative method for testing the antimicrobial susceptibilities of different microorganisms, including fastidious bacteria. Another advantage of this method is the speed of preparation, and its sensitivity for testing single isolates. However, a disadvantage of the E-test is that it is more expensive than other methods, such as agar disk diffusion and agar or broth dilution (4, 9, 10). The aim of this study was to use the E-test method to detect the susceptibility of Staphylococcus species isolated from sheep and goats with subclinical mastitis to amikacin and amoxicillin-clavulanic acid. Material and Methods Bacterial isolates. A total of 100 Staphylococcus strains isolated from milk samples of awassi dairy sheep and domestic goats suffering from subclinical mastitis were used. The milk samples were obtained from lactating sheep and goats between February 2006 and December 2007 from farms in the Hatay district. All lactating sheep and goats in the farms were screened for subclinical mastitis using CMT as described previously (23), and midstream milk samples showed a positive test result (10 ml) were collected from each side of the udder before morning milking. The teats were sterilised with 70% alcohol before milk collection, and samples were transported chilled for microbiological tests within 4 h of collection, and were processed within 1 h. The presence of Staphylococcus strains was determined by culturing 0.01 ml of each sample on 5% sheep blood agar and Staphylococcus medium plates incubated 24-36 h at 37 C. The microorganisms growing in these media were examined in terms of colony structure, Gram staining, microscopic appearance, protein-a existence, tube coagulase, and other biochemical specifications, and were diagnosed as Staphylococcus sp. and, additionally, were confirmed by API STAPH (Biomérieux, SA, France) (13, 14). Antimicrobial agents. E-test strips containing amoxicillin-clavulanic acid (XL) and amikacin (AK) were purchased from AB Biodisk. XL and AK MICs were determined by E-test according to the manufacturer s guidelines (AB BIODISK, Solna, Sweden) and the MIC range of the strips was 0.016 256 µg/ml. Antimicrobial susceptibility testing. Antimicrobial susceptibility of the isolates was determined by E-test, which was conducted according to the manufacturer s instructions on Mueller Hinton agar plates (Oxoid). Bacterial suspensions were prepared from a fresh culture and adjusted to MacFarland No. 0.5 turbidity standard. The E-test strips were placed onto the surface of inoculated Mueller Hinton agar plates (150 mm diameter) and the plates were incubated for 24 h at 37 C. After incubation, the MIC value was read directly from the scale printed on the strips at the point where the zone of growth inhibition intersected with the graduated strip. In terms of MIC interpretation, the CLSI (CLSI, 2006), (5), recommends susceptibility breakpoints for XL (susceptibility (S) 4 µg/ml, I:-, resistance (R) 8 µg/ml) and AK (S 16 µg/ml, I =32 µg/ml, R 64 µg/ml) and these breakpoints are valid for all Staphylococcus sp. The findings were evaluated as percentages among the isolates and the antimicrobial susceptibility. Results Identification of isolates. With the API STAPH system, out of all strains, 24 were identified as S. epidermidis, 17 as S. intermedius, 12 as S. xylosus, 10 as S. warneri, nine were identified as S. saprophyticus, nine as S. capitis, five as S. simulans, five as S. haemolyticus, three as S. sciuri, two as S. auricularis, two as S. hyicus, one as S. caprae, and one as S. cohnii. The distribution of isolates according to species of animals is summarised in table 1. The most commonly isolated species in sheep was S. epidermidis, and in goats S. intermedius. Antibiotic susceptibility of isolates. While the MIC of XL against goat isolates has been found to be 0.125-1 µg/ml and amikacin to be 0.25-12 µg/ml, the XL MIC values against the sheep strains ranged between 0.047 and 2 µg/ml and AK ranged from 0.75-6 µg/ml. When the obtained MIC values were evaluated according to the CLSI MIC Criteria, it was seen that all Staphylococcus strains were susceptible to AK and XL. The MIC ranges and the percentage of the susceptibility of the strains are summarised in Tables 2 and 3. E-test results of some Staphylococcus sp. susceptible to AK and XL are shown in Figs 1 and 2. Table 1 Distribution of identified Staphylococcus strains Number of isolates Strains sheep goat S.epidermidis 20 4 S.intermedius 5 12 S.xylosus 7 5 S.warneri 6 4 S.saprophyticus 8 1 S.capitis 2 7 S.simulans 1 4 S.haemolyticus - 5 S.sciuri - 3 S auricularis - 2 S.hyicus - 2 S.caprae - 1 S.cohnii 1 - Total 50 50
403 Table 2 In vitro activity of AK against 100 staphylococci strains (sheep and goat isolates) using E-test Organism (number of isolates) of isolates of sheep % S a of isolates of goat % S a S.epidermidis (24) 0.75-4 100 1-1.5 100 S.intermedius (17) 1.5-6 100 0.38-8 100 S.xylosus (12) 1.5-4 100 0.38-3 100 S.warneri (10) 1.5-3 100 1.5-4 100 S.saprophyticus (9) 0.75-4 100 12 100 S.capitis (9) 2 100 0.25-2 100 S.simulans (5) 2 100 1-2 100 S.haemolyticus (5) - - 0.75-2 100 S.sciuri (3) - - 0.25-4 100 S auricularis (2) - - 2-3 100 S.hyicus (2) - - 1.5 100 S.caprae (1) - - 1 100 S.cohnii (1) 3 100 - - Total 100 0.75-6 100 0.25-12 100 a.percentage of amikacin-susceptible strains. b.ak - amikacin c.breakpoints used for Staphylococcus sp. isolates: susceptible (S),intermediate (I), resistant (R) CLSI MIC Criteria for AK b S c 16, I =32, R 64 Table 3 In vitro activity of XL against 100 staphylococci strains (sheep and goat isolates) using E-test Organism (number of isolates) of isolates of sheep % S a of isolates of goat % S a S.epidermidis (24) 0.047-2 100 0.25-0.50 100 S.intermedius (17) 0.38-1.5 100 0.094-0.75 100 S.xylosus (12) 0.094-0.75 100 0.094-1 100 S.warneri (10) 0.094-1.5 100 0.094-0.25 100 S.saprophyticus (9) 0.047-1 100 0.125 100 S.capitis (9) 0.50-1 100 0.094-0.38 100 S.simulans (5) 0.50 100 0.032-0.75 100 S.haemolyticus (5) - - 0.19-0.38 100 S.sciuri (3) - - 0.094-0.75 100 S auricularis (2) - - 0.19-0.25 100 S.hyicus (2) - - 0.19 100 S.caprae (1) - - 0.25 100 S.cohnii (1) 0.75 100 - - Total 100 0.047-2 100 0.125-1 100 a.percentage of XL-susceptible strains. b.xl - amoxicillin/clavulanic acid c.breakpoints used for Staphylococcus sp. isolates: susceptible (S),intermediate (I), resistant (R) CLSI MIC Criteria for XL b S c 4, I -, R 8
404 Fig. 1. E-test of a Staphylococcus sp. that is sensitive to AK. Fig. 2. E-test of a Staphylococcus sp. that is sensitive to XL. Discussion Antimicrobial therapy is critically important in the control of mastitis in sheep and goats. For a successful treatment of clinical and subclinical mastitis, it is important not only to isolate the causative microorganisms and their antibiotic susceptibility, but also to provide suitable flock management, hygiene conditions, and use of teat-dips. Nonetheless, pretreatment identification of bacteria and their antimicrobial susceptibility are usually not applicable in terms of time and costs. For this reason, in a stock farming region, empirical drug treatments according to anticipatory prevalence and susceptibility study results have been applied, and antibiotic susceptibility data feature strongly in the success of the treatment. In Turkey and the Middle East, within the antibiotic susceptibility tests, the Kirby-Bauer disk diffusion method has been most frequently used. While this method has important advantages, such as being practical, easy, and having a low cost, it cannot determine MIC values. In determining MIC, methods such as agar dilution, micro-dilution broth and E-test are the most commonly used (16, 21). Knowing MIC values of an antibiotic that will be used in mastitis therapy is of high importance for anticipating the suitable therapeutic concentration of this antibiotic, and for assessing the antibiotic susceptibility trend of pathogens. All over the world, with Staphylococcus mastitis at a high rate as a form of bacterial mastitis, the effective treatment and prevention of mastitis in sheep, goat, and dairy cows is critically important (7, 17). In previous studies, it has been reported that coagulase negative staphylococci (CNS) are the most frequent microorganisms in chronic and subclinical intramammary infections (IMI) of goats and sheep; this ratio in goats may vary from 25% to 93% (1, 3, 19, 24). When McDougall et al. (17) in Vermont, USA, investigating the prevalence and incidence of subclinical mastitis in goats and sheep, determined that CNS were the most common isolates in both species. Moroni et al. (18) evaluated the antibiotic susceptibility of 70 strains of CNS isolated from the milk of dairy goats with the micro-dilution broth method, and found Staphylococcus strains to consist of S. epidermidis, S. caprae, S. lugdunensis, S. kloosii, S. simulans, S. warneri, and S. chromogenes. In the same study, amoxycillin-clavulanic acid was found to be more active against S. epidermidis, S. caprae, and S. lugdunensis species. In Israeli dairy goats, it has been reported that CNS are the main pathogen microorganisms in subclinical IMI (15). In the study, the CNS ratio (goat - 65.4%, sheep - 76.5%) in goats and sheep diagnosed with bacteriological analysis and subclinical mastitis was found to be very similar to those reported in other studies. In goat strains, S. intermedius was the most common, followed by S. capitis. In a study performed in two commercial dairy goat farms in Italy, it was discovered that 80.7% of subclinical mastitis cases developed at the start of lactation and were due to CNS species. In one herd, the infective organism was found to be mostly S. caprae (43% of infections), while in the another herd, it was mostly S. epidermidis (48% of infections) (19). In the same study, the MIC against S. caprae was found to range from 0.12 to 7.8 µg/ml, with the micro-dilution broth method, while the MIC range for S. epidermidis was found to be from 0.0015 to 1.95 µg/ml. All goat strains were susceptible to XL, with a MIC range of 0.125-1 µg/ml. In sheep, the most common CNS strain was S. epidermidis, and all Staphylococcus sp. strains were susceptible to AK and XL (MIC 0.75-6 µg/ml, 0.047-2 µg/ml, respectively). In South-Eastern Bulgaria (3), resistance of 96 Staphylococcus sp. isolated from goats with subclinical mastitis has been studied against various antibiotics with the disk diffusion method, with a determination of 83.3% sensitivity to amoxycillinclavulanic acid, and 100% sensitivity to amikacin. Pengov and Ceru (21) investigated the antimicrobial drug susceptibility of S. aureus strains isolated from bovine and ovine mammary glands with the E-test method. The antimicrobial susceptibility of cats Bordetella bronchiseptica was researched by the E-
405 test and agar dilution methods, and it was found that the use of the test was as quick and as simple as the disk diffusion tests, and was also a quantitative indicator of antimicrobial susceptibility (25). In another study, the antimicrobial susceptibility testing of Campylobacter jejuni and Campylobacter coli strains isolated from poultry was determined by the E-test and disk diffusion methods, and it was noted that the E-test was a simple method (20). The E-test in this study has not been compared to other susceptibility tests, but it was found to be a practical and easy method in terms of utilisation and evaluation in a routine laboratory to identify the MIC and drug susceptibility of isolates. In most of the studies throughout the world, the E-test is utilised successfully in the susceptibility testing of broad groups of microbial pathogens, including aerobes, anaerobes, fungi, and Mycobacterium sp. (10, 11). 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