Antimicrobial Resistance of Enterococci Isolated from Mastitic Bovine Milk Samples in Korea

Zoonoses and Public Health SHORT COMMUNICATION Antimicrobial Resistance of Enterococci Isolated from Mastitic Bovine Milk Samples in Korea H. M. Nam, S. K. Lim, J. S. Moon, H. M. Kang, J. M. Kim, K. C. Jang, J. M. Kim, M. I. Kang, Y. S. Joo and S. C. Jung National Veterinary Research and Quarantine Service, Anyang City, Gyeonggi-do, Korea Impacts The first article about antimicrobial resistance in enterococci isolated from bovine mastitic milk samples in Korea. Information on the antimicrobial resistance of enterococcal species from bovine milk may be useful for assessing the scale of the resistance problem and the needs for monitoring resistance rates in bovine isolates. In this study, enterococcal isolates belonging to minor species showed antimicrobial resistance rates as high as those of E. faecium and E. faecalis. Keywords: Enterococci; antimicrobial resistance; milk; mastitis Correspondence: H.-M. Nam. Bacteriology and Parasitology Division, National Veterinary Research and Quarantine Service, 48, Anyang 6-dong, Anyang, Gyeonggi-do, 43-824, Republic of Korea. Tel.: +82-31-467-1772; Fax: +82-31-467-1778; E-mail: namhm@nvrqs.go.kr Received for publication April 1, 9 doi: 1.1111/j.1863-2378.9.137.x Summary The aim of this study was to determine the antimicrobial resistance of various species of enterococci isolated from mastitic bovine milk samples. A total of 15 enterococci isolates were examined: Enterococcus faecalis (n = 47), Enterococcus faecium (n = 39), Enterococcus gallinarum (n = 6), Enterococcus avium (n = 6), Enterococcus hirae (n = 5) and Enterococcus durans (n = 2). All the isolates were susceptible to ampicillin, gentamicin and vancomycin, and only a single E. hirae isolate was resistant to ampicillin. In general, the most frequently observed resistance among the enterococcal isolates was to tetracycline (69.5%), followed by penicillin (64.7%), erythromycin (57.1%) and cephalothin (44.7%). A similar antimicrobial resistance pattern was observed among individual species except E. durans, which exhibited only tetracycline resistance. Resistance observed among isolates of E. hirae and E. gallinarum was almost as high as E. faecium and E. faecalis. Of 15 isolates, only six (5.7%) strains of E. faecium were susceptible to all the antimicrobials tested and about 52% (55/ 15) showed resistance to more than three antimicrobials. The most common multiple resistance pattern was penicillin, tetracycline and erythromycin, which was observed in 32 of 15 (3.4%) isolates. This study demonstrates that enterococcal isolates belonging to minor species showed antimicrobial resistance rates as high as those of E. faecium and E. faecalis, and that monitoring of antimicrobial resistance should not be restricted only to those two major species. Introduction Enterococci, one of the leading causes of nosocomial infections, pose a significant threat to public health with their intrinsic resistance to many broad-spectrum antimicrobials (Hayes et al., 4). While Enterococcus faecalis and Enterococcus faecium account for up to 9% of clinical isolates, the incidence of infection caused by minor enterococcal species shows an alarming increase (Prakash et al., 5). Considerable diversity of species was also observed in the enterococcal strains isolated from animals (Jung et al., 7), suggesting that monitoring of resistance should not be restricted to E. faecalis and E. faecium alone. Moreover, the emergence of multidrug resistance among the minor species of enterococci highlights the importance of precise identification of enterococci to ª 9 Blackwell Verlag GmbH Zoonoses Public Health. 1

Antimicrobial Resistance of Enterococci Isolated from Bovine Mastitis H. M. Nam et al. species level for characterizing the species-specific antimicrobial resistance (Prakash et al., 5). Enterococci can also cause many economically important animal diseases including bovine mastitis (Devriese et al., 1992). While data are sparse, the incidence of enterococcus as aetiological agents of bovine mastitis varied from (Østerås et al., 6) to 21.2% (Bradley et al., 7). Enterococcus spp. are used as indicator bacteria for the development of antimicrobial resistance (Danish Integrated Antimicrobial Resistance Monitoring and Research Programme (DANMAP), 3), and provide accurate information on previous antibiotic treatment of the animals (Centers for Disease Control and Prevention (CDC), 2). As bovine mastitis is the single most common cause for antimicrobial use in dairy farms, knowledge of resistance profiles of enterococci isolates from mastitic milk samples would be useful for assessing the scale of the resistance problem and monitoring changes in resistance rates of dairy enterococci isolates. However, little information is available on enterococcal pathogens isolated from milk samples, and if any, most studies focus on the two major species, E. faecalis and E. faecium (Pitkälä et al., 4). The aim of this study was, therefore, to examine the antimicrobial resistance of various species of enterococci isolated from mastitic bovine milk samples taken from dairy cattle during 4 8 in Korea. Materials and Methods Bacterial isolates During January 4 to October 8, a total of 12 58 quarter milk samples from lactating cows on 435 dairy herds nationwide were examined in the Mastitis Diagnostic Laboratory of national veterinary research and quarantine service (NVRQS) in Korea. Cows that were being treated with antimicrobials were excluded and milk samples were aseptically collected from individual mammary quarters by the herd owner or personnel from NVRQS. Bacterial examination of 2185 samples with somatic cell count of cells/ml, an indicator of udder infection, was conducted using standard laboratory techniques (National Mastitis Council Inc., 199): 1 ll of milk was streaked onto 5% blood agar plates (KOMED, Seoul, Korea) and incubated at 35 C to 37 C for 48 h. All organisms presumptively identified as enterococci by colony morphology and Gram stain were identified to the species level using the VITEK system (biomérieux, Hazelwood, MO, USA) as described by the manufacturer. Antimicrobial susceptibility test In vitro antimicrobial susceptibility testing was conducted using the disc diffusion method, according to the recommendations of the National Committee for Clinical Laboratory Standards (National Committee for Clinical Laboratory Standards (NCCLS), 2). For susceptibility testing in Enterococci spp., commercially prepared antimicrobial sensitivity discs (Becton-Dickinson, Cockeysville, MD, USA) having the following seven antimicrobial agents and concentrations were used: gentamicin (1 lg/disk), ampicillin (1 lg/disk), penicillin (1 U/disk), erythromycin (15 lg/disk), vancomycin (3 lg/disk), cephalothin (3 lg/disk) and tetracycline (3 lg/disk). Isolates were categorized as susceptible, intermediate and resistant based upon interpretive criteria developed by the National Committee for Clinical Laboratory Standards (2). Enterococusc faecalis ATCC 29212 and Staphylococcus aureus ATCC 25923 were used as quality control organisms. Test results were accepted only when the zone of inhibition for the control strains fell within the acceptable ranges. Results A total of 15 enterococci were isolated and examined for antimicrobial susceptibilities: E. faecalis (n = 47), E. faecium (n = 39), E. gallinarum (n = 6), E. avium (n = 6), E. hirae (n = 5) and E. durans (n = 2). The in vitro antimicrobial resistance profiles of all enterococcal isolates against seven antimicrobials are summarized in Fig. 1. Overall, ampicillin, gentamicin and vancomycin were effective against enterococci, while the most frequently observed resistance was to tetracycline, followed by penicillin, erythromycin and cephalothin. Table 1 summarizes the antimicrobial resistance profiles of the six enterococci species identified in this study. Although some fluctuations were noted among individual species, they generally share a similar antimicrobial resistance pattern except E. durans that exhibited only tetracycline resistance. Resistance observed among isolates of E. hirae and E. gallinarum was almost as high as E. faecium and E. faecalis. Percentage 1 8 6 Resistance of Enterococcus spp. (n = 15) Intermediate Resistant AM CF P TE E VA GM* Antimicrobials Fig. 1. Antimicrobial resistance of enterococcal isolates from mastitis milk samples from dairy cattle in Korea. AM: Ampicillin; CF: Cephalothin; P: Penicillin; TE: Tetracycline; E: Erythromycin; VA: Vancomycin; GM: Gentamycin. *Low-level gentamicin resistance (1 lg/disk). 2 ª 9 Blackwell Verlag GmbH Zoonoses Public Health.

H. M. Nam et al. Antimicrobial Resistance of Enterococci Isolated from Bovine Mastitis Table 1. Antimicrobial resistance profiles of the six enterococcal species isolated from bovine milk in Korea (n = 15) No. intermediate (I) and resistant (R) isolates E. fecalis (N = 47) E. faecium (N = 39) E. avium (N =6) E. gallinarum (N =6) E. hirae (N =5) E. durans (N =2) Antimicrobials I R I R I R I R I R I R Ampicillin 1 Cephalothin 8 26 6 16 1 2 2 1 Penicillin 31 24 3 5 5 Tetracycline 1 39 5 4 3 2 Erythromycin 16 3 3 22 1 1 1 4 3 Vancomycin 1 5 2 Gentamycin* 5 12 17 1 2 3 *Low-level gentamicin resistance. E. fecalis (n = 47) E. faecium (n = 39) Other enterococcus spp. (n = 19) Percentage of resistant strains 3 1 1 2 3 4 5 6 7 Number of antimicrobials Percentage of resistant strains 3 1 1 2 3 4 5 6 7 Number of antimicrobials Percentage of resistant strains 3 1 1 2 3 4 5 6 7 Number of antimicrobials Fig. 2. Percentage of enterococcal isolates that showed resistance to the number of antimicrobials tested. Figure 2 presents the percentage of enterococcal isolates showing resistance against the number of antimicrobials tested by species. Of 15 isolates, only six (5.7%) strains of E. faecium were susceptible to all the antimicrobials tested in this study. About 52% (55/15) of the isolates showed resistance to more than three antimicrobials, and Table 2. Resistance patterns most frequently observed among the enterococcal isolates from mastitic milk samples of dairy cattle in Korea (n = 15) Species Most frequently observed patterns of resistance No. bacteria (%) E. faecalis (N = 47) CF-P-TE-E 11 (23) 12 P-TE-E 1 (21) 21 (44) E. faecium (N = 39) P-TE-E 7 (17) 11 CF-P-E 6 (15) 13 (33) Other Enterococci P-TE-E 5 (26) 1 spp.* (N = 19) Total (n = 15) 39 (37) 33 No. patterns CF, cephalothin; P, penicillin; TE, tetracycline; E, erythromycin. *Included all enterococcal isolates other than E. fecalis and E. faecium: E. avium (N = 6), E. gallinarum (N = 6), E. hirae (N = 5), and E. durans (N = 2). the highest proportion of strains resistant to 3 antimicrobials was observed in E. faecalis (52.7%, 29/55), followed by E. faecium (34.5%, 19/55) and minor Enterococcus spp. (12.7%, 7/55). Table 2 summarizes the resistance patterns most frequently observed in E. faecalis, E. faecium and other Enterococci spp. In all species, a diversity of multiresistance phenotypes was observed and the most common multiple resistance pattern was penicillin, tetracycline and erythromycin. Combined resistance to these three agents was observed in 32 (3.4%) of 15 isolates, among which 21 (%) strains were identical in their resistance pattern. Discussion Generally, the prevalence of antimicrobial resistance observed in this study is much higher than those of the previous studies on mastitis in Finland (Pitkälä et al., 4) and Uruguay (Gianneechini et al., 2), although tetracycline resistance observed in our study is slightly lower than the 73% reported from Finland. In particular, the prevalence of penicillin resistance in our study is remarkably high compared with those reported from other countries (Gianneechini et al., 2; Pitkälä et al., 4). Except vancomycin, all the antimicrobials examined for resistance in this study were the most ª 9 Blackwell Verlag GmbH Zoonoses Public Health. 3

Antimicrobial Resistance of Enterococci Isolated from Bovine Mastitis H. M. Nam et al. representative and commonly used in animals including cattle in Korea. Unfortunately, however, we have no information on the use of antibiotics on the dairy farms included in this study. Although the paucity of domestic data makes it difficult to compare our result with those of other studies, Korea is known to be one of the hottest spot in the world with regard to penicillin resistance among clinical isolates, which was 79.7% in 1998 (Song, 1998). As penicillin is a mainstay of therapy for infections with enterococci, resistance of enterococci against this drug has important clinical implications (Song, 1998). Three antimicrobials were effective against enterococci in this study: ampicillin, gentamicin and vancomycin. Although a single E. hirae isolate showed resistance, ampicillin showed superior activity against enterococci compared with gentamicin and vancomycin, to which low-level resistance and intermediate resistance were observed in 32 (3.4%) and eight (7.6%) of the isolates, respectively. Our finding that over 95% of enterococcal isolates were susceptible to ampicillin is consistent with the previous reports for bovine mastitis from many other countries (Pitkälä et al., 4; Tenhagen et al., 6). While ampicillin is generally known to be the drug of choice to treat enterococcal infections (Guiney and Urwin, 1993), nosocomial outbreaks caused by ampicillin-resistant E. faecium were reported in many countries (Quiñones et al., 5; Lester et al., 8), suggesting the need for continued monitoring of enterococci resistant to this antimicrobial. Gentamicin also showed good activity against enterococci in this study. While 3% (32/15) of the isolates showed resistance to 1 lg gentamicin discs, this may be intrinsic low level resistance (Lopes et al., 3). Enterococci are generally considered intrinsically resistant to low-level gentamicin (Kak and Chow, 2). Thus, criteria for classifying isolates as resistant or susceptible to this agent include only high-level resistance and recommend the use of high concentration discs, with a gentamicin concentration of 1 lg (Lopes et al., 3). Similar to our findings, however, results of a previous study showed that dairy enterococci are not intrinsically resistant to gentamicin, as generally accepted (Lopes et al., 3): 42% of dairy isolates showed susceptibility for 1 lg gentamicin discs. While few data are available for comparison, no gentamicin resistance was observed among enterococci isolated from clinical and subclinical bovine mastitis cases in Uruguay (Gianneechini et al., 2). Meanwhile, 76% of clinical isolates in Korea showed high-level gentamicin resistance (Yang et al., 7). Gentamicin-resitant isolates were previously described (Lopes et al., 3) and possibility of gene transfer, probably from clinical or commensal bacteria to dairy enterococci, has been suggested (Lopes et al., 5). Although gentamycin resistance was rare in this study, continued monitoring of dairy isolates is warranted. In Korea, the glycopeptides antibiotic, avoparcin was widely used as a growth promoter for poultry and swine from 1983 to 1997, and vancomycin-resistant enterococci (VRE) was first isolated from a leukaemia patient in 1992 (Park et al., 1992). Since then, VRE have been constantly recovered from patients in numerous hospitals (Kwon et al., ; Yang et al., 7) and even in healthy people (Song et al., 5). Moreover, VRE have been isolated from various animal sources in Korea, although most of them exhibited inherent low-level resistance (Seo et al., 5; Jung et al., 7). Similarly, while none of the isolates in this study was resistant to vancomycin, intermediate resistance was observed among 7.6% (8/15) of the isolates, particularly, in E. gallinarum and E. hirae. Although few data are available for comparison, prevalence of VRE in bovine raw milk samples was reported as % in Czech Republic (Schlegelová et al., 2), 8% in Costa Rica (Araya et al., 5), and 18.8% of E. faecalis and 32.8% of E. faecium isolates in Botswana (Chingwaru et al., 3). In this study, resistance observed among isolates of E. hirae and E. gallinarum was almost as high as E. faecium and E. faecalis. It seems noteworthy that one of two E. hirae strains intermediately resistant to vancomycin was the one that solely showed resistance to ampicillin in this study. Isolates belonging to this species also had a high resistance to penicillin, tetracycline and erythromycin. Enterococcus hirae is known to cause infections in animals and is uncommonly encountered in clinical isolates from humans (Park et al., ). More attention should be paid to this species because they are relatively common, particularly, in cattle (Anderson et al., 8) and E. hirae isolates from animal sources contained vana transposon, a mobile genetic element that encodes highlevel vancomycin resistance in enterococci (van den Bogaard et al., 2). Moreover, they can be a significant source of vana determinants with the potential of transfer to minor enterococcal species from humans (Robredo et al., 1999). Meanwhile, E. gallinarum also showed relatively high percentage of resistance to many antimicrobials tested in this study, which is in agreement with a previous study on enterococci from animal sources in Korea (Jung et al., 7). Indeed, E. gallinarum is one of the most prevalent species of VRE in animal isolates (Seo et al., 5; Jung et al., 7). Although vanc1 is the low-level intrinsic vancomycin resistance phenotype, it is of concern because the incidence of human infection with E. gallinarum carrying this gene has increased in the Netherlands (van den Braak et al., 1998). Recently, some cases of human infection with them have also been reported in Korea (Kim et al., 5; Lee et al., 7). 4 ª 9 Blackwell Verlag GmbH Zoonoses Public Health.

H. M. Nam et al. Antimicrobial Resistance of Enterococci Isolated from Bovine Mastitis In conclusion, this study demonstrates that enterococcal isolates that belong to minor species showed antimicrobial resistance rates as high as those of E. faecium and E. faecalis. Particularly those species carrying genes that encode resistance to antimicrobial agents considered important in human medicine are potential public health threat. More attention should be paid to the enterococcal species other than E. faecium and E. faecalis, and monitoring of antimicrobial resistance should not be restricted only to those two major species. References Anderson, J. F., T. D. Parrish, M. Akhtar, L. Zurek, and H. Hirt, 8: Antibiotic resistance of Enterococci in American bison (Bison bison) from a nature preserve compared to that of Enterococci in pastured cattle. Appl. Environ. Microbiol. 74, 1726 173. Araya, M., G. Davidovich, M. L. Arias, and C. 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