In Vitro Activities of Antimicrobials Against Six Important Species of Gram-Negative Bacteria Isolated from Raw Milk Samples in Korea

Introduction

A wide variety of Gram-negative bacteria (GNB) have been isolated from bulk tank milk, which can be contaminated with bacteria from various sources, including teat and udder surfaces, mastitic udders, and the environment of cows (Jayarao and Wang, 1999). Many GNB are intrinsically resistant to chemically unrelated antimicrobial agents, and multidrug resistance among GNB is increasing worldwide, posing a major public health threat (Slama, 2008). The particularly problematic multidrug-resistant pathogens include Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Serratia marcescens (Slama, 2008), which are also relatively frequently isolated from raw bovine milk samples (Jayarao and Wang, 1999). In our previous study on GNB in bovine mastitis (Nam et al., 2009), Pseudomonas spp., Acinetobacter spp., Enterobacter spp., Klebsiella spp., Serratia spp., and Citrobacter spp. were found to be the most common GNB, aside from Escherichia coli. Due to their poor response to antibiotic therapy, intramammary infection with these bacteria can be associated with significant mastitis problems (Bannerman et al., 2005).

Mastitis is the primary reason for the use of antimicrobials in dairy herds (Pol and Ruegg, 2007), and the dairy farm environment is a reservoir for the development of bacterial resistance against antimicrobials used in human medicine (Burgos et al., 2005). Thus, data concerning antimicrobial activities among GNB from mastitic milk samples could be useful for assessing the scale of resistance problem and monitoring changes in resistance rates in dairy farm environments. However, limited information exists on antimicrobial resistance profile of GNB from mastitic milk, and most reports focused mainly on E. coli or grouped all coliforms together (Tenhagen et al., 2006). The objective of this study was, therefore, to provide baseline information on in vitro activities of 12 antimicrobials against six different GNB species isolated from raw milk samples of mastitic dairy cattle in the Republic of Korea (hereinafter referred to as Korea).

Materials and Methods

The GNB strains examined in this study were isolated from our previous work (Nam et al., 2009). Briefly, GNB were isolated from milk samples submitted to or collected by Mastitis Diagnostic Laboratory of National Veterinary Research and Quarantine Service in Korea from January 2003 to June 2008. Bacterial examination of the milk samples was conducted using standard laboratory techniques (NMC, 1999). The GNB bacteria initially identified by colony morphology and Gram stain were further identified to the species level using the VITEK system (bioMérieux, Hazelwood, MO) as described by the manufacturer. The bacterial identification was regarded as acceptable when the confidence level of the analysis was ≥90%. A total of 225 isolates belonging to 6 important species of GNB—A. baumannii (n = 17), Citrobacter freundii (n = 19), Enterobacter cloacae (n = 54), K. pneumoniae (n = 55), P. aeruginosa (n = 45), and S. marcescens (n = 35)—were evaluated for resistance to 12 antimicrobials in this study.

In vitro antimicrobial susceptibility testing was conducted by the disc diffusion method and interpreted according to the guidelines of the Clinical and Laboratory Standards Institute (formerly NCCLS, 2002). For susceptibility testing in GNB, commercially prepared antimicrobial sensitivity discs (BBL; Becton-Dickinson, Cockeysville, MD) having the following 12 antimicrobial agents and concentrations were used: amikacin (10 μg/disk), gentamicin (10 μg/disk), kanamycin (30 μg/disk), streptomycin (10 μg/disk), ampicillin (10 μg/disk), ticarcillin (75 μg/disk), piperacillin (100 μg/disk), cephalothin (30 μg/disk), cefazolin (30 μg/disk), chloramphenicol (30 μg/disk), tetracycline (30 μg/disk), and rifampin (5 μg/disk). Cultures were classified as sensitive, intermediate, and resistant on the basis of the diameter of the zone of inhibition. E. coli American Type Culture Collection (ATCC) 25922 strain was used as the quality-control organism. Test results were accepted only when the zone of inhibition for the control strain was within the acceptable ranges.

Results

Table 1 summarizes the in vitro susceptibilities of each bacterial species to 12 antimicrobial agents. Wide differences in susceptibility were observed among the bacterial species for all antimicrobials tested except rifampin, to which all species uniformly showed almost no susceptibility (range, 3.7% to 15.8%). Overall, most beta-lactams tested in this study were not inhibitory to GNB except piperacillin and ticarcillin: cephalothin, cefazolin, and ampicillin had almost no activity against most bacterial species with a few exceptions; K. pneumoniae showed relatively high susceptibility to cephalothin (61.8%) and cefazolin (83.6%), and C. freundii had exceptionally high level of susceptibility to ampicillin (68.4%) compared with other species. The only beta-lactams that had high activities to most species were the extended-spectrum penicillins, piperacillin, and ticarcillin, although K. pneumoniae showed exceptional low susceptibility rate to ticarcillin (27.2%). Aminoglycosides showed high activities against GNB tested in this study. Most GNB isolates were susceptible to amikacin and gentamicin, and kanamycin was also effective against most species except for P. aeruginosa. Streptomycin was found to be the least effective of all aminoglycosides tested except A. baumannii and P. aeruginosa. Although streptomycin showed lower activity than kanamycin, no marked differences were observed in resistance rates to these two agents among most species. One exception was S. marcescens, which showed remarkable difference between the susceptibility to streptomycin and kanamycin (48.5% and 85.7%). Resistances to tetracycline and chloramphenicol were highly variable among the species: while A. baumannii and E. cloacae showed over 82% susceptibility to tetracycline, S. marcescens and P. aeruginosa showed only 11.4% and 6.7% of susceptibility to this drug, respectively. Chloramphenicol was extremely active against C. freundii, S. marcescens, and E. cloacae (>92%) but showed no activity against A. baumannii and P. aeruginosa (5.9% and 17.8%).

About half (45.3%, 102/225) of GNB isolates exhibited simultaneous resistance to 5 or more of 12 antimicrobial agents tested. The vast majority of P. aeruginosa (86.6%, 39/45) and S. marcescens (65.7%, 23/35) isolates were resistant to ≥5 antimicrobials, and 29% (16/55) of K. pneumoniae showed resistance to ≥ 7 drugs. An isolate of P. aeruginosa showed susceptibility only to chloramphenicol out of 12 agents tested in this study. For C. freundii and E. cloacae, only 10.5% (2/19) and 14.8% (8/54) of the isolates showed coresistance to more than five antimicrobials, respectively.

Discussion

Although numerous studies have reported antimicrobial susceptibility patterns of mastitis pathogens worldwide (Erskine et al., 2002; Tenhagen et al., 2006), few studies have specifically documented resistance data on GNB species from dairy cattle. Most previous studies on mastitis in Korea have mainly focused on major pathogens such as Staphylococcus aureus, and comparable data are not available on antimicrobial resistance, especially for specific GNB species tested in this study. Erskine et al. (2002) have studied antimicrobial resistance of K. pnemoniae, S. marcescens, and P. aeruginosa isolates from bovine mastitis, and their findings are in accordance with the results obtained from this study. The results of this study are also generally comparable to those of our previous work (Nam et al., 2009), in which antimicrobial susceptibility was assessed for GNB genera, not for GNB species. However, P. aeruginosa and A. baumannii in this study presented much lower susceptibility against several antimicrobials than Pseudomonas spp. and Acinetobacter spp., respectively, suggesting that these two species may often be associated with multidrug resistance. When compared with GNB isolates from fecal samples of healthy dairy cattle (Sawant et al., 2007), remarkably higher resistances were observed in this study particularly to tetracycline, kanamycin, and streptomycin, the antimicrobials widely used for treatment of disease in animals.

Our finding that amikacin and gentamicin had high activities against all GNB species is similar to the previous studies on antimicrobial resistance in GNB isolated from other sources (Lima-Bittencourt et al., 2007). The lower activity of streptomycin compared with other aminoglycosides tested in this study may have been due to the wider use in large animals than other drugs. The inactivity of beta-lactams against GNB except the extended-spectrum penicillins could be related to some well-known resistance mechanisms such as intrinsic AmpC beta-lactamases (Susić, 2004). Among the antimicrobials tested, rifampin was the only drug that had almost no activity against all GNB species without exception. In combination with other antimicrobials such as colistin, rifampin has shown effective activity against multidrug-resistant strains of P. aeruginosa and A. baumannii (Giamarellos-Bourboulis et al., 2001, 2003). Although no comparable data are available for the activity of rifampin against GNB isolated from mastitic milk, the prevalence of rifampin resistance observed in this study is almost threefold higher than that found in enterobacteria isolated from freshwater streams (Lima-Bittencourt et al., 2007). The wide gap in resistance rates may be due to the differences in environmental conditions that can affect the mechanism mediating resistance and the level of exposure to antimicrobials.

This study indicates that multiple antimicrobial resistances are prevalent among GNB isolates from mastitic milk samples of dairy cattle in Korea. To understand the scale of resistance problem and predict future trends in resistance development, antimicrobial resistance patterns of the environmental pathogens are important. The data obtained from this study provide the status of antimicrobial resistance among the Gram-negative environmental bacteria that commonly exist in dairy farm environments.