Medycyna Wet. 2007, 63 (1) 65
Praca oryginalna Original paper
Mastitis is one of the most important diseases in dairy cows throughout the world, and it is responsible for great economic losses to dairy industry. Classically, the mastitis pathogens have been divided into con-tagious and environmental organisms. Concon-tagious pathogens are noted for their ability to be spread be-tween quarters and cows during the milking process. Various Gram-negative bacteria, from environmental exposure and poor hygiene, may cause clinical masti-tis (3, 7, 13). Escherichia coli and Klebsiella spp. are the commonest of these bacteria classed as environ-mental agents because they grow commonly in the cows habitat and infect the udder as opportunists (4, 6, 13).
Environmental bovine mastitis caused by E. coli bacteria has increased in many countries (3, 14, 16). Antimicrobial agents are generally used to treat E. coli mastitis, although there is no convincing evidence that antimicrobials are effective (7). Antibiotic therapy may be useful if the host response is compromised, as du-ring the puerperal period, or if the growth of bacteria in the milk is abundant (4, 7). However, the use of antimicrobial agents causes selection pressure toward antimicrobial resistance among bacteria, and resistan-ce to one antimicrobial agent can be linked with
resi-stance against other antimicrobials (1, 5, 15). The re-sistance patterns of the bacterial populations can vary between countries or even herds, which may reflect the quantitative and qualitative aspects of antimicro-bial treatment (1, 12). Therefore, we aimed to assess the in vitro resistance rates to antibiotics commonly used in veterinary medicine of the E. coli isolates isolated from cows with cases of clinical mastitis in Burdur province of Turkey.
Material and methods
Sample collection and E. coli isolates. E. coli isolates were isolated from milk samples, which were collected from cases of clinical mastitis in cow herds in Burdur province of Turkey during the years 2000-2005. For bacteriological culture, approximately 5 ml of milk was collected into sterile tubes after first cleaning the teats. All samples were transported to the laboratory ice-cooled within 2 h of the collection. From each milk sample, 0.1 ml was inoculated onto sheep blood agar and MacConkey agar (Oxoid Ltd, Hampshire, England) and plates were incubated at 37°C for 24 and 48 h. E. coli was identified by standard labora-tory techniques, including Gram staining, colony morpho-logy, production of indole, hydrogen sulphide and urease, acid from glucose, gas from glucose, L-tryptophane deami-nase and lysine decarboxylase (8). All isolates were stored at 20°C in trypticase soy broth containing 10% glycerol. Prior to testing, all isolates were serially cultured twice on
In vitro activity of chosen antibiotics against
Escherichia coli isolated from bovine mastitis cases*
)
HULYA TURUTOGLU, DILEK OZTURK
Department of Microbiology, Faculty of Veterinary Medicine, Akdeniz University, 15100 Burdur, Turkey
Turutoglu H., Ozturk D.
In vitro activity of chosen antibiotics against Escherichia coli isolated from bovine mastitis cases Summary
The objective of this study was to determine the in vitro resistance rates to 8 antibiotics of Escherichia coli isolated from cows with cases of clinical mastitis in Burdur province of Turkey during the 2000 to 2005 period. Antimicrobial susceptibility testing was performed on a total of 101 E. coli isolates. Only 9 of the isolates were susceptible to all tested antibiotics, while the remaining 92 isolates were determined to be phenotypically resistant at least to one of the antibiotics. Among the 8 antimicrobial agents tested, resistance was most frequent for trimethoprim/sulphamethoxazole, neomycin, oxytetracycline and gentamicin. The resistance rates of the isolates to enrofloxacin, danofloxacin, amoxycillin/clavulanic acid, cefuroxime, gentamicin, oxytetra-cycline neomycin and trimethoprim/sulphamethoxazole were 10.9%, 11.9%, 25.7%, 26.7%, 33.7%, 51.5%, 55.4% and 64.4%, respectively. In conclusion, the differences observed in the efficacy of the antibiotics tested to E. coli isolates indicate the importance of antibiotic susceptibility tests and periodic surveillance of the antibiotic susceptibilities of pathogenic bacteria.
Keywords: bovine mastitis, Escherichia coli
*) The research was supported by Scientific Research Council of Akdeniz University.
Medycyna Wet. 2007, 63 (1) 66
sheep blood agar base and incubated for 24 h at 37°C under aerobic conditions.
Antibiotic susceptibility testing. The antibiotic suscepti-bility test was carried out on 101 E. coli isolates. Anti-biotic susceptibility testing was performed by disk dif-fusion method on Mueller-Hinton Agar (Oxoid) according to National Committee of Clinical Laboratory Standards (2). Ten colonies from blood agar base containing 5% sheep blood incubated at 37°C for 18 h were suspended in 2 ml sterile saline to a density approximately equal to McFarland Opacity Standard No.0.5. A sterile cotton swab was dipped into the suspension and streaked on the entire surface of a Mueller-Hinton agar plate. The inoculum was allowed to dry for 5 min. The antibiotic disks were dispen-sed on the surface of the media and were incubated aerobi-cally at 37°C for 18 h. Antibiotics tested were: amoxicil-lin/clavulanic acid (Oxoid, 30 µg), cefuroxime (Oxoid, 30 µg), neomycin (Oxoid, 30 µg), enrofloxacin (Oxoid, 5 µg), danofloxacin (Mast Diagnostics, Mast Group Ltd., Merseyside, UK, 5 µg), gentamicin (Oxoid, 10 µg), trime-thoprim/sulphamethoxazole (Oxoid, 25 µg) and oxytetra-cycline (Oxoid, 30 µg). The results were recorded as resi-stant or susceptible by measurement of the inhibition zone diameter. The reference strain used for antibiotic suscepti-bility assays was E. coli strain ATCC 25922.
Results and discussion
The disk diffusion method was used to test the 101 E. coli isolates for susceptibility to 8 antibiotics com-monly used in veterinary medicine. Only 9 (8.9%) of the isolates were susceptible to all tested antibiotics, while the remaining 92 (91.1%) isolates were found phenotypically resistant at least to one of the antibio-tics. Among the 8 antimicrobial agents tested, resistan-ce was most frequent for trimethoprim/sulphametho-xazole, neomycin, oxytetracycline and gentamicin. Iso-lates obtained between 2004 and 2005 years presented more resistance to enrofloxacin (24.2%) and dano-floxacin (27.3%) from the other years. The resistance rates of the isolates to enrofloxacin, danofloxacin,
amo-xycillin/clavulanic acid, cefuroxime, gentamicin, oxy-tetracycline neomycin and trimethoprim/sulphametho-xazole were 10.9%, 11.9%, 25.7%, 26.7%, 33.7%, 51.5%, 55.4% and 64.4%, respectively (tab. 1).
In this paper, information on the in vitro efficacy of 8 antibiotics against E. coli isolates from cases of cli-nical mastitis in cow herds was presented. Resistance rates of E. coli isolates were found to be significantly higher than those reported in USA (17), Czech Re-public (14), Switzerland (9) and Finland and Israel (10). This is probably due to the uncontrolled use of anti-microbial agents in the treatment of dairy herds as de-scribed by Aarestrup (1). Because the anti-microbial drugs in Turkey were widely used in cattle herds for the prophylaxis and treatment of udder infections, but also for other infections therefore a high rate of resi-stance to these antibiotics was not unexpected. Lehto-lainen et al. (10) have reported that the in vitro anti-microbial susceptibility of the E. coli isolates from cli-nical bovine mastitis in Finland and Israel was high; only 27% of the isolates showed resistance to one or more tested antimicrobial agents. However, of the 101 E. coli isolates processed in our study, 91.1% were found phenotypically resistant at least to one of the antibiotics. This result was similar to the finding of Schlegelova et al. (14), who have reported that the highest numbers of resistance phenotypes among the mastitis pathogens found in E. coli and resistance against one or more anti-microbial drugs was found in 73% of E. coli isolates. The susceptibility of isolates used in this study to the more recently developed com-pounds (amoxicillin/clavulanic acid, enrofloxacin, danofloxacin, cefuroxime) was high compared with results from long-established antibiotics. The most common resistance rates among the long-established antibiotics were to trimethoprim/sulphamethoxazole (64.4%), neomycin (55.4%), oxytetracycline (51.5%) and gentamicin (33.7%). This result is similar to the findings reported for same antibiotics in England (15) and Switzerland (9). s c it o i b it n A ) s e t a l o s i 5 4 = n ( 1 0 0 2 -0 0 0 2 2002-2003(n=23isolates) 2004-2005(n=33isolates) Total(n=101isolates) e l b it p e c s u S Resistant Suscepitble Resistant Suscepitble Resistant Suscepitble Resistant n % n % n % n % n % n % n % n % F E C 34 75.6 11 24.4 16 69.6 7 30.4 24 72.7 19 27.3 74 73.3 27 26.7 O E N 16 35.6 29 64.4 17 73.9 6 26.1 12 36.4 21 63.6 45 44.6 56 55.4 F N E 43 95.6 12 14.4 22 95.7 1 14.3 25 75.8 18 24.2 90 89.1 11 10.9 F N D 43 95.6 12 14.4 22 95.7 1 14.3 24 72.7 19 27.3 89 88.1 12 11.9 N E G 29 64.4 16 35.6 16 69.6 7 30.4 22 66.7 11 33.3 67 66.3 34 33.7 C T O 13 28.9 32 71.1 18 78.3 5 21.7 18 54.5 15 45.5 49 48.5 52 51.5 C M A 34 75.6 11 24.4 15 65.2 8 34.8 26 78.8 17 21.2 75 74.3 26 25.7 T X S 19 42.2 26 57.8 11 47.8 12 52.2 16 18.2 27 81.8 36 35.6 65 64.4
Explanations: CEF cefuroxime; NEO neomycin; ENF enrofloxacin; DNF danofloxacin; GEN gentamicin; OTC oxytetra-cycline; AMC amoxicillin/clavulanic acid; SXT trimethoprim/sulphamethoxazole
Medycyna Wet. 2007, 63 (1) 67 Besides trimethoprim/sulphamethoxazole,
neomy-cin, gentamicin and oxytetracycline, b-lactam antibio-tics are also the most commonly used antimicrobial agents for cattle (5). Most mastitis cases are caused by Gram-positive bacteria and have mainly been treated with b-lactam antimicrobials administered by intra-mammary or parenteral routes (5, 13). In the presen-ted study, the resistance rates of the E. coli isolates to amoxycillin/clavulanic acid and cefuroxime were 25.7% and 26.7%, respectively. This result may be explained by increasing the resistance to these anti-microbial agents in E. coli isolates as reported by Lira et al. (11).
Fluoroquinolones such as ciprofloxacin, enrofloxa-cin and danofloxaenrofloxa-cin are less frequently applied in bovine mastitis cases, except the treatment of acute clinical mastitis caused by Gram-negative bacteria (13). Although no resistance to ciprofloxacin was detected in E. coli isolates from bovine mastitis in Finland and Israel (10), we found that E. coli isolates were resi-stant to enrofloxacin (10.9%) and danofloxacin (11.9%). Isolates obtained between 2004 and 2005 years presented more resistance to enrofloxacin (24.2%) and danofloxacin (27.3%) from other years. This contrary result might be due to the widespread use of fluoroquinolone in cows for the treatment and prevention of diseases in Turkey. Regular use of these antibiotics may result in the spread of resistant strains in the course of time.
In conclusion, the differences observed in the effi-cacy of the antibiotics tested to E. coli isolates show the importance of antibiotic susceptibility tests. A cri-ticism for use of antibiotics to treat mastitis is that it leads to development of resistant strains of bacteria that can be transferred to man. Therefore, periodic sur-veillance of the antibiotic susceptibilities of patho-genic bacteria isolated from dairy cows with clinical mastitis would be an important measure in detecting emergence and spreading of resistance.
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Authors address: Assoc. Prof. Dr. Hulya Turutoglu, Department of Microbiology, Faculty of Veterinary Medicine, Akdeniz University, 15100 Burdur Turkey; e-mail: hulyaturutoglu@hotmail.com
