Brief Communication
Comparative mutant prevention concentration and antibacterial activity of fluoroquinolones against Escherichia coli in diarrheic buffalo calves Supriya Beri1, Pritam K. Sidhu2, Gurpreet Kaur3, Mudit Chandra3, Satyavan Rampal1 1
Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Science University, Ludhiana, India, 2Animal Disease Research Centre, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Science University, Ludhiana, India, 3Department of Veterinary Microbiology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Science University, Ludhiana, India Owing to emerging threat of antimicrobial resistance, mutant prevention concentration (MPC) is considered as an important parameter to evaluate the antimicrobials for their capacity to restrict/allow the emergence of resistant mutants. Therefore, MPCs of ciprofloxacin, enrofloxacin, levofloxacin, moxifloxacin, and norfloxacin were determined against Escherichia coli isolates of diarrheic buffalo calves. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were also established. The MICs of ciprofloxacin, enrofloxacin, levofloxacin, moxifloxacin and norfloxacin were 0.009, 0.022, 0.024, 0.028, and 0.036 mg/ml, respectively. The MBCs obtained were very close to the MICs of respective drugs that suggested a bactericidal mode of action of antimicrobials. The MPCs (mg/ml) of ciprofloxacin (4.26MIC), moxifloxacin (4.86MIC), and norfloxacin (5.16MIC) were approximately equal but slightly lower than enrofloxacin (7.66MIC) and levofloxacin (8.56MIC) against clinical isolates of E. coli. The MPC data suggested that enrofloxacin has the potential for restricting the selection of E. coli mutants during treatment at appropriate dosing. Keywords: Buffalo calves, Diarrhea, Escherichia coli, Fluoroquinolones, Mutant prevention concentration
Escherichia coli (E. coli) is recognized as one of the most important bacterial causes of bovine diarrhea.1 No antimicrobial agent has a label claim for treating calf diarrhea by parenteral administration, but, offlabel use of broad-spectrum beta-lactams, potentiated sulfonamides, and fluoroquinolones is recommended.1,2 Since past few years, it has been noticed that strains of E. coli have become increasingly resistant to most first-line antibiotics, including extended-spectrum cephalosporins, ampicillin, aminoglycosides, and fluoroquinolones.3–7 There is a possibility that veterinary quinolones select resistant strains in food animals and these strains may have the potential to be resistant to quinolones used in human medicine.
Correspondence to: P. K. Sidhu, Animal Disease Research Centre, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Science University, Ludhiana 141004, Punjab, India. Email: psidhu25@ rediffmail.com
ß 2014 Edizioni Scientifiche per l’Informazione su Farmaci e Terapia DOI 10.1179/1973947814Y.0000000173
Resistance has been observed to fluoroquinolones exhibiting modest in vitro activity against targeted microbes. 8–11 It is possible that fluoroquinolones with potent activity (in vitro) against target microbes may delay the emergence of resistance in these pathogens. Having broad spectrum of antimicrobial activity, enrofloxacin is the most commonly used fluoroquinolone in livestock sector.6 Therefore, enrofloxacin was selected as a bench mark drug to compare the activity with other second-, third-, and fourth-generation fluoroquinolones against E. coli isolates obtained from diarrheic buffalo calves. Ciprofloxacin was selected due to its routine use in human medicine and considered as bench mark drug for determining the susceptibility of pathogens to fluoroquinolones. Owing to serious concern about antibiotic resistance, mutant prevention concentration (MPC), which is the concentration of antibiotic that prevents the selection of resistant-mutant subpopulation from a bacterial population of large size
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(.1010 cfu/ml), has gained interest.12,13 Many studies have focused on MPC of fluoroquinolones against several microorganisms: E. coli,14,15 Streptococcus pneumonia,16,17 and Pseudomonas aeruginosa.18 Other pharmacodynamic parameters, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) differ almost invariably between strains of same bacterial species, so it is important to measure the MIC and MBC in a number of strains from different sources to ensure a representative of population.19,20 To the best of our knowledge, there is no published study that compares in vitro sensitivities of fluoroquinolones of four generations against clinical E. coli isolates of diarrheic buffalo calves. Therefore, we determined MIC, MBC, and MPC of enrofloxacin against E. coli of diarrheic buffalo calves and compared it with ciprofloxacin, levofloxacin, moxifloxacin, and norfloxacin under identical laboratory conditions using a consistent standardized methodology recommended by the Clinical and Laboratory Standards Institute (CLSI).21 Pure antibiotic standards of ciprofloxacin hydrochloride, enrofloxacin, and norfloxacin were purchased from M.P. Biomedicals Pvt. Ltd (Mumbai, India); and levofloxacin and moxifloxacin hydrochloride were purchased from Sigma-Aldrich Co. (New Delhi, India). The media for isolation and identification of E. coli, namely, Mueller-Hinton broth (MHB), Mueller-Hinton agar (MHA), triple sugar iron (TSI) agar, eosin methylene blue (EMB) agar, MacConkeys lactose agar (MLA), and antibiotic discs of ciprofloxacin and enrofloxacin were purchased from Hi-media Laboratories Pvt. Ltd (Mumbai, India). The MHB, petri plates with MLA, EMB, and MHA; and TSI slants were prepared using standard laboratory methods, stored at 4uC, and used within 1 week after preparation. The pipettes were calibrated before use for 99.9% of accuracy. A total of 15 fecal samples were collected aseptically from the rectum of diarrheic buffalo calves (age: 1–6 months) from dairy farms of Ludhiana, Punjab, India, during the Year 2012. The fecal samples were kept on ice before transportation to the laboratory and then stored at 4uC till further analysis. Test organism, E. coli MTCC 739, used as reference strain, was obtained from IMTECH (Chandigarh, India). From each fecal sample, a loopful was inoculated in MHB and incubated at 37uC for 4–8 hours. This broth culture was used to isolate pure culture of E. coli. The broth culture was streaked on MLA agar plates and incubated at 37uC for 24 hours. Colonies obtained on MLA plates were subjected to confirmation of test organism by streaking on EMB plates,
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Gram’s staining, and biochemical tests (IMViC test and TSI test). The MICs of antibiotics against E. coli were determined in MHB using micro-dilution method.21,22 For standardization of bacterial count, two to four medium sized colonies of E. coli grown on MLA plate were inoculated in 9 ml of MHB and incubated at 37uC for 6 h. The inoculums size was 16108– 56108 CFU/ml before adding it to microwell plates., To these wells, 10 ml of E. coli culture was added to obtain a final inoculum of 1–56106 CFU/ml and incubated at 37uC for 18 hours. Growth and sterility controls were run with each set of MIC. The MIC procedure was performed in five over-lapping sets to increase accuracy in the concentration range of 0.001– 1.00 mg/ml of drugs. The MIC was the lowest concentration of drug that completely inhibited visible growth of E. coli compared to growth control. The MBC of each drug was determined using MLA plates with concentration range of 0.005–1.0 mg/ml.23 The MBC was recorded as the lowest concentration of drug, which inhibited complete growth of E. coli on MLA plates after 24-hour incubation. Bauer and Kirby’s disc diffusion method was followed for performing Culture Sensitivity Test of antimicrobials against E. coli isolates. The isolates were declared sensitive/resistant to antimicrobials on the basis of recommended zone of diameters of inhibition (CLSI, 2008). Diameters of zone of inhibition of .22–30 mm and .30–40 mm indicated sensitivity of ciprofloxacin and enrofloxacin, respectively, for E. coli. The E. coli isolates sensitive to fluoroquinolones were stored at 4uC for further processing to determine pharmacodynamic characteristics of antibiotics against these pathogens. Antibiotic standards (stock solutions51 mg/ml) were prepared by dissolving them in their respective solvents according to manufacturer’s guidelines and stored in small aliquots (100 ml) in eppendorf tubes at 220uC for use in future. The MPC of ciprofloxacin, enrofloxacin, levofloxacin, moxifloxacin, and norfloxacin were determined by the method of Blondeau and co-workers.16 Briefly, for each experiment, a natural isolate of E. coli was freshly grown from stock stored at 4uC. A starter culture of bacterial count of §1010 CFU/ml was achieved by inoculating 10 colonies of E. coli in 200 ml of MHB and incubating it overnight at 37uC for 16 hours. One milliliter of this culture was diluted 1 : 10 in pre-warmed MHB and incubated again at 37uC for 6 hours. It was then centrifuged at 10 000 rev/min for 20 minutes at 4uC to get a pellet of bacteria. The supernatant was discarded and pellet was re-suspended in fresh MHB and final inoculums obtained was .1010 CFU/ml. A 100 ml of this culture was used to inoculate MLA plates, already prepared
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Table 1 Mean MIC, MBC, and MPC (mg/ml) of fluoroquinolones against E. coli isolates (n55) in diarrheic buffalo calves
Table 2 The MBC/MIC and MPC/MIC ratios of fluoroquinolones against E. coli isolates (n55) in diarrheic buffalo calves
Drug
MIC
MBC
MPC
Drug
Ciprofloxacin Enrofloxacin Levofloxacin Moxifloxacin Norfloxacin
0.009 0.022 0.024 0.028 0.036
0.016 0.036 0.038 0.038 0.050
0.038 0.168 0.204 0.136 0.184
Ciprofloxacin Enrofloxacin Levofloxacin Moxifloxacin Norfloxacin
with known concentrations of antibiotic. The plates were incubated at 37uC for 48 hours and were examined every 24 hours for growth of pathogen. The MPC was recorded in the lowest multiples of MIC of antibiotics that allowed no growth of bacteria till 48 hours of incubation. The drug concentrations (mg/ml) used for MPC were in the range of 16MIC–646MIC. Each experiment was done in triplicate on different days to take into account the inter-day and intra-day variability. All isolates of E. coli obtained from fecal samples were initially screened using disk-diffusion susceptibility tests (data not shown). Five isolates fully susceptible to ciprofloxacin, enrofloxicin, levofloxacin, moxifloxacin, and norfloxacin were used in the study. The MIC, MBC, and MPC results have been detailed in Table 1. The MIC and MBC of enrofloxacin against clinical isolates of E. coli ranged from 0.02–0.03 and 0.02–0.04 mg/ml, respectively. The ciprofloxacin showed range of 0.008–0.009 and 0.01–0.02 mg/ml, respectively, for MIC and MBC. For norfloxacin, the MICs varied between 0.02 and 0.05 mg/ml and MBCs were 0.04–0.06 mg/ml. The ranges of MIC and MBC for levofloxacin and moxifloxacin were 0.02–0.04 and 0.02–0.05 mg/ml, respectively. (Table 1). The MPCs of ciprofloxacin, moxifloxacin and norfloxacin against E. coli were 4–56MIC of respective antibiotics. The MPC of enrofloxacin and levofloxacin was 7.6–8.56MIC. The ratios of MBC/ MIC and MPC/MIC for all fluoroquinolones have been given in Table 2. Antimicrobial resistance will continue to have a large impact on the livestock industry if not investigated and controlled by implementation of strategies to prevent its development. Determination of MPC is important for reduction of incidence of fluoroquinolone resistance.12,16,24 It has been suggested that drug exposure below the MPC may promote selection of resistant mutants. i.e. drug concentrations falling in mutant selection window (MSW), between MIC and MPC, may enrich and amplify resistant mutants.13,24,25 The MPC data of enrofloxacin against E. coli of diarrheic buffalo calves are not available. However, ciprofloxacin, levofloxacin, norfloxacin, and moxifloxacin were included in
MBC/MIC
MPC/MIC
1.78 1.64 1.58 1.38 1.39
4.22 7.64 8.50 4.86 5.11
study for consistent comparison of fluoroquinolone’s MPCs and five E. coli isolates were selected to determine inter-strain variability. The MPC of enrofloxacin (7.66MIC) was comparable to the value of 8.56MIC obtained for levofloxacin (Tables 1 and 2). The ciprofloxacin showed lowest MPC (4.26MIC) which was closer to moxifloxacin (4.76MIC) and norfloxacin (5.16MIC). The present findings supported the published data in which MPC range for ciprofloxacin and enrofloxacin against susceptible E. coli isolates was 4–166MIC.15,26 No significant difference in MPC values of fluoroquinolones for reference and clinical strains of E. coli was observed. Similar findings for enrofloxacin and ciprofloxacin have been recorded by Pasquali and Manfreda.26 The MPC/MIC ratio defines the concentration range in which resistant mutant subpopulations are selected and amplified, with the lower values suggesting a better ability of antibiotic to prevent emergence of mutants.16,18 In the current study, MPC/MIC ratios indicated that ciprofloxacin, moxifloxacin, and norfloxacin represent lower selective pressure for proliferation of resistant mutants of E. coli than enrofloxacin (Table 2). Credito et al. reported a slightly lower MPC/MIC ratio (2–4) for levofloxacin and moxifloxacin against Streptococcus pneumoniae.27 This indicated that MPC/MIC ratio of the antimicrobial may vary with bacterial species. It was consistent with the hypothesis put forward by the previous workers that antibacterial activity of drugs varies with the species and strain of bacteria.28–31 Similar MPC/ MIC ratio of enrofloxacin and levofloxacin indicated that both drugs are similar in preventing the growth of first-step mutants of E. coli in diarrheic calves (Table 2). The higher MPC/MIC ratio of enrofloxacin than ciprofloxacin, moxifloxcain and norfloxacin may be attributed to the common use of enrofloxacin in veterinary medicine. However, high prevalence of E. coli resistance unrelated to antibiotic consumption has been documented in man.31,32 MPC is a useful parameter for therapeutics as well when it is considered in conjunction with the pharmacokinetic (PK) profile of the drug. Although PK studies were not conducted in diarrheic buffalo calves, low range of MPC/MIC ratio suggested that
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the MPC concept can be applicable to these antimicrobials. Earlier, PK studies of enrofloxacin were conducted in healthy buffalo calves in our laboratory.33 Enrofloxacin concentration (0.176 mg/ ml) higher than MPC of E. coli was maintained for the period of 5 min (0.52 mg/ml) to 18 hours (0.182 mg/ml) after drug administration. This indicated that the drug concentrations will not be in MSW at least for the first 18 hours or 75% time of the 24-hour dosage interval and there will be remote chances that enrofloxacin exert selective pressure for growth and amplification of resistant E. coli mutants. Considering lower MPC/MIC ratios of ciprofloxacin, norfloxacin, and moxifloxacin than enrofloxacin, these drugs may exert similar or better efficacy than enrofloxacin not only in terms of treatment, but also in preventing the risk of resistance emergence. Importantly, metabolic conversion of enrofloxacin to ciprofloxacin was 79% in buffalo calves.33 Considering simultaneous presence of both enrofloxacin and ciprofloxacin in vivo, the microbiologically active drug concentrations will not fall in MSW for 24 hours. Since it is well known that in combination therapy bactericidal drugs act synergistically, therefore, it is expected that enrofloxacin when used for treatment may deliver greater antibacterial activity with least risk of resistant mutant’s emergence. Given that E. coli MPC90 was not determined due to small number of isolates, the findings should be used carefully in clinical conditions. Nevertheless, it is a first report measuring the comparative MPC for fluoroquinolones of three generations against E. coli of diarrheic buffalo calves. The low MIC of enrofloxacin illustrated the sensitivity of E. coli to the antibacterial effects of drug despite its common use in animal health (Table 1). The lowest MIC of enrofloxacin compared to MICs of levofloxacin, moxifloxacin, and norfloxacin indicated its capacity to sustain sensitivity to E. coli. However, ciprofloxacin was two times sensitive than enrofloxacin against E. coli. Similar observations have been reported by Singer et al.,32 when these authors determined MICs of sixteen antibiotics of different classes against E. coli isolates (209) collected from fecal samples of cows. The ciprofloxacin MIC50 (0.015 mg/ ml) was lowest followed by ceftiofur and gentamicin than other fifteen antibiotics.32 The MIC50 of enrofloxacin have been previously reported as §0.0625 mg/ml for E. coli (n5195) isolated from calves suffering from neonatal diarrhea.34 In our study, MIC of enrofloxacin was three times lesser than previously reported values. But we could not determine MIC50 of enrofloxacin because the number of tested strains was small. However, MIC of a drug may vary with the geographical location and species of pathogen. High sensitivity pattern of enrofloxacin was similar to previous
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reports of 1999 and it confirmed that the drug still holds a good future for the treatment of E. coli infections of veterinary origin.34 Higher sensitivity of enrofloxacin than norfloxacin and similar activity to moxifloxacin against E. coli demonstrated that enrofloxacin will be as effective as new generation fluoroquinolones for the treatment of diarrhea in buffalo calves. The present findings were consistent with the previous investigation, where low MIC50 (0.03 mg/ml) of enrofloxacin compared to marbofloxacin (0.06 mg/ml) was obtained for E. coli of diseased dogs.24 The MBC: MIC ratios of ,2 against E. coli confirmed that these drugs are bactericidal and activity is by concentration-dependent mechanism (Table 2). Similarly, very close values of MIC and MBC for danofloxacin, enrofloxacin, and marbofloxacin against Mannheimia haemolytica were reported in calves.22,23,33 The study confirmed that a bactericidal action against E. coli is likely with clinical dose rates of enrofloxacin in diarrheic buffalo calves. It is concluded that enrofloxacin and levofloxacin exhibited slightly lower efficacy than ciprofloxacin, moxifloxacin and norfloxacin in restricting the emergence of resistant E. coli sub-population. The susceptibility of E. coli isolates to fluoroquinolones indicated by MIC and MBC was in the order: ciprofloxacin. enrofloxacin.levofloxacin.moxifloxacin.norfloxacin. The enrofloxacin, being an exclusively veterinary fluoroquinolone, appears to be effective for the treatment of bovine diarrhea in field conditions due to its comparable therapeutic efficacy to ciprofloxacin, levofloxacin and moxifloxacin. Additionally, it has the advantage of minimizing the risk of resistance emergence during treatment when used at appropriate dose rates in buffalo calves. However, use of small number of strains is limitation of the study and further investigations with greater number of E. coli strains are required to draw valid conclusions.
Disclaimer Statements Contributors All the authors contributed equally in the execution of experiments and writing of this manuscript. Funding None. Conflicts of interest None to declare. Ethics approval Not required.
References 1 Constable PD. Antimicrobial use in the treatment of calf diarrhea. J Vet Intern Med. 2004;18:8–17. 2 Aiello SE, Mays A. 1998. Intestinal diseases in ruminants. In: The Merck veterinary manual. 8th ed. Whitehouse Station, NJ: Merck & Co. 1998. p. 258–60. 3 Everett MJ, Jin YF, Ricci V, Piddock LJ. Contributions of individual mechanisms to fluoroquinolone resistance in 36 Escherichia coli strains isolated from humans and animals. Antimicrob Agents Chemother. 1996;40:2380–6.
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4 Bradford PA, Petersen PJ, Fingerman IM, White DG. Characterization of expanded spectrum cephalosporin resistance in E. coli isolates associated with bovine calf diarrhoeal disease. J Antimicrob Chemother. 1999;44:607–10. 5 Garau J, Xercavins M, Rodriguez-Carballeira M, Gomez-Vera JR, Coll I, Vidal D, et al. Emergence and dissemination of quinolone-resistant Escherichia coli in the community. Antimicrob Agents Chemother. 1999;43:2736–41. 6 Webber M, Piddock LJ. Quinolone resistance in Escherichia coli. Vet Res. 2001;32:623. 7 Seiffert SN, Hilty M, Perreten V, Endimiani. An extendedspectrum cephalosporin-resistant Gram-negative organisms in livestock: an emerging problem for human health? Drug Resist Updat. 2013;16:22–45. 8 Urban C, Rahman N, Zhao X, Mariano N, Segal-Maurer S, Drlica K, et al. Fluoroquinolone-resistant Streptococcus pneumoniae associated with levofloxacin therapy. J Infect Dis. 2001;184:794–8. 9 Davidson R, Cavalcanti R, Brunton JL, Bast DJ, de Azavedo JC, Kibsey P, et al. Resistance to levofloxacin and failure of treatment of pneumococcal pneumonia. N Engl J Med. 2002;346:747–50. 10 Kays MB, Denys GA. Fluoroquinolone susceptibility, resistance and pharmacodynamics versus clinical isolates of Streptococcus pneumoniae from Indiana. Diagn Microbiol Infect Dis. 2001;40:193–8. 11 Ross JJ, Worthington MG, Gorbach SL. Resistance to levofloxacin and failure of treatment of pneumococcal pneumonia. N Engl J Med. 2002;347:65–7. 12 Hansen GT, Blondeau JM. Comparison of the minimum inhibitory, mutant prevention and minimum bactericidal concentrations of ciprofloxacin, levofloxacin and garenoxacin against enteric Gram-negative urinary tract infection pathogens. J Chemother. 2005;17:484–92. 13 Olofsson SK, Marcusson LL, Lindgren PK, Hughes D, Cars O. Selection of ciprofloxacin resistance in Escherichia coli in an in vitro kinetic model: relation between drug exposure and mutant prevention concentration. J Antimicrob Chemother. 2006;57:1116–21. 14 Linde HJ, Lehn N. Mutant prevention concentration of nalidixic acid, ciprofloxacin, clinafloxacin, levofloxacin, norfloxacin, ofloxacin, sparfloxacin or trovafloxacin for Escherichia coli under different growth conditions. J Antimicrob Chemother. 2004;53,:252–7. 15 Marcusson LL, Olofsson SK, Komp LP, Cars O, Hughes D. Mutant prevention concentrations of ciprofloxacin for urinary tract infection isolates of Escherichia coli. J Antimicrob Chemother. 2005;55:938–43. 16 Blondeau J, Zhao X, Hansen G, Drlica, K. Mutant prevention concentrations (MPC) of fluoroquinolones with clinical isolates of Streptococcus pneumoniae. Antimicrob Agents Chemother. 2001;45:433–8. 17 Smith HJ, Walters M, Hisanaga T, Zhanel GG, Hoban DJ. Mutant prevention concentrations for single-step fluoroquinolone-resistant mutants of wild-type, efflux-positive, or ParC or GyrA mutation-containing Streptococcus pneumoniae isolates. Antimicrob Agents Chemother. 2004;48:3954–8. 18 Hansen GT, Zhao X, Drlica K, Blondeau JM. Mutant prevention concentration for ciprofloxacin and levofloxacin with Pseudomonas aeruginosa. Int J Antimicrob Agents. 2006;27:120–4.
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19 Lees P, Shojaee AF. Rational dosing of antimicrobial drugs: animals versus humans. Int J Antimicrob Agents. 2002;19:269– 84. 20 McKellar QA, Sanchez, Bruni SF, Jones DG. Pharmacokinetic/ pharmacodynamic relationships of antimicrobial drugs used in veterinary medicine. J Vet Pharmacol Ther. 2004;27:503–14. 21 Clinical Laboratory Standards Institute (CLSI). Development of in vitro susceptibility testing criteria and quality control parameters for veterinary antimicrobial agents. Approved guideline, 3rd edition M37-A3. Wayne, PA: CLSI; 2008. 22 Sidhu PK, Landoni MF, Aliabadi MH, Toutain PL, Lees P. Pharmacokinetic and pharmacodynamic modelling of marbofloxacin administered alone and in combination with tolfenamic acid in calves. J Vet Pharmacol Ther. 2011;34:376–87. 23 Shojaee AF, Lees P. Pharmacokinetic-pharmacodynamic integration of danofloxacin in the calf. Res Vet Sci. 2003;74:247–59. 24 Gebru E, Choi MJ, Lee SJ, Damte D, Park SC. Mutantprevention concentration and mechanism of resistance in clinical isolates and enrofloxacin/marbofloxacin-selected mutants of Escherichia coli of canine origin. J Med Microbiol. 2011;60:1512–22. 25 Blondeau JM. New concepts in antimicrobial susceptibility testing: the mutant prevention concentration and mutant selection window approach. Vet Dermatol. 2009;20:383–96. 26 Pasquali F, Manfreda G. Mutant prevention concentration of ciprofloxacin and enrofloxacin against Escherichia coli, Salmonella Typhimurium and Pseudomonas aeruginosa. Vet Microbiol. 2007;119:304–10. 27 Credito K, Kosowska-Shick K, McGhee P, Pankuch GA, Appelbaum PC. Comparative study of the mutant prevention concentrations of moxifloxacin, levofloxacin and gemifloxacin against Pneumococci. Antimicrob Agents Chemother. 2010;54:2673–7. 28 Toutain PL. Antibiotic treatment of animals–a different approach to rational dosing. Vet J. 2003;165:98–100. 29 Blondeau JM, Borsos S, Blondeau LD, Blondeau BJ, Hesje CE. Comparative minimum inhibitory and mutant prevention drug concentrations of enrofloxacin, ceftiofur, florfenicol, tilmicosin and tulathromycin against bovine clinical isolates of Mannheimia haemolytica. Vet Microbiol. 2012;160:85–90. 30 Martinez MN, Papich MG, Drusano GL. Dosing regimen matters: the importance of early intervention and rapid attainment of the pharmacokinetic/pharmacodynamic target. Antimicrob Agents Chemother. 2012;56: 2795–805. 31 Bartoloni A, Bartalesi F, Mantella A, Dell’Amico E, Roselli M, Strohmeyer M, et al. High prevalence of acquired antimicrobial resistance unrelated to heavy antimicrobial consumption. J Infect Dis. 2004;189:1291–4. 32 Singer RS, Patterson SK, Wallace RL. Effects of therapeutic ceftiofur administration to dairy cattle on Escherichia coli dynamics in the intestinal tract. Appl Environ Microbiol. 2008;74:6956–62. 33 Balaje RM, Sidhu PK, Kaur G, Rampal S. Mutant prevention concentration and PK–PD relationships of enrofloxacin for Pasteurella multocida in buffalo calves. Res Vet Sci. 2013;95(3):1114–24. 34 Orden JA, Ruiz-Santa-Quiteria JA, Garcia S, Cid D, De La Fuente R. In vitro activities of cephalosporins and quinolones against Escherichia coli strains isolated from diarrheic dairy calves. Antimicrob Agents Chemother. 1999;43:510–3.
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Comparative mutant prevention concentration and antibacterial activity of fluoroquinolones against Escherichia coli in diarrheic buffalo calves Supriya Beri1, Pritam K. Sidhu2, Gurpreet Kaur3, Mudit Chandra3, Satyavan Rampal1 1
Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Science University, Ludhiana, India, 2Animal Disease Research Centre, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Science University, Ludhiana, India, 3Department of Veterinary Microbiology, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Science University, Ludhiana, India Owing to emerging threat of antimicrobial resistance, mutant prevention concentration (MPC) is considered as an important parameter to evaluate the antimicrobials for their capacity to restrict/allow the emergence of resistant mutants. Therefore, MPCs of ciprofloxacin, enrofloxacin, levofloxacin, moxifloxacin, and norfloxacin were determined against Escherichia coli isolates of diarrheic buffalo calves. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were also established. The MICs of ciprofloxacin, enrofloxacin, levofloxacin, moxifloxacin and norfloxacin were 0.009, 0.022, 0.024, 0.028, and 0.036 mg/ml, respectively. The MBCs obtained were very close to the MICs of respective drugs that suggested a bactericidal mode of action of antimicrobials. The MPCs (mg/ml) of ciprofloxacin (4.26MIC), moxifloxacin (4.86MIC), and norfloxacin (5.16MIC) were approximately equal but slightly lower than enrofloxacin (7.66MIC) and levofloxacin (8.56MIC) against clinical isolates of E. coli. The MPC data suggested that enrofloxacin has the potential for restricting the selection of E. coli mutants during treatment at appropriate dosing. Keywords: Buffalo calves, Diarrhea, Escherichia coli, Fluoroquinolones, Mutant prevention concentration
Escherichia coli (E. coli) is recognized as one of the most important bacterial causes of bovine diarrhea.1 No antimicrobial agent has a label claim for treating calf diarrhea by parenteral administration, but, offlabel use of broad-spectrum beta-lactams, potentiated sulfonamides, and fluoroquinolones is recommended.1,2 Since past few years, it has been noticed that strains of E. coli have become increasingly resistant to most first-line antibiotics, including extended-spectrum cephalosporins, ampicillin, aminoglycosides, and fluoroquinolones.3–7 There is a possibility that veterinary quinolones select resistant strains in food animals and these strains may have the potential to be resistant to quinolones used in human medicine.
Correspondence to: P. K. Sidhu, Animal Disease Research Centre, College of Veterinary Science, Guru Angad Dev Veterinary and Animal Science University, Ludhiana 141004, Punjab, India. Email: psidhu25@ rediffmail.com
ß 2014 Edizioni Scientifiche per l’Informazione su Farmaci e Terapia DOI 10.1179/1973947814Y.0000000173
Resistance has been observed to fluoroquinolones exhibiting modest in vitro activity against targeted microbes. 8–11 It is possible that fluoroquinolones with potent activity (in vitro) against target microbes may delay the emergence of resistance in these pathogens. Having broad spectrum of antimicrobial activity, enrofloxacin is the most commonly used fluoroquinolone in livestock sector.6 Therefore, enrofloxacin was selected as a bench mark drug to compare the activity with other second-, third-, and fourth-generation fluoroquinolones against E. coli isolates obtained from diarrheic buffalo calves. Ciprofloxacin was selected due to its routine use in human medicine and considered as bench mark drug for determining the susceptibility of pathogens to fluoroquinolones. Owing to serious concern about antibiotic resistance, mutant prevention concentration (MPC), which is the concentration of antibiotic that prevents the selection of resistant-mutant subpopulation from a bacterial population of large size
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(.1010 cfu/ml), has gained interest.12,13 Many studies have focused on MPC of fluoroquinolones against several microorganisms: E. coli,14,15 Streptococcus pneumonia,16,17 and Pseudomonas aeruginosa.18 Other pharmacodynamic parameters, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) differ almost invariably between strains of same bacterial species, so it is important to measure the MIC and MBC in a number of strains from different sources to ensure a representative of population.19,20 To the best of our knowledge, there is no published study that compares in vitro sensitivities of fluoroquinolones of four generations against clinical E. coli isolates of diarrheic buffalo calves. Therefore, we determined MIC, MBC, and MPC of enrofloxacin against E. coli of diarrheic buffalo calves and compared it with ciprofloxacin, levofloxacin, moxifloxacin, and norfloxacin under identical laboratory conditions using a consistent standardized methodology recommended by the Clinical and Laboratory Standards Institute (CLSI).21 Pure antibiotic standards of ciprofloxacin hydrochloride, enrofloxacin, and norfloxacin were purchased from M.P. Biomedicals Pvt. Ltd (Mumbai, India); and levofloxacin and moxifloxacin hydrochloride were purchased from Sigma-Aldrich Co. (New Delhi, India). The media for isolation and identification of E. coli, namely, Mueller-Hinton broth (MHB), Mueller-Hinton agar (MHA), triple sugar iron (TSI) agar, eosin methylene blue (EMB) agar, MacConkeys lactose agar (MLA), and antibiotic discs of ciprofloxacin and enrofloxacin were purchased from Hi-media Laboratories Pvt. Ltd (Mumbai, India). The MHB, petri plates with MLA, EMB, and MHA; and TSI slants were prepared using standard laboratory methods, stored at 4uC, and used within 1 week after preparation. The pipettes were calibrated before use for 99.9% of accuracy. A total of 15 fecal samples were collected aseptically from the rectum of diarrheic buffalo calves (age: 1–6 months) from dairy farms of Ludhiana, Punjab, India, during the Year 2012. The fecal samples were kept on ice before transportation to the laboratory and then stored at 4uC till further analysis. Test organism, E. coli MTCC 739, used as reference strain, was obtained from IMTECH (Chandigarh, India). From each fecal sample, a loopful was inoculated in MHB and incubated at 37uC for 4–8 hours. This broth culture was used to isolate pure culture of E. coli. The broth culture was streaked on MLA agar plates and incubated at 37uC for 24 hours. Colonies obtained on MLA plates were subjected to confirmation of test organism by streaking on EMB plates,
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Gram’s staining, and biochemical tests (IMViC test and TSI test). The MICs of antibiotics against E. coli were determined in MHB using micro-dilution method.21,22 For standardization of bacterial count, two to four medium sized colonies of E. coli grown on MLA plate were inoculated in 9 ml of MHB and incubated at 37uC for 6 h. The inoculums size was 16108– 56108 CFU/ml before adding it to microwell plates., To these wells, 10 ml of E. coli culture was added to obtain a final inoculum of 1–56106 CFU/ml and incubated at 37uC for 18 hours. Growth and sterility controls were run with each set of MIC. The MIC procedure was performed in five over-lapping sets to increase accuracy in the concentration range of 0.001– 1.00 mg/ml of drugs. The MIC was the lowest concentration of drug that completely inhibited visible growth of E. coli compared to growth control. The MBC of each drug was determined using MLA plates with concentration range of 0.005–1.0 mg/ml.23 The MBC was recorded as the lowest concentration of drug, which inhibited complete growth of E. coli on MLA plates after 24-hour incubation. Bauer and Kirby’s disc diffusion method was followed for performing Culture Sensitivity Test of antimicrobials against E. coli isolates. The isolates were declared sensitive/resistant to antimicrobials on the basis of recommended zone of diameters of inhibition (CLSI, 2008). Diameters of zone of inhibition of .22–30 mm and .30–40 mm indicated sensitivity of ciprofloxacin and enrofloxacin, respectively, for E. coli. The E. coli isolates sensitive to fluoroquinolones were stored at 4uC for further processing to determine pharmacodynamic characteristics of antibiotics against these pathogens. Antibiotic standards (stock solutions51 mg/ml) were prepared by dissolving them in their respective solvents according to manufacturer’s guidelines and stored in small aliquots (100 ml) in eppendorf tubes at 220uC for use in future. The MPC of ciprofloxacin, enrofloxacin, levofloxacin, moxifloxacin, and norfloxacin were determined by the method of Blondeau and co-workers.16 Briefly, for each experiment, a natural isolate of E. coli was freshly grown from stock stored at 4uC. A starter culture of bacterial count of §1010 CFU/ml was achieved by inoculating 10 colonies of E. coli in 200 ml of MHB and incubating it overnight at 37uC for 16 hours. One milliliter of this culture was diluted 1 : 10 in pre-warmed MHB and incubated again at 37uC for 6 hours. It was then centrifuged at 10 000 rev/min for 20 minutes at 4uC to get a pellet of bacteria. The supernatant was discarded and pellet was re-suspended in fresh MHB and final inoculums obtained was .1010 CFU/ml. A 100 ml of this culture was used to inoculate MLA plates, already prepared
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Table 1 Mean MIC, MBC, and MPC (mg/ml) of fluoroquinolones against E. coli isolates (n55) in diarrheic buffalo calves
Table 2 The MBC/MIC and MPC/MIC ratios of fluoroquinolones against E. coli isolates (n55) in diarrheic buffalo calves
Drug
MIC
MBC
MPC
Drug
Ciprofloxacin Enrofloxacin Levofloxacin Moxifloxacin Norfloxacin
0.009 0.022 0.024 0.028 0.036
0.016 0.036 0.038 0.038 0.050
0.038 0.168 0.204 0.136 0.184
Ciprofloxacin Enrofloxacin Levofloxacin Moxifloxacin Norfloxacin
with known concentrations of antibiotic. The plates were incubated at 37uC for 48 hours and were examined every 24 hours for growth of pathogen. The MPC was recorded in the lowest multiples of MIC of antibiotics that allowed no growth of bacteria till 48 hours of incubation. The drug concentrations (mg/ml) used for MPC were in the range of 16MIC–646MIC. Each experiment was done in triplicate on different days to take into account the inter-day and intra-day variability. All isolates of E. coli obtained from fecal samples were initially screened using disk-diffusion susceptibility tests (data not shown). Five isolates fully susceptible to ciprofloxacin, enrofloxicin, levofloxacin, moxifloxacin, and norfloxacin were used in the study. The MIC, MBC, and MPC results have been detailed in Table 1. The MIC and MBC of enrofloxacin against clinical isolates of E. coli ranged from 0.02–0.03 and 0.02–0.04 mg/ml, respectively. The ciprofloxacin showed range of 0.008–0.009 and 0.01–0.02 mg/ml, respectively, for MIC and MBC. For norfloxacin, the MICs varied between 0.02 and 0.05 mg/ml and MBCs were 0.04–0.06 mg/ml. The ranges of MIC and MBC for levofloxacin and moxifloxacin were 0.02–0.04 and 0.02–0.05 mg/ml, respectively. (Table 1). The MPCs of ciprofloxacin, moxifloxacin and norfloxacin against E. coli were 4–56MIC of respective antibiotics. The MPC of enrofloxacin and levofloxacin was 7.6–8.56MIC. The ratios of MBC/ MIC and MPC/MIC for all fluoroquinolones have been given in Table 2. Antimicrobial resistance will continue to have a large impact on the livestock industry if not investigated and controlled by implementation of strategies to prevent its development. Determination of MPC is important for reduction of incidence of fluoroquinolone resistance.12,16,24 It has been suggested that drug exposure below the MPC may promote selection of resistant mutants. i.e. drug concentrations falling in mutant selection window (MSW), between MIC and MPC, may enrich and amplify resistant mutants.13,24,25 The MPC data of enrofloxacin against E. coli of diarrheic buffalo calves are not available. However, ciprofloxacin, levofloxacin, norfloxacin, and moxifloxacin were included in
MBC/MIC
MPC/MIC
1.78 1.64 1.58 1.38 1.39
4.22 7.64 8.50 4.86 5.11
study for consistent comparison of fluoroquinolone’s MPCs and five E. coli isolates were selected to determine inter-strain variability. The MPC of enrofloxacin (7.66MIC) was comparable to the value of 8.56MIC obtained for levofloxacin (Tables 1 and 2). The ciprofloxacin showed lowest MPC (4.26MIC) which was closer to moxifloxacin (4.76MIC) and norfloxacin (5.16MIC). The present findings supported the published data in which MPC range for ciprofloxacin and enrofloxacin against susceptible E. coli isolates was 4–166MIC.15,26 No significant difference in MPC values of fluoroquinolones for reference and clinical strains of E. coli was observed. Similar findings for enrofloxacin and ciprofloxacin have been recorded by Pasquali and Manfreda.26 The MPC/MIC ratio defines the concentration range in which resistant mutant subpopulations are selected and amplified, with the lower values suggesting a better ability of antibiotic to prevent emergence of mutants.16,18 In the current study, MPC/MIC ratios indicated that ciprofloxacin, moxifloxacin, and norfloxacin represent lower selective pressure for proliferation of resistant mutants of E. coli than enrofloxacin (Table 2). Credito et al. reported a slightly lower MPC/MIC ratio (2–4) for levofloxacin and moxifloxacin against Streptococcus pneumoniae.27 This indicated that MPC/MIC ratio of the antimicrobial may vary with bacterial species. It was consistent with the hypothesis put forward by the previous workers that antibacterial activity of drugs varies with the species and strain of bacteria.28–31 Similar MPC/ MIC ratio of enrofloxacin and levofloxacin indicated that both drugs are similar in preventing the growth of first-step mutants of E. coli in diarrheic calves (Table 2). The higher MPC/MIC ratio of enrofloxacin than ciprofloxacin, moxifloxcain and norfloxacin may be attributed to the common use of enrofloxacin in veterinary medicine. However, high prevalence of E. coli resistance unrelated to antibiotic consumption has been documented in man.31,32 MPC is a useful parameter for therapeutics as well when it is considered in conjunction with the pharmacokinetic (PK) profile of the drug. Although PK studies were not conducted in diarrheic buffalo calves, low range of MPC/MIC ratio suggested that
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the MPC concept can be applicable to these antimicrobials. Earlier, PK studies of enrofloxacin were conducted in healthy buffalo calves in our laboratory.33 Enrofloxacin concentration (0.176 mg/ ml) higher than MPC of E. coli was maintained for the period of 5 min (0.52 mg/ml) to 18 hours (0.182 mg/ml) after drug administration. This indicated that the drug concentrations will not be in MSW at least for the first 18 hours or 75% time of the 24-hour dosage interval and there will be remote chances that enrofloxacin exert selective pressure for growth and amplification of resistant E. coli mutants. Considering lower MPC/MIC ratios of ciprofloxacin, norfloxacin, and moxifloxacin than enrofloxacin, these drugs may exert similar or better efficacy than enrofloxacin not only in terms of treatment, but also in preventing the risk of resistance emergence. Importantly, metabolic conversion of enrofloxacin to ciprofloxacin was 79% in buffalo calves.33 Considering simultaneous presence of both enrofloxacin and ciprofloxacin in vivo, the microbiologically active drug concentrations will not fall in MSW for 24 hours. Since it is well known that in combination therapy bactericidal drugs act synergistically, therefore, it is expected that enrofloxacin when used for treatment may deliver greater antibacterial activity with least risk of resistant mutant’s emergence. Given that E. coli MPC90 was not determined due to small number of isolates, the findings should be used carefully in clinical conditions. Nevertheless, it is a first report measuring the comparative MPC for fluoroquinolones of three generations against E. coli of diarrheic buffalo calves. The low MIC of enrofloxacin illustrated the sensitivity of E. coli to the antibacterial effects of drug despite its common use in animal health (Table 1). The lowest MIC of enrofloxacin compared to MICs of levofloxacin, moxifloxacin, and norfloxacin indicated its capacity to sustain sensitivity to E. coli. However, ciprofloxacin was two times sensitive than enrofloxacin against E. coli. Similar observations have been reported by Singer et al.,32 when these authors determined MICs of sixteen antibiotics of different classes against E. coli isolates (209) collected from fecal samples of cows. The ciprofloxacin MIC50 (0.015 mg/ ml) was lowest followed by ceftiofur and gentamicin than other fifteen antibiotics.32 The MIC50 of enrofloxacin have been previously reported as §0.0625 mg/ml for E. coli (n5195) isolated from calves suffering from neonatal diarrhea.34 In our study, MIC of enrofloxacin was three times lesser than previously reported values. But we could not determine MIC50 of enrofloxacin because the number of tested strains was small. However, MIC of a drug may vary with the geographical location and species of pathogen. High sensitivity pattern of enrofloxacin was similar to previous
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reports of 1999 and it confirmed that the drug still holds a good future for the treatment of E. coli infections of veterinary origin.34 Higher sensitivity of enrofloxacin than norfloxacin and similar activity to moxifloxacin against E. coli demonstrated that enrofloxacin will be as effective as new generation fluoroquinolones for the treatment of diarrhea in buffalo calves. The present findings were consistent with the previous investigation, where low MIC50 (0.03 mg/ml) of enrofloxacin compared to marbofloxacin (0.06 mg/ml) was obtained for E. coli of diseased dogs.24 The MBC: MIC ratios of ,2 against E. coli confirmed that these drugs are bactericidal and activity is by concentration-dependent mechanism (Table 2). Similarly, very close values of MIC and MBC for danofloxacin, enrofloxacin, and marbofloxacin against Mannheimia haemolytica were reported in calves.22,23,33 The study confirmed that a bactericidal action against E. coli is likely with clinical dose rates of enrofloxacin in diarrheic buffalo calves. It is concluded that enrofloxacin and levofloxacin exhibited slightly lower efficacy than ciprofloxacin, moxifloxacin and norfloxacin in restricting the emergence of resistant E. coli sub-population. The susceptibility of E. coli isolates to fluoroquinolones indicated by MIC and MBC was in the order: ciprofloxacin. enrofloxacin.levofloxacin.moxifloxacin.norfloxacin. The enrofloxacin, being an exclusively veterinary fluoroquinolone, appears to be effective for the treatment of bovine diarrhea in field conditions due to its comparable therapeutic efficacy to ciprofloxacin, levofloxacin and moxifloxacin. Additionally, it has the advantage of minimizing the risk of resistance emergence during treatment when used at appropriate dose rates in buffalo calves. However, use of small number of strains is limitation of the study and further investigations with greater number of E. coli strains are required to draw valid conclusions.
Disclaimer Statements Contributors All the authors contributed equally in the execution of experiments and writing of this manuscript. Funding None. Conflicts of interest None to declare. Ethics approval Not required.
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19 Lees P, Shojaee AF. Rational dosing of antimicrobial drugs: animals versus humans. Int J Antimicrob Agents. 2002;19:269– 84. 20 McKellar QA, Sanchez, Bruni SF, Jones DG. Pharmacokinetic/ pharmacodynamic relationships of antimicrobial drugs used in veterinary medicine. J Vet Pharmacol Ther. 2004;27:503–14. 21 Clinical Laboratory Standards Institute (CLSI). Development of in vitro susceptibility testing criteria and quality control parameters for veterinary antimicrobial agents. Approved guideline, 3rd edition M37-A3. Wayne, PA: CLSI; 2008. 22 Sidhu PK, Landoni MF, Aliabadi MH, Toutain PL, Lees P. Pharmacokinetic and pharmacodynamic modelling of marbofloxacin administered alone and in combination with tolfenamic acid in calves. J Vet Pharmacol Ther. 2011;34:376–87. 23 Shojaee AF, Lees P. Pharmacokinetic-pharmacodynamic integration of danofloxacin in the calf. Res Vet Sci. 2003;74:247–59. 24 Gebru E, Choi MJ, Lee SJ, Damte D, Park SC. Mutantprevention concentration and mechanism of resistance in clinical isolates and enrofloxacin/marbofloxacin-selected mutants of Escherichia coli of canine origin. J Med Microbiol. 2011;60:1512–22. 25 Blondeau JM. New concepts in antimicrobial susceptibility testing: the mutant prevention concentration and mutant selection window approach. Vet Dermatol. 2009;20:383–96. 26 Pasquali F, Manfreda G. Mutant prevention concentration of ciprofloxacin and enrofloxacin against Escherichia coli, Salmonella Typhimurium and Pseudomonas aeruginosa. Vet Microbiol. 2007;119:304–10. 27 Credito K, Kosowska-Shick K, McGhee P, Pankuch GA, Appelbaum PC. Comparative study of the mutant prevention concentrations of moxifloxacin, levofloxacin and gemifloxacin against Pneumococci. Antimicrob Agents Chemother. 2010;54:2673–7. 28 Toutain PL. Antibiotic treatment of animals–a different approach to rational dosing. Vet J. 2003;165:98–100. 29 Blondeau JM, Borsos S, Blondeau LD, Blondeau BJ, Hesje CE. Comparative minimum inhibitory and mutant prevention drug concentrations of enrofloxacin, ceftiofur, florfenicol, tilmicosin and tulathromycin against bovine clinical isolates of Mannheimia haemolytica. Vet Microbiol. 2012;160:85–90. 30 Martinez MN, Papich MG, Drusano GL. Dosing regimen matters: the importance of early intervention and rapid attainment of the pharmacokinetic/pharmacodynamic target. Antimicrob Agents Chemother. 2012;56: 2795–805. 31 Bartoloni A, Bartalesi F, Mantella A, Dell’Amico E, Roselli M, Strohmeyer M, et al. High prevalence of acquired antimicrobial resistance unrelated to heavy antimicrobial consumption. J Infect Dis. 2004;189:1291–4. 32 Singer RS, Patterson SK, Wallace RL. Effects of therapeutic ceftiofur administration to dairy cattle on Escherichia coli dynamics in the intestinal tract. Appl Environ Microbiol. 2008;74:6956–62. 33 Balaje RM, Sidhu PK, Kaur G, Rampal S. Mutant prevention concentration and PK–PD relationships of enrofloxacin for Pasteurella multocida in buffalo calves. Res Vet Sci. 2013;95(3):1114–24. 34 Orden JA, Ruiz-Santa-Quiteria JA, Garcia S, Cid D, De La Fuente R. In vitro activities of cephalosporins and quinolones against Escherichia coli strains isolated from diarrheic dairy calves. Antimicrob Agents Chemother. 1999;43:510–3.
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