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Antimicrobial Resistance in Bacteria Isolated From Dairy Herds in Chile

B. San Martín, MV, DMV*

J. Kruze, MV, PhD

M. A. Morales, MV, MsSc*

H. Agüero, MV, MsSc*

D. Iragüen, MV*

S. Espinoza, MV, MsSc*

B. León, MV

C. Borie, MV, MsSc*


*Laboratory of Pharmacology and Laboratory of Microbiology, Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile, POB 2, Correo 15, La Granja, Santiago, Chile

Facultad de Ciencias, Universidad Austral de Chile, POB 167, Valdivia, Chile

COOPRINSEM, POB 827, Osorno, Chile


KEY WORDS: Antimicrobial agent,
bacterial resistance, dairy cow


Minimum inhibitory concentration (MIC) was determined in a total of 1,419 strains of Escherichia coli, streptococci, and staphylococci isolated from lactating cows suffering from clinical mastitis in Chile during the 2000 to 2001 period. Antimicrobial agents tested were penicillin, amoxicillin, ampicillin, cefoperazone, gentamicin, sulfamethoxazole, oxytetracycline, cefquinome, trimethoprim, enrofloxacin, florfenicol, ceftiofur, lincomicyn, and pirlimycin. Staphylococcus aureus showed the highest level of resistance to lincomycin (38.9%), amoxicillin (38.1%), penicillin (28.8%), ampicillin (26.0%), and cefquinome (24.7%). The corresponding values for cuagulase-negative staphylococci (CNS) strains were 56.9%, 42.3%, 31.5%, and 26.8% against penicillin, lincomycin, amoxicillin, and ampicillin, respectively. Streptococcal strains were highly resistant only to lincomycin (61.9%), and E.coli was sensitive to most antimicrobials assayed, with the exception of oxytetracycline and enrofloxacin (20.6% and 19.3% resistance, respectively). The study found that 34.9% of staphylococcal strains were betalactamase producers; none of the E. coli strains showed extended spectrum betalactamase (ESBL). Finally, due to the high resistance levels detected in the present study, we believe that it is necessary for Chile to set up permanent resistance surveillance programs.


Antimicrobial agents represent one of the main therapeutic tools both in human and veterinary medicine to control and, in some cases, to eradicate a wide range of bacterial infectious diseases. However, currently, the judicious use of these drugs is of great global concern.1–4

Bovine mastitis is one of the most important bacterial diseases in dairy cattle throughout the world, and it is responsible for great economic losses to milk producers as well as to the milk processing industries. In the United States, the annual losses due to mastitis have been estimated at 1.8 billion dollars, and the average cost of clinical mastitis is about $185 per cow/year. These losses include reduced milk production, discarded milk, replacement cost, extra labor, treatment, and veterinary services.5

Many factors can influence the development of mastitis; however, the inflammation of the mammary gland is usually a consequence of invasion and colonization in the secretory tissue by one or more microorganisms, especially Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, and Escherichia coli.1,6 To successfully control these intramammary infections and to avoid increases of bacterial resistance and treatment failure, veterinarians should be aware of the antimicrobial activity characteristics against these major mastitis pathogens before administering in vitro susceptibility tests. Increased resistance of S. aureus and cuagulase-negative staphylococci (CNS) isolated from bovine mastitis cases to antimicrobial agents was reported by Gentilini et al.7 and Myllys et al.3

Antimicrobial agents are the main therapeutic tool used in Chile to control bacterial diseases, representing about 45% of total drugs used in livestock.1 However, little information is available concerning sensitivity patterns in veterinary medicine, including mastitis pathogens. Some regional studies conducted in dairy herds have shown 6.2% of Streptococcus spp resistant to penicillin; a higher proportion of Staphylococcus aureus organisms have shown resistance to penicillin and ampicillin (26% and 21%, respectively), but were sensitive to cefotaxime and cefradine.8,9

Conversely, Borie et al.10 found E. coli strains highly resistant to tetracycline (62.6%) but highly sensitive to gentamicin, cefoperazone, cefotaxime, neomycin, and sulfadiazine/trimethoprim (more than 92%). More recently, San Martín et al.11 found that a number of E. coli, S. aureus, and Staphylococcus spp strains were sensitive to sulfadiazine/trimethoprim.

Considering these results and the recommendations of the World Health Organization (2000), the aim of this study was to develop a systematic and programmed evaluation protocol of bacterial resistance of the main mastitis pathogens isolated from dairy cows in Chile during a 2-year period. These results will allow veterinarians to evaluate updated information on bacterial resistance, providing a more rational use of antimicrobial agents.


Bacterial Isolates

During a 2-year period (2000–2001), 2,914 quarter milk samples were aseptically collected from dairy cows with clinical mastitis from different dairy herds in Chile. All milk samples were collected before milking using sterile, disposable 15 mL screw cap tubes after standardized procedures recommended by the National Mastitis Council (NMC).5 Isolation and bacterial identification were performed according to NMC5 and International Dairy Federation12 recommendations. Within 8 hours after collection, 25 mL of each milk sample was plated onto trypticase agar plates (BBL® Becton Dickinson and Company, Cockeysville, MI) containing 5% sheep blood and 0.1% esculin. After 24 to 48 hours incubation at 37˚C, all plates were examined under ultraviolet light for bacterial growth and esculin splitting. Staphylococcal isolates were identified according to morphologic and hemolytic properties and coagulase and DNAse tests. Streptococci were identified on morphologic and hemolytic characteristics, CAMP test reaction, and biochemical tests (SVA Strep, Uppsala, Sweden). Identification of coliform bacteria was based on morphology and biochemical tests (API 20E, BioMerieux, Chile).

Antibacterial Agents

The antibacterial agents selected for this study included 1) antimicrobial agents used for mastitis in Chile: penicillin (Sigma Chemical), amoxicillin (Sigma Chemical), ampicillin (Sigma Chemical), cefoperazone (Sigma Chemical), lincomycin (Pharmacia, Chile), and pirlimycin (Pharmacia); and 2) antimicrobial agents used to treat other infections in bovine species: sulfamethoxazole (Sigma Chemical), oxytetracycline (Sigma Chemical), cefquinome (Intervet, Chile), trimethoprim (Intervet), enrofloxacin (Bayer, Chile), florfenicol (Schering-Plough Animal Health, Chile), ceftiofur (Pharmacia), and gentamicin (Sigma Chemical).

Antibacterial Sensitivity Test

Determination of minimum inhibitory concentration (MIC) was performed following NCCLS recommendations.13 S. aureus ATCC 29213 and E. coli ATCC 25922 were used as control organisms. Stock solutions were prepared in solvent as described in the NCCLS13 or according to manufacturer and stored at 4° to 8°C until used (1 month maximum). Stepwise 2-fold dilution of each antibacterial drug was prepared on the test day to cover the expected ranges of MICs.

Bacterial isolates were grown overnight at 37˚C in Mueller Hinton broth (BBL) supplemented with 5% (v/v) desfribinated sheep blood for streptococci strains. Bacterial inocula were adjusted with sterile saline to the 0.5 McFarland´s standard. Using a Steer’s replicator, 104 cfu/spot of each test strain was inoculated on the Mueller Hinton agar plate with different concentrations of the antimicrobial agents.

Chromogenic Cephalosporin Disc Method

The Cefinase disc test (BBL) was used as a screening test for detection of b-lactamase enzymes on staphylococcal strains that showed resistance to any b-lactam or cephalosporin. The test was performed according to manufacturer directions. A yellow to red color change within 1 hour on the area where the culture was applied was considered a positive reaction. S. aureus ATCC 29213 was included as the positive control.

Detection of Extended-Spectrum

The presence of extended-spectrum b-lactamase (ESBL) enzyme was determined on all E. coli strains that showed resistance to at least one cephalosporin in study. This was done using the inhibitor-potentiated disc-diffusion test based on the standard disc-diffusion method described by the NCCLS.13 All bacterial isolates were grown overnight at 37˚C in Mueller Hinton broth (BBL) supplemented with 0.5% bovine serum. Bacterial inocula were adjusted with sterile saline to the 0.5 McFarland´s standard and plated onto Mueller-Hinton agar plates (BBL). Paper discs containing standard concentrations of cefotaxime, 30 g, cefotaxime-clavulanic acid, 30/10 g, ceftazidime, 30 g, and ceftazidime-clavulanic acid, 30/10 g (Becton and Dickinson Microbiology Systems) were simultaneously placed on the same culture medium. A more than 5-mm increase in a zone diameter for either antimicrobial agent tested in combination with clavulanic acid versus its zone when tested alone was considered a positive result for ESBL. E. coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as negative and positive control strains, respectively.

Statistical Analysis

A percentage analysis of each bacterial species was performed on the total tested strains. For resistance analysis, it was considered that every single strain reacts individually to different antimicrobial concentrations, expressing the results as absolute MIC values (mg/mL). MIC was defined as the lowest concentration of antimicrobial agent that completely prevented the visible growth of the organism; MIC50 and MIC90 were defined as the concentration of the antimicrobial agents able to inhibit the growth of 50% and 90% of the population of microorganisms, respectively. The MIC breakpoints employed were those published by NCCLS14,13 and Schering-Plough Animal Health, Chile. Presence of the b-lactamase was expressed as percentage of positive strains.


A total of 1,419 isolates were identified from 2,914 milk samples cultured. S. aureus, CNS, and E. coli were the three most frequently isolated, representing 44.7%, 24.2%, and 15.7% of total isolates, respectively.

All isolates were tested for bacterial resistance. MIC values of the control strains were within the expected ranges of all antimicrobials assayed. MIC ranges of each antimicrobial tested as well as MIC50 MIC90 values and percentage of resistance of all tested strains are presented in Tables 1 through 4.

According to breakpoint analysis, a high percentage of bacterial resistance was observed among gram-positive microorganisms against some antimicrobials tested. Staphylococcus aureus strains were resistant to amoxicillin (38.1%), ampicillin (26.0%), cefquinome (24.7%), penicillin (28.8%), and lincomycin (38.9%). For other antimicrobials tested (cefoperazone, ceftiofur, pirlimicyn, and sulfamethoxazole and trimethoprim), the values were below 9%. CNS showed high resistance rates to penicillin (56.9%), lincomycin (42.3%), amoxicilllin (31.5%), and ampicillin (26.8%). Resistance values for all other antimicrobials tested were below 8%. A high resistance rate was observed among Streptococcus spp isolates, in particular, against lincomycin (61.9%) and enrofloxacin (38.1%). Resistance values for all other antimicrobials varied between 10% and 37%.

E. coli showed low resistance levels to all antimicrobials tested compared with gram-positive bacteria. Resistance to ceftiofur, enrofloxacin, gentamicin, and oxytetracycline ranged between 11% and 21%. For all other antimicrobials, resistance varied between 1% and 7%, with the exception of sulfamethoxazole and trimethoprim, which were 100% sensitive strains. Regarding b-lactamase production, 34.9% of all Staphylococcus spp resistant to at least one cephalosporin or b-lactamic agent gave a positive result to a cefinase test. ESBL producer strains were not found in E. coli isolates.


Frequency and distribution of mastitis pathogens isolated in the present study are very similar to those previously reported in Chile.8,15,16 However, it is interesting to note that the isolation rate of CNS has been increasing during recent years compared with previous reports. This trend had also been described in other countries such as Finland, Brazil, and Belgium.3,17,18 The role of CNS as a mastitis pathogen is not clearly established. Although it is well known that these microorganisms can easily colonize the teat canal, increasing somatic cell count in milk and lowering milk quality, they also contribute to improving the white cell barrier of the mammary gland impairing invasion and colonization of other major mastitis pathogens.19,20

To analyze the resistance of the isolated strains to amoxicillin, cefoperazone, cefquinome, ceftiofur, enrofloxacin, gentamicin, lincomycin, and sulfamethoxazole and trimetoprim, we used the breakpoints for other animal infections caused by the bacteria we studied. This was necessary because the NCCLS13 only provides this information in bovine mastitis for penicillin, ampicillin, oxytetracycline, and pirlimycin. To analyze the isolated strains to florfenicol, we considered the breakpoints given by Schering Plough Animal Health, Chile.

The high level of resistance documented in this study among gram positive bacteria probably reflects the lack of an organized antimicrobial resistance surveillance program for animal foods in Chile as has been implemented in the European Community and other countries.2,21,22 The World Health Organization has stated that antimicrobial resistance is a serious, complex hazard of international concern and recommends that such programs be established world wide for both human and veterinary medicine.4

Analyzing the resistance of each strains isolated, the high level of CNS resistance to b-lactam is alarming. This situation was already described in Finland3 and Sao Paulo, Brazil.17 Though CNS are considered minor pathogens and can be treated successfully with antibiotics,23 the result in this study must be considered. These organisms may be a reservoir of resistant strains and become an important clinical hazard, a situation that has already been observed in other places.3,17

Also, our results for Streptococcus species are interesting. A number of international reports have shown that this genus is highly sensitive to b-lactam and cephalosporin.3,24 However, this was not the case in our study: a high proportion of Streptococcus species were resistant to this group of drugs. This situation can be explained by the extensive use of these drugs in Chile for many years. A high resistance level of Streptococcus species was also found against enrofloxacin; although little information is available about resistance to this drug. It is important to note that Myllys et al.3 found 7% of Streptococcus strains isolated from bovine mastitis in Finland were resistant.

The high resistance to lincomycin seen among gram-positive bacteria may be a consequence of its extended use in Chile for the treatment of bovine mastitis for more than 20 years. It is well known that the defense mechanisms of Streptococcus and Staphylococcus against macrolides and lincosamines are plasmid or transposons encoded that can be strongly induced, resulting in high resistance levels.25,26

Sensitivity of E. coli strains to most antibiotics tested was greater than most gram-positive organisms, and a high resistance level was only observed against oxytetracycline and enrofloxacin.

The high percentage of b-lactamase-producing Staphylococcus strains detected by the Cefinase test (34.9%), correlates well with the high MIC values obtained against the b-lactamic and cephalosporin agents. Similar results were reported for Staphylococcus aureus isolated from bovine mastitis in Sweden, Switzerland, the United States, Ireland,1 Finland,3 and Australia.24 However, they are much lower than those reported in Argentina,27 England, and Germany.1 According to De Oliveira et al,1 the percentage of b-lactamase-producing strains would have been higher after induction with penicillin or oxacillin. Therefore, we suggest that induction procedures should be incorporated as routine testing in our country to improve the detection of this type of enzyme.

In human medicine, the prevalence of ESBL of 1% to 4.8% among isolates of E. coli has been reported by other authors.28,29 In veterinary medicine, the ESBL has been report in E. coli isolates associated with bovine calf diarrheal disease.30 In our study, ESBL was not detected among isolates from bovine mastitis.

According to the results reported here, it is possible to conclude that veterinary medicine experts in Chile are not aware of the worldwide hazard of bacterial resistance as denounced by the World Health Organization.4 This situation further reinforces the urgent need for establishing a rational, organized control program for antimicrobial usage in animal health. To avoid further misuse of antibiotics in veterinary prescription, a permanent national bacterial-resistance surveillance program must be implemented as soon as possible. To this end, Aarestrup31 indicated that highest level of bacterial resistance is seen in countries in which there are no restriction policies on antimicrobial use.


Staphylococcus aureus remains the most frequent mastitis pathogen in Chile, followed by the environmental mastitis-causing organisms CNS and E. coli. Bacterial resistance is greater among gram-positive bacteria, especially against b-lactamic agents and lincomycin. A high proportion of Staphylococcus strains produce b-lactamase. E. coli organisms that are resistant to third-generation cephalosporins are not ESBL producers. The results in the present study clearly show that our country is at risk for the world hazard of bacterial resistance.


The authors gratefully acknowledge all private veterinarians who took part in this research project by either arranging dairy farm visits or collaborating in the sampling routine. The financial support from FONDECYT Chile (Research Project N1000–782) is also fully acknowledged.


1. De Oliveira AP, Watts J, Salmon S, Aarestrup F: Antimicrobial susceptibility of Staphylococcus aureus isolated from bovine mastitis in Europe and the United States. J Dairy Sci 83:855–862, 2000.

2. Martel JL, Tardy F, Brisabois A, et al: The French antibiotic resistance monitoring programmes. Int J Antimicrob Agents 14:275–283, 2000.

3. Myllys V, Asplund K, Brofeldt E, et al: Bovine mastitis in Finland in 1998 and 1995: Changes in prevalence and antimicrobial resistance. Acta Vet Scand 39:119–126, 1998.

4. World Health Organization: Overcoming antimicrobial resistance, in Report on Infectious Diseases. Geneve, Switzerland: World Health Organization, 2000, p. 67.

5. National Mastitis Council: Current concepts of bovine mastitis. 4th ed. Arlington, VA: National Mastitis Council, 1996.

6. Fox LK, Gay JM. Contagious mastitis. Vet Clin North Am 9:475–487, 1993.

7. Gentilini E, Denamiel G, Tirante L, et al: Bovine mastitis: b–lactamase production. Staphylococcus aureus antibiotic resistance evolution. Proceedings of the 3rd International Mastitis Seminar, Tel Aviv, Israel, 1995:84–85.

8. San Martín B, Borie C, Zurich L. Estudio de resistencia bacteriana frente a diferentes antibióticos utilizados en mastitis clínica bovina. Monogr Med Vet 13:49–52, 1991.

9. San Martín B, Borie C, Gutiérrez ME, Zurich L. Estudio de sensibilidad y concentraciones mínimas inhibitorias de cefalosporinas sobre cepas de Staphylococcus aureus aisladas de mastitis clínica bovina. Av Cs Vet 7:57–60, 1992.

10. Borie C, San Martín B, Guerra D, Zurich L. Estudio de sensibilidad frente a diferentes antibióticos y concentraciones mínimas inhibitorias de 3 cefalosporinas en cepas de Escherichia coli aisladas de mastitis séptica bovina. Av Cs Vet 8:134–137, 1993.

11. San Martín B, Cañon H, Iragüen D, et al: Depletion study of trimethoprim and sulphadiazine in milk and its relationship with mastitis pathogenic bacteria strains minimum inhibitory concentrations (MICs) in dairy cows. J Vet Pharmacol Ther 24:1–6, 2001.

12. International Dairy Federation: Isolation and identification of mastitis bacteria. IDF Document No. 132, Brussels, Belgium, 1981;19.

13. National Committee for Clinical Laboratory Standards: Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; Approved Standard. NCCLS Document M31–A, Vol 19, No 11, 1999.

14. National Committee for Clinical Laboratory Standards: Methods for dilution antimicrobial susceptibility test for bacteria that grow aerobically. Approved Standard. NCCLS Document M7–A3, Vol. 13, Number 25, 1993.

15. Kruze J, Chauhán E, Gonzalez V, Santos A: Mastitis clínica: I. Estudio bacteriológico en rebaños bovinos de leche de la provincia de Osorno. Proceedings VI Congreso Nacional de Medicina Veterinaria, Santiago, Chile. 1986:SA–025.

16. León B: Frecuencia de aislamiento de los principales agentes de mastitis en el sur de Chile. In: II Seminario Calidad de Leche Bovina. Osorno, Chile: Colegio Médico Veterinario de Chile, Consejo Regional Osorno, 1997;34–44.

17. Costa EO, Benites NR, Guerra JL, Melville PA: Antimicrobial susceptibility of Staphylococcus spp. isolated from mammary parenchymas of slaughtered dairy cows. J Vet Med B Infect Dis Vet Public Health 47:99–103, 2000.

18. Devriese LA, Laevens H, Haesebrouck F, Hommez J: A simple identification scheme for coagulase negative staphylococci from bovine mastitis. Res Vet Sci 57:240–244, 1994.

19. Matthews KR, Harmon RJ, Langlois BE: Effect of naturally occuring coagulase-negative staphylococci infections on new infections by mastitis pathogens in the bovine. J Dairy Sci 74:1855–1859, 1991.

20. Nickerson SC, Boddie RL: Effect of naturally occuring coagulase-negative staphylococcal infections on experimental challenge with major mastitis pathogens. J Dairy Sci 77:2526–2536, 1994.

21. Aarestrup FM, Bager F, Jensen NE, et al: Resistance to antimicrobial agents used for animal therapy in pathogenic, zoonotic and indicator bacteria isolated from different food animals in Denmark: A baseline study for the Danish Integrated Antimicrobial Resistance Monitoring Programme (DANMAP). APMIS 106:745–747, 1998.

 22.        Bager F: DANMAP: monitoring antimicrobial resistance in Denmark. Int J Antimicrob Agents 14:271–274, 2000.

23. Pyörälä S, Pyörälä E: Efficacy of bovine clinical mastitis therapy during lactation. Proceedings of the XVII Nordic Veterinary Congress. Reykjavik, Iceland: The Icelandic Veterinary Association, 1994:42–45.

24. Sobiraj A, Kron A, Schollmeyer U, Failing K: Federal investigations on the distribution and in vitro resistance udder pathogenic bacteria in the milk of cows with subclinical mastitis. Tierärztl Prax 25:108–115, 1997.

25. Matsuoka M: Study of macrolide, lincosamide, and streptogramin B antibiotic resistance in Staphylococcus aureus. Yakugaku Zasshi 120:374–386, 2000.

26. Zhong P, Cao Z, Hammond R, et al: Induction of ribosome methylation in MLS-resistant Streptococcus pneumoniae by macrolides and ketolides. Microb Drug Resist 5:183–188, 1999.

27. Gentilini E, Denamiel G, Llorente P, et al: Antimicrobial susceptibility of Staphylococcus aureus isolated from bovine mastitis in Argentina. J Dairy Sci 83:1224–1227, 2000.

28. Pai H, Lyu S, Lee JH, et al: Survey of extended-spectrum beta-lactamases in clinical isolates of Escherichia coli and Klebsiella pneumoniae: Prevalence of TEM-52 in Korea. J Clin Microbiol 37:1758–1763, 1999.

29. Stobberingh EE, Arends J, Hoogkamp-Korstanje JA, et al: Occurrence of extended-spectrum beta-lactamases (ESBL) in Dutch hospitals. Infection 27:348–354, 1999.

30. 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 44:607–610, 1999.

31. Aarestrup FM: Association between the consumption of antimicrobial agents in animal husbandry and the occurrence of resistant bacteria among food animals. Int J Antimicrob Agents 12:279–285, 1999.



Table 1. Minimun Inhibitory Concentrations and Percentage of Resistance of 635 Staphylococcus aureus Strains Isolated From Clinical Bovine Mastitis Against Different Antimicrobial Agents


                                                                                    Range (µg/ml)


  agent                          MIC50           MIC90                  Range                 Breakpoint       % Resistance


  Amoxicillin                     0.125              1.0                   0.06–4.0                     ³ 0.5                     38.1


  Ampicillin                       0.125              1.0                   0.06–4.0                     ³ 0.5                     26.0


  Cefoperazone                0.5                2.0                  0.25–16.0                    ³ 8.0                      6.1


  Cefquinome                    2.0               16.0                 0.25–32.0                    ³ 8.0                     24.7


  Ceftiofur                         0.5                2.0                 0.125  16.0                   ³ 8.0                      6.1


  Enrofloxacin                 0.125              0.5                   0.06–8.0                     ³ 2.0                      2.5


  Florfenicol                       0.5                2.0                 0.25–128.0                  ³ 32.0                     4.9


  Gentamicin                      1.0                4.0                  0.25–32.0                   ³ 16.0                     3.3


  Lincomycin                     0.5                8.0                 0.125–16.0                   ³ 2.0                     38.9


  Oxytetracycline              2.0                4.0                   0.5–64.0                    ³ 16.0                     6.8


  Penicillin                         0.25               1.0                  0.062–2.0                   ³ 0.25                    28.8


  Pirlimycin                         0.5                2.0                 0.125–16.0                   ³ 4.0                      8.3


  Trimethoprim              0.25/4.75       1.0/19.0     0.25/4.75 to 8.0/152.0      ³ 4.0/76.0                  6.9




Table 2. Minimun Inhibitory Concentrations of 343 Coagulase-Negative Staphylococcus (CNS) Strains Isolated From Clinical and Subclinical Bovine Mastitis Against Different Antimicrobial Agents


                                                                                    Range (µg/ml)


   agent                         MIC50           MIC90                  Range                 Breakpoint       % Resistance


  Amoxicillin                      0.25               2.0                  0.125–4.0                    ³ 0.5                     31.5

  Ampicillin                       0.125              1.0                   0.06–4.0                     ³ 0.5                     26.8

  Cefoperazone               0.25               1.0                 0.125–16.0                   ³ 8.0                      2.6

  Cefquinome                   0.25               2.0                 0.125–32.0                   ³ 8.0                      7.6

  Ceftiofur                        0.25               2.0                 0.125–16.0                   ³ 8.0                      2.6

  Enrofloxacin                  0.25               1.0                  0.125–8.0                    ³ 2.0                      2.9

  Gentamicin                     0.50               4.0                  0.25–64.0                   ³ 16.0                     4.1

  Lincomycin                     1.0                4.0                  0.25–16.0                    ³ 2.0                     42.3

  Oxitetracycline               2.0                8.0                  0.25–32.0                   ³ 16.0                     1.9

  Penicillin                         0.25               1.0                   0.06–4.0                    ³ 0.25                    56.9

  Trimethoprim              0.25/4.75       1.0/19.0     0.125/2.38 to 4.0/76.0      ³ 4.0/76.0                  0.9





Table 3. Minimun Inhibitory Concentrations of 218 Streptococcus Strains Isolated From Clinical and Subclinical Bovine Mastitis Against Different Antimicrobial Agents


                                                                                    Range (µg/ml)


  agent                         MIC50         MIC90                 Range                 Breakpoint       % Resistance


  Amoxicillin                       2.0               16.0                  0.5–64.0                     ³ 8.0                     31.2

  Ampicillin                         2.0               16.0                  0.5–64.0                     ³ 8.0                     34.9

  Cefoperazone                1.0                8.0                  0.25–32.0                    ³ 8.0                     20.2

  Cefquinome                    1.0                8.0                  0.25–32.0                    ³ 8.0                     18.8

  Ceftiofur                         1.0                8.0                  0.25–32.0                    ³ 8.0                     14.2

  Enrofloxacin                   0.5                4.0                   0.25–8.0                     ³ 2.0                     38.1

  Florfenicol                       2.0               32.0                0.25 (128.0                  ³ 32.0                    11.0

  Lincomycin                     1.0                4.0                   0.25–8.0                     ³ 1.0                     61.9

  Oxitetracycline               1.0               16.0                 0.25–64.0                    ³ 8.0                     27.5

  Penicillin                          1.0                8.0                  0.25–64.0                    ³ 4.0                     36.7

  Pirlimycin                         0.5                2.0                 0.062–1 6.0                  ³ 4.0                     10.1

  Trimethoprim               0.50/9.0        4.0/76.0     0.125/2.38-32.0/608.0      ³ 4.0/76.0                 17.4



Table 4. Minimun Inhibitory Concentrations of 223 Escherichia coli Strains Isolated From Clinical and Subclinical Bovine Mastitis Against Different Antimicrobial Agents


                                                                                    Range (µg/ml)


  agent                         MIC50         MIC90                 Range                 Breakpoint       % Resistance


  Cefoperazone                0.5                2.0                 0.125–32.0                   ³ 8.0                      3.6

  Cefquinome                    0.5                2.0                 0.125–32.0                   ³ 8.0                      6.3

  Ceftiofur                         1.0                8.0                 0.125–32.0                   ³ 8.0                     11.2

  Enrofloxacin                  0.25               4.0                 0.062–16.0                   ³ 2.0                     19.3

  Florfenicol                       1.0                8.0                  0.25–64.0                   ³ 32.0                     6.7

  Gentamicin                      2.0               32.0                 0.5 -128.0                   ³ 16.0                    15.7

  Oxitetracycline               2.0               32.0               0.25 ³ 128.0                 ³ 16.0                    20.6

  Trimethoprim             0.125/2.38       0.5/9.5      0.063/1.20 to 2.0/38.0      ³ 4.0/76.0                  0.0



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