In vitro sensitivity of porcine respiratory pathogens to tiamulin/amoxicillin combination

N. Prapasarakul 1, P. Klakai 1, N. Taechakriengkri 1, J. Vasuvat 1, A. Sae-Ue 1, W. Neramitmansook 2, M. Makhanon 3

1 3

Faculty of Veterinary Science, Chulalongkorn University. 2National Institute of Animal Health, Department of Livestock Development. Novartis Ltd Animal Health BU, Bangkok, Thailand.

Introduction
Haemophilus parasuis (HP), Actinobacillus pleuropnuemoniae (APP), Streptococcus spp., and Pasteurella multocida (PM) are common causes of severe forms of pneumonia in swine9. In general, bacterial respiratory infection can be effectively controlled by antimicrobial treatment. Tiamulin provides outstanding activity against swine dysentery and chronic pneumonia3. The synergistic activity of tiamulin and chlortetracycline against PM, APP and Bordetella bronchiseptica was reported previously1. Amoxicillin, a beta lactam antibiotic, inhibits cell wall synthesis in active phase of bacteria6. It is commonly mixed into the feed for post weaning pigs. Drug synergism between pleuromutilin antibiotics and amoxicillin has not been investigated until now. The aims of this study were to determine

minimal inhibitory concentrations (MICs) of antimicrobials to respiratory pathogens isolated from porcine pneumonic lungs and to determine the combined effect of tiamulin and amoxicillin.

performed in broth micro-dilution assay8. Polystyrene microplates were used for the test. The suspension was diluted until 5x105 CFU for the inoculum5. Antimicrobial agents: Two antimicrobial agents - tiamulin hydrogen fumarate (Novartis, Switzerland), amoxicillin (Sigma, USA) - were used for determination of MICs and their combined effect by using chessboard titration technique2,8. Duplicate two-fold dilutions of antibiotics were made from 0.5 256 mcg/ml. Sensitivity of the bacteria strains to each antimicrobial and combination of antimicrobials was tested in duplicate. The fractional inhibitory concentration (FIC) index was determined using the previously described protocol8.

Materials and Methods

P. multocida, Streptococcus spp., APP and H. parasuis were isolated from pneumonic pig lungs in Thailand during 2005. Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC27853 were used as control strains. H. parasuis was detected by published polymerase chain reaction7. MICs of each antibacterial agent were determined for each bacterial isolate according to the CLSI (former NCCLS) method4. The sensitivity test was

Results and Discussion

Twenty eight strains of the pathogens (5x P. multocida, 5x Streptococcus spp.,7x A. pleuropneumoniae, 11x H. parasuis) were isolated from pneumonic lungs and used for the test. The MIC values of tiamulin and amoxicillin to each pathogen are summarized in Table 1. 3 of 11 H. parasuis and 2 of 7 of APP isolates, showed susceptibility to tiamulin (breakpoint 16), and all Streptococcus and P. multocida isolates (breakpoint 4) were sensitive to tiamulin5. On the other hand, amoxicillin was effective only against the H. parasuis isolates. For APP and HP, the MIC concentration of tiamulin and amoxicillin used in combination ranged from 0.5-8 and 0.015-4 mcg/ml, respectively. The FIC indices show synergism between tiamulin and amoxicillin against all tested APP and H. parasuis strains. However, all tested P. multocida and Streptococcus

Table 1. MICs of amoxicillin and tiamulin for respiratory pathogens

amoxicillin Bacteria species N MIC 50 (mcg/ml) 4 32 64 128 Range (mcg/ml) 2-128 16-64 32-128 128 tiamulin MIC 50 (mcg/ml) 8 32 4 4 Range (mcg/ml) 1-128 16-128 2-16 4-16

H.parasuis A.pleuropneumonia Streptococcus spp. P.multocida

11 7 5 5

Table 2. Synergism between tismulin and amoxicillin by using the broth microdilution assay
Result by Bacteria species N Synergism (FIC0.5) 11 7 0 0 Indifference (0.5FIC4) 0 0 5 5 Antagonism (FIC>4) 0 0 0 0

H.parasuis A.pleuropneumonia Streptococcus spp. P.multocida

11 7 5 5

spp., isolates showed indifferent levels of MICs. Antagonistic effects were not observed during the experiment (Table 2).The results indicate that amoxicillin acts synergistically together with tiamulin against pathogens like APP and H. parasuis. Single application of tiamulin provides satifactory effect against Streptococcus spp., and P. multocida In conclusion, combined use of tiamulin and amoxicillin may be useful in the treatment of complex infections based on APP and HP. The recommendation not to combine amoxicillin with a drug inhibiting bacterial protein synthesis6, has to be reconsidered based on the trial results (Table 2). However, the mechanism should be proven in further studies.

References
1. Burch DG. et al., 1986 Vet. Rec. 119, 108-112. 2. Jones RM. et al., 2002. J. Clin. Microbiol. 40, 461-465. 3. Friendship R.M. 2000 Antimicrob Use in swine. In: Antimicrob Therapy. P. 602-617. 4. NCCLS, 2000. M7-A5, Wayne, Pa. 5. McDermott PF. et al., 2001. J Clin. Microbiol. 39. 4283-4287.

6. Mogenet, L., 2002 Antibiotherapy. 27-73.

Rational

7. Oliveira S. et al., 2001 J. Vet. Diagn. Invest. 13, 495-501. 8. Rand, KH. et al., 1993. Antimicrob Chemother. 37, 613-615. 9. Taylor, D.J. 1999. Disease of swine. P. 345-354.

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