Penetration of the antibiotic fosfomycin into swine intestinal mucosa colonized with Lawsonia intracellularis

PEREZ, D S; SORACI, A L; MARTINEZ, G; FERNANDEZ PAGGI, M B; RICCIO, M B; DIEGUEZ, S; TAPIA, M O

Nantes

Congreso; 13th International Congress of the European Association for Veterinary Pharmacology and Toxicology; 2015

European Association for Veterinary Pharmacology and Toxicology

Penetration of the antibiotic fosfomycin into swine intestinalmucosa colonized with Lawsonia intracellularisD. PE´REZ, A. SORACI, G. MARTINEZ, M. B. FERNANDEZPAGGI, M. B. RICCIO, S. DIEGUEZ & M. O. TAPIAFisiopatologıa, Facultad de Cs. Veterinarias, UNCPBA, Tandil,Buenos Aires, ArgentinaINTRODUCTION/OBJECTIVEFosfomycin (FOS) is an antibiotic (ATB) used for the treatmentof lung and enteric infections of pigs. Intracellular fluids of enterocytescan act as biophase for Lawsonia intracellularis (LAW),causative agent of porcine proliferative enteropathy (PPE),which presence could modify ATB penetration. The aim of thisstudy was to determine FOS penetration into swine intestinalmucosa colonized with LAW.MATERIALS AND METHODSFour healthy pigs in grow-finish stage; live attenuated vaccine(Enterisol Ileitis, Boehringer-Ingelheim); primers for LAW PCRdetection (GATAATCTACCTTCGAGACGG; TGACCTCAGTGTCAGTTATCGT,Invitrogen); calcium FOS. Explants were producedfrom ileum of euthanized animals. As a positive controlfor the PCR, LAW DNA was obtained from the vaccine. Explants(0.5 ml of vaccine; 24 h of incubation; 37°C) wereincubated with 580 lg ml1 of calcium FOS (0.5?6 h). Then,they were washed to remove extracellular FOS, deproteinized(1 ml of methanol) and sonicated (30 min) to release intracellularATB. Tubes were centrifuged (6 min; 4°C; 10 000 rpm),supernatants were evaporated to dryness (60°C), dry extractswere dissolved in 200 ll of HPLC water and 1 ml of Folchreagent (hexane-ethanol; 1:0.2) was added for lipids removal.Samples were shaken (20 min), centrifuged (6 min), hexanephase was discarded, 40 ll of each sample were taken and carriedto 800 ll with HPLC water, filtered and analyzed by HPLCMS/MS.RESULTSIntracellular concentration of FOS ranged between 3.75 and24.81 lg ml1 (Tmax: 4 h). On previous studies on healthyswine intestinal explants, we have found that a low concentrationof ATB enters into the enterocytes (5.84?12.99 lg ml1;Tmax: 2 h), which could be attributed to the soluble nature ofFOS. When comparing intracellular concentrations of FOSfound in explants with LAW versus those found on healthypigs intestinal explants, a higher proportion is present in explantswith the bacteria (4%) than in those explants withoutLAW (2%). However, differences were not statistically signifi-cant (p > 0.05).CONCLUSIONSAlthough FOS concentrations are not too high, they exceedthe MIC90 for E. coli (0.5 lg ml1) and Salmonella (4 lg ml1).There are no studies indicating FOS MIC90 for LAW. Nevertheless,MIC90 of various ATB for LAW ranges between 0.125and 128 lg ml1. Further studies should be carried out todetermine FOS MIC90 for LAW to discern the usefulness of thi