Antibacterial Activity Of Antimicrobial Peptide PMAP-36 Combined With Tetracycline Against Porcine Extraintestinal Pathogenic Escherichia Coli In Vitro And In Vivo

Extraintestinal pathogenic Escherichia coli(ExPEC)is an important zoonotic pathogen that can infect mammals and birds widely.In recent years,the isolation rate of ExPEC in pig farms in China has increased significantly,and most of the isolated strains are drug-resistant strains,which brings significant challenges to traditional treatment methods.Therefore,there is an urgent need to find new treatment strategies.Antimicrobial peptides(AMPs)are a class of small molecular peptides that are widely present in nature and play an essential role in combating microbial infections without inducing drug resistance.PMAP-36 belongs to the cathelicidin family of porcine antimicrobial peptides with broad antimicrobial and immunomodulatory activity.As a new treatment method,combining AMPs and traditional antibiotics is expected to improve the antibacterial effect and reduce the amount of antibiotics used.However,whether PMAP-36 can combine antibiotics to treat porcine ExPEC infection is still unclear.In this study,the antibacterial activity of PMAP-36 combined with tetracycline against porcine ExPEC was evaluated in vitro and in vivo.1.Synergistic antibacterial effect of antimicrobial peptides and traditional antibiotics on porcine ExPEC in vitroThe antimicrobial peptides CATH-1,CATH-2,CATH-3,and CATH-B1 from chicken,CRAMP from mice and PMAP-36,and PR-39 from swine were detected by broth microdilution method.The minimal inhibitory concentration(MIC)and minimal bactericide concentration(MBC)of seven antimicrobial peptides against porcine ExPEC strain PCN033 and neonatal meningitis ExPEC strain RS218 were determined.The results showed that the two pig-derived AMPs were the most bactericidal,with PMAP-36 and PR-39 having an MBC of 10μM against PCN033 and 5μM against RS218.The fractional bactericide concentration index(FBCI)of PMAP-36 and PR-39 combined with tetracycline or gentamicin against PCN033 and RS218 were measured by checkerboard method.PMAP-36 and tetracycline,gentamicin showed synergistic antibacterial effects against the multidrug-resistant strain PCN033 in vitro,with a 4-fold reduction in concentration with PMAP-36,a 16-fold reduction with tetracycline and an 8-fold reduction with gentamicin,with FBCI of 0.3125 and 0.375 respectively.2.Antibacterial effect of PMAP-36 combined with tetracycline on porcine ExPEC in vitroFurthermore,the synergistic bactericidal effect of PMAP-36 in combination with tetracycline and gentamicin was investigated.The results of time-killing curve showed that tetracycline and gentamicin at sub-MBC could not kill PCN033,but PCN033 was eliminated within 20 min when combined with PMAP-36.Bacterial morphology was observed by scanning electron microscopy,and it was found that the combination treatment of PMAP-36 and tetracycline significantly destroyed bacterial morphology compared with PCN033 alone.In addition,the combination of tetracycline and MAP-36showed lower cytotoxicity and hemolytic activity.PCN033 was passaged continuously for 30 passages under the condition of tetracycline and PMAP-36 alone or in combination,and the results showed that tetracycline alone caused a 3-fold increase in resistance to tetracycline in PCN033,while combination treatment did not change its resistance to tetracycline.Notably,PMAP-36 reduced the ability of PCN033 to invade and adhere to peritoneal macrophages,while the combination with tetracycline did not show a synergistic effect on this effect.These results showed that the combination of PMAP-36and tetracycline could enhance the bactericidal effect of porcine ExPEC and reduced the haemolytic activity and cytotoxicity of PMAP-36 on mammalian cells.At the same time,the combination of PMAP-36 and tetracycline could slow the development of tetracycline resistance of PCN033.3.Efficacy of PMAP-36 combined with tetracycline in the treatment of porcine ExPEC infection in miceTo comprehensively investigate the antibacterial activity of PMAP-36 in combination with tetracycline against porcine ExPEC.C57BL/6 wild-type mice were intraperitoneally infected with 1×10~7 CFU of porcine ExPEC strain PCN033 to establish an intraperitoneal infection model.The efficacy of PMAP-36 combined with tetracycline in treating porcine ExPEC infection was evaluated in vivo.The survival of mice treated with single drug and the combined drug was recorded.The bacterial colonization in liver,spleen,lung,blood and peritoneal lavage fluid(PLF)of mice in each treatment group was compared.HE staining was used to observe the pathological changes of lung and spleen.ELISA was used to detect the levels of inflammatory factors in tissues and PLF.The expression level of tight junction protein Occludin in lung tissue was detected by Western blot.The recruitment of immune cells in PLF was detected by flow cytometry,and the secretion and expression of chemokines were detected by ELISA.The results showed that the combined treatment of PMAP-36 and tetracycline significantly increased the survival rate of mice infected with porcine ExPEC(60%,6/10),compared with 0%(10/10)and20%(2/10)of mice treated with tetracycline or PMAP-36 alone.Compared with the single-drug treatment group,the combined-drug treatment group significantly reduced the bacterial colonization of organs,blood and PLF.The combined treatment could dramatically improve the symptoms of inflammatory cell infiltration in lung,alveolar wall thickening,inflammatory cell infiltration in spleen,splenic sarcomere atrophy,and cell necrosis in mice.The levels of inflammatory factors(IL-1β,Il-1β,IL-6,IL-12 and TNF-α)in liver,spleen,lung and PLF in the combined treatment group were lower than those in the single treatment group.There was no significant difference in the protein expression of tight junction protein Occludin among the treatment groups.The number of immune cells in the PLF of the combination group was significantly higher than that of the single drug group.Still,there was no difference in the expression levels of chemokines CXCL1 and CXCL2.These results indicate that PMAP-36 combined with tetracycline can protect mice from systemic infection with porcine ExPEC and has an excellent synergistic therapeutic effect,which provides some experimental basis for the combined application of these two drugs in clinical practice.