The Potential Molecular Mechanism Of Subtherapeutic Fluoroquinolones Exacerbate Leptospirosis

Leptospirosis,caused by pathogenic spirochetes,is responsible for a worldwide zoonotic disease.Animals may get this disease when their damaged skin and mucous membrane direct contact with urine or indirectly from contaminated water.The clinical symptoms of leptospirosis may range from jaundice,hemoglobinuria,abortion,to a severe infection with renal and hepatic failure,and even death.The epidemic of this disease harms the human health and makes heavy losses in animal husbandry.It is reported that treatment of chronically infected mice with subtherapeutic dose ciprofloxacin served to exacerbate infection by regulating the innate immunity and virulence of pathogen,for example staphylococcus and colibacillus.In our previous study,we found that the subtherapeutic dose norfloxacin induced worse result of infected hamsters.Lots of longer leptospira were founded when together with leptospira and subtherapeutic dose norfloxacin,whose concentration was belowed the MIC to leptospira.Whether the subtherapeutic dose norfloxacin changed the virulence of leptospira or not? If so,how about other quinolone antibiotics? What is their regulatory mechanism? Finding answers of these questions will help to illuminate the pathogenic mechanism of leptospira.In this study,we constructed the acute infection model by intraperitoneal injection of Leptospira interrogans serovar Icterohaemorrhagiae(56601).The survival rates of hamsters were record after treatment with norfloxacin and ciprofloxacin in various doses during the21-day test period.The expression levels of TNF-? and IL-1? in blood,liver,kidney,and lung were detected by RT-q PCR.The number of leptospira in liver,kidney,and lung was detected by q PCR.The histopathology was also studied.These works will evaluate the efficacy of subtherapeutic dose norfloxacin and ciprofloxacin against leptospirosis.In vitro,leptospira were treated with low-concentration norfloxacin.Then using dark-field microscopy and laser scanning confocal microscope observed the feature of leptospira.RT-q PCR was used to quantify the expression of outer virulence proteins(Kdp A,Kdp B,Lip L21,Lip L41,Lip L71,Lip L32,Fla A,Fla B,Lig A,Lig B)of leptospira.Hamsters were injected the low concen-trations norfloxacin-treated leptospira.Then the survival rates of hamsters were record.The risk of usingsubtherapeutic dose Fluoroquinolones and its mechanism were studied.The results showed that leptospira at the dose of 102~106induced stable half died,and the LD100 of leptospira 56601 was 107.Treatment with subtherapeutic dose norfloxacin and ciprofloxacin made a lower survival rates than untreated hamsters.Using the q PCR,the leptospira burden in liver,kidney and lung were detected,and leptospira burden was more in subtherapeutic dose norfloxacin group.Meanwhile,treatment with subtherapeutic dose norfloxacin aggravated the pathological change of liver,kidney and lung in hamsters,delayed the gene expression of TNF-? and IL-1? in blood,liver,kidney and lung in hamsters.The MIC of norfloxacin against the leptospira 56601 was 1-2?g/ml.The longer leptospires were founded when norfloxacin was at the concentration of 0.25?g/ml.The reference genes of leptospira,Lip L41 and Lip L71,were filtered by ge Norm.And the low concentrations norfloxacin improved the gene expression of Fla B and Lig B using 2-(?) method.In conclusion,our results confirmed the risk of using subtherapeutic dose Fluoroquinolones in leptospirosis.The subtherapeutic dose Fluoroquinolones can delay the inflammatory response of host and regulate the expression of virulence protein of leptospira.These will contribute to the risk happen.