The Study Of Susceptibility Testing In Vitro Methods And Drug-resistant Spectral Analysis Of Bartonella Bacterial

BackgroundBartonella is a newly discovered microorganism which can cause a variety of diseases of animals and human. More and more diseases and clinical symptoms were confirmed to be associated with Bartonella infection. The extensive pathogenicity of Bartonella caused great concern and attention. At present, Bartonella species have consisted of 22 species and 3 subspecies, at least 9 of which can cause human diseases, mainly B.bacilliformis, B.henselae, B.quintana, B.elizabethae, B.grahamii, B.clarrdgeriae and B.vinsonii subsp berkhoffii, B. vinsoniisubsp arupensis. Bartonella spp. are not only the main pathogens of a cat-scratch disease (CSD), but also have relations with the Carrion's disease, endocarditis, bacillary angiomatosis, and Chronic Bartonella hyperlipidemia. Sometimes they can cause retinitis, encephalitis, glomerulonephritis and pneumonia. CSD is more prevalent in clinical practice. Cat-scratch disease is usually self-healing in immunocompetent individual but for the person with immune deficiency (especially AIDS), infections will lasting very long time, even the whole life, and clinical symptoms often repeated. Due to the extensive use of broad-spectrum antibiotics and the emerging of new pathogens, drug-resistance of bacterial has become a hot spot of global concern. Rational use of antibiotics is not only the premise of reducing the drug-resistant strains, but also the key to the successful treatment of infections. So it is imperative to establish a rapid and accurate susceptibility testing methods in vitro, to accurately detect the sensitivity of bacteria to antibiotics, as well as to monitor bacterial drug resistance and guide clinical regular medication.OBJECTIVE:To establish and perfect the susceptibility test methods of Bartonella in vitro-E test method (second gold standard); the E test method and gold standard agar dilution method were compared to determine the optimal susceptibility test methods (the optimal concentration of bacteria, the optimal medium and time to incubate) of Bartonella in vitro. The method was also used to detect the antibiotics sensitivity and minimum inhibitory concentration (MIC) of Bartonella isolates from different hosts (cats, dogs, human, mouse). At the same time, we will also analyze the drug-resistant spectrum to provide reference for guide clinical use of drugs and drug resistance monitoring.Methods:E test method, susceptibility test methods of Bartonella in vitro were established. At first, sensitivity and the minimum inhibitory concentration (MIC) to 22 antimicrobial compounds of international standards strains of Bartonella bacteria (B.henselae, B.grahami) were detected using chocolate medium and 5% sheep blood agar medium of soybean trypsin experiment, which will determine the optimum medium. Different concentrations of inoculation were used to determine the optimum inoculation concentration, and results were observed at the 7th and 14th day to determine the optimum incubation time. This method was compared to the agar dilution method (gold standard) to evaluate the reliability. The sensitivity and the minimum inhibitory concentration (MIC) of Bartonella strains from different hosts (cats, dogs, monkeys, mouse)to 22 antimicrobial compounds were detected using the established E test method.Results:we established susceptibility test methods of Bartonella in vitro-E test method and confirm the trypsin containing 5% sheep blood agar was the optimal medium of soybean, the optimal inoculation concentration is 2.0MCF, for most of the Bartonella bacteria, the 7th day is the optimal time to incubate, and 14th day was the best time for a few Bartonella species which grew especially slowly (B.bacilliformis, B.clarrdgeriae). According to the comparison with the gold standard agar dilution method (gold standard), both methods had high degree of consistency. Furthermore, E test is easy to operate and well repetive. E test method was confirmed to be the optimum susceptibility testing method of Bartonella in vitro. With this method, the rest 6 Bartonella strains (B.clarrdgeriae B.bacilliformis, B.elizabethae, B.d), and 26 isolates from different hosts were detected, Bartonella strains were sensitive to Doxycycline, Azithromycin, Erythromycin, Clarithromycin, Amoxicillin, Penicillin G,Tobramycin, Gentamicin, Netilmicin, Ceftazidime,Rifampicin, Amoxicillin-clavulanate, and Ceftriaxone. The MIC values below the minimum detection concentration of E test strips (0.002 or 0.016). However, difference of MIC to the same antibiotic among different species of Bartonella was significant. B.henselae (ATCC49882) is Recommended as a quality control strains of susceptibility testing of Bartonella in vitro.Conclusion:The E test method was confirmed to be the optimal susceptibility testing method of Bartonella in vitro. It can provide a new, simple and effective method for monitoring the drug-resistance of Bartonella. The testing capacity of E test method was significantly higher than agar dilution method. Furthermore, some Bartonella strains were detected using E test method, which would provide scientific information for the clinical medication and the establishment of Bartonella drug-sensitive standard.