Combination Susceptibility Testing Of Various Antimicrobials In Vitro And Effects Of Sub-MIC Antimicrobials On Bacterial Biofilms Formation

In recent years, antimicrobials used in animal can be excessive. For example, using of antimicrobials as daily preventive agents and animal grown promoter; Not according to the clinical guidelines; Abuse of antimicrobials for unknown etiology, but it’s ineffective to pure fungi infection; Adopting kinds of antimicrobials in combination blindly for serious and mixed infection. All these undoubtedly increase bacterial resistance and result to the use of antimicrobials are in subinhibitory concentration (sub-MIC). While some researches indicated that antibacterial agents may interfere biofilms formation at sub-MIC, so as to avoid causing chronic infection. For this reason, the study focuses on two aspects: how to select right antibacterial combinations for therapy and how to take sub-MIC for preventive drugs correctly.OBJECTIVES:the trials based on the original classification of antimicrobials, adding quinolones, rifamycins, lincosamide and herbal extracts-berberine, study their interactions between drugs on clinical isolate of Staphylococcus aureus, Escherichia coli and Pasteurella, representing Gram-positive bacteria and Gram-negative bacteria. In addition, we studied the effects of sub-MIC antimicrobials on clinical isolate of bacteria biofilms formation. That gives the hope through the experiment can provide certain theoretics for veterinary clinical using, improve the improper antimicrobial combinations. While also warning people that the use of sub-MIC for biofilms formation must be based on laboratory research results.METHODS: the sensitivities of12kinds of drugs on3species of bacteria were evaluated by determining the minimal inhibitory concentration (MIC) with the method of broth two-fold dilution and the interactions between them were evaluated by checkerboard micro-dilution method and expressed as fractional inhibitory concentration (FIC) index. The biofilm-forming ability of isolates and influences of specific antibiotics at sub-MIC on the biofilms formation were detected by microtiter plate assay with optical density (OD) index.RESULTS:(1) Antibacterial combinations of Staphylococcus aureus, representing Gram-positive bacteria, are as following: the results of original four types antibacterial combinations are consistent with previous reports. Enrofloxacin in combination with ceftiofur showed an additive effect, also rifampin in combination with ceftiofur or doxycycline, berberine in combination with florfenicol or sulfadimidine; when enrofloxacin with doxycycline, florfenicol or sulfamethazine, rifampin with kanamycin or florfenicol, lincomycin with tylvaosin or colistin sulfate, were combined antagonistic effect. Rests of the combinations were expressed indifferent effect, while the FIC of berberine in combination with tylvaosin or ceftiofur are1.5, indicating that combinations can be applied to reduce the toxicity and economic costs with fewer dose.(2) Antibacterial combinations of Escherichia coli and Pasteurella, representing Gram-negative bacteria, are as following:the results of original four types antibacterial combinations are consistent with previous reports except frist-class antimicrobial in combination with third-class are not all antagonist effect, when FIC was1.5, it’s suggested that they can be used together with fewer dose. Enrofloxacin in combination with ceftiofur, colistin sulphate, sulfamethazine or berberine showed synergistic/additive effect, with florfenicol or rifampicin performed antagonistic/indifferent effect. Rifampin with colistin sulphate, doxycycline, florfenicol or berberine was combined additive effect, with kanamycin, lincomycin or enrofloxacin showed antagonistic/indifferent effect. Berberine with amoxicillin or florfenicol matched indifferent effect, and the FICs were1.5, with others all showed additive/synergistic effect. Lincomycin with colistin sulphate or sulfamethazine showed synergistic/additive effect, with amoxicillin, kanamycin, tylvaosin, florfenicol or rifampicin expressed antagonistic effect. Rests of the combinations were indifferent effect.(3) Compared with the control group, Staphylococcus aureus has a strong ability to form biofilms. Experimental conditions were screened, and identified to culture2days in TSB at37℃as optimal conditions. Influences of specific antibiotics (kanamycin, colistin sulphate, tylvaosin, clarithromycin, enrofloxacin, berberine and lincomycin) at sub-MIC on the biofilms formation in Staphylococcus aureus were researched. Results show that only tylvaosin significantly enhanced biofilm formation, while clarithromycin expressed opposite effect, suggesting that the same type drugs with different structures showed diffrent effect. The amount of biofilm formation reached the minimal in1/2MIC of enrofloxacin, with a dose-dependent effect. Berberine and lincomycin showed significant inhibition effect on higher sub-MIC.