| Staphylococcus aureus is the main pathogen of chicken staphylococcal disease.There are many types of this disease,causing huge economic losses to the chicken industry.Clinical manifestations include acute septicemia,arthritis,chick umbilitis and periostitis.Bacitracin(BAC),a polypeptide antibiotic,is a secondary metabolite produced by Bacillus licheniform.Bacitracin methylene disalicylate(BMD),a second-generation bacitracin product,can be used to treat diseases such as S.aureus infection and necrotizing enteritis in chickens.The characteristics and mechanism of bacitracin-induced bacterial resistance are currently unclear in the treatment.Therefore,this study explored the resistance selection of bacitracin on S.aureus in vivo and in vitro and analyzed the expression levels of bacitracin resistance genes in different resistant S.aureus,in order to more clearly and thoroughly reveal the resistance mechanism of S.aureus to bacitracin.In this study,a strain of S.aureus FS127 that is bacitracin-sensitive and pathogenic was initially screened out using drug susceptibility tests and animal pathogenicity tests.The drug susceptibility test results of 169 clinical isolates of S.aureus showed that S.aureus showed high resistance to bacitracin,with a resistance rate of 98.82%.Under laboratory conditions,1/2 MIC bacitracin was used to induce resistance of the strain FS127 in vitro,the drug resistance rate of S.aureus was calculated on bacitracin-containing plates at different drug concentrations and the resistance induction characteristics were analyzed;the growth curves and susceptibility to six antibiotics,including gentamicin,ciprofloxacin,tetracycline,doxycycline,chloramphenicol and penicillin,were determined for different resistance levels of S.aureus.Under clinical conditions,a model of S.aureus FS127-infected chickens was first established;then the clinically recommended drug dose of 200mg/L(water)was used as the middle-dose administration group;a low-dose administration group(50 mg/L),a high-dose administration group(500 mg/L)and a model group were set at the same time.The experiment was divided into two cycles in which the chickens were continually administered BMD for 7 d and then stopped for 7 d.Cloacal swabs were collected on days 0,2,6,10,13,17,20,24,27,and 31 of the experiment,which were used to preliminarily screened S.aureus through CHRO Magar plates containing 0×MIC,2×MIC,8×MIC bacitracin;simultaneously calculate the colonization and drug resistance levels of S.aureus;the susceptibility of intestinal isolates of S.aureus to bacitracin and the other 6 antibiotics was detected by micro-broth dilution method.The expression levels of related drug resistance genes Vra D,Bra D,Bra R and Bac A in S.aureus with different resistance levels induced in vivo and in vitro were detected by RT-PCR with and without drug stimulation.The results of an in vitro drug resistance induction test showed that after 1/2 MIC of bacitracin was cultured for 1 d,4 d,and 5 d,the resistant strains with MIC values of 4μg/m L,16 μg/m L,and 32 μg/m L appeared;continued to cultivate for 25 d,the highest resistance MIC value of the strains was still 32 μg/m L;after induction,resistance strains were passaged without drug for 10 d and the MIC of strains did not change,indicating that their drug resistance was stable;the growth curves and susceptibility to other six antibiotics of induced-resistant strains with different resistance levels were no significant differences.Under clinical conditions,the results of the treatment test of BMD on S.aureus showed that compared with the model group,the three different administration groups had a certain inhibitory effect on S.aureus in the first treatment period and the inhibitory effect was positively linked with the administration dose;in the second treatment period,the emergence and increasing proportion of drug-resistant bacteria led to the weakening of the drug’s inhibitory effect in the later stage of treatment and bacterial colonization recovered after drug withdrawal,especially colonization of S.aureus in the high-dose group was ultimately higher than that in the other three groups.The proportion of intestinal resistant S.aureus was positively connected with BMD administration dose and administration time according to statistical data of BAC-resistant S.aureus.The ratio of BAC-resistant bacteria in the low-dose group ranged from 2% to 35.7%,the ratio of BAC-resistant bacteria in the medium-dose group ranged from 2.3% to 50.3% and the ratio of BAC-resistant bacteria in the high-dose group ranged from 2.3% to 64.5%;the earliest emergence time of BACresistant bacteria in different dose groups was 3 d after treatment and the maximum MIC value of induced S.aureus was 32 μg/m L,indicating that BMD can easily induce the resistance of S.aureus during the treatment process.Except for the enhanced susceptibility to chloramphenicol,there was no significant difference in the susceptibility of in vivoinduced resistant bacteria to other antibiotics.According to the test results,this study recommends the clinical dosing regimen of the combination of medium-dose bacitracin and chloramphenicol.The results of quantitative RT-PCR showed that: under the stimulation of bacitracin,the expression trends of TCS/ABC transporter genes Vra D,Bra D and Bra R in various resistance levels of strains showed a consistent up-regulation and the transcript level was Vra D>Bra D>Bra R under the stimulation of bacitracin;the transcript levels of the three genes in strains with varying resistance levels were not statistically different in the absence of drug administration;the expression of Bac A in different strains was positively correlated with the resistance level of the strains regardless of the presence of drug stimulation;for the same strain,the transcription level of Bac A was higher under drug stimulation than without drug,however it was lower than that of Vra D and Bra D.Differences in resistance levels between strains may be caused by the differential expression of Bac A or may be caused by the synergistic effect of Bac A and TCS/ABC transporters,which needs to be further explored.In summary,bacitracin can induce BAC-resistance of S.aureus in vivo and in vitro.In chickens,bacitracin methylene disalicylate promoted the generation of BAC-resistant bacteria and the resistance rate was positively correlated with the drug dose and dosing time;Bac A plays an important role in the differential resistance of S.aureus to bacitracin.Therefore,clinical monitoring of BAC-resistant S.aureus should be strengthened in order to use bacitracin products more scientifically and rationally in production. |
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