摘要
为分析临床分离的52株猪源支气管败血波氏杆菌(Bb)在纸片扩散法(K-B)药敏试验中出现的庆大霉素(GEN)异质性耐药(HR)情况,本研究采用微量肉汤稀释法检测52株Bb分离株对GEN的最小抑菌浓度(MIC);采用K-B法检测分离株对GEN的敏感性;通过观察抑菌环内是否有菌落初步筛选疑似GEN HR菌株;采用菌落谱性分析法(PAP)测定初筛菌株的MIC与最大不抑菌浓度(MNIC)的比值,以及耐药亚群菌株的发生频率,验证初筛的HR菌株。结果显示:52株猪源Bb对GEN耐药的菌株占13.46%(7/52),多数分离株的MIC值为2μg/mL~8μg/mL,对GEN表现为敏感或中介,占全部菌株的86.54%(45/52),其中Bb48株的MIC为2μg/mL,抑菌环直径为16 mm属于GEN敏感菌株。通过观察纸片法药敏试验中的抑菌环内是否存在菌落,初步筛选对GEN HR的菌株。结果显示,有7株疑似HR菌株;PAP试验结果显示,6株疑似HR菌株的MIC/MNIC比值均小于8,为假阳性HR菌株,只有Bb48株的MIC/MNIC值为16且耐药亚群发生频率为1.3×10^(-6),按照标准判定其为GEN HR Bb菌株。从PAP试验验证的Bb48株中有菌落生长的最高浓度GEN平板上随机挑3株耐药亚群菌株,37℃培养12 h,每小时测一次菌落数,共测12 h连同Bb48株一起绘制生长曲线;将上述试验选取的3株耐药亚群菌株连续传50代,每5代测其MIC值判定耐药亚群菌株的耐药遗传稳定性。生长曲线结果显示Bb48株及其耐药亚群菌株的生长速率均无显著差异(P>0.05);耐药遗传稳定性试验结果显示,将Bb48株中的3株耐药亚群菌株传代,其中两株菌各代次的MIC均为32μg/mL,1株菌的MIC为8μg/mL~32μg/mL。表明耐药亚群菌株对GEN的耐药性能够有效遗传。上述结果表明,52株猪源Bb中的Bb48为GEN HR菌株,Bb48及其耐药亚群菌株的生长速率基本一致,且对GEN的耐药性能够稳定遗传。本研究首次得到了罕见的对GEN HR的Bb菌株,提示临床中应合理使用GEN,并监测HR菌株的出现,对临床使用GEN治疗猪源Bb感染具有重要指导意义。
To analyze the heteroresistance to Gentamicin(GEN)in clinical isolates of 52 porcine-origin Bordetella bronchiseptica(Bb)strains in antibiotic sensitivity tests by the Kirby-Bauer(K-B),the present study was conducted to measure the minimum inhibitory concentration(MIC)of 52 isolates using the micro-broth dilution method;the K-B was used to detect the GEN susceptibility of the isolates,while the suspected heteroresistant strains were initially screened by observing whether there were colonies in the inhibition ring;the population analysis profiling(PAP)was used to determine whether the MIC/maximun non-inhibitory concentration(MNIC)values of the primary strains and whether the frequency of antibiotic-resistant subgroups in heteroresistant strains to verify the heteroresistant strains.The results showed that 52 strains of B.bronchiseptica were resistant to GEN at 13.46%(7/52),and most isolates had MIC values in the range of 2μg/mL-8μg/mL,appearing to be sensitive to GEN or intermediary,accounting for 86.54%(45/52)of all isolates,of which the MIC of Bb48 was 2μg/mL and the diameter of the inhibitory ring of Bb48 was 16 mm,which belonged to the GEN-sensitive strains.Observation and preliminary screening of the presence of colonies within the inhibition ring in the K-B test showed 7 suspected heteroresistant strains,PAP test showed that the MIC/MNIC ratios of the 6 suspected heteroresistant strains were less than 8,which were false-positive strains,only the MIC/MNIC value of strain Bb48 was 16 and the frequency of resistant subgroups was 1.3×10^(-6),which was determined as GEN heteroresistant strain of B.bronchiseptica according to the criteria.Three strains of resistant subpopulation were randomly picked from the GEN plate with the highest concentration of colony growth in Bb48 strains verified by PAP test,and cultured at 37℃for 12 hours.The number of colonies was measured every hour,and the growth curve was plotted together with the Bb48 strains for 12 hours.The 3 resistant subpopulation strains selected in the above test were continuously passaged for 50 generations,and the MIC values were measured every 5 generations to determine the genetic stability of the 3 strains in resistant subpopulation.The results of growth curve analysis showed that the growth rate of antibiotic-resistant subgroup strains in Bb48 was not statistically different from that of parental strains(P>0.05);the results of antibiotic resistance genetic stability test showed that the MIC of antibiotic-resistant subgroup strains in Bb48 consistently maintained at about 32μg/mL after passage,indicating that the antibiotic resistance could be effectively inherited.Combining the above experimental results,among the 52 strains of B.bronchiseptica,Bb48 was verified to be a GEN heteroresistant strain,and the growth rate of the resistant subgroup in Bb48 was basically the same as that of the parent strain,and the resistance to GEN could be stably inherited.In this study,a rare Bb strain with heteroresistance to GEN was obtained for the first time,suggesting that GEN should be used reasonably in clinical practice and the emergence of heterogeneous resistant strains should be monitored,which has important guiding significance for the clinical use of GEN in the treatment of porcine Bb infection.
作者
樊浩然
高树基
王海堃
袁朔
张小玲
赵战勤
张慧
汪洋
FAN Hao-ran;GAO Shu-ji;WANG Hai-kun;YUAN Shuo;ZHANG Xiao-ling;ZHAO Zhan-qin;ZHANG Hui;WANG Yang(Henan Provincial Engineering Research Center for Detection and Prevention and Control of Emerging Infectious Diseases in Livestock and Poultry,College of Animal Science and Technology,Henan University of Science and Technology,Luoyang 471000,China;China Animal Health and Epidemiology Center,Qingdao 266032,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2023年第8期801-806,共6页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金(32172852、31902309)
河南省杰出青年科学基金(222300420005)。
