Peláez T, Cercenado E, Alcalá L, Marín M, Martín-López A, Martínez-Alarcón J, Catalán P, Sánchez-Somolinos M, Bouza E
Microbiology and Infectious Disease Service, Hospital General Universitario Gregorio Marañón, Madrid, Spain.
J Clin Microbiol. 2008 Sep;46(9):3028-32. doi: 10.1128/JCM.00524-08. Epub 2008 Jul 23.
At our institution, the prevalence of clinical isolates of Clostridium difficile with resistance to metronidazole is 6.3%. We observed that initial metronidazole MICs of 16 to 64 mg/liter against toxigenic, primary fresh C. difficile isolates, as determined by agar dilution, decreased to 0.125 mg/liter after the isolates were thawed. In this study, we examined the possibility of heterogeneous or inducible resistance. Totals of 14 metronidazole-resistant and 10 metronidazole-susceptible clinical isolates of toxigenic C. difficile were studied. The isolates were investigated for the presence of nim genes by PCR. After the isolates were thawed, susceptibility testing was done by agar dilution, by disc diffusion using a 5-mug metronidazole disc, and by the Etest method. An experiment for determining the effect of prolonged exposure to metronidazole was applied to all resistant isolates and to susceptible control strains. None of the isolates presented the nim genes. All initially metronidazole-resistant C. difficile isolates became susceptible after thawing; however, they presented slow-growing subpopulations within the inhibition zones of both the disk and the Etest strip. All metronidazole-susceptible isolates remained homogeneously susceptible by both methods. After prolonged exposure in vitro to metronidazole, no zone of inhibition was found around the 5-microg disk in any of the metronidazole-resistant isolates, and the MICs as determined by the Etest method ranged from 0.125 to >256 mg/liter, with colonies growing inside the inhibition zone. Our results indicate that (i) resistance to metronidazole was not due to the presence of nim genes, (ii) resistance to metronidazole in toxigenic C. difficile isolates is heterogeneous, and (iii) prolonged exposure to metronidazole can select for in vitro resistance. We recommend routine performance of the disk diffusion method (5-microg metronidazole disk) with primary fresh C. difficile isolates in order to ensure that metronidazole-heteroresistant populations do not go undetected.
在我们机构，对甲硝唑耐药的艰难梭菌临床分离株的流行率为6.3%。我们观察到，通过琼脂稀释法测定，针对产毒的、原发性新鲜艰难梭菌分离株，初始甲硝唑最低抑菌浓度（MIC）为16至64毫克/升，分离株解冻后该浓度降至0.125毫克/升。在本研究中，我们研究了异质性或诱导性耐药的可能性。共研究了14株对甲硝唑耐药和10株对甲硝唑敏感的产毒艰难梭菌临床分离株。通过聚合酶链反应（PCR）检测分离株中nim基因的存在情况。分离株解冻后，通过琼脂稀释法、使用5微克甲硝唑纸片的纸片扩散法以及Etest法进行药敏试验。对所有耐药分离株和敏感对照菌株进行了一项确定延长暴露于甲硝唑影响的实验。所有分离株均未呈现nim基因。所有最初对甲硝唑耐药的艰难梭菌分离株解冻后均变为敏感；然而，它们在纸片和Etest条带的抑菌圈内呈现生长缓慢的亚群。两种方法检测时，所有对甲硝唑敏感的分离株均保持均匀敏感。在体外长时间暴露于甲硝唑后，任何对甲硝唑耐药的分离株在5微克纸片周围均未发现抑菌圈，通过Etest法测定的MIC范围为0.125至>256毫克/升，抑菌圈内有菌落生长。我们的结果表明：（i）对甲硝唑的耐药性并非由于nim基因的存在；（ii）产毒艰难梭菌分离株对甲硝唑的耐药性是异质性的；（iii）长时间暴露于甲硝唑可在体外选择出耐药性。我们建议对原发性新鲜艰难梭菌分离株常规进行纸片扩散法（5微克甲硝唑纸片）检测，以确保不会漏检对甲硝唑异质性耐药的群体。
