Sahm D F, Free L, Smith C, Eveland M, Mundy L M
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Clin Microbiol. 1997 Aug;35(8):2026-30. doi: 10.1128/jcm.35.8.2026-2030.1997.
Surveillance cultures for vancomycin-resistant enterococci (VRE) and subsequent characterization of the isolates can be extremely burdensome and difficult. Therefore, efficient and reliable schemes for the characterization of surveillance isolates are needed. In this study, a commercial agar (bile esculin azide agar with 6 microg of vancomycin per ml [BEAA]; Remel, Lenexa, Kans.) was used in the initial screening step to establish relatively rapid (i.e., in < or = 24 h from the time of isolation) phenotype-based and PCR-based schemes for the detection and characterization of VRE. The phenotype-based scheme included Gram staining of growth on BEAA and subculture of cocci on sheep blood agar plates for vancomycin disk diffusion and pyrazinamidase (PYR) testing. For the PCR scheme, inocula for van gene detection were taken directly from the BEAA plates. The phenotypic approach was applied to 378 surveillance cultures that yielded growth on BEAA. Gram staining quickly eliminated gram-positive bacilli from further testing, and a negative PYR test classified 25 additional isolates as probable pediococci. A positive PYR test reliably identified 121 single-patient VRE isolates that included 83 Enterococcus faecium, 33 E. gallinarum, and 5 E. casseliflavus strains. The vancomycin inhibition zone size clearly distinguished VanA and VanB strains from VanC strains within 24 h of BEAA isolation. All VanA and VanB strains failed to produce zones of >6 mm, while only one VanC strain produced a zone of < 15 mm. Challenging this phenotypic scheme with 47 stock VRE isolates produced similar findings. In direct PCR analyses, false-negative vanA and vanB results occurred with 12% of the specimens. Many of the false-negative reactions also failed to produce an internal control product, which underscores the need for including control primers when a PCR scheme is used. In summary, the phenotype- and the PCR-based schemes provide efficient methods for characterizing VRE within 24 h of isolation.
对耐万古霉素肠球菌（VRE）进行监测培养以及随后对分离株进行鉴定可能极其繁琐且困难。因此，需要高效且可靠的方案来鉴定监测分离株。在本研究中，一种商用琼脂（每毫升含6微克万古霉素的胆汁七叶苷叠氮化物琼脂[BEAA]；Remel公司，莱尼克斯，堪萨斯州）被用于初始筛选步骤，以建立相对快速（即从分离之时起≤24小时）的基于表型和基于PCR的方案，用于检测和鉴定VRE。基于表型的方案包括对BEAA上生长的细菌进行革兰氏染色，并将球菌在羊血琼脂平板上进行传代培养以进行万古霉素纸片扩散试验和吡嗪酰胺酶（PYR）检测。对于PCR方案，直接从BEAA平板上获取用于检测van基因的接种物。将表型方法应用于378份在BEAA上生长的监测培养物。革兰氏染色迅速将革兰氏阳性杆菌排除在进一步检测之外，而PYR试验阴性将另外25株分离株归类为可能的片球菌。PYR试验阳性可靠地鉴定出121株单患者VRE分离株，其中包括83株粪肠球菌、33株鹑鸡肠球菌和5株黄色肠球菌菌株。在BEAA分离后24小时内，万古霉素抑菌圈大小能清楚地区分VanA和VanB菌株与VanC菌株。所有VanA和VanB菌株产生的抑菌圈均不大于6毫米，而只有1株VanC菌株产生的抑菌圈小于15毫米。用47株VRE标准菌株对该表型方案进行验证得到了类似的结果。在直接PCR分析中，12%的标本出现vanA和vanB的假阴性结果。许多假阴性反应也未能产生内部对照产物，这突出了使用PCR方案时包含对照引物的必要性。总之，基于表型和基于PCR的方案为在分离后24小时内鉴定VRE提供了有效的方法。
