Phenotypic methods for screening carbapenem-resistant Enterobacteriaceae and assessment of their antimicrobial susceptibility profile

Abstract INTRODUCTION: In this study, we used phenotypic methods to screen carbapenem-resistant Enterobacteriaceae (CREs) and evaluated their antimicrobial sensitivity profile. METHODS: One hundred and seventy-eight CREs were isolated at a university hospital in south Brazil in a one-year period. Samples were assessed using disk diffusion tests with inhibitors of β-lactamases such as phenylboronic acid (AFB), cloxacillin (CLOXA), and ethylenediaminetetraacetic acid (EDTA). Strains with differences in zone diameters ≥ 5mm for disks supplemented or not were considered producers of carbapenemases. RESULTS: Klebsiella pneumoniae was the most prevalent CRE, which appeared in 80.3% cases (n = 143). Among clinical materials, the rectal swab was responsible for 43.4% of the isolations (n = 62), followed by urine (18.9%; n = 27). Among the CREs identified in this study, the growth of 56.7% (n = 101) isolates, which were putative producers of Klebsiella pneumoniae carbapenemase (KPC), were inhibited by AFB, whereas 7.3% (n = 13) isolates were inhibited by both AFB and CLOXA and were considered as putative producers of plasmid-mediated AmpC; approximately 3.4% (n = 6) were inhibited by EDTA, which possibly produced metallo-β-lactamase. Lastly, 32.6% (n = 58) cases showed negative results for AFB, CLOXA, and EDTA sensitivity, and represented another class of β-lactamases and/or mechanism of resistance. CONCLUSIONS: Phenotypic screening of CREs is important for clinical laboratories that monitor outbreaks of resistant microbes. Phenotypic tests that use carbapenemase inhibitors and enhancers such as AFB, CLOXA, and EDTA are necessary since they are good screening methods for the detection of carbapenemases.

摘要：
引言：本研究采用表型检测方法筛选碳青霉烯类耐药肠杆菌科（carbapenem-resistant Enterobacteriaceae, CREs），并对其抗菌药物敏感性谱进行评估。
方法：本研究于巴西南部一所大学附属医院，在为期一年的时间内分离得到178株碳青霉烯类耐药肠杆菌科（CREs）。采用纸片扩散法，结合β-内酰胺酶抑制剂苯硼酸（phenylboronic acid, AFB）、氯唑西林（cloxacillin, CLOXA）以及乙二胺四乙酸（ethylenediaminetetraacetic acid, EDTA）对样本进行检测。当添加与未添加抑制剂的纸片抑菌圈直径差值≥5mm时，判定该菌株为碳青霉烯酶产生菌。
结果：肺炎克雷伯菌是本次研究中最常见的CRE，检出率为80.3%（n=143）。在临床标本类型中，直肠拭子分离占比最高，达43.4%（n=62），其次为尿液标本，占比18.9%（n=27）。本研究鉴定的CRE菌株中，56.7%（n=101）的菌株可被苯硼酸（AFB）抑制，推测为肺炎克雷伯菌碳青霉烯酶（Klebsiella pneumoniae carbapenemase, KPC）产生菌；7.3%（n=13）的菌株可同时被AFB与CLOXA抑制，判定为质粒介导的AmpC酶产生菌；约3.4%（n=6）的菌株可被EDTA抑制，推测产金属β-内酰胺酶。另有32.6%（n=58）的菌株对AFB、CLOXA及EDTA均无敏感性反应，代表另一类β-内酰胺酶及/或耐药机制。
结论：对于开展耐药菌暴发监测的临床实验室而言，碳青霉烯类耐药肠杆菌科的表型筛查具有重要意义。采用碳青霉烯酶抑制剂与增效剂（如AFB、CLOXA及EDTA）的表型检测方法，可作为碳青霉烯酶检测的有效筛查手段，具备较高的临床应用价值。