Department of Microbiology and Molecular Biology, Brigham Young University, Provo, UT 84602, USA.
Chemical Engineering Department, Brigham Young University, Provo, UT, USA.
J Med Microbiol. 2021 Dec;70(12). doi: 10.1099/jmm.0.001465.
Antibiotic resistance, particularly in cases of sepsis, has emerged as a growing global public health concern and economic burden. Current methods of blood culture and antimicrobial susceptibility testing of agents involved in sepsis can take as long as 3-5 days. It is vital to rapidly identify which antimicrobials can be used to effectively treat sepsis cases on an individual basis. Here, we present a pentaplex, real-time PCR-based assay that can quickly identify the most common beta-lactamase genes ( carbapenemase (KPC); New Delhi metallo-beta-lactamase (NDM); cefotaximase-Munich (CTX-M); cephamycin AmpC beta-lactamases (CMY); and Oxacillinase-48 (OXA-48)) from pathogens derived directly from the blood of patients presenting with bacterial septicemia. To develop an assay which can rapidly identify the most common beta-lactamase genes in Carbapenem-resistant bacteria (CREs) from the United States. Septicemia caused by carbapenem-resistant bacteria has a death rate of 40-60 %. Rapid diagnosis of antibiotic susceptibility directly from bacteria in blood by identification of beta-lactamase genes will greatly improve survival rates. In this work, we develop an assay capable of concurrently identifying the five most common beta-lactamase and carbapenemase genes. Primers and probes were created which can identify all subtypes of carbapenemase (KPC); New Delhi metallo-beta-lactamase (NDM); cefotaximase-Munich (CTX); cephamycin AmpC beta-lactamase (CMY); and oxacillinase-48 (OXA-48). The assay was validated using 13 isolates containing various PCR targets from the Centre for Disease Control Antimicrobial Resistance Isolate Bank Carbapenemase Diversity Panel. Blood obtained from volunteers was spiked with CREs and bacteria were separated, lysed, and subjected to analysis via the pentaplex assay. This pentaplex assay successfully identified beta-lactamase genes derived from bacteria separated from blood at concentrations of 4-8 c.f.u. ml. This assay will improve patient outcomes by supplying physicians with critical drug resistance information within 2 h of septicemia onset, allowing them to prescribe effective antimicrobials corresponding to the resistance gene(s) present in the pathogen. In addition, information supplied by this assay will lessen the inappropriate use of broad-spectrum antimicrobials and prevent the evolution of further antibiotic resistance.
抗生素耐药性，尤其是在败血症的情况下，已成为日益严重的全球公共卫生问题和经济负担。目前用于败血症的血液培养和抗菌药物敏感性试验的方法可能需要长达 3-5 天。快速确定哪些抗菌药物可以用于个体患者的有效治疗败血症是至关重要的。在这里，我们提出了一种五重实时 PCR 检测方法，可以快速识别直接从患有细菌性败血症的患者血液中分离的病原体中最常见的β-内酰胺酶基因（碳青霉烯酶（KPC）；新德里金属β-内酰胺酶（NDM）；头孢噻肟酶-慕尼黑（CTX-M）；头孢菌素 AmpCβ-内酰胺酶（CMY）；和 Oxacillinase-48（OXA-48））。开发一种能够快速鉴定美国耐碳青霉烯肠杆菌科（CRE）中最常见的β-内酰胺酶基因的检测方法。由耐碳青霉烯类药物的细菌引起的败血症的死亡率为 40-60%。通过鉴定β-内酰胺酶基因，直接从血液中的细菌快速诊断抗生素敏感性将大大提高存活率。在这项工作中，我们开发了一种能够同时鉴定五种最常见的β-内酰胺酶和碳青霉烯酶基因的检测方法。设计了引物和探针，可识别所有类型的碳青霉烯酶（KPC）；新德里金属β-内酰胺酶（NDM）；头孢噻肟酶-慕尼黑（CTX-M）；头孢菌素 AmpCβ-内酰胺酶（CMY）；和 Oxacillinase-48（OXA-48）。该检测方法使用来自疾病控制与预防中心抗菌药物耐药性分离库的耐碳青霉烯类药物多样性面板的 13 个包含各种 PCR 靶标。从志愿者中获得的血液中混入了 CRE 并分离出细菌，裂解后，通过五重检测方法进行分析。该五重检测方法成功地从血液中分离的细菌中鉴定出β-内酰胺酶基因，其浓度为 4-8 c.f.u. ml。该检测方法将通过在败血症发作后 2 小时内为医生提供关键的耐药性信息，从而改善患者的预后，使他们能够根据病原体中存在的耐药基因开出有效的抗菌药物。此外，该检测方法提供的信息将减少广谱抗菌药物的不当使用，并防止进一步产生抗生素耐药性。
