Rapid Identification Of Antibiotic Resistant Bactacteria Using An Enzyme Thermal Sensor

Antibiotic resistance(AR)has become a serve threat to human health.Misuse and overuse of antibiotics is a major cause of AR due to lack of timely and accurate result of AR diagnostic test during clinical treatment.Producing ?-lactamase is a major mechanism leading to AR for gram-negative bacteria.Among more than one thousand kinds of ?-lactamase,extended spectrum ?-lactamase(ESBLs)and carbapenemase are listed as two key targets for clinical surveillance and detection because they show potent antibiotic resistant activity to the 3rd and 4th generation of cephalosporin and carbapenem which are broad-spectrum antibiotics.Currently,ESBLs NDP assay,Carba NP assay and phenotypic assays are major methods to detect ESBL and carbapenemase producing bacteria,however,these methods yet show weakness in sensitivity and turnaround time of detection.Although the penicillinase thermal sensor showed a high efficacy to detect illegal exogenous addition of penicillinase in milk,but this sensor can not be applied into detect ESBLs and carbapenemase producing bacteria due to its intrinsic weakness in hydrolysis of cephalosporins and carbapenems.In this study,New Delhi metallo-b-lactamase-1(NDM-1)that is able to broad-spectrum hydrolyze cephalosporins and carbapenems was used to establish a NDM-1 thermal sensor,and sensitivity and efficacy of the NDM-1thermal sensor in detection of ESBLs,carbapenemase as well as ESBLs/carbapenemase producing bacteria was evaluated in combination with b-lactamase inhibitors.It was observed that the NDM-1 thermal sensor,compared to the penicillinase thermal sensor,show more efficacy to quantitate cephalosporins and carbapenems ranging from 32.5 to 1000 mg/L.The ?-lactamase inhibitors “avibactam(AVI)and EDTA” can facilitate the NDM-1 thermal sensor to identify class of the tested b-lactamases without apparent effect on detection signal.Hence,the NDM-1 thermal sensor can accurately identify individual CTX-M-14 or NDM-1,combined CTX-M-14 and NDM-1 as well as one bacteria co-producing SHV-12 and NDM-1.Onthe other hand,the NDM-1 thermal sensor showed a higher sensitivity to detect ESBLs and carbapenemase than the spectrometer;the NDM-1 thermal sensor showed at least 10 times more sensitivity to detect an ESBL producing clinical bacteria than the ESBLs NDP assay.In addition,detection to the tested antibiotics of the NDM-1 thermal sensor still maintained around 80%activity after more than one thousand tests.Finally,13 strains of ESBLs and carbapenems resistant bacteria identified by phenotypic assay and molecular assay were selected as the test subjects for clinical retrospective study.To further evaluate the effectiveness of NDM-1 thermal sensor in detecting ESBLs and carbapenems resistant bacteria.The detection of 13 clinical AR bacteria by the NDM-1 thermal sensor and the phenotypic assay and molecular assay show 100% consistence.These data indicate that the NDM-1 thermal sensor established in this study is a sensitive,rapid,accurate and stable device suitable for rapid detection of ESBL and carbapenemase producing bacteria.