FARPA-based tube array coupled with quick DNA extraction enables ultra-fast bedside detection of antibiotic-resistant pathogens.

Rapid and accurate detection of pathogens and antimicrobial-resistant (AMR) genes of the pathogens are crucial for the clinical diagnosis and effective treatment of infectious diseases. However, the time-consuming steps of conventional culture-based methods inhibit the precise and early application of anti-infection therapy. For the prompt treatment of pathogen-infected patients, we have proposed a novel tube array strategy based on our previously reported FARPA (FEN1-aided recombinase polymerase amplification) principle for the ultra-fast detection of antibiotic-resistant pathogens on site. The entire process from "sample to result" can be completed in 25 min by combining quick DNA extraction from a urine sample with FARPA to avoid the usually complicated DNA extraction step. Furthermore, a 36-tube array made from commercial 384-well titre plates was efficiently introduced to perform FARPA in a portable analyser, achieving an increase in the loading sample throughput (from several to several tens), which is quite suitable for the point-of-care testing (POCT) of multiple pathogens and multiple samples. Finally, we tested 92 urine samples to verify the performance of our proposed method. The sensitivities for the detection of E. coli, K. pneumoniae, E. faecium, and E. faecalis were 92.7%, 93.8%, 100% and 88.9%, respectively. The specificities for the detection of the four pathogens were 100%. Consequently, our rapid, low-cost and user-friendly POCT method holds great potential for guiding the rational use of antibiotics and reducing bacterial resistance.

Multiplexed and Rapid AST for Escherichia coli Infection by Simultaneously Pyrosequencing Multiple Barcodes Each Specific to an Antibiotic Exposed to a Sample.

Antimicrobial susceptibility testing (AST) is an effective way to guide antibiotic selection. However, conventional culture-based phenotypic AST is time-consuming. The key point to shorten the test is to quantify the small change in the bacterial number after the antibiotic exposure. To achieve rapid AST, we proposed a combination of multiplexed PCR with barcoded pyrosequencing to significantly shorten the time for antibiotic exposure. First, bacteria exposed to each antibiotic were labeled with a unique barcode. Then, the pool of the barcoded products was amplified by PCR with a universal primer pair. Finally, barcodes in the amplicons were individually and quantitatively decoded by pyrosequencing. As pyrosequencing is able to discriminate as low as 5% variation in target concentrations, as short as 7.5 min was enough for cultivation to detect the susceptibility of Escherichia coli to an antibiotic. The barcodes enable more than six kinds of drugs or six kinds of concentrations of a drug to be tested at a time. The susceptibility of 6 antibiotics to 43 E. coli-positive samples from 482 clinical urine samples showed a consistency of 99.3% for drug-resistant samples and of 95.7% for drug-sensitive samples in comparison with the conventional method. In addition, the minimum inhibitory concentration (MIC) of 29 E. coli samples was successfully measured. The proposed AST is dye free (pyrosequencing), multiplexed (six antibiotics), fast (a half-working day for reporting the results), and able to detect the MIC, thus having a great potential for clinical use in quick antibiotic selection.

Risk factors for and clinical outcomes of ceftazidime-avibactam-resistant carbapenem-resistant Klebsiella pneumoniae nosocomial infections: a single-center retrospective study.

PURPOSE: The emergence of ceftazidime-avibactam (CZA) resistance in carbapenem-resistant Klebsiella pneumoniae (CRKP) has been increasingly reported in recent years. We aimed to identify the risk factors of CZA-resistant CRKP infection and assess clinical outcomes of the patients. METHODS: The study retrospectively analyzed the clinical and microbiological data of patients with CRKP infection to identify risk factors, clinical features, and outcomes using multivariate logistic regression analysis. RESULTS: A total of 103 patients with CRKP infection were enrolled in this study. Multivariate analysis showed previous renal replacement therapy (OR 3.966, 95% CI 1.301-12.090, P = 0.015) was an independent risk factor for CZA-resistant CRKP infection. The 28-day mortality was higher in patients infected with CZA-resistant CRKP (27.9%) than those with CZA-susceptible CRKP (7.1%) (P = 0.009). CZA-resistant CRKP infection (OR 20.308, 95% CI 1.461-282.293, P = 0.025), and mechanical ventilation (OR 14.950, 95% CI 1.034-216.212, P = 0.047) were independent predictors for 28-day mortality in patients with CRKP infection. Lower level of platelet count (OR 0.987, 95% CI 0.975-0.999, P = 0.032) on the day of CRKP infection onset was related to 28-day mortality. Kaplan-Meier curves showed that the CZA-resistant CRKP group had a shorter survival time than the CZA-susceptible CRKP group. CONCLUSION: The prevalence and mortality of CZA-resistant CRKP are still increasing. Strengthening the hospital infection control of renal replacement therapy and mechanical ventilation may help to prevent CZA-resistant CRKP.