Sensitive and Specific Loop-Mediated Isothermal Amplification Assays for Detection of Salmonella, CTX-M-1 Group Genes, mph(A), and ermB in Stool and Blood Samples Based on Orange to Green Visible Dye.

Salmonella is a globally prevalent foodborne bacterium, and ceftriaxone and azithromycin have been regarded as drugs of choice for treating Salmonella infections, particularly in children. With the growing incidence of ceftriaxone and azithromycin resistance in Salmonella, there is an urgent requirement for a rapid and dependable gene testing approach to enhance the efficacy of treating Salmonella infections. Utilizing the orange to green visible dye approach, this study developed loop-mediated isothermal amplification (LAMP) assays for the sensitive and specific detection of Salmonella, ceftriaxone and azithromycin resistance genes (including CTX-M-1 group, mph(A), and ermB genes) in stool and blood samples. The specificity and sensitivity of primers during the LAMP assays for detection of Salmonella, CTX-M-1 group, mph(A), and ermB genes were determined in this study. The detection threshold for Salmonella was found to be 1.5 × 103 colony-forming units (CFU)/mL, while it was 1.5 × 102 CFU/mL for CTX-M-1 group genes (including blaCTX-M-3, blaCTX-M-15, and blaCTX-M-55), 1.5 × 102 CFU/mL for mph(A), and 1.5 × 102 CFU/mL for ermB, showing 10-103-fold, 103-fold, and 105-fold increased sensitivity compared with the polymerase chain reaction assay, respectively. Results indicated that the LAMP primers designed for Salmonella, CTX-M-1 group, mph(A), and ermB genes possess high specificity (100%) and sensitivity (over 94%). This novel approach advocates its application in detecting Salmonella, CTX-M-1 group, mph(A), and ermB genes.

Sensitive and rapid detection of tet(X2) ~ tet(X5) by loop-mediated isothermal amplification based on visual OTG dye.

The emergence of tigecycline-resistant tet(X2/X3/X4/X5) genes poses a new threat to the efficacy of anti-infective therapy and the safety of our food and environment. To control the transfer of such genes, a sensitive and rapid molecular method is warranted to detect tet(X2/X3/X4/X5) genes in clinical isolates. Herein, we established a loop-mediated isothermal amplification (LAMP) assay to rapidly detect tet(X2/X3/X4/X5) genes, and the results were assessed by chromogenic visualization. The specificity and sensitivity of the primers during the LAMP assay for the simultaneous detection of tet(X2/X3/X4/X5) genes were determined in this study. All 48 clinical strains without tet(X2/X3/X4/X5) genes yielded negative results during the LAMP assay, substantiating the high specificity of the LAMP primers. The detection thresholds of this assay were 1.5 × 102 CFU/ml and 0.2 fg/uL corresponding to a 10 to 100-fold and 100-fold increase in sensitivity compared to polymerase chain reaction (PCR) assays. Out of 52 bacterial strains tested, using PCR as a reference, our research revealed that the LAMP assay demonstrated a sensitivity and specificity of 100%. To sum up, our novel approach has huge prospects for application in the simultaneous detection of tet(X2/X3/X4/X5) genes and can be applied to detect other drug-resistance genes.

Clinical distribution of carbapenem genotypes and resistance to ceftazidime-avibactam in Enterobacteriaceae bacteria.

Introduction: Bacterial resistance is a major threat to public health worldwide. To gain an understanding of the clinical infection distribution, drug resistance information, and genotype of CRE in Dongguan, China, as well as the resistance of relevant genotypes to CAZ-AVI, this research aims to improve drug resistance monitoring information in Dongguan and provide a reliable basis for the clinical control and treatment of CRE infection. Methods: VITEK-2 Compact automatic analyzer was utilized to identify 516 strains of CRE collected from January 2017 to June 2023. To determine drug sensitivity, the K-B method, E-test, and MIC methods were used. From June 2022 to June 2023, 80 CRE strains were selected, and GeneXpert Carba-R was used to detect and identify the genotype of the carbapenemase present in the collected CRE strains. An in-depth analysis was conducted on the CAZ-AVI in vitro drug sensitivity activity of various genotypes of CRE, and the results were statistically evaluated using SPSS 23.0 and WHONET 5.6 software. Results: This study identified 516 CRE strains, with the majority (70.16%) being K.pneumoniae, followed by E.coli (18.99%). Respiratory specimens had highest detection rate with 53.77% identified, whereas urine specimens had the second highest detection rate with 17.99%. From June 2022 to June 2023, 95% of the strains tested using the CRE GeneXpert Carba-R assay possessed carbapenemase genes, of which 32.5% were blaNDM strains and 61.25% blaKPC strains. The results showed that CRE strains containing blaKPC had a significantly higher rate of resistance to amikacin, cefepime, and aztreonam than those harboring blaNDM. Conclusions: The CRE strains isolated from Dongguan region demonstrated a high resistance rate to various antibiotics used in clinical practice but a low resistance rate to tigecycline. These strains produce Class A serine carbapenemases and Class B metals ß-lactamases, with the majority of them carrying blaNDM and blaKPC. Notably, CRE strains with blaKPC and blaNDM had significantly lower resistance rates to tigecycline. CAZ-AVI showed a good sensitivity rate with no resistance to CRE strains carrying blaKPC. Therefore, CAZ-AVI and tigecycline should be used as a guide for rational use of antibiotics in clinical practice to effectively treat CRE.