Recombinase-aided amplification assay for rapid detection of imipenem-resistant Pseudomonas aeruginosa and rifampin-resistant Pseudomonas aeruginosa

Abstract

The indiscriminate use of antibiotics has resulted in a growing resistance to drugs in Pseudomonas aeruginosa. The identification of antibiotic resistance genes holds considerable clinical significance for prompt diagnosis. In this study, we established and optimized a Recombinase-Aided Amplification (RAA) assay to detect two genes associated with drug resistance, oprD and arr, in 101 clinically collected P. aeruginosa isolates. Through screening for the detection or absence of oprD and arr, the results showed that there were 52 Imipenem-resistant P. aeruginosa (IRPA) strains and 23 Rifampin-resistant P. aeruginosa (RRPA) strains. This method demonstrated excellent detection performance even when the sample concentration is 10 copies/μL at isothermal conditions and the results could be obtained within 20 minutes. The detection results were in accordance with the results of conventional PCR and Real-time PCR. The detection outcomes of the arr gene were consistently with the resistance spectrum. However, the antimicrobial susceptibility results revealed that 65 strains were resistant to imipenem, while 49 strains sensitive to imipenem with oprD were identified. This discrepancy could be attributed to genetic mutations. In summary, the RAA has higher sensitivity, shorter time, and lower-cost instrument requirements than traditional detection methods. In addition, to analyze the epidemiological characteristics of the aforementioned drug-resistant strains, we conducted Multilocus Sequence Typing (MLST), virulence gene, and antimicrobial susceptibility testing. MLST analysis showed a strong correlation between the sequence types ST-1639, ST-639, ST-184 and IRPA, while ST-261 was the main subtype of RRPA. It was observed that these drug-resistant strains all possess five or more virulence genes, among which exoS and exoU do not coexist, and they are all multidrug-resistant strains. The non-coexistence of exoU and exoS in P.aeruginosa is related to various factors including bacterial regulatory mechanisms and pathogenic mechanisms. This indicates that the relationship between the presence of virulence genes and the severity of patient infection is worthy of attention. In conclusion, we have developed a rapid and efficient RAA (Recombinase-Aided Amplification) detection method that offers significant advantages in terms of speed, simplicity, and cost-effectiveness (especially in time and equipment aspect). This novel approach is designed to meet the demands of clinical diagnostics.

Keywords: ARR; OprD; Pseudomonas aeruginosa; antimicrobial susceptibility testing; rapid detection; recombinase-aided amplification.

Figure 1

Technical route of this study. (I) Clinical isolates are collected and cultured. (II) Antimicrobial susceptibility testing and PCR for drug-resistant genes. (III) Selection of genes. (IV) High temperature denaturation extraction of bacterial DNA. (V-VI) Detection of oprD/arr by RAA. The blue curve represents the template DNA, the blue circle represents the probe, the red circle represents the primer, and the orange represents the recombinant enzyme medium. The reaction results can be detected with a constant temperature instrument.

Figure 2

Selection of specific regions and primer positions for RAA assay. (A) Primer and probe set to amplify the oprD gene. (B) Primer and probe set to amplify the arr gene. Probes are indicated by blue, primer are indicated by orange.

Figure 3

The optimization of temperature and probe concentration of RAA assay for IRPA and RRPA detection. (A, B) The RAA analysis of oprD gene at different temperature (A) and different probe concentration (B). (C, D) The RAA analysis of arr gene at different temperature (C) and different probe concentration (D). The results showed that their optimal temperature is 39°C and the optimal probe amplification concentration is 10 μM. NC, Negative Control.

Figure 4

The sensitivity of RAA and PCR assay for IRPA and RRPA detection. The sensitivity of RAA analysis using the primer and probe set RAA-oprD-primer1 (A), RAA-arr-primer2 (B). The plasmid ranged from 1×10⁰ copies/μL to 1 × 10⁷ copies/μL. NC, Negative Control. The sensitivity of PCR for IRPA and RRPA detection. (C, D) The sensitivity of RAA and conventional PCR detection.

Figure 5

The specificity of RAA assay for IRPA (A) and RRPA (B) detection. Only the recombinant plasmids produced amplification signals, whereas the negative control and control bacterial samples produced negative amplification signals. NC, Negative Control.

Figure 6

The detection performance of RAA assay in clinical samples for IRPA (A) and RRPA (B). Both IRPA and RRPA are multidrug-resistant strains. NC, Negative Control.

Figure 7

Molecular characteristics of IRPA (A) and RRPA (B) isolates. The result shows that the most common type is ST1639 for IRPA (A), and ST261 for RRPA (B).