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Pseudomonas aeruginosa is a major opportunistic pathogen in hospital-acquired infections and exhibits increasing antibiotic resistance. A rapid and sensitive molecular method for its detection in clinical samples is needed to guide therapeutic treatment and to control P. aeruginosa outbreaks. In this study, we established a polymerase spiral reaction (PSR) method for rapid detection of P. aeruginosa by targeting the toxA gene, which regulates exotoxin A synthesis. Real-time turbidity monitoring and a chromogenic visualization using hydroxynaphthol blue were used to assess the reaction. All 17 non- P. aeruginosa strains tested negative, indicating the high specificity of the PSR primers. The detection limit was 2.3 pg/µl within 60 min at isothermal temperature (65°C), 10-fold more sensitive than conventional PCR. Then, the PSR assay was applied to a clinical surveillance of P. aeruginosa in three top hospitals in Beijing, China. Of the 130 sputum samples collected from ICU patients with suspected multi-resistant infections, 37 P. aeruginosa isolates were identified from the positive samples. All clinical strains belonged to 10 different P. aeruginosa multilocus sequence typing groups and exhibited high resistance to carbapenems, cephalosporins, and aminoglycosides. Interestingly, of the 33 imipenem-resistant isolates, 30 (90.9%) had lost the outer membrane porin oprD gene. Moreover, isolate SY-95, containing multiple antibiotic resistance genes, possessed the ability to hydrolyze all antibiotics used in clinic and was susceptible only to polymyxin B. Our study showed the high level of antibiotic resistance and co-occurrence of resistance genes in the clinical strains, indicating a rapid and continuing evolution of P. aeruginosa. In conclusion, we developed a P. aeruginosa PSR assay, which could be a useful tool for clinical screening, especially in case of poor resources, or for point-of-care testing.

Acinetobacter baumannii is an important opportunistic pathogen associated with a variety of nosocomial infections. A rapid and sensitive molecular detection in clinical isolates is quite needed for the appropriate therapy and outbreak control of A. baumannii. Group 2 carbapenems have been considered the agents of choice for the treatment of multiple drug resistant A. baumannii. But the prevalence of carbapenem-resistant A. baumannii (CRAB) has been steadily increasing in recent years. Here, we developed a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of A. baumannii in clinical samples by using high-specificity primers of the blaOXA-51 gene. Then we investigated the OXA-carbapenemases molecular epidemiology of A. baumannii isolates in 2 comprehensive hospitals in Beijing. The results showed that the LAMP assay could detect target DNA within 60 min at 65°C. The detection limit was 50 pg/μl, which was about 10-fold greater than that of PCR. Furthermore, this method could distinguish A. baumannii from the homologous A. nosocomialis and A. pittii. A total of 228 positive isolates were identified by this LAMP-based method for A. baumannii from 335 ICU patients with clinically suspected multi-resistant infections in 2 hospitals in Beijing. The rates of CRAB are on the rise and are slowly becoming a routine phenotype for A. baumannii. Among the CRABs, 92.3% harbored both the blaOXA-23 and blaOXA-51 genes. Thirty-three pulsotypes were identified by pulsed-field gel electrophoresis, and the majority belonged to clone C. In conclusion, the LAMP method developed for detecting A. baumannii was faster and simpler than conventional PCR and has great potential for both point-of-care testing and basic research. We further demonstrated a high distribution of class D carbapenemase-encoding genes, mainly OXA-23, which presents an emerging threat in hospitals in China.

Klebsiella pneumoniae is a wide-spread nosocomial pathogen. A rapid and sensitive molecular method for the detection of K. pneumoniae in clinical samples is needed to guide therapeutic treatment. In this study, we first described a loop-mediated isothermal amplification (LAMP) method for the rapid detection of capsular polysaccharide synthesis regulating gene rcsA from K. pneumoniae in clinical samples by using two methods including real-time turbidity monitoring and fluorescence detection to assess the reaction. Then dissemination of K. pneumoniae strains was investigated from ICU patients in three top hospitals in Beijing, China. The results showed that the detection limit of the LAMP method was 0.115 pg/µl DNA within 60 min under isothermal conditions (61°C), a 100-fold increase in sensitivity compared with conventional PCR. All 30 non- K. pneumoniae strains tested were negative for LAMP detection, indicating the high specificity of the LAMP reaction. To evaluate the application of the LAMP assay to clinical diagnosis, of 110 clinical sputum samples collected from ICU patients with clinically suspected multi-resistant infections in China, a total of 32 K. pneumoniae isolates were identified for LAMP-based surveillance of rcsA. All isolates belonged to nine different K. pneumoniae multilocus sequence typing (MLST) groups. Strikingly, of the 32 K. pneumoniae strains, 18 contained the Klebsiella pneumoniae Carbapenemase (KPC)-encoding gene blaKPC-2 and had high resistance to β-lactam antibiotics. Moreover, K. pneumoniae WJ-64 was discovered to contain blaKPC-2 and blaNDM-1 genes simultaneously in the isolate. Our data showed the high prevalence of blaKPC-2 among K. pneumoniae and co-occurrence of many resistant genes in the clinical strains signal a rapid and continuing evolution of K. pneumoniae. In conclusion, we have developed a rapid and sensitive visual K. pneumoniae detection LAMP assay, which could be a useful tool for clinical screening, on-site diagnosis.