Survey and rapid detection of Klebsiella pneumoniae in clinical samples targeting the rcsA gene in Beijing, 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

Immunization with Individual Proteins of the Lrp/AsnC Family Induces Protection Against Brucella melitensis 16M Challenges in Mice

Brucellosis is one of the most common zoonoses worldwide. Subunit vaccines are promising for the prevention of human brucellosis. In our previous protective antigen screening studies, we identified a new protective antigen, BMEI0357, which belongs to the Lrp/asnC protein family, a conserved transcriptional regulator in bacteria that is absent in eukaryotes. In the present study, the Brucella genome annotation was screened and a total of 6 proteins were identified as members of the Lrp/AsnC family. Lrp/AsnC proteins have 2 domains that are conserved among the family members. However, sequence similarities between these proteins ranged from 9% to 50%, indicating high sequence heterogeneity. To test whether proteins of this family have similar characteristics, all 6 proteins were cloned and expressed in Escherichia coli. The recombinant proteins were purified and their protective efficacy was evaluated in BALB/c mice challenged with Brucella melitensis 16M. The results show that all 6 Lrp/AsnC proteins could induce a protective immune response against Brucella melitensis 16M. Antibodies against the Lrp/AsnC proteins were detected in the immunized mice. However, levels of antibodies against these proteins were relatively variable in human brucellosis sera. Taken together, our results show that these 6 proteins of the Lrp/AsnC family in Brucella could induce protective immune responses in mice