Abstract
Klebsiella pneumoniae is an important pathogenic bacterium commonly associated with human healthcare and community-acquired infections. In recent years, K. pneumoniae has become a significant threat to global public and veterinary health, because of its high rates of antimicrobial resistance (AMR). Early diagnosis of K. pneumoniae infection and detection of any associated AMR would help to accelerate directed therapy and reduce the risk of the emergence of multidrug-resistant isolates. In this study, we identified three target genes (yhaI, epsL, and xcpW) common to K. pneumoniae isolates from both China and Europe and designed loop-mediated isothermal amplification (LAMP) assays for the detection of K. pneumoniae in clinical samples. We also designed LAMP assays for the detection of five AMR genes commonly associated with K. pneumoniae. The LAMP assays were validated on a total of 319 type reference strains and clinical isolates of diverse genetic backgrounds, in addition to 40 clinical human sputum samples, and were shown to be reliable, highly specific, and sensitive. For the K. pneumoniae–specific LAMP assay, the calculated sensitivity, specificity, and positive and negative predictive values (comparison with culture and matrix-assisted laser desorption/ionization–time of flight mass spectrometry) were all 100% on clinical isolates and, respectively, of 100%, 91%, and 90%, and 100% when tested on clinical sputum samples, while being significantly faster than the reference methods. For the bla KPC and other carbapenemases’ LAMP assays, the concordance between the LAMP results and the references methods (susceptibility tests) was 100%, on both pure cultures (n = 125) and clinical samples (n = 18). In conclusion, we developed highly sensitive and specific LAMP assays for the clinical identification of K. pneumoniae and detection of carbapenem resistance.
Highlights
Klebsiella pneumoniae is a Gram-negative bacterium belonging to the Enterobacteriaceae family within the Enterobacterales order (Adeolu et al, 2016)
To assess the feasibility of blood samples testing with our diagnostics, the limit of bacterial copies per loop-mediated isothermal amplification (LAMP) reaction was determined in spiked defibrinated sheep blood samples, using a protocol previously described in other studies (Goto et al, 2010; Manajit et al, 2018)
Both LAMP and polymerase chain reaction (PCR) assays, performed on the DNA directly extracted from the sputum samples, showed that 11 of 12 carbapenem-resistant K. pneumoniae isolates carried the blaKPC gene (13, 25–27, 31 and 35–40; Figure 5 and Supplementary Table S7) and that one isolate harbored the blaNDM gene (28; Figure 5 and Supplementary Table S7)
Summary
Klebsiella pneumoniae is a Gram-negative bacterium belonging to the Enterobacteriaceae family within the Enterobacterales order (Adeolu et al, 2016). With the advent of high-throughput whole genome sequencing and core genome multilocus sequence typing in the last decade, several genomic studies investigated the population structure of K. pneumoniae and the evolution of AMR clones (Bialek-Davenet et al, 2014; Holt et al, 2015) These studies demonstrated the direct transfer of AMR plasmids between K. pneumoniae and other Enterobacterales in isolates recovered from hospital environments (Wyres and Holt, 2016), and the wide association of K. pneumoniae with carbapenemaseproducing genes blaKPC, blaOXA-48-like, and blaNDM-1, and the extended-spectrum beta-lactamase (ESBL) gene blaCTX-M-15 (Munoz-Price et al, 2013; Pitout et al, 2015; Zowawi et al, 2015; Wyres and Holt, 2016). All the designed assays were shown to be reliable, highly specific, and sensitive
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