Development of Loop-Mediated Isothermal Amplification Rapid Diagnostic Assays for the Detection of Klebsiella pneumoniae and Carbapenemase Genes in Clinical Samples

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.

Keywords: Klebsiella pneumoniae (K. pneumoniae); LAMP (loop mediated isothermal amplification); antimicrobial resistance (AMR); carbapenemases genes; rapid diagnostics.

FIGURE 1

Identification of discriminatory gene targets for K. pneumoniae. This matrix was generated using BLAST (Abricate) using an 80% identity and coverage threshold to identify potential variation. Intensity of the blue heat map indicates identity relative to query sequences (Supplementary File S2) (blue = 100%, white = 0%/absence). Genes encoded by both Klebsiella species are also listed, for reference (bottom 4). The genomes are ordered according to the branching produced using core genome SNP as identified using ParSNP and FastTree. The green nodes are K. pneumoniae genomes, and the red nodes, K. oxytoca genomes. The colors indicate the six sequence types (STs) identified in the Klebsiella included in the study.

FIGURE 2

K. pneumoniae LAMP detection in pure cultures. (A) Representative fluorometric LAMP results, targeting the yhaI gene, performed on type strain (TS1) and clinical isolates (CS1–12) of K. pneumoniae from the United Kingdom, with H₂O as a negative control. (B) Representative colorimetric LAMP results, targeting the yhaI gene, performed on type strains (TS) and clinical isolates (CS) of K. pneumoniae from the United Kingdom (TS1–3) and from China (TS4 and CS13–18), as well as on type strains of E. coli, E. cloacae, Pseudomonas aeruginosa, and P. putida, with H₂O as a negative control.

FIGURE 3

Carbapenemase-producing genes LAMP and PCR testing on clinical isolates. (A) Representative colorimetric LAMP results targeting the bla _KPC gene performed on 30 of the 120 clinical isolates of K. pneumoniae collected at Ruijin Hospital (Shanghai, China); 19 isolates were bla _KPC-positive (yellow; 1, 4, 6, 8–11, 13–16, 22–28, and 30), and 11 were negative (pink). (B) Representative results of endpoint PCR targeting the bla _KPC gene performed on the same 30 clinical isolates of K. pneumoniae than the ones tested by LAMP. Nineteen isolates were bla _KPC-positive (amplification band at 240 bp; 1, 4, 6, 8–11, 13–16, 22–28, and 30), and 11 were negative (no amplification band). The marker (M) used for these experiments was a 100-bp DNA ladder. (C) Representative colorimetric LAMP results targeting the bla _NDM (a), bla _OXA-48-like (b), bla _IMP (c), and bla _VIM, (d) carbapenemase-producing genes, obtained on 28 of the 120 clinical isolates of K. pneumoniae collected at Ruijin Hospital (Shanghai, China). For each AMR gene, the positive control (+) was a clinical isolate, collected from the Ruijin Hospital (Shanghai, China) and confirmed to be positive by both PCR and sequencing. Two K. pneumoniae clinical isolates were positive (yellow; 31 and 37) for bla _NDM (a), three were positive (yellow; 38, 39, and 43) for bla _OXA-48-like (b), one was positive (yellow; 47) for bla _IMP (c), and two were positive (yellow; 54 and 55) for bla _VIM (d) genes. (D) Representative PCR amplification results targeting the bla _NDM (a), bla _OXA-48-like (b), bla _IMP (c), and bla _VIM (d) genes, obtained on the same 28 clinical isolates of K. pneumoniae than the ones tested by LAMP. For each AMR gene, the positive control (+) was a clinical isolate, collected from the Ruijin Hospital (Shanghai, China) and confirmed to be positive by both PCR and sequencing. Two K. pneumoniae clinical isolates were positive (amplification band at 621 bp; 31 and 37) for bla _NDM (a), three were positive (amplification band at 438 bp; 38, 39 and 43) for bla _OXA-48-like (b), one was positive (amplification band at 232 bp; 47) for bla _IMP (c), and two were positive (amplification band at 390 bp; 54 and 55) for bla _VIM (d) genes. The marker (M) used for these experiments was a 100-bp DNA ladder.

FIGURE 4

K. pneumoniae LAMP and PCR detection in clinical sputum samples. (A) Colorimetric LAMP results, targeting the yhaI gene, obtained on DNA extracted from 40 clinical sputum samples; 20 of the samples were positive (yellow; 5, 11, 13, 20, and 25–40), and 20 were negative (pink; 1–4, 6–10, 12, 14–19, and 21–24). DNA extracted from K. pneumoniae HS11286 was used as positive control (+), and the negative control (−) was molecular H₂O. (B) PCR results, for yhaI gene amplification, obtained on DNA extracted 40 clinical sputum samples (same than A); 19 of the samples were positive (amplification bands; 5, 11, 13, and 25–40), and 21 were negative (no band; 1–4, 6–10, 12, and 14–24). DNA extracted from K. pneumoniae HS11286 was used as positive control (+) and the negative control (−) was molecular H₂O. The marker (M) was a 100-bp DNA ladder.

FIGURE 5

Carbapenemase-producing genes LAMP and PCR testing on clinical sputum samples. (A) Colorimetric LAMP results, targeting the bla _KPC gene, obtained on DNA extracted from the 18 clinical sputum samples that contained K. pneumoniae (according to MALDI-TOF); 11 of the samples were bla _KPC -positive (yellow; 13, 25–27, 31, and 35–40), and 7 were negative (pink). DNA extracted from K. pneumoniae HS11286 was used as a positive control (+), and the negative control (−) was molecular-grade H₂O. (B) PCR results, for bla _KPC amplification, obtained on DNA extracted from the 18 clinical sputum samples that contained K. pneumoniae (same as A); 11 of the samples were bla _KPC -positive (amplification bands; 13, 25–27, 31, and 35–40), and 7 were negative (no band). DNA extracted from K. pneumoniae HS11286 was used as positive control (+), and the negative control (−) was molecular-grade H₂O. The marker (M) was a 100-bp DNA ladder. (C) Colorimetric LAMP results, targeting the bla _NDM (a), bla _OXA-48like (b), bla _IMP (c), and bla _VIM (d) genes, obtained on the DNA extracted from sample 28. Positive controls (+) and negative controls (−) were used for each assay. Sample 28 was positive (yellow) for bla _NDM (a) and negative (pink) for the other carbapenemase-producing genes (b–d). (D) PCR results, targeting the bla _NDM (a), bla _OXA-48like (b), bla _IMP (c), and bla _VIM (d) genes, obtained on the DNA extracted from sample 28. Positive controls (+) and negative controls (−) were used for each assay. Sample 28 was positive (amplification band at 621 bp) for bla _NDM (a) and negative (no band) for the other carbapenemase-producing genes. The marker (M) was a 100-bp DNA ladder.