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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 blaKPC 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.

Klebsiella pneumoniae is the most common cause of nosocomial infections. There are two main types of K.pneumoniae (Kp) - classical K.pneumoniae (cKp) and hypervirulent K.pneumoniae (hvKp). In 2015, an outbreak of nosocomial infections caused by Kp was recorded in China for the first time, showing signs of hypervirulence and multidrug resistance (production of blaKPC-2). The purpose of this work is to assess the frequency of rectal colonization of patients admitted to a planned treatment in a cancer hospital with virulent and multidrug resistant strains of Kp and Pseudomonas aeruginosa. The study included 168 patients, 156 isolates of gram-negative bacteria were isolated from their rectal samples. Only one P.aeruginosa isolate turned out to be a blaVIMproducer. 30 isolates of Klebsiella spp.: Kp n=25; K.oxytoca n=3; K.planticola n=1; K.variicola n=1. Producers of carbapenemases have not been identified among them. Hypervirulence markers were detected in two of the three K.oxytoca isolates: in one (string-test «-») - all 5 genes (iucA,prmpA,prmpA2, iroB,peg-344), as well as two additional virulence genes, terB and irp2; in the second (string-test «+») - rmpA and irp2. A hypermucoid phenotype was observed in six Kp isolates and one K.oxytoca. Markers of hypervirulence, presumably with plasmid localization, were found in the Kp isolates: iucA (aerobactin), n=3; prmpA (hypermucoid phenotype regulator), n=3; iroB (salmohelin), n=2; and also, peg-344 (inner membrane conveyor), n=4; virulence determinants of presumably chromosomal localization: terB, n=1 and irp2, n=8. TerB and irp2 were also found in the K.variicola isolate. It is obvious that rectal carriage of bacteria is a real problem as it may be a constant reservoir and source of virulence genes, along with resistance genes.

This study aims to investigate the prevalence of clinically relevant carbapenemases genes ( bla KPC , bla NDM and bla OXA-48 ) in water samples collected over one-year period from hospital (H), raw and treated wastewater of two wastewater treatment plants (WWTPs) as well as along the Zenne River (Belgium). The genes were quantified in both particle-attached (PAB) and free-living (FLB) bacteria. Our results showed that absolute abundances were the highest in H waters. Although absolute abundances were significantly reduced in WWTP effluents, the relative abundance (normalized per 16S rRNA) was never lowered through wastewater treatment. Particularly, for the PAB the relative abundances were significantly higher in the effluents respect to the influents of both WWTPs for all the genes. The absolute abundances along the Zenne River increased from upstream to downstream, peaking after the release of WWTPs effluents, in both fractions. Our results demonstrated that bla KPC , bla NDM and bla OXA-48 are widely distributed in the Zenne as a consequence of chronic discharge from WWTPs. To conclude, the levels of carbapenemases genes are significantly lower than other genes conferring resistance to more widely used antibiotics (analyzed in previous studies carried out at the same sites), but could raise up to the levels of high prevalent resistance genes. [ABSTRACT FROM AUTHOR]

Antibiotic resistance is arguably the most serious health-related issue of the current time. This is even more so with carbapenem-resistant Enterobacteriaceae, for such microorganisms are resistant to the carbapenems (the 'antibiotics of last resort'). One of the most important considerations is in the detection of bacteria that carry the carbapenem-resistant gene. For this, molecular-based phenotypic and genetic-based polymerase chain reaction (PCR) methods are available. In contrast to phenotypic methods, molecular-genetic techniques, such as PCR, are considered to have the potential for improved detection of carbapenem-resistant genes by virtue of specificity, accuracy and rapidity. The tendency in PCR techniques is to develop towards the real-time systems equipped with multiplexing functionality. However, as shown in our study, standard PCR with electrophoresis detection continues widely to be used for the detection and identification of the carbapenemase gene. Therefore, despite progress in PCR technology, methods deployed for the detection of serious hospital-acquired infections around the world are arguably neither the most accurate nor the most efficient. This issue is of concern for pharmaceutical scientists in relation to the use and development of PCR technology and in relation to new drug development. [ABSTRACT FROM AUTHOR]

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Poirier, Kuang, Siedler, Borah, Mehat, Liu, Tai, Wang, van Vliet, Ma, Jenkins, Clark, La Ragione, Qu and McFadden.)