Cloning of a gene encoding a unique haemolysin from Klebsiella pneumoniae and its potential use as a species-specific gene probe

Chuang, Yin-Ching; Su, Jer-Horng; Lin, Chun-Chu; Chang, Mau Song

doi:10.1006/mpat.2002.0499

Cited by 31 publications

(20 citation statements)

References 27 publications

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“…K. pneumoniae isolate 675920 was isolated in 2015 from a sputum specimen from an elderly male with pulmonary infection in a public hospital in Lianyungang City, China. Bacterial species identification was performed by 16S rDNA gene sequencing [28] and by PCR detection of K. pneumoniae-specific khe gene [29]. The MLST scheme for K. pneumoniae was derived from the PasteurMLST database (http://bigsdb.pasteur.…”

Section: Bacterial Strain and Identificationmentioning

confidence: 99%

WITHDRAWN: Structure genomics of two chimera plasmids p675920-1 and p675920-2 coexisting in a multi-drug resistant Klebsiella pneumoniae isolate

Feng¹,

Yin²,

Zhan³

et al. 2018

Oncotarget

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This study dealt with detailed genomic characterization of two multidrug resistant (MDR) plasmids p675920-1 and p675920-2 from a single clinical Klebsiella pneumoniae isolate 675920. p675920-1 was essentially a hybrid of the IncFII plasmid pHN7A8 and the IncR plasmid pKPC-LK30, and functioned as an IncFII plasmid with inactivation of the IncR replication gene. The backbone of p675920-2 was a hybrid of a novel replicon, three maintenance regions (22.0-, 2.7-, 2.6-kb in length, respectively) as found in pKPYL2, p10164-3 and pK1HV, respectively, and the entire 25.9-kb conjugal transfer region of pKPYL2. p675920-1 and p675920-2 carried a large number of resistance genes, which contributed to resistance to at least seven classes of antibiotics (β-lactams, quinolones, aminoglycosides, fosfomycins, sulphonamides, trimethoprims, and tetracyclines) and one kind of heavy mental (mercury). All of these resistance genes are associated with mobile elements such as insertion sequences, insertion sequence-based transposition units, and transposons, which constituted a total of three novel MDR regions, two in p675920-1 and another in p675920-2. Coexistence of two MDR plasmids p675920-1 and p675920-2 made K. pneumoniae 675920 tend to become extensively drug-resistant.

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Section: Bacterial Strain and Identificationmentioning

confidence: 99%

WITHDRAWN: Structure genomics of two chimera plasmids p675920-1 and p675920-2 coexisting in a multi-drug resistant Klebsiella pneumoniae isolate

Feng¹,

Yin²,

Zhan³

et al. 2018

Oncotarget

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“…identified this genus correctly. Only one K. pneumoniae strain was not identified by the probe specific for the khe gene [48]. This can be explained by the diversity of khe alleles among K. pneumoniae strains.…”

Section: Discussionmentioning

confidence: 95%

Rapid Identification of Carbapenemase Genes in Gram-Negative Bacteria with an Oligonucleotide Microarray-Based Assay

et al. 2014

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Rapid molecular identification of carbapenemase genes in Gram-negative bacteria is crucial for infection control and prevention, surveillance and for epidemiological purposes. Furthermore, it may have a significant impact upon determining the appropriate initial treatment and greatly benefit for critically ill patients. A novel oligonucleotide microarray-based assay was developed to simultaneously detect genes encoding clinically important carbapenemases as well as selected extended (ESBL) and narrow spectrum (NSBL) beta-lactamases directly from clonal culture material within few hours. Additionally, a panel of species specific markers was included to identify Escherichia coli, Pseudomonas aeruginosa, Citrobacter freundii/braakii, Klebsiella pneumoniae and Acinetobacter baumannii. The assay was tested using a panel of 117 isolates collected from urinary, blood and stool samples. For these isolates, phenotypic identifications and susceptibility tests were available. An independent detection of carbapenemase, ESBL and NSBL genes was carried out by various external reference laboratories using PCR methods. In direct comparison, the microarray correctly identified 98.2% of the covered carbapenemase genes. This included blaVIM (13 out of 13), blaGIM (2/2), blaKPC (27/27), blaNDM (5/5), blaIMP-2/4/7/8/13/14/15/16/31 (10/10), blaOXA-23 (12/13), blaOXA-40-group (7/7), blaOXA-48-group (32/33), blaOXA-51 (1/1) and blaOXA-58 (1/1). Furthermore, the test correctly identified additional beta-lactamases [blaOXA-1 (16/16), blaOXA-2 (4/4), blaOXA-9 (33/33), OXA-10 (3/3), blaOXA-51 (25/25), blaOXA-58 (2/2), CTX-M1/M15 (17/17) and blaVIM (1/1)]. In direct comparison to phenotypical identification obtained by VITEK or MALDI-TOF systems, 114 of 117 (97.4%) isolates, including Acinetobacter baumannii (28/28), Enterobacter spec. (5/5), Escherichia coli (4/4), Klebsiella pneumoniae (62/63), Klebsiella oxytoca (0/2), Pseudomonas aeruginosa (12/12), Citrobacter freundii (1/1) and Citrobacter braakii (2/2), were correctly identified by a panel of species specific probes. This assay might be easily extended, adapted and transferred to point of care platforms enabling fast surveillance, rapid detection and appropriate early treatment of infections caused by multiresistant Gram-negative bacteria.

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“…Mapping of the presence/absence of the sequence region expected to be amplified by the primers was performed across Klebsiella phylogenetic diversity ( Figure S1 ). Regarding the targets proposed for the entire Kp complex, this in-silico analysis revealed that only khe and tyrB (24, 25) presented both a high sensitivity and a high specificity. We further analysed in silico the primers target sites for these two genes using primer-blast.…”

Section: Resultsmentioning

confidence: 96%

“…The tyrB and khe genes were previously proposed as targets for the specific detection of Kp (24, 38). As shown above, tyrB and khe targets were not totally specific for the Kp complex.…”

Section: Resultsmentioning

confidence: 99%

“…Some molecular methods (without need of sequencing) have been proposed over the years for the rapid detection of Kp (21–25). They target the 16S-23S rRNA internal transcribed spacer sequence (ITS) (21), coding sequences of tyrB (25), khe (24, 26), chromosomal beta-lactamase ( bla ) genes (27, 28) or other molecular targets (22). Some of these targets are described as able to detect the Kp complex (21), while others were designed for specific members of the Kp complex, such as Kp1 and Kp3 (22, 23).…”

Section: Introductionmentioning

confidence: 99%

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The ZKIR Assay, a novel Real-Time PCR Method for the Detection of Klebsiella pneumoniae and Closely Related Species in Environmental Samples

Barbier

Rodrigues

Depret

et al. 2019

Preprint

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Klebsiella pneumoniae (Kp) is of growing public health concern due to the emergence of strains that are multidrug-resistant, virulent, or both. Taxonomically, Kp includes seven phylogroups, with Kp1 (K. pneumoniae sensu stricto) being medically prominent. Kp can be present in environmental sources such as soils and vegetation, which could act as reservoirs of animal and human infections. However, the current lack of screening methods to detect Kp in complex matrices limits research on Kp ecology. Here we analysed 4222 genome sequences and found that existing molecular detection targets lack specificity for Kp. A novel real-time PCR method, the ZKIR assay, was developed and used to detect Kp in 96 environmental samples. Results were compared to a culture-based method using SCAI agar medium coupled to MALDI-TOF mass spectrometry identification. Whole-genome sequencing of environmental Kp was performed. The ZKIR assay was positive for the 48 tested Kp reference strains, whereas 88 non-Kp strains were negative. The limit of detection of Kp in spiked soil microcosms was 1.5 × 10-1 CFU g-1 after enrichment for 24 h in LB supplemented with ampicillin, and 1.5 × 103 to 1.5 × 104 CFU g-1 directly after soil DNA extraction. The ZKIR assay was more sensitive than the culture method. Kp was detected in 43% of environmental samples. Genomic analysis of the isolates revealed a predominance of phylogroups Kp1 (65%) and Kp3 (32%), a high genetic diversity (23 MLST sequence types), a quasi-absence of antibiotic resistance or virulence genes, and a high frequency (50%) of O-antigen type 3. This study shows that the ZKIR assay is an accurate, specific and sensitive novel method to detect the presence of Kp in complex matrices, and indicates that Kp isolates from environmental samples differ from clinical isolates.IMPORTANCEThe Klebsiella pneumoniae species complex (Kp) includes human and animal pathogens, some of which are emerging as hypervirulent and/or antibiotic resistant strains. These pathogens are diverse and classified into seven phylogroups, which may differ in their reservoirs and epidemiology. Proper management of this public health hazard requires a better understanding of Kp ecology and routes of transmission to humans. So far, detection of these microorganisms in complex matrices such as food or the environment has been difficult due to a lack of accurate and sensitive methods. Here, we describe a novel method based on real-time PCR, which enables detection of all Kp phylogroups with high sensitivity and specificity. We used this method to detect Kp isolates from environmental samples, and show based on genomic sequencing that they differ in antimicrobial resistance and virulence gene content, from human clinical Kp isolates. The ZKIR PCR assay will enable rapid screening of multiple samples for Kp presence and will thereby facilitate tracking the dispersal patterns of these pathogenic strains across environmental, food, animal and human sources.

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Cloning of a gene encoding a unique haemolysin from Klebsiella pneumoniae and its potential use as a species-specific gene probe

Cited by 31 publications

References 27 publications

WITHDRAWN: Structure genomics of two chimera plasmids p675920-1 and p675920-2 coexisting in a multi-drug resistant Klebsiella pneumoniae isolate

WITHDRAWN: Structure genomics of two chimera plasmids p675920-1 and p675920-2 coexisting in a multi-drug resistant Klebsiella pneumoniae isolate

Rapid Identification of Carbapenemase Genes in Gram-Negative Bacteria with an Oligonucleotide Microarray-Based Assay

The ZKIR Assay, a novel Real-Time PCR Method for the Detection of Klebsiella pneumoniae and Closely Related Species in Environmental Samples

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