ST11 is the main clone of Klebsiella pneumoniae prevailing in China(10, 11). Many literatures have reported that ST11 Klebsiella pneumoniae carries blaKPC−2 gene(12, 13), and only a small number of reported strains carry both blaNDM−5 and blaKPC−2 genes(14, 15). Prof. Wang Hui pointed out that the Escherichia coli producing KPC-2 and NMD-1 at the same time is the KPC-2 producing Escherichia coli that accidentally obtained the plasmid of NDM-1(16). This article also partially supports this theory. Klebsiella pneumoniae KPN-hnqyy, KP69 and KP19-2029 have similar genomes (Fig. 2), but KPN-hnqyy produces both KPC-2 and NDM-5 carbapenemases, while KP69 and KP19-2029 only contains KPC-2 carbapenemase, so it is guessed that ST11 Klebsiella pneumoniae KPN-hnqyy that produces both KPC-2 and NDM-5 carbapenemase may be derived from similarly KPC-2 carrying Klebsiella pneumoniae KP69 and KP19-2029 that have obtained blaNDM−5 gene cluster accidentally. This was further verified by comparing the chromosomes of similar strains and their respective plasmids carrying blaKPC−2 gene cluster. The chromosomal genomes of Klebsiella pneumoniae KPN-hnqyy, KP69 and KP19-2029 were similar (Fig. 2), their plasmids of different types also contained similar IncFII and IncR drug resistance gene fusion regions (similarity > 99.9%) (Fig. 3), indicating that in such type ST11 Klebsiella pneumoniae, this drug resistance gene fusion region and chromosomal genome of the plasmid may be relatively stable, but there may be some recombination hotspots at both ends of the drug resistance gene region, resulting in the easy integration of different exogenous sequences and leading to a large range of recombination in the backbone region of the plasmid. In this study, the backbone IncN regions of plasmids pKPN-hnqyy-kpc and pL22-2 are similar (similarity > 99%) (Fig. 2), both ends are connected to the IS26 transposase gene, but the chromosomal genome similarity of their corresponding strains is 67.21% (Fig. 2), so it can be inferred that the IncN replicon region of plasmid pKPN-hnqyy-kpc may be derived from different strains by means of IS26-mediated gene recombination.
At present, it is reported abroad that the metastatic elements carrying the blaKPC−2 gene are mainly Tn4401 transposon(17), which contains two insertion sequences, ISKpn6 and ISKpn7, in their surrounding sequences (Fig. 4). However, it was found in China that the metastatic element carrying blaKPC−2 gene usually contained an insertion element of Tn3 upstream, and ISKpn7 was replaced with ISKpn27 downstream containing an insertion sequence of Tn1721/Tn1722 to form a Tn3-△Tn4401-Tn1721/Tn1722 complex transposable element carrying the blaKPC−2 gene(18). In this study, in the environment of blaKPC−2 gene, on the basis of the above complex transposable element, IS26 family transposase was inserted at both ends, and only a short sequence of Tn3 recombinase gene remained upstream, and the gene of some Tn1721/Tn1722 transposable elements was truncated downstream (Fig. 4), indicating that this sequence may evolve from a similar sequence of Tn3-△Tn4401-Tn1721/Tn1722. The effect of this transposable element sequence change on the efficiency of blaKPC−2 gene transfer and the expression level of blaKPC−2 gene is unknown.
The blaNDM−5 gene is mostly carried by transferable plasmids such as IncX or IncF plasmids(19, 20). In this study, the pKPN-hnqyy-ndm plasmid is relatively special, temporarily unclassifiable, and the conjugation test is negative. Importantly, its plasmid backbone part contains two phage-associated insertion sequences, which are also found in the chromosomal genome of this strain. Phage is one of the important vectors for horizontal gene transfer and can mediate the transfer of various resistance genes such as bla KPC and blaNDM(11). It has been documented that plasmids carrying the blaKPC−2 gene can acquire phages and form a complex of plasmids and phages(21, 22). In this study, the relationship between blaNDM−5 gene and phage and the effect of inserting phage fragments in the plasmid on the plasmid stability and conjugation efficiency remain unknown. The blaNDM−5 gene environment contains a tandem resistance gene cassette composed of aadA2, dfrA12 and sul1 genes, and similar sequences are also found in plasmids of different species from a variety of bacteria(23), and it is typically characterized by IS26 insertion elements at both ends of the gene cassette. IS26, as one of the most important members of the IS6 insertion sequence family, can not only affect the horizontal transfer, integration, rearrangement, and expression of related genes within or between strains, but also affect the integration of plasmids(24, 25). Different from the viewpoint of Prof. Wang Hui et al.(16), the NDM-5 carbapenemase gene herein may not be obtained by plasmid conjugation, but by the transfer of IS26 transposable elements.
The Klebsiella pneumoniae KPN-hnqyy in this study was first found in stool screening of a child with hematological diseases, and was subsequently isolated from its pancreatic juice, blood, ascites, and stool samples 6 months after its discharge from hospital, indicating that this bacterium can colonize the patient's intestine for a long time and may become a pathogenic bacterium. Typically, in the absence of antibiotic pressure, if the plasmid of the strain contains a resistance gene, it allows the bacterium to pay an "adaptation price", resulting in the natural disappearance of the resistance plasmid at the end of treatment or after the colonizer is transferred to other hosts(26). However, the ST11 Klebsiella pneumoniae in this study colonized the patient's intestine for a long time, which may be due to mutations in the chromosome of the strain or the weakening of the "adaptation price" by the expression of plasmids. At present, ST11-type CRKP carrying blaKPC−2is increasing at an alarming rate(27), especially the emergence of CRKP carrying both blaKPC−2 and NMD−5 genes in this study, and it can colonize the intestine for a long time; thereafter, it may disseminate into the surrounding environment through feces, which undoubtedly increases the difficulty of CRKP treatment, prevention and control.
Klebsiella pneumoniae positive for both KPC-2 and NDM-5 carbapenemases in this study is type ST11, and the blaKPC−2 gene is located on the IncR/IncN/IncF fusion particle and is present in a structure evolved from Tn3-△Tn4401-Tn1721/Tn1722 with both ends inserted into the IS26 transposable element; the blaNDM−5 gene is located on a special plasmid containing a phage insert and is present in a tandem resistance gene cassette containing multiple resistance genes composed of the IS26 transposable element at both ends. The Klebsiella pneumoniae KPN-hnqyy that carries both NDM-5 and KPC-2 may be derived from the KPC-2 carrying Klebsiella pneumoniae KP69 and KP19-2029 strains accidentally obtain the NDM-5 gene by the transfer of IS26 transposable elements. The wide transmission of Klebsiella pneumoniae ST11 carrying the blaKPC-2 gene in China and its ability to obtain other carbapenemase genes through transposable element IS26 are well worth attention.