Dissemination of NDM-1-Producing Enterobacteriaceae Mediated by the IncX3-Type Plasmid

<div><p>The emergence and spread of NDM-1-producing Enterobacteriaceae have resulted in a worldwide public health risk that has affected some provinces of China. China is an exceptionally large country, and there is a crucial need to investigate the epidemic of <i>bla</i>_NDM-1-positive Enterobacteriaceae in our province. A total of 186 carbapenem-resistant Enterobacteriaceae isolates (CRE) were collected in a grade-3 hospital in Zhejiang province. Carbapenem-resistant genes, including <i>bla</i>_KPC, <i>bla</i>_IMP, <i>bla</i>_VIM, <i>bla</i>_OXA-48 and <i>bla</i>_NDM-1 were screened and sequenced. Ninety isolates were identified as harboring the <i>bla</i>_KPC-2 genes, and five <i>bla</i>_NDM-1-positive isolates were uncovered. XbaI-PFGE revealed that three <i>bla</i>_NDM-1-positive <i>K</i>. <i>pneumoniae</i> isolates belonged to two different clones. S1-PFGE and southern blot suggested that the <i>bla</i>_NDM-1 genes were located on IncX3-type plasmids with two different sizes ranging from 33.3 to 54.7 kb (n=4) and 104.5 to 138.9 kb (n=1), respectively, all of which could easily transfer to <i>Escherichia coli</i> by conjugation and electrotransformation. The high-throughput sequencing of two plasmids was performed leading to the identification of a smaller 54-kb plasmid, which had high sequence similarity with a previously reported pCFNDM-CN, and a larger plasmid in which only a 7.8-kb sequence of a common gene environment around <i>bla</i>_NDM-1 (<i>bla</i>_NDM-1-<i>trpF</i>- <i>dsbC</i>-<i>cutA1</i>-<i>groEL</i>-Δ<i>InsE</i>,) was detected. PCR mapping and sequencing demonstrated that four smaller <i>bla</i>_NDM-1 plasmids contained a common gene environment around <i>bla</i>_NDM-1 (IS<i>5</i>-<i>bla</i>_NDM-1-<i>trpF</i>- <i>dsbC</i>-<i>cutA1</i>-<i>groEL</i>). We monitored the CRE epidemic in our hospital and determined that KPC-2 carbapenemase was a major risk to patient health and the IncX3-type plasmid played a vital role in the spread of the <i>bla</i>_NDM-1 gene among the CRE.</p></div

This is the Fig 2 Location of the <i>bla</i>_NDM-1 gene and the IncX3 type of <i>bla</i>_NDM-1 plasmid.

<p>This is the Fig 2 Location of the <i>bla</i>_NDM-1 gene and the IncX3 type of <i>bla</i>_NDM-1 plasmid.</p

Table_3.DOCX

<p>Tigecycline is one of the last resort treatments for carbapenem-resistant Klebsiella pneumoniae (CRKP) infections. Tigecycline resistance often occurs during the clinical treatment of CRKP, yet its mechanism has still not been clearly elucidated. This study presents an analysis of a tigecycline resistance mechanism that developed in clinical isolates from a 56-year-old female patient infected with CRKP during tigecycline treatment. Consecutive clonal consistent K. pneumoniae isolates were obtained during tigecycline treatment. Whole genome sequencing of the isolates was performed, and putative single nucleotide polymorphisms and insertion and deletion mutations were analyzed in susceptible and resistant isolates. The identified gene of interest was examined through experiments involving transformations and conjugations. Four isolates, two of which were susceptible and two resistant, were collected from the patient. All of the isolates belonged to Sequence Type 11 (ST11) and were classified as extensively drug resistant (XDR). One amino acid substitution S251A in TetA was identified in the tigecycline-resistant isolates. Subsequent transformation experiments confirmed the contribution of the TetA variant (S251A) to tigecycline resistance. The transfer capacity of tigecycline resistance via this mutation was confirmed by conjugation experiments. Using southern blot hybridization and PCR assays, we further proved that the tetA gene was located on a transferable plasmid of ca. 65 kb in an Escherichia coli EC600 transconjugant. Our results provide direct in vivo evidence that evolution in the tetA gene can lead to tigecycline treatment failure in CRKP clinical strains that carry tetA. Moreover, the transfer capacity of tigecycline resistance mediated by mutated tetA is a threat.</p

Table_2.DOCX

<p>Tigecycline is one of the last resort treatments for carbapenem-resistant Klebsiella pneumoniae (CRKP) infections. Tigecycline resistance often occurs during the clinical treatment of CRKP, yet its mechanism has still not been clearly elucidated. This study presents an analysis of a tigecycline resistance mechanism that developed in clinical isolates from a 56-year-old female patient infected with CRKP during tigecycline treatment. Consecutive clonal consistent K. pneumoniae isolates were obtained during tigecycline treatment. Whole genome sequencing of the isolates was performed, and putative single nucleotide polymorphisms and insertion and deletion mutations were analyzed in susceptible and resistant isolates. The identified gene of interest was examined through experiments involving transformations and conjugations. Four isolates, two of which were susceptible and two resistant, were collected from the patient. All of the isolates belonged to Sequence Type 11 (ST11) and were classified as extensively drug resistant (XDR). One amino acid substitution S251A in TetA was identified in the tigecycline-resistant isolates. Subsequent transformation experiments confirmed the contribution of the TetA variant (S251A) to tigecycline resistance. The transfer capacity of tigecycline resistance via this mutation was confirmed by conjugation experiments. Using southern blot hybridization and PCR assays, we further proved that the tetA gene was located on a transferable plasmid of ca. 65 kb in an Escherichia coli EC600 transconjugant. Our results provide direct in vivo evidence that evolution in the tetA gene can lead to tigecycline treatment failure in CRKP clinical strains that carry tetA. Moreover, the transfer capacity of tigecycline resistance mediated by mutated tetA is a threat.</p

Table_1.DOCX

<p>Tigecycline is one of the last resort treatments for carbapenem-resistant Klebsiella pneumoniae (CRKP) infections. Tigecycline resistance often occurs during the clinical treatment of CRKP, yet its mechanism has still not been clearly elucidated. This study presents an analysis of a tigecycline resistance mechanism that developed in clinical isolates from a 56-year-old female patient infected with CRKP during tigecycline treatment. Consecutive clonal consistent K. pneumoniae isolates were obtained during tigecycline treatment. Whole genome sequencing of the isolates was performed, and putative single nucleotide polymorphisms and insertion and deletion mutations were analyzed in susceptible and resistant isolates. The identified gene of interest was examined through experiments involving transformations and conjugations. Four isolates, two of which were susceptible and two resistant, were collected from the patient. All of the isolates belonged to Sequence Type 11 (ST11) and were classified as extensively drug resistant (XDR). One amino acid substitution S251A in TetA was identified in the tigecycline-resistant isolates. Subsequent transformation experiments confirmed the contribution of the TetA variant (S251A) to tigecycline resistance. The transfer capacity of tigecycline resistance via this mutation was confirmed by conjugation experiments. Using southern blot hybridization and PCR assays, we further proved that the tetA gene was located on a transferable plasmid of ca. 65 kb in an Escherichia coli EC600 transconjugant. Our results provide direct in vivo evidence that evolution in the tetA gene can lead to tigecycline treatment failure in CRKP clinical strains that carry tetA. Moreover, the transfer capacity of tigecycline resistance mediated by mutated tetA is a threat.</p

Additional file 1: of Dissemination of blaNDM-5 gene via an IncX3-type plasmid among non-clonal Escherichia coli in China

cg-MLST of blaNDM-5-positive isolates. (DOCX 61 kb

Chromosomal integration and plasmid fusion occurring in ST20 carbapenem-resistant <i>Klebsiella pneumoniae</i> isolates coharboring <i>bla</i>_NDM-1 and <i>bla</i>_IMP-4 induce resistance transmission and fitness variation

To investigate the epidemiology of ST20 carbapenem-resistant Klebsiella pneumoniae (CRKP) in China, and further explore the genomic characteristics of blaIMP-4 and blaNDM-1 coharboring isolates and plasmid contributions to resistance and fitness. Seven ST20 CRKP isolates were collected nationwide, and antimicrobial susceptibility testing was performed. Antimicrobial resistance genes, virulence genes, and plasmid replicons were identified via whole-genome sequencing, and clonality assessed via core-genome multilocus sequence typing. Furthermore, we found four dual-metallo-β-lactamases (MBL)-harbouring isolates, the gene location was detected by Southern blotting, and plasmid location analysis showed that blaIMP-4 was located on a separate plasmid, a self-conjugative fusion plasmid, or the bacterial chromosome. These isolates were subjected to long-read sequencing, the presence of blaIMP-4 in different locations was identified by genomic comparison, and transposon units were detected via inverse PCR. We subsequently found that blaIMP-4 on the fusion plasmid and bacterial chromosome was formed via intact plasmid recombination by the IS26 and ltrA, respectively, and the circular transposon unit was related to cointegration, however, blaIMP-4 in different locations did not affect the gene stability. The blaNDM-1-harbouring plasmid contributed to the increased resistance to β-lactams and shortened survival lag time which was revealed in plasmid cured isolates. In summary, the K. pneumoniae ST20 clone is a high-risk resistant clone. With the use of ceftazidime/avibactam, MBL-positive isolates, especially dual-MBL-harbouring isolates, should be given additional attention.</p