Detection of the Novel Imp-38 among Carbapenemase-producing Enterobacteriaceae in a University Hospital, China

Introduction: This study set out to investigate the molecular epidemiology of carbapenemase-producing Enterobacteriaceae isolates collected from Xiang Ya Hospital, Hunan, China. Methodology: The carbapenemase genes from Enterobacteriaceae isolates were determined by PCR and sequencing. Relatedness of Klebsiella pneumoniae isolates was evaluated by pulsed-field gel electrophoresis. Results: Twenty-four out of 738 non-repetitive Enterobacteriaceae isolates harbored carbapenemase genes including IMP-38, a novel IMP-type metallo-enzyme. Nine IMP-38-producing isolates were shown to originate from the same clone and caused a small outbreak in the neonatal ward. Conclusions: IMP-38, a novel IMP-type metallo-enzyme, was one of the predominant types of carbapenemase in the clinical carbapenem-resistant Enterobacteriaceae isolates in our hospital.


Introduction
Carbapenems, such as imipenem and meropenem, have a broad activity of spectrum and stability against β-lactamases [1,2].Carbapenem-resistant Enterobacteriaceae are being increasingly reported worldwide, representing a significant threat to public health; great efforts toward detection and infection control strategies are required.A variety of carbapenemases has been reported in Enterobacteriaceae, including the Ambler class A βlactamases of KPC-type, the metallo-β-lactamases (MBL) (Ambler class B) of VIM-, IMP-, and NDMtypes, and the carbapenem-hydrolysing class D βlactamases of the OXA-48-type [3].
In our previous study, we showed that Enterobacteriaceae clinical isolates collected from three affiliated hospitals of Central South University carried a high incidence (33.4%) of ESBLs, predominately the CTX-M-type [4].In this study, we investigated the molecular epidemiology and the antimicrobial-resistant mechanisms of carbapenemases in Enterobacteriaceae clinical isolates.

Bacterial strains and plasmid
A total of 738 non-repetitive clinical Enterobacteriaceae isolates were collected from Xiang Ya Hospital, Hunan, China, between March 2010 and February 2011 (Table 1).The isolates were confirmed by Vitek2 GN card (bioMérieux, Marcy l'Etoile, France), an automatic microorganism identification system.PUC19 (Thermo Fisher Scientific, Waltham, MA, USA) was used as a vector to clone the amplified bla IMP-38 gene.

Antimicrobial susceptibility testing
Carbapenem-resistant Enterobacteriaceae isolates were screened using an agar diffusion method with imipenem, meropenem, and ertapenem disks (10 µg for each antibiotic) first.The minimal inhibitory concentrations (MICs) of the following antibiotics in carbapenemase-producing isolates were then determined by agar dilution test: imipenem, meropenem, ertapenem, cefotaxime, cefepime, amikacin, ciprofloxacin, aztreonam, ceftriaxone, gentamicin, piperacillin/tazobactam, tetracycline, and polymyxin B (Oxoid Basingstoke, UK).Escherichia coli ATCC 25922 was used as the internal quality control strain.The results of both tests were interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines [5].

Carbapenemase phenotypic screening
The modified Hodge test was used to determine phenotypes of KPC-type carbapenemase-producing isolates.Class B carbapenemase-producing strains were detected by imipenem and the EDTA synergy test [6].

PCR amplification and sequencing for carbapenemase genes
Extracted whole-cell lysates or plasmids isolated from ertapenem-resistant isolates were used as the templates for PCR amplification to determine various carbapenemase genes, including bla KPC , bla IMP , bla VIM , bla NDM , and bla OXA-48 [7][8][9][10].PCR products were then subjected to direct sequencing with an automatic sequencer (model 3730; Applied Biosystems, Foster City, CA, USA).

Pulsed-field gel electrophoresis (PFGE) typing
PFGE was performed to evaluate clonal relationships between some carbapenemase-producing K. pneumoniae strains.The procedures included digestion with XbaI restriction enzyme at 37°C for 12 hours and electrophoresis at 120°C, 6 V/cm, 0.5-70 seconds for 24 hours.The XbaI restriction patterns of genomic DNA of the isolates were analyzed and interpreted according to the criteria described by Tenover et al. [11].

Cloning of the bla IMP-38 gene
The entire bla IMP-38 gene was amplified using two pairs of primers flanked with specific restriction sites.The PCR products were cloned into pUC19 vector for sequencing.

Phenotypic testing for carbapenemase
Among the 738 strains, 27 strains were positive by the modified Hodge test, 20 were positive by the EDTA-disk synergy test (Table 1).

Determination of carbapenemase genes
Twenty-four out of forty-four ertapenem-resistant isolates were found to carry carbapenemase genes, among which four were KPC type-positive and twenty were IMP-type positive strains.The plasmids isolated from these carbapenemase-producing strains were found to carry carbapenemase genes.Sequencing analysis showed that eleven strains contained IMP-4, among which five were K. pneumoniae, three were E. cloacae, one was E. coli, one was C. freundii, and one was K. oxytoca.In addition, KPC-2 was found in four K. pneumonia isolates.IMP-38, a novel IMP-type metallo-enzyme, was found in nine K. pneumoniae isolates.The gene showed a contig of 874 bp with an open reading frame of 741 nucleotides encoding a putative protein of 246 amino acids.It differed from bla IMP-4 [12] by one point mutation (A640G), which led to one amino acid substitution (Ser214Gly).Since this one amino acid substitution has not been described yet in IMP-type enzymes produced by clinical strains, it was designated as IMP-38.

PFGE typing
The PFGE patterns of the nine IMP-38-positive K. pneumoniae isolates showed homologous appearance, with differences of only one to three bands; they can, therefore, be considered to be five subclones of one pulsed-field-type clone.Among them, KP8, KP9, KP12, KP13, and KP15 belonged to the same subclone (Figure 1A).The PFGE patterns of the four KPC-2positive K. pneumoniae isolates were identical (Figure 1B).

Discussion
The metallo-β-lactamases are the most molecularly diverse carbapenemases, showing the greatest clinical threat.During a one-year period in our hospital, 738 non-repetitive clinical Enterobacteriaceae isolates were collected, among which 24 were identified as carbapenemase-producing isolates, with 6 strains showing resistance to impenem and 15 to meropenem.Therefore, carbapenemase phenotypic screening of Enterobacteriaceae isolates with decreasing susceptibility should be enhanced in order to avoid missing the carbapenemase-producing isolates.
IMP-4, the first MBL found in a plasmid from a C. youngae isolate in 2001 in China, now appears to be the most dominant variant in China [13].In the present study, we detected twenty IMP-producing isolates, including eleven IMP-4-positive and nine IMP-38positive isolates.Our study showed that bla IMP genes were disseminated in different enterobacterial species.The IMP-38 was identified as differing from IMP-4 by a point mutation, suggesting that IMP-38 may originate from IMP-4.The nine IMP-38-positive K. pneumoniae isolates belonged to the same clone and were collected from the trachea cannula of nine newborn patients who suffered from respiratory distress syndrome in the neonatal ward in October 2010.Our finding drew great concerns from clinicians of the neonatal ward and the hospital's infection control department.Strengthening disinfection of the ward and medical facilities, as well as quarantining the nine neonates, effectively controlled the spread of the IMP-38 carrying clone.
The worldwide spread of KPC-producing isolates has become a serious problem in the past five years, ever since it was first described in the northeastern United States [2].A high prevalence rate of KPC-2 Enterobacteriaceae isolates has been reported in Zhejiang province, China, and sporadic reports have been made in Shanghai City, Jiangsu, and Jiangxi provinces [14][15][16].In this study, four KPC-2producing K. pneumoniae isolates with identical origination were detected.They were isolated from three non-adjacent departments in our hospital -the integrated ward, ICU, and orthopedics department.These data -the first report in Hunan provincesuggest that clone dissemination is the way KPCpossessing K. pneumoniae isolates are spread in the hospital.
In summary, carbapenem resistance in our hospital has mainly appeared in K. pneumoniae clinical isolates and has mostly resulted from production of IMP and KPC types of metallo-beta-lactamases. IMP-38 is a novel IMP-type metallo-enzyme identified as one of the predominant types of carbapenemase in the clinical carbapenem-resistant Enterobacteriaceae isolates in our hospital.

Nucleotide sequence
The sequence of bla IMP-38 has been deposited to GenBank under the accession number of HQ875573.

Table 1 .
Carbapenemase phenotypes and genotypes of Enterobacteriaceae isolates