Identification of plasmid IncQ1 and NTEKPC-IId harboring bla KPC-2 in isolates from Klebsiella pneumoniae infections in patients from Recife-PE, Brazil

Abstract INTRODUCTION: This study investigated the genetic environment of bla KPC-2 in Klebsiella pnemoniae multi-drug resistant clinical isolates. METHODS: Four carbapenemase gene isolates resistant to carbapenems, collected from infected patients from two hospitals in Brazil, were investigated using polymerase chain reaction and plasmid DNA sequencing. RESULTS: The bla KPC-2 gene was located between ISKpn6 and a resolvase tnpR in the non-Tn4401 element (NTEKPC-IId). It was detected on a plasmid belonging to the IncQ1 group. CONCLUSIONS To our knowledge, this is the first report of the presence of the bla KPC-2 gene in the NTEKPC-IId element carried by plasmid IncQ1 from infections in Brazil.

Klebsiella pneumoniae is one of the pathogens responsible for healthcare-associated infections (HAIs). Infections can, depending on the anatomic site affected and the patient's immune status,lead to a range of adverse clinical outcomes, including death, as this gram-negative bacterium carries several antibiotic resistance and virulence genes 1 .
The most relevant antibiotic-resistant genes in this bacterial species are those that encode carbapenemases, (KPC). K. pneumoniae isolates from different countries, including Brazil, have been found to contain the KPC encoding gene bla KPC-2 . This gene has been found to be located on plasmids of different sizes and nucleotide sequences and belong to different incompatibility groups (Incs), the most prevalent being IncL/M, IncFII and IncN 2,3 . Nicoletti et al. (2015) 4 identified the IncQ plasmid in K. pneumoniae carrying carbapenemase BKC-1 from São Paulo, Brazil. IncQ1 is a stable and mobilizable plasmid that can be transferred among a wide range of gram negative bacteria through conjugative plasmids present in the same bacterial cell, which facilitates its transmission in a hospital environment. There are few studies in Brazil that characterize the plasmid Incs of K. pneumoniae, mainly because some of these are non-typeable plasmids, such as the ones studied by Pereira et al (2013) 5 .
These plasmids may also harbor different isoforms of the Tn4401 transposon. Nine variants of Tn4401 (a-i) have been described 6 , of which variants "a" and "b" are the most common. Non-Tn4401 (NTE -KPC ) 7,8 elements that can carry bla KPC-2 have also been described, including those recently detected in two colonization isolates in Brazil 8 .
The aim of this study was to investigate the genetic environment of the bla KPC-2 gene from clinical isolates of K. pneumoniae resistant to carbapenems, thus helping to understand the dissemination of carbapenem resistance. This may help develop new strategies to prevent the spread of these resistance genes in the hospital environment.
Four multi-drug resistant (MDR) clinical isolates of K. pneumoniae (K3R2, K4R2, K6R2, and K1E) were selected, following isolation, from infections (peritoneal fluid, blood cultures,  and cerebrospinal fluid) in patients from two hospitals in Recife-PE, Brazil in 2016. The isolates were identified biochemically using the automated VITEK® 2 method. The isolates were stored in 20% glycerol at -70°C. For the analyses, the isolates were cultured in BHI (Brain Heart Infusion) broth or Luria Bertani (LB) for 18 hours at 37°C.
The genomic DNA of the isolates was extracted by the Wizard Genomic DNA purification kit (Promega-Brazil). The bla KPC-2 gene was investigated usingpolymerase chain reaction (PCR) using previously described primers and amplification conditions 2 . Negative and positive controls were included in each PCR. The amplified products were electrophoresed in 1% agarose gel under constant voltage of 100 V in 0.5 × TBE buffer (Tris-base, boric acid, and ethylenediamine tetra-acetic acid, EDTA-pH8,0).
The enterobacterial repetitive intergenic consensus (ERIC)-PCR method was used to determine the clonal relationship of the isolates using previously described primers and amplification conditions 2 . The amplified products were electrophoresed in 1.5% agarose gel under a constant voltage of 100V in 0.5 × TBE buffer.
Plasmid DNA was extracted using the Plasmid Mini Kit (Qiagen), was quantified using the NanoDrop spectrophotometer and Qubit fluorometric platform (ThermoFisher Scientific). The libraries were built using the Nextera XT Library Preparation (Illumina) and were quantified via real-time PCR using the Library Quantification kit -Illumina/Universal (Kapa Biosystems). Sequencing was performed using MiSeq equipment (Illumina) with the MiSeq 500-cycle cartridge Nano kit V2 (Illumina). The data were processed using the Trimmomatic tool 10 , and de-novo assemblies were performed using the Velvet tool 11 . The annotated plasmid DNA sequences were visualized using Artemis Sanger software 12 .
The IncQ plasmid was identified using PCR with the primers and the amplification conditions described by Götz et al. (1996) 13 .
The PCR products were electrophoresed on 1.0% agarose gel in TBE buffer. The IncQ plasmid was also identified using in silico PCR; bioinformatics tools used included sequence manipulation suite (SMS) (http://www.bioinformatics.org/sms2/index.html) and primer-basic local alignment search tool (BLAST), using the primers for determination of all different plasmid incompatibility groups, as defined by Carattoli et al (2005) 14 .
The isolates of K. pneumoniae were MDR, with resistance to β-lactams, and especially to carbapenems ( Table 1), and they were suspected of being producers of KPC. Using PCR, the presence of the bla KPC-2 gene in the four K. pneumoniae isolates analyzed was confirmed. The ERIC-PCR genotyping test showed that all the isolates presented distinct clonal profiles, with a maximum of 40% similarity, and therefore they did not present a clonal relationship ( Table 1).The plasmid DNA was sequenced to a depth of approximately 238 times. The analysis of the plasmid DNA sequences from all isolates using the Resfinder and GenBank databases confirmed the presence of the bla KPC-2 (882 bp) antibioticresistance gene. The gene was identified with 100% similarity when compared with a sequence deposited in GenBank (CP023186.1). The bla KPC-2 gene was observed in similar genetic locations of all isolates and was inserted between the ΔISKpn6 insert sequence and a resolvase tnpR; between 402-558 bp upstream of the bla KPC-2 gene, a truncated bla TEM gene was an evidence that bla KPC-2 was inserted into a non-Tn4401 (variant NTE KPC -IId) (Figure 1). We found deletions in tnpA and a total deletion of the ISKpn7 insert sequence (Figure 1).
In the same consensus sequence where the non-Tn4401 was located, plasmid mobility proteins (mobA, mobB, and mobC) and replication proteins (repA, repB, and repC) were found with a 100% similarity to the reference pool of the IncQ1 RSF1010 (M28829.1).
The oriV, oriT, and repB genes were also found inserted into the same consensus sequence of the replication and mobilization genes as the bla KPC-2 gene. Thus, this result suggests that all the isolates have the genes encoding the plasmid IncQ1 and the bla KPC-2 gene in the same consensus sequence (Figure 1 and Table 2).
PlasmidFinder confirmed the presence of a plasmid belonging to the variant incompatibility group IncQ1. The in silico PCR with all the isolates, tested positive for the IncQ1 replicons (oriV-436bp, oriT-191bp, and repB-1160pb). Comparative analysis among the four K. pneumoniae isolates of this study and reference sequences  for the IncQ1 and IncQ-like plasmids deposited in GenBank showed 98% to 100% similarity to the oriV gene (M21475.1), to the oriT gene (X04830.1), and to the repB gene (M28829.1).
The PCR for the IncQ1 replicons also confirmed this result, with the repB, oriV, and oriT genes of the plasmid IncQ1 amplified in all isolates analyzed. In addition to IncQ1, the isolates also presented other plasmids, but these were not typable given the total size of the DNA sequence ( Table 2).
Antimicrobial resistance genes are spread among enterobacteria due to the horizontal transfer of mobile genetic elements. The bla KPC-2 gene is found associated with several different plasmids 1,7 . However, little was known about the plasmid genetic environment of this gene in clinical isolates of K. pneumoniae in Brazil, and especially in Recife-PE, where the first reports of KPC in Brazil came from.
The bla KPC-2 gene is often found inserted into transposon Tn440, which has different isoforms, but it has also been found in a non-Tn4401 mobile element (NTE KPC ) in China, Argentina, Brazil, and Russia 7,15,16 . NTE KPC has been separated into three groups based on the absence or presence of the bla TEM gene, where the second group, NTE KPC -II, includes the variant with a truncated bla TEM gene 15,16 .
The non-Tn4401 variant of the present study resembles the NTE KPC-IId variant (Figure 1). These findings corroborate the results obtained by our research group with Klebsiella aerogenes in Recife-PE, Brazil, which had 100% similarity with a sequence deposited in GenBank (MG786907, MH000708).
IncQ and IncQ-like plasmids have been found in different bacterial species such as Escherichia coli, Salmonella typhimurium, Salmonella enterica serovar, Pseudomonas aeruginosa, and Enterobacter cloacae from locations in Canada, Italy, the United Kingdom, and Germany 17 .
This report demonstrates the presence of the bla KPC-2 gene in the non-Tn4401 element (NTE KPC-IId), which is carried by small, mobilizable, and promiscuous plasmids of the type IncQ1, in four clinical MDR isolates of infection by K. pneumoniae in Northeast Brazil. This data indicates that this type of plasmid may have been responsible for spreading the bla KPC-2 gene among K. pneumoniae in patients from hospitals in Recife, Brazil.
The study by Pereira et al. (2013) 5  Collectively, these results reveal the dynamics of the genetic environment of the bla KPC-2 gene and emphasize the continuous recombination and evolution of plasmids and transposons. This may make the spread of different resistance genes in K. pneumoniae isolates more likely, introducing additional difficulties to the development of possible measures to control the spread of this form of bacterial resistance.