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

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 .

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 , .

Nicoletti et al. (2015) 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) .

These plasmids may also harbor different isoforms of the Tn4401 transposon. Nine variants of Tn4401 (a-i) have been described , of which variants "a" and "b" are the most common. Non-Tn4401 (NTE-KPC) , elements that can carry bla KPC-2 have also been described, including those recently detected in two colonization isolates in Brazil .

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.

Susceptibility to different classes of antimicrobials was assessed using the automated Bactec 9120 (Phoenix BD) system. The antimicrobials tested were amikacin, ampicillin, ampicillin/sulbactam, cefalotin, ceftazidime, cefepime, cefoxitin, ciprofloxacin, ceftriaxone, cefuroxime, colistin, gentamycin, ertapenem, imipenem, meropenem, and tigecycline. The interpretation was performed according to the criteria of the Clinical and Laboratory Standards Institute (CLSI 2017) .

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 . 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 . 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 , and de-novo assemblies were performed using the Velvet tool . The annotated plasmid DNA sequences were visualized using Artemis Sanger software .

The IncQ plasmid was identified using PCR with the primers and the amplification conditions described by Götz et al. (1996) . 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) .

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) antibiotic-resistance 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 NTEKPC-IId) (Figure 1). We found deletions in tnpA and a total deletion of the ISKpn7 insert sequence (Figure 1).

TABLE 1:

Source of isolation, origin, ERIC-PCR profile, and resistance profile of clinical MDR isolates of K. pneumoniae, Recife-PE, Brazil, 2016.

Isolates	Origin	ERIC-PCR profile	Resistance profile
K3R2	Peritoneal fluid	P1	AMP, APS, CFL, CFZ, CFP, CXM, CAZ, CRO, CFX, CIP, ERT, IPM, MER
K4R2	Blood culture	P2	AMP, APS, CFL, CFZ, CFP, CXM, CAZ, CRO, CFX, CIP, ERT, IPM, MER
K6R2	Cerebrospinal fluid	P3	AMP, APS, CFL, CFZ, CFP, CXM, CAZ, CRO, CFX, CIP, ERT, IPM, MER
K1E	Blood culture	P5	AMP, APS, CFL, CFZ, CFP, CAZ, CRO, CFX, CIP, ERT, GEN, IPM, MER

K: Klebsiella pneumoniae; R2: public hospital; E: private hospital; P: profile; AMP: ampicillin; APS: ampicillin/sulbactam; CFL: cefalotin; CFZ: cefazolin; CFP: cefepime; CFX: cefoxitin, CAZ: ceftazidime; CRO: ceftriaxone; CXM: cefuroxime; CIP: ciprofloxacin; ERT: ertapenem; GEN: gentamycin, IPM: imipenem; MER: meropenem.

FIGURE 1:

Comparison among Tn4401, non-Tn4401 (NTE KPC -IId), and IncQ1 carrying the bla KPC-2 gene in Klebsiella pneumoniae. Tn4401 with bla KPC-2 gene between ISKpn6 and ISKpn7; NTEKPC-IId with deletion of the ISKpn7 and truncated bla TEM gene; K3-R2 isolate with bla KPC-2 gene between truncated bla TEM gene and ΔISKpn6 as well as mobility and replication genes encoding the plasmid IncQ1. Protein-coding sequences are represented by the arrows and labeled with gene name. Vertical lines represent gaps schematizing the termination of one contig and the beginning of another contig in the isolate. Gray fill represents homologous shared regions. The direct repeat sequence of NTE KPC -IId is represented by a circle.

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).

TABLE 2:

Plasmid sequence characteristics of K. pneumoniae isolates and comparative analysis with plasmid RSF1010-IncQ (accession number: M28829.1).

Isolates	K3R2	K4R2	K6R2	K1E	IncQ (M28829.1)
Reference	This study	This study	This study	This study	Scholz et al. (1989)
Size of the sequenced plasmid DNA	27,508 bp	27,325 bp	59,776 bp	84,800 bp	8,684 bp
GC	57.95%	58.13%	55.35%	53.84%	61%
CDS	31	31	70	94	40
Inc	IncQ1 and NTP	IncQ1 and NTP	IncQ1 and NTP	IncQ1 and NTP	IncQ1 and NTP
Resistance genes	bla KPC-2	bla KPC-2	bla KPC-2	bla KPC-2	str sul
Mobility and replication	mobA	mobA	mobA	mobA	mobA
Proteins	mobB	mobB	mobB	mobB	mobB
	mobC	mobC	mobC	mobC	mobC
	repA	repA	repA	repA	repA
	repB	repB	repB	repB	repB
	repC	repC	repC	repC	repC
	repF	repF	repF	repF	repF

K: Klebsiella pneumoniae; R2: public hospital; E: private hospital; GC: guanine and cytosine; bp: base pairs; CDS: coding sequence; Inc: plasmid incompatibility group; KPC: Klebsiella pneumoniae carbapenemase; streptomycin; sulfonamide; mob: mobility protein; rep: replication proteins; NTP: not typable.

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 , . 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 (NTEKPC) in China, Argentina, Brazil, and Russia , , . NTEKPC has been separated into three groups based on the absence or presence of the bla TEM gene, where the second group, NTEKPC-II, includes the variant with a truncated bla TEM gene , .

The non-Tn4401 variant of the present study resembles the NTEKPC-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 .

This report demonstrates the presence of the bla KPC-2 gene in the non-Tn4401 element (NTEKPC-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) used K. pneumoniae isolates from Recife-Pernambuco, Brazil, but it was not possible to type the plasmids. Cerdeira et al. (2019) found the bla KPC-2 gene in the NTEKPC-IId gene carried by IncQ1plasmids in two colonization isolates of K. pneumoniae in Brazil (uninformed locality).

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.