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. 2023 Nov 16;18(11):e0292288. doi: 10.1371/journal.pone.0292288

Decoding the genetic structure of conjugative plasmids in international clones of Klebsiella pneumoniae: A deep dive into blaKPC, blaNDM, blaOXA-48, and blaGES genes

Shadi Aghamohammad 1,#, Mahshid Khazani Asforooshani 1,2,#, Yeganeh Malek Mohammadi 1, Mohammad Sholeh 1, Farzad Badmasti 1,*
Editor: Farah Al-Marzooq3
PMCID: PMC10653425  PMID: 37971980

Abstract

Carbapanem-resistant Klebsiella pneumoniae is a globally healthcare crisis. The distribution of plasmids carrying carbapenemase genes among K. pneumoniae poses a serious threat in clinical settings. Here, we characterized the genetic structure of plasmids harboring major carbapenemases (e.g. blaKPC, blaNDM, blaOXA-48-like, and blaGES) from K. pneumoniae using bioinformatics tools. The plasmids carrying at least one major carbapenemase gene were retrieved from the GenBank database. The DNA length, Inc type, and conjugal apparatus of these plasmids were detected. Additionally, allele types, co-existence, co-occurrence of carbapenemase genes, gene repetition, and sequence types of isolates, were characterized. There were 2254 plasmids harboring carbapenemase genes in the database. This study revealed that blaKPC-2, blaNDM-1, blaOXA-48, and blaGES-5 were the most prevalent allele types. Out of 1140 (50%) plasmids were potentially conjugative. IncFII, IncR, IncX3, and IncL replicon types were predominant. The co-existence analysis revealed that the most prevalent of other resistance genes were blaTEM-1 (related to blaKPC), blaOXA-232 (related to blaOXA-48), bleMBL (related to blaNDM), and aac (6′)-Ib4 (related to blaGES). The co-occurrence of carbapenemases was detected in 42 plasmids while 15 plasmids contained carbapenemase gene repetitions. Sequence alignments highlighted that plasmids carrying blaKPC and blaOXA-48-like were more homogeneous whereas the plasmids carrying blaNDM were divergent. It seems that K. pneumoniae utilizes diversity of genetic flexibility and recombination for resistance against carbapenems. The genetic structure of the plasmids showed that class I and III, Tn3 family, Tn5403 family derivatives, and Tn7-like elements were strongly associated with carbapenemases. The mobilizable plasmids carrying carbapenemases play an important role in the spread of these genes. In addition, gene repetition maybe is related to carbapenem heteroresistance. According to MST (minimum spanning tree) results, the majority of plasmids belonged to sequence type (ST) 11, ST14, and ST12. These international clones have a high capacity to acquire the carbapenemase-containing plasmids.

1. Introduction

Klebsiella pneumoniae is one of the most important opportunistic pathogens found in nosocomial and community-acquired infections as well as in asymptomatic fecal carriages [1, 2]. K. pneumoniae isolates play an important role in causing serious infections, including pneumonia, bloodstream infections, urinary tract infections, surgical site, and burn wound infections [3]. In addition, the presence and spread of resistance genes pose a challenge to successful treatment. Extended-spectrum beta-lactamase (ESBL)-producing and carbapenemase-producing K. pneumoniae (CPK) isolates are repeatedly associated with failure of antibiotic therapy [4]. The mortality rate caused by carbapenem-resistant K. pneumoniae (CRK) isolates is significantly twice as high compared with infections caused by carbapenem-susceptible isolates [5]. Several reasons, including severe co-morbidities, higher virulence of CRK isolates, improper use of antibiotics along with high level of toxicity, are associated with the increase in mortality rates [6].

Various classes of carbapenemase, according to Ambler classification, are detected in K. pneumoniae isolates, including class B, metallo-beta-lactamases (New Delhi Metallo-beta-lactamase—NDM), class D carbapenemases (e.g. OXA-48), and class A carbapenemase of K. pneumoniae (e.g. KPC) [7]. Although CPK isolates are common in different regions, KPC is endemic in the United States and some European countries, including Greece and Italy. Whereas, MBLs (metallo-beta-lactamases, including NDM-1) and OXA-48-like are found mainly in Asian countries such as Turkey, India, Pakistan, and the Middle East region [8]. It seems that the presence of carbapenem resistance genes on conjugative plasmids could lead to their worldwide dissemination and therefore the high prevalence of CPK isolates could be a serious problem for the health care system all over the world. Also, the coexistence of carbapenemase-encoding genes with other resistance genes such as aminoglycoside-modifying genes in K. pneumoniae isolates exacerbates the problem of antibiotic resistance as a challenge in the curing of infectious [9, 10]. The harboring of these resistance genes on mobile genetic elements (MGEs), including class 1 integron, transposons, and insertion sequences are usually carried on conjugative plasmids, contribute to expansion of antimicrobial resistance (AMR).

Resistance genes are usually associated with specific clonal groups. Strains of multidrug-resistant K. pneumoniae isolates are generally found in sequence type (ST) 147, ST15, and ST258. In addition, virulence genes are also carried in several specific STs, including ST147, ST15, ST48, ST101, and ST383. Plasmids harboring carbapenem-resistance genes, including blaKPC belonging to IncFIIk/IncR, are typically found in ST11 and play an important role in the spread of resistance genes in Asian countries, including China [11]. Moreover, ST11 is highly related to hypermucoviscous isolates of K. pneumoniae. Isolates of carbapenem-resistant K. pneumoniae ST11 can acquire the virulence of large plasmids from hypervirulent isolates [12]. In other words, virulence plasmids from hypervirulent K. pneumoniae isolates can be transferred to resistant isolates [13] and Therefore, the spread of these high virulence and resistance capacity plasmids among prevalent STs such as ST11 could be complicated and potentially life-threatening for patients, especially those hospitalized in intensive care units (ICUs) for a long time [14].

According to resistant K. pneumoniae, various epidemiological studies have been conducted so far. However, it is still important to analysis that led to the decipher of the genetic structures of plasmids harboring AMR genes. Also the investigation of the MGEs associated with the resistant plasmids and prevalent STs, play an important role in the spread of antibiotic resistance among bacteria. Characterization of these MGEs can give rise to a better insight of how antimicrobial resistance might widely spread. In this study, we detected and compared the different allele types of the major carbapenemase genes, including blaKPC, blaNDM, blaOXA-48, and blaGES from K. pneumoniae using bioinformatics tools. In addition, we characterized the genetic properties of carbapenemases harboring plasmids including replicon types, conjugation ability, the co-existence (e.g. linkage of carbapenemases with other antimicrobial resistance genes), co-occurrence (e.g. having at least two carbapenemase genes in one strain), gene repetition, alignment and phylogenetic relatedness.

2. Materials and methods

2.1. Preparation of initial dataset

The complete nucleotide sequences of the plasmids containing each of the four carbapenemase genes, including blaKPC, blaNDM, blaOXA-48, and blaGES were retrieved from the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/). To get all completed plasmids and partial DNA fragments carrying carbapenemase genes two types of BLASTn including microbial BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=MicrobialGenomes) and standard BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_SPEC=GeoBlast&PAGE_TYPE=BlastSearch) were performed, respectively. Further analyses which have been conducted in the current study, are presented in Fig 1.

Fig 1. The flowchart conducted in the current study.

Fig 1

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Two BLAST approaches including microbial BLAST and standard BLAST had been applied to retrieve all completed plasmids and DNA fragments carrying carbapenemase genes. All tools and functions have been shown in this pipeline.

2.2. Allele types determination of carbapenemase genes

The allele types of the mentioned carbapenemase genes located on the completed plasmids and partial DNA fragments were detected using the beta-lactamase database (http://bldb.eu/). The criteria were 100% identity and 100% coverage. In addition, the prevalence of each allele type was calculated.

2.3. Detection of other AMR genes on retrieved DNAs

The Comprehensive Antibiotic Resistance Database (CARD) (https://card.mcmaster.ca/home) was used to detect the presence of antimicrobial resistance genes against carbapenem, extended-spectrum beta-lactams, fluoroquinolones, aminoglycosides, chloramphenicol, tetracycline, macrolide, and other antibiotics [15]. The co-existence (gene linkage) of other antimicrobial resistance genes with the major carbapenemase genes in the plasmids was characterized. Moreover, the availability of the at least two major carbapenemase genes in an isolate were considered as co-occurrence.

2.4. Genetic evaluation of plasmid harboring carbapenemase genes

The conjugal apparatus including oriT, relaxase, type IV coupling protein (T4CP), and type IV secretion system (T4SS) was detected by oriTfnder tool (https://tool-mml.sjtu.edu.cn/oriTfinder/oriTfinder.html) [16]. The incompatibility (Inc) group of plasmids was identified by the Center for Genomic Epidemiology (CGE) web tool PlasmidFinder 2.1 (https://cge.food.dtu.dk/services/PlasmidFinder/) [17]. The ClustAGE software package (http://vfsmspineagent.fsm.northwestern.edu/cgi-bin/clustage_plot.cgi) was applied to compare the similarity/heterogeneity of plasmids carrying the predominant carbapenemase genes, including blaNDM-1, blaOXA-48-like, and blaKPC-2 and the results were depicted by EvolView (www.evolgenius.info/evolview) [18]. In addition, the data on the geographical regions, isolation sources, years and hosts of all isolates harboring crabapenemase genes were extracted and summarized.

2.5. Clonal relatedness of strains harboring carbapenemase genes

The distribution of the major carbapenemase-encoding genes among the different STs was assessed. For each plasmid, the ST of the associated chromosome was determined using seven housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB) via the PubMLST database (https://pubmlst.org/) [19]. The clonal relatedness of STs was characterized using PHYLOViZ version 2.0 to generate a minimum spanning tree (MST) for all STs [20].

3. Results

3.1. The distributions and the allele types of the carbapenemase genes

Two thousand two hundred and fifty-four (2254), including 1132 plasmids harboring blaKPC, 495 plasmids containing blaNDM, 617 plasmids with blaOXA-48-like, and 10 plasmids with blaGES were retrieved from GenBank database in microbial BLAST. In addition, 362, 132, 124, and 11 DNA partial fragments carrying blaKPC, blaNDM, blaOXA-48-like, and blaGES, respectively were found according to the standard BLAST. Moreover, all data on the geographical regions, isolation sources, years and hosts of harboring crabapenemases of isolates have been shown in Table 1 and S1 File.

Table 1. The additional data on the geographical regions, isolation sources, years and hosts of all isolates harboring crabapenemases.

Data Gene bla GES blaOXA-48-like bla NDM bla KPC
No. of accession numbers 9 595 616 1129
Available Biosample 2 582 410 794
Geographic location South Africa (1) Spain (219)
Netherlands (131)
Switzerland (42)
India (41)
China (28)
Germany (19)
Russia (9)
USA (9)
Australia (7)
Other countries (46)
Missing data (31)		 China (122)
Bangladesh (57)
USA (29)
Thailand (24)
Vietnam (18)
India (11)
UK (10)
South Korea (9)
Italy (8)
Russia (7)
Canada (6)
Myanmar (6)
Nepal (6)
Spain (6)
Switzerland (6)
Other countries (35)
Missing data (50)
 China (382)
USA (101)
Spain (58)
Italy (36)
Brazil (29)
Germany (23)
Japan (18)
Other countries (60)
Missing data (87)
Isolation source Trachea (1) Hospital (201)
Urine (33)
Blood (32)
Rectal swab (17)
Sputum (11)
Trachea (10)
Wound (8)
Endotracheal (8)
Pus (7)
Stool (5)
Abdominal (4)
Skin (3)
Intestine (3)
Outdoors (2)
Nasal (2)
Other (48)
Missing data (188)
 Blood (57)
Urine (37)
Wound (36)
Sputum (32)
Rectal (12)
Hospital (8)
Feces (8)
Trachea (8)
River (5)
Pus (5)
Stool (4)
Respiratory (4)
Nasal (2)
Other (53)
Missing data (139)
 Sputum (106)
Blood (87)
Urine (58)
Hospital (48)
Rectal swab (30)
Broncho aspirate (11)
Lavage (8)
Stool (6)
Wound (6)
Trachea (5)
Abdominal (4)
Other (114)
Missing data (311)
Collection date Missing data (1) 2018 (276)
2019 (90)
2017 (49)
2014 (40)
2015 (22)
2016 (20)
2020 (17)
2013 (15)
2022 (6)
2021 (4)
2012 (4)
2011 (1)
Massing data (38)		 2017 (85)
2016 (53)
2019 (44)
2018 (34)
2015 (30)
2013 (22)
2014 (20)
2021 (18)
2012 (14)
2020 (11)
2022 (6)
2011 (3)
2010 (3)
200 (1)
Missing data (66)		 2018 (154)
2017 (96)
2019 (78)
2016 (51)
2015 (46)
2014 (41)
2013 (37)
2020 (34)
2021 (31)
2012 (28)
2022 (23)
Other years (58)
Missing data (117)
Host Homo sapiens (1) Homo sapiens (521)
Dog (7)
Cat (2)
Flies (2)
Canis (1)
Missing data (49)		 Homo sapiens (314)
Canis (2)
Chicken (1)
Dog (1)
Cattel (1)
Cat (1)
Swine (1)
Houseflies (1)
Animal (1)
Missing data (87)		 Homo sapiens (634)
Equus (1)
Dog (1)
Pig (1)
Missing data (157)
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The size of the complete plasmids ranged from 726 bp to 583,215 bp. The most prevalent alleles found in carbapenemase genes were blaKPC-2 (997/1494), blaNDM-1 (415/627), blaOXA-48 (316/741), and blaGES-5 (11/21). All allele types are shown in Fig 2 and S2 File. One thousand one hundred forty (1140/2254, 50.5%) plasmids were potentially conjugative and carried all four conjugal components including oriT, relaxase, type IV coupling protein (T4CP), and type IV secretion system (T4SS). The blaKPC was mostly located on the plasmids with IncFII/IncR replicon types (332/1132, 29.4%). The blaNDM gene was mainly related to the plasmids with the IncX3 replicon type (111/495, 22.6%). While, 434/617, 70.3% of plasmids with blaOXA-48 had the IncL replicon type.

Fig 2. The prevalence of allele types of the major carbapenemase genes.

Fig 2

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A) The different of allele types of blaKPC gene, B) The proportions of all allele types observed in blaNDM gene, C) The frequency of blaOXA-48-like allele types, D) All allele types detected in blaGES gene.

3.2. The co-existence of other antimicrobial resistance genes in the plasmids

Different antimicrobial resistance genes against various classes of antibiotics, including extended-spectrum beta-lactams, carbapenems, quinolones, aminoglycosides, chloramphenicol, tetracycline, macrolide, fosfomycin and sulfonamides along with genes encoding efflux pump proteins and antiseptic-resistance genes, were found in plasmids carrying a least one carbapenemase gene. The most prevalent co-existed genes in plasmids harboring blaKPC, blaOXA, blaNDM, and blaGES were blaTEM-1, blaOXA-232, bleMBL, and aac (6′)-Ib4, respectively. See Fig 3 and S3 File.

Fig 3. The prevalence of other antimicrobial resistance in plasmids containing major carbapenemase genes.

Fig 3

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The co-existence rate of other antibiotic resistance genes detected in plasmids harboring blaKPC gene (A), blaOXA-48 gene (B), blaNDM gene (C), and blaGES gene (D).

3.3. The co-occurrence of carbapenemase genes

Analysis of the data retrieved from the GenBank database revealed that the co-occurrence of carbapenemase genes in different plasmids but in the same strains. This coincidence was found in forty-two genomes. The co-occurrence of carbapenemase genes with predominant allele types was as follows. A number of seventeen plasmids had blaNDM-1 and blaKPC-2. Four plasmids had blaOXA-48/blaNDM-1, and three plasmids had blaOXA-48/blaKPC-2. The rest of the plasmids having the co-occurrence genes has been shown in Table 2. Also four plasmids, including NZ_CP094991, NZ_CP104796.1, NZ_CP086664.1, and NZ_CP090126.1 simultaneously contained three carbapenemase genes, including blaOXA-48/blaNDM-1/blaKPC-2, blaOXA-181/blaNDM-1/blaNDM-4, blaOXA-48/blaNDM-1/blaKPC-2, and blaNDM-1/blaKPC-2/blaKPC-2, respectively. See Table 2. The conjugal plasmids were various among the strains. For example, in NZ_CP050376.1 and NZ_CP041082.1 the plasmids were potentially conjugative, whereas in some other strains, e.g. CP065949.1 and NZ_CP024038.1 one plasmid was potentially conjugative and another plasmid was not conjugative. In strains with plasmids containing three carbapenemase genes, the plasmids were potentially conjugative or at least were mobilizable (only lacked oriT).

Table 2. Genomic data on STs, Carbapenemase genes, other resistance genes and conjugal apparatus among whole genome sequencing of K. pneumoniae with co-occurrence of carbapenemase genes.

Genome accession number ST No. of plasmids Accession number of plasmids carrying carbapenemases Carbapenem genes Other resistant genes Size of plasmids Prediction of Inc availability Conjugation data* Genetic environment
oriT/Relax T4SS T4CP
CP065949.1 11 5 NZ_CP065954 bla NDM-1 N/D 63769 IncL 1 1 1 IS transposase
NZ_CP065952 bla KPC-2 blaCTX-M-65, catII from Escherichia coli K-12 100684 IncFII (pHN7A8), IncR 0 0 0 Tn3 family transposase, IS
NZ_CP068572 11 3 NZ_CP068574 bla OXA-48 blaCTX-M-14, aph(3’’)-Ib, aph(3’)-Vib, aph(6)-Id 72218 IncL 1 1 1 Tn3 family transposase,IS transposase
NZ_CP068575 bla KPC-2 bla TEM-1 86878 RepB (R1701) 0/1 1 1 IS transposase
NZ_CP061957.1 11 3 NZ_CP061958 bla OXA-48 blaCTX-M-14, blaTEM-1, aac(3)-Iie, aph(3’’)-Ib, aph(3’)-Vib, aph(6)-Id 109135 IncL, IncR 1 1 1 Tn3 family transposase, Tn3-like element Tn5403 family, IS
NZ_CP061960 bla KPC-2 blaCTX-M-65, blaSHV-12 144349 IncFII (pHN7A8),IncR 0 0 0 Tn3 family transposase, Tn3-like element TnAs1, IS
NZ_CP029689.1 11 6 NZ_CP030135 bla OXA-48 N/D 65500 IncL 1 1 1 IS transposase
NZ_CP030134 bla KPC-2 bla CTX-M-65 60307 IncR 0 0 0 IS transposase
NZ_CP050371.1 11 4 NZ_CP050375 bla OXA-181 N/D 51140 ColKP3, IncX3 0/1 1 1 Tn3 family transposase, Tn3-like element IS3000 family, IS transposase
NZ_CP050374 bla NDM-1 blaCTX-M-15, rmtF, aac(6’)-Ib, arr-2, BRP(MBL), aac(3)-IIe 193462 IncFIB (pQil), IncFII (K), RepB (R1701) 0 0 0 Class 1 integron, Tn3 family transposase, Tn3-like element Tn3, IS
NZ_CP071279.1 14 5 NZ_CP071281 bla OXA-48 blaCTX-M-14, aph(3’’)-Ib, aph(6)-Id 68932 IncM1 1 1 1 Tn3 family transposase,IS transposase
NZ_CP071280 bla NDM-1 aph(3’)-VI, mphE, msrE, armA, sul1, qacEdelta1, aadA2, dfrA12 269326 IncFIB (pNDM-Mar), IncHI1B (pNDM-MAR) 0/1 1 1 Class 1 integron, Tn3 family transposase, Tn3-like element Tn3, IS
NZ_CP097237.1 14 5 NZ_CP097241 bla OXA-232 N/D 6141 ColKP3 0 0 0 N/D
NZ_CP097238 bla NDM-1 dfrA14, blaOXA-1, qnrB1, aph(3’)-VI, mphE, msrE, armA, sul1, qacEdelta1, aadA2, dfrA12, aac(6’)-Ib-cr6 283371 IncFIB (pNDM-Mar), IncHI1B (pNDM-MAR) 0/1 1 1 Class 1 integron, Tn3-like element IS3000 family transposase, Tn3 family transposase, IS
NZ_CP078033.1 14 4 NZ_CP078037 bla OXA-232 N/D 6141 ColKP3 0 0 0 N/D
NZ_CP078034 bla NDM-1 blaCTX-M-15, blaOXA-9, blaTEM-1, sul2, dfrA14, blaOXA-1, qnrB1, mphE, msrE, armA, sul1, qacEdelta1, aadA2, dfrA12, aac(6’)-Ib10, aadA, aph(3’’)-Ib, aph(6)-Id, aac(6’)-Ib-cr6 319619 IncFIB (pNDM-Mar), IncHI1B (pNDM-MAR), IncR 0/1 1 1 Class 1 integron, Tn3 family transposase, Tn3-like element Tn3, IS
NZ_CP012753.1 14 2 NZ_CP012755 bla OXA-232 N/D 6141 ColKP3 0 0 0 N/D
NZ_CP012754 bla NDM-1 aph(3’)-VI, qnrB1, dfrA12, aadA2, qacEdelta1, sul1, armA, msrE, mphE 273628 IncFIB (pNDM-Mar), IncHI1B (pNDM-MAR) 0/1 1 1 Class 1 integron, Tn3 family transposase, Tn3-like element Tn3, IS
NZ_CP006798.1 14 4 NZ_CP006802 bla OXA-232 N/D 6141 ColKP3 0 0 0 N/D
NZ_CP006799 bla NDM-1 aph(3’)-VI, qnrB1, blaOXA-1, dfrA14, dfrA12, aadA2, qacEdelta1, sul1, armA, msrE, mphE, AAC(6’)-Ib-cr6 283371 IncFIB (pNDM-Mar), IncHI1B (pNDM-MAR) 0/1 1 1 Class 1 integron, Tn3 family transposase, Tn3-like element IS3000, IS
NZ_CP050376.1 15 5 NZ_CP050381 bla OXA-244 N/D 71402 IncFII (pCoo) 1 1 1 IS transposase
NZ_CP050380 bla NDM-1 sul2, armA, msrE, mphE, qnrs1, aph(3’)-VI, dfrA5, qacEdelta1, mphA, aph(3’)-Ia 353810 IncFIB (pNDM-Mar), IncHI1B (pNDM-MAR) 1 1 1 Class 1 integron, Tn3 family transposase, Tn3-like element Tn3, IS
NZ_CP104796.1 16 6 NZ_CP104800 bla OXA-181 qnrS1 51478 ColKP3, IncX3 0/1 1 1 IS transposase
NZ_CP104795 bla NDM-1 bleMBL, blaSHV-12 53814 IncX3 0/1 1 1 IS transposase
NZ_CP104797 bla NDM-4 bleMBL, bleMBL 193355 IncFIB (pKPHS1) 0/1 1 1 Tn3-like element Tn5403, IS
NZ_CP080362.1 16 6 NZ_CP080367 bla OXA-181 N/D 51479 ColKP3, IncX3 0/1 1 1 Tn3 family transposase, Tn3-like element IS3000 family, IS transposase
NZ_CP080366 bla NDM-4 bleMBL,blaTEM-1, rmtB 85191 IncFII (Yp) 0/1 1 1 Tn3-like element Tn3, IS
NZ_CP058940.1 16 7 NZ_CP058945 bla OXA-181 N/D 50126 ColKP3, IncX3 0/1 1 1 Tn3 family transposase, Tn3-like element IS3000 family, IS transposase
NZ_CP058942 bla NDM-5 blaTEM-1, rmtB, sul1, dfrA12, ErmB, mphA, qacJ, aadA2 99664 IncFII 1 1 1 Class 1 integron, Tn3 family transposase, Tn3-like element TnAs1, IS
NZ_CP041927.1 16 4 NZ_CP041931 bla OXA-181 N/D 51479 ColKP3, IncX3 0/1 1 1 Tn3 family transposase, Tn3-like element IS3000 family, IS transposase
NZ_CP041930 bla NDM-4 bleMB, blaTEM-1, rmtB 86019 IncFII (Yp) 0/1 1 1 Tn3 family transposase, Tn3-like element Tn5403, IS
NZ_CP024038.1 16 6 NZ_CP024042 bla OXA-232 N/D 6141 ColKP3 0 0 0 N/D
NZ_CP024039 bla NDM-1 dfrA12, aadA2, qacEdelta1, sul1, tet(B), tetR 125285 IncFIA 1 1 1 Class 1 integron, Tn3 family transposase, Tn3-like element IS3000, IS
NZ_CP086447.1 101 7 NZ_CP086451 bla OXA-48 N/D 63589 IncL 1 1 1 IS transposase
NZ_CP086448 bla NDM-1 sul2, cmy-16, sul1, qnrA6, qacEdelta1, aadA2, dfrA12, mphE, msrE, armA, aph(3’)-VI, blaOXA-10, cmlA5, arr-2, dfrA14, floR, tet(A), aph(6)-Id, aph(3’’)-Ib 189866 IncC 1 1 1 Class 1 integron, Tn3 family transposase, IS
NZ_CP050360.1 147 10 NZ_CP050368 bla OXA-181 N/D 6812 ColKP3 0 0 0 Tn3-like element Tn5403 family
NZ_CP050367 bla NDM-5 dfrA12,qacEdelta1,sul1,rmtB,TEM-1,aadA2,mphA,ermB 103085 IncFII 1/1 1 1 Tn3-like element TnAs1 family transposase,Tn3 family transposase,class 1 integron, IS
NZ_CP077779.1 377 5 NZ_CP077782 bla OXA-48 N/D 63589 IncL 1 1 1 IS transposase
NZ_CP077784 bla NDM-1 sul1, qacEdelta1, dfrA5, aph(3’)-VI, qnrs1, mphE, msrE, armA, sul2, mphA 348342 IncFIB (pNDM-Mar), IncHI1B (pNDM-MAR) 1 1 1 Class 1 integron, Tn3 family transposase, Tn3-like element TnAs1, IS
NZ_CP091813.1 383 5 NZ_CP091815 bla OXA-48 blaCTX-M-14, aph(3’’)-Ib, aph(3’)-Vib, aph(6)-Id 72224 IncL 1/1 1 1 Tn3 family transposase, IS
NZ_CP091814 bla NDM-5 aph(3’)-VI, qnrS1, blaCTX-M-15, blaTEM-1, sul2, armA, msrE, mphE, dfrA5, qacEdelta1, sul1, mphA, aac(6’)-Ib10, aadA, aph(3’)-Ia 376754 IncFIB (pNDM-Mar), IncHI1B (pNDM-MAR) 1 1 1 Class 1 integron, Tn3 family transposase, Tn3-like element TnAs1, IS
CP034200.2 383 2 CP034202 bla OXA-48 blaCTX-M-14, aph(6)-Id, aph(3’)-VIb, aph(3’’)-Ib 72057 IncL 1 1 1 Tn3 family transposase, IS transposase
CP034201 bla NDM-5 qnrS1, blaCTX-M-15, blaTEM-1, sul1, qacEdelta1, dfrA5, mphE, msrE, armA, sul2, aph(3’)-Via, aac(6’)-Ib10, aadA, aph(3’)-Ia 372826 IncFIB (pNDM-Mar), IncHI1B (pNDM-MAR) 1 1 1 Tn3-like element TnAs3, Tn3-like element Tn3, IS
NZ_CP094991 39 5 NZ_CP094992 bla OXA-48 catI, blaOXA-1, blaTEM-1, qnrS1, blaCTX-M-15, ant(2’’)-Ia, qacEdelta1, sul1, aac(6’)-Ib-cr6, aadA 326663 IncHI1B (pNDM-MAR) 1 1 1 Class 1 integron integrase, IS transposase
NZ_CP094993 bla NDM-5 arr-3, cmlA5, blaOXA-10, qacEdelta1, sul1, armA, msrE, dfrA12, aadA2, bleMBL, blaOXA-1, ant(3’’)-IIa, mphE, aac(6’)-Ib-cr6, aac(3)-IIe 174840 IncC 1 1 1 Class 1 integron, Tn3 family transposase, IS
NZ_CP094994 bla KPC-2 N/A 102252 IncFIB (pQil), IncFII (K) 1 1 1 Tn3-like element Tn4401, IS
NZ_CP086664.1 39 6 NZ_CP086665 bla OXA-48 catI, blaOXA-1, blaTEM-1, qnrS1, blaCTX-M-15, ant(2’’)-Ia, qacEdelta1, sul1, aac(6’)-Ib-cr6, aadA 323074 IncHI1B (pNDM-MAR) 1 1 1 Class 1 integron integrase IntI1, Tn3 family transposase,IS transposase
NZ_CP086668 bla KPC-2 N/A 102252 IncFIB (K), IncFIB (pQil), IncFII(K) 1 1 1 Tn3-like element Tn4401, IS
NZ_CP086666 bla NDM-1 arr-3, cmlA5, blaOXA-10, qacEdelta1, sul1, armA, msrE, dfrA12, aadA2, bleMBL, blaOXA-1, ant(3’’)-IIa, mphE, aac(6’)-Ib-cr6, aac(3)-IIe 174841 IncC 1 1 1 Class 1 integron, Tn3 family transposase, IS
NZ_CP041082.1 101 7 NZ_CP041085 bla OXA-48 N/D 63499 IncL 1 1 1 IS transposase
NZ_CP041083 bla NDM-1 sul2, cmy-16, blaCTX-M-15, mphE, msrE, qacEdelta1, blaOXA-10, cmlA5, arr-2, floR, tet(A), aph(6)-Id, aph(3’’)-Ib, armA, aph(3’)-VI 179254 IncC 1 1 1 Class 1 integron, Tn3 family transposase, IS
NZ_CP101776.1 11 7 NZ_CP101780 bla NDM-1 bleMBL, blaSHV-12 53988 IncX3 0/1 1 1 Tn3, IS
NZ_CP101782 bla KPC-2 blaCTX-M-65, blaTEM-1, rmtB, blaSHV-12 111949 IncFII (pHN7A8), 1/0 1 0 Tn3, IS
NZ_CP090203.1 11 3 NZ_MZ546616 bla NDM-1 cmy-6, arr-3, dfrA27, qacEdelta1, sul1, rmtC, bleMBL, aadA16 140306 IncC 1 1 1 Class 1 integron, Tn3 family transposase, IS
NZ_MZ546615 bla KPC-2 blaCTX-M-65, blaTEM-1, rmtB, blaSHV-12 126203 IncFII (pHN7A8) 1/0 1 0 Tn3,IS
NZ_CP092656.1
 11 3 NZ_CP092653 bla NDM-1 mphE, msrE, armA, sul1, qacEdelta1, aadA5, dfrA1, bleMBL, fosA3, blaSHV-12, qnrB4, dha-1, qnrB2, mphA 355489 N/A 0/1 1 1 Class 1 integron, Tn3 family transposase, Tn3-like element TnAs1, IS
NZ_CP092655 bla KPC-2 dfrA14, arr-3, qnrS1, blaTEM-1, blaCTX-M-3, aac(6’)-Ib10 71683 IncU 1 1 1 Class 1 integron, Tn3 family transposase, Tn3 like, IS
NZ_CP091846.1 11 4 NZ_CP091847 bla NDM-1 mphE, msrE, armA, sul1, qacEdelta1, aadA5, dfrA1, bleMBL, fosA3, blaSHV-12, qnrB4, dha-1, qnrB2, mphA 349120 N/A 0/1 1 1 Class 1 integron, Tn3 family transposase, Tn3-like element TnAs1, IS
NZ_CP091849 bla KPC-2 dfrA14, arr-3, qnrS1, blaTEM-1, blaCTX-M-3, aac(6’)-Ib10 72015 IncU 1 1 1 Class 1 integron, Tn3 family transposase, Tn3 like, IS
NZ_CP039819.1 11 3 NZ_CP039821 bla NDM-1 dfrA14, bleMBL 63046 IncN 1 1 1 Class 1 integron, IS
NZ_CP039820 bla KPC-2 blaCTX-M-65, fosA3, blaTEM-1, rmtB, catII from E. coli K-12 172001 IncFII (pHN7A8), IncR 1 1 1 Tn3 family transposase, IS
NZ_CP039808.1
 11 4 NZ_CP039811 bla NDM-1 bleMBL, blaSHV-12 53097 IncX3 0/1 1 1 Tn3 family transposase, Tn3-like element IS3000, IS
NZ_CP039810 bla KPC-2 blaCTX-M-65, blaTEM-1, rmtB 153556 IncFII (pHN7A8), IncR 1 1 1 Tn3-like element TnAs1 family, IS
CP034327.1 11 4 CP034323 bla NDM-1 ble MBL 53144 IncX3 0/1 1 1 Tn3 family transposase, Tn3-like element IS3000, IS
CP034324 bla KPC-2 blaSHV-12, rmtB, blaTEM-1, FosA3, blaCTX-M-65, catII from E. coli K-12 159467 IncFII (pHN7A8), IncR 1 1 1 Tn3 family transposase, IS transposase
CP034327.1 11 4 CP034323 bla NDM-1 ble MBL 53144 IncX3 0/1 1 1 Tn3 family transposase, Tn3-like element IS3000, IS
CP034324 bla KPC-2 blaSHV-12, rmtB, blaTEM-1, fosA3, blaCTX-M-65, catII from E. coli K-12 159467 IncFII (pHN7A8), IncR 1 1 1 Tn3 family transposase, IS transposase
CP109983.1 15 8 NZ_CP109986 bla NDM-1 ble MBL 86272 IncFII (Yp) 1/1 1 1 IS transposase
NZ_CP109990 bla KPC-2 N/D 7329 ND 0/0 0 0 IS transposase
NZ_CP090126.1 15 6 NZ_CP090129 bla NDM-1 bleMBL, blaSHV-12 53096 IncX3 0/1 1 1 Tn3 family transposase, Tn3-like element IS3000, IS
NZ_CP090128 bla KPC-2 mphE, msrE, armA, sul1, qacEdelta1 88164 IncFII (Yp) 1 1 1 Class 1 integron, Tn3 family transposase, IS
NZ_CP090127 bla KPC-2 N/D 103807 IncX6 1 1 1 Tn3-like element Tn3, IS
NZ_CP039813.1 15 5 NZ_CP039817 bla NDM-1 N/D N/A 51995 1 1 1 Tn3-like element Tn5403, IS
NZ_CP039815 bla KPC-2 bla CTX-M-15 97386 IncFII (Yp) 1 1 1 Tn3-like element Tn3 family transposase, IS
NZ_CP039802.1 15 5 NZ_CP039806 bla NDM-1 N/D 51995 IncN2 1 1 1 Tn3-like element Tn5403, IS
NZ_CP039805 bla KPC-2 bla CTX-M-15 97386 IncFII (Yp) 1 1 1 Tn3-like element Tn3 family transposase, IS
NZ_CP026586.1 86 4 NZ_CP026590 bla NDM-1 dfrA14, qnrs1, bleMBL 49215 IncN 0/1 1 1 Class 1 integron, IS
NZ_CP026589 bla KPC-2 blaCTX-M-65, blaTEM-1, rmtB, fosA3 89247 IncFII (pHN7A8) 1 1 0 Tn3 family transposase, IS
NZ_CP091048.1 464 7 NZ_CP091052 bla NDM-1 bla SHV-12 59349 IncX3 0/1 1 1 Tn3 family transposase, Tn3-like element IS3000, IS
NZ_CP091049 bla KPC-2 aac(3)-IId, arr-3, dfrA27, qacEdelta1, sul1, qnrB4, aadA16 248847 N/A 0/1 1 1 Class 1 integron, Tn3 family transposase, Tn3-like element TnAs1, IS
NZ_CP084986.1 2667 3 NZ_CP084987 bla NDM-1 arr-3, dfrA27, qacEdelta1, sul1, bleMBL, mphA, aac(3)-IId, blaTEM-1, aadA16, aph(6)-Id, aph(3’’)-Ib, sul2 362034 IncQ1 0/1 1 1 Class 1 integron, Tn3 family transposase, IS
NZ_CP084988 bla KPC-2 N/A 142244 RepB (R1701) 1 1 1 Tn3-like element ISPa38, Tn3-like element Tn3, Tn3-like element Tn5403, IS
NZ_CP063147.1 2667 3 NZ_CP063149 bla NDM-1 blaTEM-1, aac(3)-IId, mphA, sul1, bleMBL, qacEdelta1, qnrB6, arr-3, sul2, aph(3’’)-Ib, aph(6)-Id, aadA16, dfrA27 375474 IncQ1 0/1 1 1 Class 1 integron, Tn3 family transposase, IS
NZ_CP063148 bla KPC-2 N/D 159093 RepB (R1701) 1 1 1 Tn3-like element Tn5403 family, Tn3-like element Tn3 family, Tn3-like element ISPa38 family, IS
NZ_CP039828.1 3493 3 NZ_CP039829 bla NDM-1 mphE, msrE, sul1, qacEdelta1, arr-3, blaTEM-1, bleMBL, catB3, blaOXA-1, aadA16, dfrA27, aac(6’)-Ib9, qacG, aac(6’)-Ib-cr6, catII from Escherichia coli K-12 317231 N/A 0/1 1 1 Class 1 integron, Tn3 family transposase, Tn3-like element TnAs1, IS
NZ_CP039831 bla KPC-2 N/D 175540 IncFIA (HI1) 1 1 1 Tn3-like element Tn4401 family transposase, Tn3-like element Tn4401 family resolvase TnpR, Tn3-like element Tn5403 family, Tn3-like element ISPa38 family, IS
NZ_CP039823.1 3493 4 NZ_CP039824 bla NDM-1 mphE, msrE, sul1, qacEdelta1, arr-3, blaTEM-1, catB3, blaOXA-1, aadA16, dfrA27, aac(6’)-Ib9, qacG, aac(6’)-Ib-cr6, catII from E. coli K-12 318848 N/D 0/1 1 1 Class 1 integron, Tn3 family transposase, Tn3-like element Tn3, IS
NZ_CP039826 bla KPC-2 N/D 175540 IncFIA (HI1) 1 1 1 Tn3-like element Tn4401 family transposase, Tn3-like element Tn4401 family resolvase TnpR, Tn3-like element Tn5403 family, Tn3-like element ISPa38 family, IS
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*1 means presence and 0 means absence

3.4. The gene repetition in the plasmids

According to this study, there were 15 plasmids with carbapenemase gene repetitions. See Table 3. In eleven plasmids harboring blaKPC-2 (NC_011383.1, CP107423.1, NZ_OM144977.1, NZ_OL891656.1, NZ_CP066901.1, NZ_CP097691.1, NZ_CP097674.1, and NZ_MT920901.1), blaNDM-1, (NZ_CP098375.1) and blaGES-24 (LC623933.1, and LC620536.1), the carbapenemase gene had two copy numbers. In addition, in four plasmids, including NZ_MZ512197.1, NZ_CP064771.1, NZ_CP008933.1, and NZ_CP098375.1, three copy numbers of genes had been detected. No repetition was found in plasmids carrying blaOXA-48. The plasmids containing blaKPC with two or three copy numbers mostly belonged to ST11. They had IncFII replicon type and was potentially conjugative. While plasmids with blaNDM-1 were associated with ST2816 and ST15, had mostly IncFIB, and were potentially conjugative or mobilizable. Both plasmids harboring blaGES-24 belonged to ST12, IncFII, and IncCol of plasmids and they didn’t have conjugal systems.

Table 3. Gene repetition of carbapenemases which has been found from the plasmids.

Gene Accession number Repetition
bla KPC NC_011383.1 2
CP107423.1 2
NZ_OM144977.1 2
NZ_OL891656.1 2
NZ_CP066901.1 2*
NZ_MZ512197.1 3*
NZ_CP097691.1 2
NZ_CP097674.1 2
NZ_CP064771.1 3
NZ_MT920901.1 2*
bla NDM CP030858.1 3
NZ_CP008933.1 3
NZ_CP098375.1 2
bla GES LC623933.1 2
LC620536.1 2
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*Gene duplication was detected in DNA sequence with point mutations and frameshifts.

3.5. The genetic structure of carbapenemase genes

Class 1 integrons, and transposons including Tn3 family transposase, Tn3-like elements, Tn5403 family transposase, Tn7-like elements, and various insertion sequences (IS) were found in plasmids harboring carbapenemase genes. Forty-three 43/2250 (1.9%), 8 (0.25%), and 3 (0.13%) had intI1, intI2, and intI3, respectively. Four hundred and twelve (412/2254, 18.2%) plasmids carried class 1 integrons along with Tn3 family transposase and Tn3-like elements, and 1036/2254 (45.9%) had the other genetic elements, including Tn3 and Tn3-like families.

The blaNDM-1 gene was found between IS30 and Tn3-like transposase genes. Mapping of this transposon revealed that bleMBL, tat, cutA, groES, and groEL were located adjacent to blaNDM-1. The blaOXA-48 was located between IS1-like and IS4-like families and lysR was also a neighbor. Mapping of blaKPC-2 showed that this carbapenemase gene was flanked by the transposase family ISKpn27 and transposase family ISKpn6. Class 1 integron and class 3 integron were found in plasmids containing blaGES-5 and blaGES-24, respectively. Other genes, including aac (6)-Ia, cat, ant, DUF86, invA, and blaA were found adjacent to blaGES-24 and dfrB1, blaOXA-10, aac(6’)-Ib4 and qacE delta1 were seen near to blaGES-5. The highly prevalent genetic structures associated with the carbapenemase genes have been shown in Fig 4.

Fig 4. The genetic environments of major carbapenemase genes associated with class 1 integrons, insertion sequences and transposons.

Fig 4

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A) The genetic features of blaNDM-1 flanked by Tn3 like elements. The blaNDM-1 comes together by other resistance genes, including tat and bleMBL, localized on the Tn3-like elements. B) The genetic environment of blaOXA-48 is between IS1-like and IS4-like families and lysR is also a neighbor. C) The blaKPC-2 flanks between ISKpn6 and ISKpn27. The core structure of ISKpn27/ISKpn7-dnaA-blaKPC-2-ISKpn6 is highly epidemic in KPC-producing K. pneumoniae isolates. D) The blaGES-5 associated with intI1, E) While genetic features of blaGES-24 related to intI3.

3.6. Plasmid analysis

The sequence comparisons showed that plasmids carrying blaKPC and blaOXA-48 appear to be more homogeneous and conserved, whereas the plasmids carrying blaNDM were more heterogenic and distributed in the circular dendrogram. See Fig 5.

Fig 5. The circular sequence alignment of eighty-five plasmids harboring multiple major carbapenemase genes.

Fig 5

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Data shows that plasmids carrying blaKPC are more convergent. On the hand, blaNDM encoding plasmids are divergent and have different genetic characteristics (molecular weight, replicon typing, conjugal apparatus and other antimicrobial genes).

The plasmids harboring blaKPC which were located in the same cluster mostly had IncFII replicon types and were mainly potentially conjugative. In addition, the plasmids with blaOXA-232 had the ColKP3 replicon type and were all non-conjugative, whereas blaOXA-48 had the IncL replicon type and were all potentially conjugative. Among plasmids harboring blaNDM, the conjugation pattern and Inc type were different. Plasmids in the same cluster had the same replicon type (e.g., IncC) but a different Inc type in comparison to plasmids that were in a different cluster (which had IncFIB replicon type). It should also be noted that some of these plasmids that were in one cluster, were all mobilized. While the others in the same cluster had a different conjugal pattern.

3.7. Clonal relatedness of strains harboring carbapenemase genes

According to the data, most of the blaKPC (270/1132, 23.8%) and blaNDM (33/495, 6.6%) genes were located on plasmids belonging to ST11. The blaOXA-48 (64/617, 10.3%) was almost exclusively carried on plasmids belonging to ST14, and blaGES (3/10, 30%) was associated with ST12. Some STs were associated with only one carbapenemase gene. For example, ST258 was associated with blaKPC, ST1 with blaNDM, and ST231 associated with blaOXA-48-like. On the other hand, some other STs, including ST37, ST35, ST16, ST392, ST147, ST17, ST23, ST101, ST307, ST11, ST14, and ST437 were multi-harboring carbapenemase genes and had at least one carbapenemase gene, including blaKPC, blaNDM, and blaOXA-48. See Fig 6.

Fig 6. The minimum spanning tree (MST) of STs carrying the plasmids containing major carbapenemase genes (e.g. blaKPC, blaNDM, blaOXA-48 and blaGES) with similarity cut off ≥4 allelic types based on multi-locus sequence typing (MLST) scheme.

Fig 6

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ST11, ST14, ST437, ST23, ST307, ST101, ST147, ST16, ST17, ST35, and ST37 are multi-harboring carbapenemases STs in different strains.

Plasmids containing blaKPC that belonged to ST11 were mainly blaKPC-2 (258/270, 95.5). However, blaKPC-33 (2/270, 0.7%), blaKPC-12 (4/270, 1.48%), blaKPC-3 (2/270, 0.7%), blaKPC-71 (1/270, 0.3%), blaKPC-74 (1/270, 0.3%), and blaKPC-93 (2/270, 0.7%) were also found sporadic. These plasmids were mainly on IncFII and IncR replicon types and 107/270 (39.6%) of plasmids were potentially conjugative. Plasmids with blaNDM belonging to ST11 were mainly blaNDM-1 (24/33, 72.7%), however, blaNDM-5 (9/33, 27.2%) was also found. These plasmids were mainly on IncX3 and IncC replicon types and 18/33 (54.5%) of plasmids were potentially conjugative while 13/33 (39.3%) were mobilized. Plasmids with blaOXA-48-like belonging to ST14 were mainly blaOXA-10 (46/64, 71.8%), however, blaOXA-232 (14/64, 21.8%), blaOXA-48 (2/64, 0.03%), and blaOXA-9 (2/64, 0.03%) were also found. It should be noted that in all 46 plasmids with blaOXA-10 had a co-existence with blaOXA-48. These plasmids were mainly on InL replicon types and 45/64 (70.3%) of plasmids were potentially conjugative. Plasmids with blaGES belonging to ST12 were blaGES-24. These plasmids had IncCol and IncFII replicon types. None of these plasmids were potentially conjugative.

4. Discussion

Carbapenem-resistant K. pneumoniae is one of the most challengeable causes of community-acquired and nosocomial infections which can increase morbidity and mortality rate [21]. Multidrug-resistant K. pneumoniae isolates complicate treatment, and carbapenems are one of the last-line agents to combat these infections. Therefore, carbapenem resistance could make the situation worse [22]. The presence of carbapenemase genes on conjugative plasmids also contributes to the higher dissemination rates [12]. According to the latest report of CDC and WHO K. pneumoniae is considered as urgent priority and the presence of resistance genes, including blaKPC2 and blaKPC3 along with blaNDM and blaOXA-48-like as the most prevalent carbapenemase genes could be a worrying issue in public health [23]. In the current study, bioinformatics tools were used to obtain more information about the genetic characteristics of K. pneumoniae plasmids harboring carbapenemase genes.

According to the MST (minimum spanning tree) results, predominated STs in each carbapenemase gene, including blaKPC, blaNDM, blaOXA, and blaGES, were ST11, ST14, and ST12. Apart from the predominant STs, some others, including ST37, ST35, ST16, ST392, ST147, ST17, ST23, ST101, ST307, ST11, ST14, and ST437, were multi-harbor sequence types and associated with plasmids containing at least one of the three carbapenemase genes, including blaKPC, blaNDM, blaOXA-48-like.

ST11 is one of the most common ST in K. pneumoniae isolates. Recently, outbreaks of ST11 carbapenem-resistant hypervirulent K. pneumoniae have been reported in Asian countries, such as China, and ST11 also accounts for 12% of carbapenem-resistant K. pneumoniae in Europe. Apart from the wide distribution of ST11 and its isolation from human samples, ST11 could also be isolated from non-human environments, according to Campos-Madueno et al [24]. ST11 is a single-locus variant of ST258, is one of the most common member of CG258, and is more distributed compared to ST258 and ST512 [25–27]. The presence of ESBL encoding genes, including blaCTX-M-65 and blaTEM-1, which were also seen in this study, is prevalent in ST11 [26]. According to the current study, plasmids harboring blaKPC and blaNDM belonging to ST11. Plasmids with blaKPC, especially blaKPC-2, mostly had IncFII and IncR replicon types and 39.6% were potentially conjugative. Plasmids with blaNDM had IncX3 and IncC replicon types and 54.5% were potentially conjugative. IncFII is one of the most prevalent plasmids found in carbapenem-resistant isolates and it is restricted to the Enterobacteriaceae family [28]. IncX3 is also found mainly in Enterobacteriaceae, has high transmissibility, and facilitates the spread of blaNDM among K. pneumonae [29, 30]. Regarding the current data, the conjugative plasmids harboring carbapenemase and ESBL genes belonging to ST11 could be notable as they could affect the dissemination of resistance genes. On the other hand, due to the genetic structure, blaNDM-1 was accompanied by other resistance genes, including tat and bleMBL, localized on the Tn3-like elements. The Tn3 family is one of the most important mobile genetic elements with the ability to spread a variety of passenger genes, including those conferring resistance to several classes of antibiotics, including carbapenem and colistin resistance [31]. Mapping of the structure containing blaKPC also revealed ISKpn6 and ISKpn27 elements. The core structure of ISKpn27/ISKpn7-dnaA-blaKPC-2-ISKpn6 is highly epidemic in KPC-producing K. pneumoniae isolates [32]. The presence of insertion sequences and transposons with high transmission capacity harboring carbapenemase genes in plasmids associated with ST11 K. pneumoniae isolates could render these isolates be lethal and cause life-threatening infections.

In the current study, ST14 was another predominant sequence type associated with plasmids containing blaOXA-48. ST14 is one of the major STs carrying multiple resistance genes and is common and associated with pediatric and neonatal infections [33]. Available studies indicate that ST14 K. pneumoniae isolates can lead to fatal infections. According to this study, plasmids containing OXA gene (e.g., blaOXA-48) and associated with ST14 mostly had IncL replicon type and were conjugative. In addition, mapping of the construct of the cassette containing OXA revealed that this gene is flanked by IS1 and IS4 families. Insertion of these elements into K. pneumoniae isolates with notable virulence factors, including ompK36 results in higher resistance to carbapenems and an increase in the probability of the treatment failures. On the other hand, the adjacency of blaOXA-48 with lysR can worsen the situation since this gene plays an important role in mediating antibiotic resistance and increasing the virulence of K. pneumoniae isolates [34]. In this study, ST12 is another predominant ST associated with plasmids carrying blaGES-24 with a notable genetic map. The co-existence of blaGES-24 with resistance genes, including aac (6)-Ia, cat, and blaA genes leads to high resistance to multiple antibiotic classes, maybe increase the rate of treatment failure.

One of the most outstanding findings of the current study is the multi-harbor STs. ST101, ST147, and ST16 are three important STs involved in the spread of K. pneumoniae isolates. Overall, the data from the current study showed that the plasmids associated with ST147 and ST101 had mostly plasmid with IncL (with blaOXA) and IncFIB and IncFII replicon types (containing blaKPC and blaNDM), that were almost potentially conjugative or at least mobilizable. The predominant alleles were blaOXA-48, blaKPC-2, and blaNDM-1, however, blaOXA-10, blaOXA-181, blaOXA-232, blaNDM-5, blaNDM-7, blaNDM-29 and blaNDM-9 could also be found in plasmids associated with ST147. These STs are highly associated with lethal infections that according to available studies ST101 could be assumed as a global threat since it plays a major role in the dissemination of resistance genes, including colistin-resistance [35]. ST16 is also a major clone associated with the spread of blaNDM-1 and blaOXA232 and is thought to be one of the most widespread and high-risk clones worldwide [36]. The results of our study also confirmed these data; however, evaluation of the plasmids examined revealed that ST16 might be associated with plasmids containing blaNDM-4 and blaNDM-5, blaOXA-48, and blaOXA181. In addition, according to this study, ST16 was associated with IncFII, IncFIA/B, IncL, and Inc ColKP3. Shukla et al, also reported that ST16 carried mainly ColKP3 [37]. Importantly, IncFII, IncFIA/B, and IncL were mostly conjugative or mobilizable. Whereas ColKP3 was mostly non-conjugative. The presence of non-conjugative plasmids in K. pneumoniae ST16 may be a noteworthy clue affecting the distribution of carbapenem-resistance genes. Taken together, the presence of these STs with high transmission capacity and multiple carbapenemase genes is a big concern in public health.

Our study also revealed remarkable points regarding the co-existence and co-occurrence of resistance genes. The blaTEM-1, blaOXA-232, bleMBL, and aac(6’)-Ib4 were the most abundant genes in the same plasmids harboring the carbapenem resistance genes. Several genes responsible for resistance to different classes of antibiotics, including aminoglycosides, extended-spectrum beta-lactamase, and carbapenem, were found to coexist with blaKPC, blaOXA-48, blaNDM, and blaGES. The co-existence of bleMBL and blaNDM was predictable, as bleMBL is always downstream of blaNDM [38]. The presence of bleMBL responsible for bleomycin resistance (as a glycopeptide antibiotic) with blaNDM could increase the rate of treatment failure. In addition, the presence of two or three plasmids with carbapenem resistance genes among different strains seems to be a critical point. Especially because these plasmids were mainly potentially conjugative and carried transposons such as the Tn3 family and integron class I. Tn3 is one of the most widespread transposase families responsible for the spread of resistance genes [31]. Therefore, these strains with different plasmids, each containing a different resistance gene. Plasmid analysis revealed a high similarity between plasmids containing blaKPC-2 and blaOXA-48, while blaNDM seems to be heterogeneous. This could be a remarkable point as it shows that blaNDM could be placed in different plasmids with different sequences, which would worsen the situation of antimicrobial resistance.

5. Conclusion

The co-existence of different classes of resistance genes, co-occurrence of various carbapenemase genes on separate plasmids, and gene repetition in a plasmid were notable findings of this study. Assessment of genetic characteristics of the plasmids also revealed that blaNDM-harboring heterogenic plasmids had a high capacity for dissemination. multi-harbor carbapenemases STs could highly affect the exacerbation of the antimicrobial resistance in this bacterium. K. pneumoniae appears to employ multiple genetic strategies for resistance against carbapenem antibiotics. First, gene repetition and locating carbapenemase genes associated with class 1 and 3 integrons, ISKpn and Tn3 plays important roles in DNA recombination. In addition, the placement of these DNA fragments on transferable plasmids paves the way for widespread expansion of antimicrobial resistance. Finally, the successful and international clones (ST11, ST14, ST437, ST23, ST307, ST101, ST147, ST16, ST17, ST35 and ST37) with high ability to capture carbapenemase-containing plasmids are actually the last circle of this journey which has dramatically increased antibiotic resistance all over the world. It seems that K. pneumoniae is collecting various resistance genes and virulence factors with all its genome capacity. Such genetic flexibility of a superbug is not only astonishing but also a very serious health threat. In the future, new methods (e.g. vaccination, novel drug targets and antibiotics, and new combination therapy such as antibodies and antibiotics) should be used to fight against K. pneumoniae.

Supporting information

S1 File. All information of BioSamples from isolates harboring carbapenemases.

(ZIP)

Click here for additional data file. (98.7KB, zip)
S2 File. The number of allele types of carbapenemase genes in a plasmid.

(XLSX)

Click here for additional data file. (13KB, xlsx)
S3 File. The co-existence of antimicrobial resistance genes in a plasmid.

(XLSX)

Click here for additional data file. (16KB, xlsx)
S4 File. The accession numbers of all retrieved plasmids.

(XLSX)

Click here for additional data file. (42.3KB, xlsx)

Acknowledgments

The authors would like to thank the personnel at the Bacteriology Department of the Pasteur Institute of Iran to commence on the manuscript for improvement.

Data Availability

The accession numbers of the datasets generated and analyzed during the current study are available in S4 File. All plasmid sequences can be retrieved from the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) or Batch Entrez (https://www.ncbi.nlm.nih.gov/sites/batchentrez).

Funding Statement

The author(s) received no specific funding for this work.

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Decision Letter 0

Farah Al-Marzooq

31 Jul 2023

PONE-D-23-18985Genetic structure of blaKPC, blaNDM, blaOXA-48, and blaGES genes associated with international clones and conjugative plasmids from Klebsiella pneumoniaePLOS ONE

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Reviewer #1: Dear authors

Thank you for your interesting study and well-written/organized paper. I read the manuscript and tried to understand what the study's aims were and how the work was carried out. Using bioinformatics methods, investigators sought to learn more about the genetic characteristics of K.pneumoniae plasmids harboring carbapenemase genes. The research was interesting, and the paper was well-written. However, I have a few minor observations:

• Page 8-15; lines 188-190: Table 1. Genomic data on STs, Carbapenemase genes, other resistance genes, and conjugal apparatus among whole genome sequencing of K.pneumoniae.

COMMENT: It would be greatly appreciated if the authors could indicate the source of /sample type (human specimen, animal specimen, food, environmental, or other sources) for these isolates. This information is crucial for comprehending horizontal gene transfer and its occurrence in humans, animals and other sources.

• Page 22; line 369: “In the future, new methods should be used to fight against K. pneumoniae”.

COMMENT: In order to combat K. pneumoniae in a more effective manner, what novel strategies or approaches do you recommend? Could you perhaps write out in detail your thoughts on this matter?

• Page 22; lines 370-372: “The authors would like to thank the personnel at the Bacteriology Department of the Pasteur Institute of Iran for their help. This research was supported by the Pasteur Institute of Iran.”

COMMENT 1: What kinds of assistance did this staff offer? They should be mentioned explicitly, and they should be acknowledged appropriately.

COMMENT 2: Please provide the sponsor grant number

• Page 22; lines 385-386: “No specific funding was received for this study”.

COMMENT: If you did not receive any funding for this study, why did you state "This research was supported by the Pasteur Institute of Iran"? it must be revised.

• ADDITIONAL COMMENT: Please use K. pneumoniae (italic) instead of K. pneumoniae.

Reviewer #2: This manuscript describes the results of the bioinformatics analysis of the genetic structure of plasmids harbouring carbapenemases in Klebsiella pneumoniae. The plasmids analyzed were completed plasmids and the genes studied were blaKPC, blaNDM, blaOXA-100 and blaGES.

A total of 2254 plasmids harboring carbapenemase genes from GenBank database were analyzed.

The objective of the manuscript is interesting and the results could be useful but the bioinformatics strategy to detect and select the carbapenem resistance genes in plasmids of Klebsiella pneumonia is not sufficiently robust and the results are not studied in deep to extract new findings and significant conclusions.

MAJOR POINTS

- The manuscript is in a very preliminary state.

- The strategy of selection of reference sequences is not very appropriate.

- The method for detecting the carbapenem resistance genes is not correctly designed and probably produces a bias in the detection of genes in the analyzed Klebsiella pneumonia genomes.

In the section “Preparation of initial dataset” of the “Materials and methods” I understand that the authors use BLASTN search for detecting the plasmids harbouring carbapenemase genes. Given that many plasmids have different hosts, a different codon usage, or minimal differences of nucleotide sequence that do not affect the function could jeopardize the detection of functional carbapenemase genes if you only use a strategy based on nucleotide sequence detection. It would be better to use protein sequences of the carbapenemases as query and Genbank sequences of nucleotides as subject database using TBLASTN. TBLASTN searches translated nucleotide databases using a protein query.

Thus, in line Line 287 the authors conclude: “According to the current study, plasmids harboring blaKPC and blaNDM belonging to ST11”. I wonder if the reason of that is that the reference genes used to select the plasmids to be analyzed come from ST11 isolates. That is one of the reason why it is better to do a TBLASTN to search the reference sequences of proteins in nucleotide databases avoiding to have a bias and allowing to select proteins functionally similar but with different nucleotide sequence.

- As a research article the manuscript does not present any important new finding neither some new interpretation of the dataset analyzed. Considered as a review lacks many of the aspects required for a review about carbapenem resistance genes in Klebsiella pneumonia.

- The discussion about the publications related with this manuscript is poor. For example these publications are not included in the references of the manuscript:

Campos-Madueno EI, Moser AI, Jost G, Maffioli C, Bodmer T, Perreten V, Endimiani A. Carbapenemase-producing Klebsiella pneumoniae strains in Switzerland: human and non-human settings may share high-risk clones. J Glob Antimicrob Resist. 2022 Mar;28:206-215.

Karampatakis T, Tsergouli K, Behzadi P. Carbapenem-Resistant Klebsiella pneumoniae: Virulence Factors, Molecular Epidemiology and Latest Updates in Treatment Options. Antibiotics (Basel). 2023 Jan 21;12(2):234. doi: 10.3390/antibiotics12020234. PMID: 36830145; PMCID: PMC9952820.

Karaiskos I, Galani I, Papoutsaki V, Galani L, Giamarellou H. Carbapenemase producing Klebsiella pneumoniae: implication on future therapeutic strategies.Expert Rev Anti Infect Ther. 2022 Jan;20(1):53-69. doi: 10.1080/14787210.2021.1935237. Epub 2021 Jun 3.PMID: 34033499 Review.

- Important analysis as the distribution of the plasmids with carbapenem resistance genes in different hosts and in different human tissues are not analyzed.

- Geographic provenance of the genomes is not included in the analysis

- Some study of the plasmids with carbapenemase genes in close species that share microenvironments is not included. This manuscript is focused on Klebsiella pneumonia but it would be needed to discuss if these carbapenem resistance genes and/or the plasmids that harbour them are also present in other bacterial species sharing host and microenvironment as it could be Escherichia coli, Salmonella or other enterobacteria.

Minor points:

Line 103:

“In addition, we characterized the genetic features of plasmids harboring carbapenemase genes including replicon types, conjugation ability, the coexistence of carbapenem with other antimicrobial resistance genes, co-occurrence of carbapenemase genes in one strain, gene repetition, and phylogenetic relatedness.”

Edit this sentence to clarify and explain in more detail what features of the plasmids harboring carbapenemase genes you had characterized in this work.

Line 107:

“The complete nucleotide sequences of plasmids containing four carbapenemase genes, including blaKPC, blaNDM, blaOXA-48, and blaGES were retrieved from the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/).”

I understand that your criterion of selection of plasmids was to have any of those 4 types of carbapenemase genes (blaKPC, blaNDM, blaOXA-48, and blaGES) not to have all the four types in a plasmid. It that is the case it is not clear in this sentence.

Line 125:

oriTfnder tool (https://bioinfomml.sjtu.edu.cn/oriTf inder/)

Please correct the name of the tool and the url

Line 147:

Indicate which is the plasmid with 5,803,733 bp

Line 164:

“plasmids carrying four mentioned carbapenemase genes.”

This sentence is confusing because I understand that there are not 4 carbapenemase genes in each selected plasmid.

Line 276

“According to the MST results, STs predominated in each carbapenemase gene, including 276 blaKPC, blaNDM, blaOXA, and blaGES, were ST11, ST14, and ST12. Apart from the predominant 277 STs, some others, including ST37, ST35, ST16, ST392, ST147, ST17, ST23, ST101, ST307, 278 ST11, ST14, and ST437, were multi-harbor sequence types and associated with plasmids 279 containing three carbapenemase genes, including blaKPC, blaNDM, blaOXA.”

Please, add (Multilocus Sequence Typing) after MST and rewrite this sentence because its meaning is not clear.

Line 293

K.pneumonae -> K. pneumoniae

In general please put K. pneumoniae in italics (it is not my case but microbiologists suffer a lot if you don’t)

Line 320

“One of the most outstanding findings of the current study is the multi-harbor STs. STs 101, 320 147, and 16 are three important sequence types involved in the spread of K.pneumoniae isolates. Overall, the data from the current study showed that the plasmids associated with ST147 and ST101 had mostly IncL (with blaOXA) and IncFIB and IncFII replicon types (containing blaKPC and blaNDM), that were almost potentially conjugative or at least mobilizable. The predominant alleles were blaOXA-48, blaKPC-2, and blaNDM-1, however, blaOXA-10, blaOXA-181, blaOXA-232, blaNDM-5, blaNDM-7, blaNDM-29 and blaNDM-9 could also be found in plasmids associated with ST147”

This sentence needs to be rewritten.

Line 344

“…plasmids harboring the carbapenem/ genes.” → plasmids harboring the carbapenem resistance genes

Line 361

“The co-existence of resistance genes belonging to different antibiotic clases,….” → The co-existence of different antibiotic classes resistance genes,….

Line 365

“multi-harbor carbapenemases and international STs (e.g. ST11, ST23, ST14, and ST12) could highly affect the exacerbation of the antimicrobial resistance in K. pneumoniae.”

IMHO this sentence has not sense.

Finally, I want to encourage the authors to continue working on it because I think they can do a better job on this topic, which is very important and needs a deep analysis.

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Reviewer #1: Yes: Dr.Melese Abate Reta (PhD)

Reviewer #2: No

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PLoS One. 2023 Nov 16;18(11):e0292288. doi: 10.1371/journal.pone.0292288.r002

Author response to Decision Letter 0

24 Aug 2023

View Letter

PONE-D-23-18985

Genetic structure of blaKPC, blaNDM, blaOXA-48, and blaGES genes associated with international clones and conjugative plasmids from Klebsiella pneumoniae

PLOS ONE

Dear Dr. Farah Al-Marzooq,

We would like to thank the reviewers for their precious time to review our submitted manuscript and their helpful and valuable comments. We believe the comments and revisions have made our manuscript a more valuable paper. The comments have been added to the manuscript and the questions have been answered. The changes, as suggested, were track changed in the manuscript.

Best regards,

Dr. Farzad Badmasti

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RE: Thank you so much you have checked it.

2. Please amend your list of authors on the manuscript to ensure that each author is linked to an affiliation. Authors’ affiliations should reflect the institution where the work was done (if authors moved subsequently, you can also list the new affiliation stating “current affiliation:….” as necessary).

RE: It was done.

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RE: Thank you for your comment. The Figure has been reproduced. There is no copy-write content in it.

Additional Editor Comments:

Please, revise the manuscript considering all the reviewers' comments , and try to answer all the queries raised by them

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Reviewers' comments:

Reviewer's Responses to Questions

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Reviewer #1: Yes

Reviewer #2: Partly

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Reviewer #2: Yes

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Reviewer #1: Dear authors

Thank you for your interesting study and well-written/organized paper. I read the manuscript and tried to understand what the study's aims were and how the work was carried out. Using bioinformatics methods, investigators sought to learn more about the genetic characteristics of K.pneumoniae plasmids harboring carbapenemase genes. The research was interesting, and the paper was well-written. However, I have a few minor observations:

• Page 8-15; lines 188-190: Table 1. Genomic data on STs, Carbapenemase genes, other resistance genes, and conjugal apparatus among whole genome sequencing of K.pneumoniae.

COMMENT: It would be greatly appreciated if the authors could indicate the source of /sample type (human specimen, animal specimen, food, environmental, or other sources) for these isolates. This information is crucial for comprehending horizontal gene transfer and its occurrence in humans, animals and other sources.

RE: First, we would like to thank you for your very careful review of our paper and for your subsequent comments, corrections, and suggestions as well. The additional data on the source, geographical region, and the year of isolation has been added to the manuscript as Table 1.

• Page 22; line 369: “In the future, new methods should be used to fight against K. pneumoniae”.

COMMENT: In order to combat K. pneumoniae in a more effective manner, what novel strategies or approaches do you recommend? Could you perhaps write out in detail your thoughts on this matter?

RE: It was done.

• Page 22; lines 370-372: “The authors would like to thank the personnel at the Bacteriology Department of the Pasteur Institute of Iran for their help. This research was supported by the Pasteur Institute of Iran.”

COMMENT 1: What kinds of assistance did this staff offer? They should be mentioned explicitly, and they should be acknowledged appropriately.

RE: Thank you for your precise observation. The correction has been done.

COMMENT 2: Please provide the sponsor grant number

• Page 22; lines 385-386: “No specific funding was received for this study”.

COMMENT: If you did not receive any funding for this study, why did you state "This research was supported by the Pasteur Institute of Iran"? it must be revised.

RE: Thank you for your comment. The correction has been done.

• ADDITIONAL COMMENT: Please use K. pneumoniae (italic) instead of K. pneumoniae.

RE: The corrections have been done.

Reviewer #2: This manuscript describes the results of the bioinformatics analysis of the genetic structure of plasmids harbouring carbapenemases in Klebsiella pneumoniae. The plasmids analyzed were completed plasmids and the genes studied were blaKPC, blaNDM, blaOXA-100 and blaGES.

A total of 2254 plasmids harboring carbapenemase genes from GenBank database were analyzed.

The objective of the manuscript is interesting and the results could be useful but the bioinformatics strategy to detect and select the carbapenem resistance genes in plasmids of Klebsiella pneumonia is not sufficiently robust and the results are not studied in deep to extract new findings and significant conclusions.

MAJOR POINTS

- The manuscript is in a very preliminary state.

RE: First, we would like to thank you for your very careful review of our paper. In this study, we attempted to present a correct and accurate genetic map of plasmids containing carbapenem resistance genes. One of our major goals was to accurately represent the distribution of four major carbapenem resistance genes in the predominant sequences types, to show the homogeneity of the plasmid, and to show the diversity of allele types. To the best of our knowledge, the representation of all this information in the genomic layer has been done in fewer articles. Furthermore, the manuscript has been enhanced by incorporating supplementary information pertaining to the origin, geographic location, and year of isolation. We really do believe that this manuscript helps investigators who have been working on cabapenemas in K. pneumoniae to design a project and finding new genetic features.

- The strategy of selection of reference sequences is not very appropriate.

RE: In this study four Refseq accession numbers of major cabapenemases were consider to seek all completed plasmids and DNA fragments using BLASTn. These BLASTs were done based an standard criteria. For example, Microbial BLAST (database: All genomes; Organism: K. pneumoniae; Optimize for: Highly similar sequences; Max target sequences: 5000; Expect threshold: 0.05; Word size: 28; Gap Costs: linear).

- The method for detecting the carbapenem resistance genes is not correctly designed and probably produces a bias in the detection of genes in the analyzed Klebsiella pneumonia genomes.

In the section “Preparation of initial dataset” of the “Materials and methods” I understand that the authors use BLASTN search for detecting the plasmids harbouring carbapenemase genes. Given that many plasmids have different hosts, a different codon usage, or minimal differences of nucleotide sequence that do not affect the function could jeopardize the detection of functional carbapenemase genes if you only use a strategy based on nucleotide sequence detection. It would be better to use protein sequences of the carbapenemases as query and Genbank sequences of nucleotides as subject database using TBLASTN. TBLASTN searches translated nucleotide databases using a protein query.

Thus, in line Line 287 the authors conclude: “According to the current study, plasmids harboring blaKPC and blaNDM belonging to ST11”. I wonder if the reason of that is that the reference genes used to select the plasmids to be analyzed come from ST11 isolates. That is one of the reason why it is better to do a TBLASTN to search the reference sequences of proteins in nucleotide databases avoiding to have a bias and allowing to select proteins functionally similar but with different nucleotide sequence.

RE: As mentioned in the title and also different parts of the manuscript, this study aimed to show the genetic map (DNA layer) of the plasmids harboring carbapenem resistance genes. Finding the dominant sequence carrying resistance genes and trying to determine the degree of heterogeneity of plasmids as well as characterizing a genomic content were the main goals of this study, and all these goals can be realized in the DNA level. For example, the determination of allele type of carabapenemases has been established based on single nucleotide polymorphisms (SNPs) in DNA layer. MLST and ST are based on SNP as well. We think there is no need to be assessed in protein layer. It should also be noted that there seems we were not clear about the sequence types. Actually, ST11 is not the reference ST of our study on which the rest of the data are analyzed, but ST11 was one of the dominant STs in this study.

- As a research article the manuscript does not present any important new finding neither some new interpretation of the dataset analyzed. Considered as a review lacks many of the aspects required for a review about carbapenem resistance genes in Klebsiella pneumonia.

RE: As mentioned above, the main objective of the current study is to reveal the precise genetic map of plasmids harboring carbapenem genes. The accurate drawing of the genetic map of the factors that play a prominent role in the transmission of carbapenem resistance as an important topic that has not yet been fully addressed. In addition, the identification of the predominant STs that may play an important role in the spread of resistance genes, along with the type of alleles they may carry, is an important issue that is prominent in AMR. There are some new findings in this study

1- Predominant allele type of each carbapenemase

2- Evaluation of conjugal apparatus in the plasmids harboring major carbapenemases

3- Detection of co-existence and co-occurrence among the plasmids

4- Determination of all genetic maps, divergence and convergence among the plasmids

5- Detection of gene repetition among carbapenemases

6- Revealing of all STs harboring carbapenemases at a glance

- The discussion about the publications related with this manuscript is poor. For example these publications are not included in the references of the manuscript:

Campos-Madueno EI, Moser AI, Jost G, Maffioli C, Bodmer T, Perreten V, Endimiani A. Carbapenemase-producing Klebsiella pneumoniae strains in Switzerland: human and non-human settings may share high-risk clones. J Glob Antimicrob Resist. 2022 Mar;28:206-215.

Karampatakis T, Tsergouli K, Behzadi P. Carbapenem-Resistant Klebsiella pneumoniae: Virulence Factors, Molecular Epidemiology and Latest Updates in Treatment Options. Antibiotics (Basel). 2023 Jan 21;12(2):234. doi: 10.3390/antibiotics12020234. PMID: 36830145; PMCID: PMC9952820.

Karaiskos I, Galani I, Papoutsaki V, Galani L, Giamarellou H. Carbapenemase producing Klebsiella pneumoniae: implication on future therapeutic strategies.Expert Rev Anti Infect Ther. 2022 Jan;20(1):53-69. doi: 10.1080/14787210.2021.1935237. Epub 2021 Jun 3.PMID: 34033499 Review.

RE: The mentioned references have been added to the manuscript.

- Important analysis as the distribution of the plasmids with carbapenem resistance genes in different hosts and in different human tissues are not analyzed.

- Geographic provenance of the genomes is not included in the analysis

RE: The additional data on the source, geographical region, and the year of isolation has been added to the manuscript.

- Some study of the plasmids with carbapenemase genes in close species that share microenvironments is not included. This manuscript is focused on Klebsiella pneumonia but it would be needed to discuss if these carbapenem resistance genes and/or the plasmids that harbour them are also present in other bacterial species sharing host and microenvironment as it could be Escherichia coli, Salmonella or other enterobacteria.

RE: The purpose of the present study was to fully and accurately investigate the genetic structure of plasmids carrying the carbapenem resistance gene in Klebsiella pneumoniae as a very important nosocomial pathogen. Considering the high number of plasmids from other genus is complex and requires a very extended and progressed analysis. In fact, the main aim of the current study was to focus on the fully evaluation of genetically aspects of plasmids harboring carbapenemase genes in just one species. The analysis of other bacteria, including Escherichia and Salmonella, and the data collection and comparison is very interesting and appealing. However, it could be done in a different study.

Minor points:

Line 103:

“In addition, we characterized the genetic features of plasmids harboring carbapenemase genes including replicon types, conjugation ability, the coexistence of carbapenem with other antimicrobial resistance genes, co-occurrence of carbapenemase genes in one strain, gene repetition, and phylogenetic relatedness.”

Edit this sentence to clarify and explain in more detail what features of the plasmids harboring carbapenemase genes you had characterized in this work.

RE: It was done.

Line 107:

“The complete nucleotide sequences of plasmids containing four carbapenemase genes, including blaKPC, blaNDM, blaOXA-48, and blaGES were retrieved from the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/).”

I understand that your criterion of selection of plasmids was to have any of those 4 types of carbapenemase genes (blaKPC, blaNDM, blaOXA-48, and blaGES) not to have all the four types in a plasmid. It that is the case it is not clear in this sentence.

RE: Thank you for your comment. The correction was done.

Line 125:

oriTfnder tool (https://bioinfomml.sjtu.edu.cn/oriTf inder/)

Please correct the name of the tool and the url

RE: The correction has been done.

Line 147:

Indicate which is the plasmid with 5,803,733 bp

RE: The correction has been done.

Line 164:

“plasmids carrying four mentioned carbapenemase genes.”

This sentence is confusing because I understand that there are not 4 carbapenemase genes in each selected plasmid.

RE: Thank you for your comment. The correction has been done.

Line 276

“According to the MST results, STs predominated in each carbapenemase gene, including 276 blaKPC, blaNDM, blaOXA, and blaGES, were ST11, ST14, and ST12. Apart from the predominant 277 STs, some others, including ST37, ST35, ST16, ST392, ST147, ST17, ST23, ST101, ST307, 278 ST11, ST14, and ST437, were multi-harbor sequence types and associated with plasmids 279 containing three carbapenemase genes, including blaKPC, blaNDM, blaOXA.”

Please, add (Multilocus Sequence Typing) after MST and rewrite this sentence because its meaning is not clear.

RE: It was done.

Line 293

K.pneumonae -> K. pneumoniae

RE: The correction has been done.

In general please put K. pneumoniae in italics (it is not my case but microbiologists suffer a lot if you don’t)

RE: The correction has been done.

Line 320

“One of the most outstanding findings of the current study is the multi-harbor STs. STs 101, 320 147, and 16 are three important sequence types involved in the spread of K.pneumoniae isolates. Overall, the data from the current study showed that the plasmids associated with ST147 and ST101 had mostly IncL (with blaOXA) and IncFIB and IncFII replicon types (containing blaKPC and blaNDM), that were almost potentially conjugative or at least mobilizable. The predominant alleles were blaOXA-48, blaKPC-2, and blaNDM-1, however, blaOXA-10, blaOXA-181, blaOXA-232, blaNDM-5, blaNDM-7, blaNDM-29 and blaNDM-9 could also be found in plasmids associated with ST147”

This sentence needs to be rewritten.

RE: It was done.

Line 344

“…plasmids harboring the carbapenem/ genes.” → plasmids harboring the carbapenem resistance genes

RE: The correction has been done.

Line 361

“The co-existence of resistance genes belonging to different antibiotic clases,….” → The co-existence of different antibiotic classes resistance genes,….

RE: The correction has been done.

Line 365

“multi-harbor carbapenemases and international STs (e.g. ST11, ST23, ST14, and ST12) could highly affect the exacerbation of the antimicrobial resistance in K. pneumoniae.”

IMHO this sentence has not sense.

RE: It was re-written.

Finally, I want to encourage the authors to continue working on it because I think they can do a better job on this topic, which is very important and needs a deep analysis.

Attachment

Submitted filename: Review letter.docx

Click here for additional data file. (30KB, docx)

Decision Letter 1

Farah Al-Marzooq

18 Sep 2023

Decoding the genetic structure of conjugative plasmids in international clones of Klebsiella pneumoniae: A deep dive into blaKPC, blaNDM, blaOXA-48, and blaGES genes

PONE-D-23-18985R1

Dear Dr. Badmasti,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Farah Al-Marzooq, MD, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The manuscript was improved after revision

Reviewers' comments:

Acceptance letter

Farah Al-Marzooq

7 Nov 2023

PONE-D-23-18985R1

Decoding the genetic structure of conjugative plasmids in international clones of Klebsiella pneumoniae: A deep dive into blaKPC, blaNDM, blaOXA-48, and blaGES genes

Dear Dr. Badmasti:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Kind regards,

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on behalf of

Dr. Farah Al-Marzooq

Academic Editor

PLOS ONE

Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 File. All information of BioSamples from isolates harboring carbapenemases.

    (ZIP)

    Click here for additional data file. (98.7KB, zip)
    S2 File. The number of allele types of carbapenemase genes in a plasmid.

    (XLSX)

    Click here for additional data file. (13KB, xlsx)
    S3 File. The co-existence of antimicrobial resistance genes in a plasmid.

    (XLSX)

    Click here for additional data file. (16KB, xlsx)
    S4 File. The accession numbers of all retrieved plasmids.

    (XLSX)

    Click here for additional data file. (42.3KB, xlsx)
    Attachment

    Submitted filename: Review letter.docx

    Click here for additional data file. (30KB, docx)

    Data Availability Statement

    The accession numbers of the datasets generated and analyzed during the current study are available in S4 File. All plasmid sequences can be retrieved from the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) or Batch Entrez (https://www.ncbi.nlm.nih.gov/sites/batchentrez).

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