Genetic characterization of bla_NDM-1-positive Klebsiella quasipneumoniae subsp. similipneumoniae from a human carrier

Abstract

Carbepenem-resistant Klebsiella quasipneumoniae have increasingly been reported across many regions, yet data on the carbapenemase-producing isolates remain scarce. This study aimed to characterize the bla_NDM-1-positive Klebsiella sp. strain M42D recovered from a human carrier in Thailand. Strain M42D exhibited high-level resistance to carbapenems but remained susceptible to colistin. Whole genome sequencing was conducted using the Illumina and PacBio platforms. A complete genome sequence identified strain M42D as K. quasipneumoniae subsp. similipneumoniae (Kqps) ST334. The bla_NDM-1 gene was determined to be transferable and located on the IncN2 pKquM42Da (41.2 kb). Mutations in OmpK36 and OmpK37, involved in carbapenem resistance, were observed. In silico analysis of 361 Kqps genomes showed that 9.1% of isolates, mostly from humans in Southeast Asia, carried a carbapenemase gene. Among these, bla_NDM-1 was present in six isolates, with only strain M42D belonging to ST334. Core genome single-nucleotide polymorphism (SNP) analysis demonstrated that M42D clustered with other Kqps ST334 isolates with 142–186 SNP differences, suggesting that they are evolutionarily distinct strains. This study reports the first bla_NDM-1-harboring Kqps ST334 isolate, highlighting the need for further genomic surveillance of Kqps from different origins, which may help to understand the antibiotic resistance, and dissemination of Kqps within the community.

1. Introduction

Klebsiella quasipneumoniae is a member of Klebsiella pneumoniae complex and exists as a commensal bacterium in the intestinal tract of humans and animals. In addition, it is prevalent in agricultural and environmental settings. Further taxonomic characterizations have subdivided K. quasipneumoniae into two subspecies, K. quasipneumoniae subsp. quasipneumoniae (KpII-A) and K. quasipneumoniae subsp. similipneumoniae (KpII-B) [1].

Many studies have shown that K. quasipneumoniae possesses several virulence factors capable of causing infections [2, 3]. Diseases caused by K. quasipneumoniae are similar to those of K. pneumoniae, including urinary tract, respiratory, and bloodstream infections [4]. K. quasipneumoniae is generally considered less virulent than K. pneumoniae, however, a previous study revealed its ability to evade immune responses by residing and persisting within macrophages [3]. Colonization of K. quasipneumoniae in the mouse intestine has also been shown to cause mucosal damage [5]. Moreover, the persistence of K. quasipneumoniae in hospital environments, for more than three years, followed by transmission to patients, has been documented [2, 6]. Due to an increasing emergence of multidrug-resistant isolates, it is of great concern that K. quasipneumoniae may cause severe and life-threatening diseases, particularly in neonates and immunocompromised patients.

Recently, reports on K. quasipneumoniae and its associated diseases have increasingly been published. A 5-year retrospective genomic study in Singapore revealed that K. quasipneumoniae accounted for 32.5% of clinical Klebsiella spp. isolates tested [7]. In addition, the clinical features and 30-day mortality rates of patients with K. quasipneumoniae infections were found to be comparable to those of patients with K. pneumoniae infections [4]. Septicemia and subsequent death due to K. quasipneumoniae have been reported in neonates in China and Nigeria [6, 8], as well as in adult patients in Saudi Arabia [9], Hong Kong [10], and the USA [4]. Additionally, K. quasipneumoniae can cause diseases even in patients with no underlying conditions, with fatal outcomes documented in some instances [4]. In Thailand, recovery of K. quasipneumoniae from human clinical specimens and rectal swab samples from hospitalized patients has been documented [11, 12]. Beyond human sources, K. quasipneumoniae has been detected in healthy swine [13, 14], and in aquatic environment (BioSample: SAMEA3531892). However, the incidence of infections caused by K. quasipneumoniae may be underestimated due to frequent misidentification as K. pneumoniae in routine clinical microbiological testing [15].

Resistance to multiple antibiotic classes, such as β-lactams, aminoglycosides, tetracyclines and quinolones, has been consistently documented in K. quasipneumoniae and poses an increasing public health threat across many regions. Approximately 50% of K. quasipneumoniae isolates collected worldwide have been identified as extended-spectrum β-lactamase producers, conferring resistance to third-generation cephalosporins [1]. Of particular concern, resistance to carbapenem and colistin, last resort antibiotics used for treating multidrug-resistant Gram-negative bacterial infections, has been increasingly observed in K. quasipneumoniae isolates [16]. Klebsiella spp. capable of producing carbapenemases are recognized as critical pathogens associated with serious diseases and elevated risks of treatment failure. Several carbapenemase genes, such as bla_KPC, bla_IMP, bla_VIM, bla_NDM and bla_OXA, have been detected in K. quasipneumoniae isolates from humans, animals, and water resources [2, 9, 16–18]. Reports of K. quasipneumoniae isolates harboring combinations of carbapenemase, such as NDM + OXA-48/181, NDM + KPC-2, NDM + VIM + OXA-48, are especially alarming [16, 18, 19]. Interestingly, K. quasipneumoniae exhibits a propensity to acquire plasmids and several resistance genes, including carbapenemase genes, from other bacterial species, potentially rendering carbapenem ineffective for treatment [1, 2].

In Thailand, antibiotic-resistant K. quasipneumoniae isolates have been detected, yet data on the carbapenemase-producing isolates remain scare. This study aimed to phenotypically and genotypically characterized the bla_NDM-1-positive Klebsiella sp. strain M42D, recovered from a rectal swab sample of a hospitalized patient in Thailand.

2. Materials and methods

2.1. Bacterial strain

The bla_NDM-1-positive Klebsiella spp. strain M42D was isolated from a rectal swab sample of a 60-year-old male patient admitted in a hospital in Phitsanulok Province, Northern Thailand, in January 2015. He was diagnosed with pneumonia and treated with ceftriaxone. Bacterial identification was initially performed using the RapID™ ONE System (ThermoFisher Scientific, MA, USA) and 16S rDNA sequencing [20].

2.2. Antibiotic susceptibility testing

Susceptibility to cefotaxime, ceftazidime, colistin (Sigma-Aldrich, MO, USA), imipenem (JW Pharmaceutical, Gyeonggi-do, Korea), ertapenem (MSD, Bangkok, Thailand), meropenem, ciprofloxacin (M&H manufacturing, Samutprakarn, Thailand), and levofloxacin (OLIC, Ayutthaya, Thailand) was assessed using broth microdilution method recommended by the Clinical and Laboratory Standards Institute (CLSI M100, 33rd ed., 2023) [21]. Twofold serial dilution of antibiotics in 200 µL cation-adjusted Mueller Hinton Broth (Oxoid, Hamshire, UK) in a microtitre plate was prepared. Bacterial suspensions were inoculated into each well at a final concentration of 5 × 10⁵ CFU mL⁻¹. The plates were incubated at 37 °C for 16–20 h. Minimum Inhibitory Concentrations (MICs) were defined as the lowest concentration, which inhibited visible growth of the organism.

Susceptibility to amikacin, piperacillin/tazobactam, and ceftazidime/avibactam was evaluated using the MIC Test Strips (Liofilchem, Roseto degli Abruzzi, Italy). Briefly, three to five colonies were adjusted to 0.5 McFarland turbidity (1.5 × 10⁸ CFU mL⁻¹) in normal saline and spread on Mueller Hinton Agar (MHA) (Oxoid, Hamshire, UK). MIC strips were placed on MHA and incubated at 37 °C for 16–20 h. Escherichia coli ATCC25922 was used as a control strain. MICs of all antibiotics were interpreted according to the CLSI guidelines [21].

2.3. Conjugation experiment

Conjugative transfer of bla_NDM-1 was investigated by broth mating assay, using sodium azide-resistant E. coli J53 as the recipient. Klebsiella spp. strain M42D and E. coli J53 were grown in Tryptic Soy Broth (TSB) in a shaker incubator (150 rpm) at 37 °C to mid-log phase (OD600 = 0.5–0.7), and adjusted to 0.5 McFarland turbidity (1.5 × 10⁸ CFU mL⁻¹). Cultures of donor and recipient cells were mixed at a 1:1 ratio (total volume 2 mL) and incubated at 37 °C overnight without shaking. Ten-fold serial dilutions of the mating mixtures were prepared and 100 µL were spread on Tryptic Soy Agar (TSA) supplemented with sodium azide (150 μg mL⁻¹) and meropenem (0.5 μg mL⁻¹) and incubated at 37 °C overnight. Enumeration of donors and recipients were done by plating serial dilutions on TSA containing meropenem (0.5 μg mL⁻¹) and sodium azide (150 μg mL⁻¹), respectively. Conjugation frequency was calculated as the number of transconjugants divided by the number of donor cells.

The presence of bla_NDM-1 in the transconjugants was confirmed by PCR using primers: 5′-ATGATGACTCAGAGCATT CG-3′ and 5′-TTATTGCATCAGAAACCGTG-3′ (812 bp). The PCR conditions were 5 min of initial denaturation at 94 °C, followed by 30 cycles at 94 °C for 45 s, 52 °C for 30 s, and 72 °C for 45 s and a final extension at 72 at 94 °C for 5 min. PCR products were analyzed by 1% agarose gel electrophoresis. MICs for the transconjugants were determined using the broth microdilution method. The IncN2 replicon-type plasmid was identified by PCR using previously described primers [22].

2.4. Genomic DNA isolation and complete genome sequencing

Genomic DNA was extracted from overnight cultures using the TIANamp Genomic DNA Kit (Tiangen®, Beijing, China) and quantified using a Nanodrop Spectrophotometer (Thermo Fisher Scientific, MA, USA). Complete genome sequencing was performed using a hybrid approach combining Illumina and Pacific Biosciences (PacBio) technologies (Macrogen, Seoul, Korea). For short-read sequencing, a DNA library was prepared using the Nextera XT DNA Library Prep Kit Reference Guide. The resulting library was sequenced using the NextSeq system according to the manufacturer's protocol (Illumina). For long-read sequencing, a DNA library was constructed using the PacBio Microbial Library according to the manufacturer's protocol prior to sequencing on the Sequel II (PacBio, Menlo Park, CA). The complete genome sequence of strain M42D was assembled by integrating reads from both Illumina and PacBio data sets using Unicycler v0.4.8 with default parameters [23].

2.5. Bioinformatics analysis

The complete genome of strain M42D, encompassing chromosomal and plasmid DNA sequences, was subjected to bioinformatics analysis. Species identification was carried out through average nucleotide identity (ANI) based on ANIb (BLAST-based ANI) using the online server tool JSpeciesWS. According to the established ANI-based species delineation criteria, an ANI value of ≥95% confirms species-level identity, whereas values below this threshold suggest taxonomic distinction [24]. Multilocus sequence typing (MLST) was performed using MLST v2.0.9, based on the K. pneumoniae scheme from the PubMLST database [25]. The capsular type (KL type) was identified using Kaptive 2.0 [26]. Virulence-associated genes were analyzed using a VFanalyzer [27]. ResFinder v4.5.0 and PlasmidFinder v4.5.0 were used to identify genes responsible for genotypic antibiotic resistance and plasmid replicon types, respectively [28, 29].

The M42D genome was annotated using the RAST tool kit (RASTtk) [30] prior to subsystem analysis, as previously recommended [31]. Among a collection of K. quasipneumoniae subsp. similipneumoniae genomes, the genomes most similar to M42D were identified using the genome distance estimation tool, Mash [32]. Core-genome content of M42D and the selected genomes were identified using Roary v.3.12.0 [33] and a phylogenetic tree based on the identified core-genes was constructed using FastTree v2.1.10 [34]. Single-nucleotide polymorphisms (SNP) and SNP distances of aligned core-genes between genomes were determined using SNP-sites v2.5.1 [35].

2.6. Data availability statement

The genome and related data of K. quasipneumoniae subsp. similipneumoniae strain M42D are available under BioProject and BioSample accession numbers, PRJNA1140749 and SAMN42848706, respectively. Publicly available genomes used for the comparative genomic analysis were retrieved from the NCBI database (Supplementary Table 1).

3. Results

3.1. Susceptibility test and transferability of bla_NDM-1

The bla_NDM-1-positive Klebsiella sp. strain M42D exhibited resistance to cefotaxime, ceftazidime (MIC ≥ 256 μg mL⁻¹each), piperacillin/tazobactam (MIC ≥ 256/4 μg mL⁻¹), ceftazidime/avibactam (MIC ≥ 256/4 μg mL⁻¹), imipenem (MIC = 64 μg mL⁻¹), ertapenem (MIC = 32 μg mL⁻¹), meropenem (MIC = 16 μg mL⁻¹) and ciprofloxacin (MIC = 2 μg mL⁻¹). However, the strain remained susceptible to amikacin, levofloxacin, and colistin (Table 1).

Table 1.

Antibiotic susceptibility and predicted antibiotic resistance genes responsible for resistance of Klebsiella sp. strain M42D

Antibiotics	MIC (μg mL−1)a	Putative ARGs and their locations on M42D genome
IPM	64	pKquM42Da: blaNDM-1
MEM	16
ETP	32
TZP	≥ 256/4	pKquM42Db: blaTEM-1
CAZ	> 256	chromosome: blaOKP-B-17 pKquM42Db: blaCTX-M-15
CTX	> 256
CZA	≥ 256/4
AMK	2	pKquM42Db: aac(6′)-Ib-cr and aadA16
CIP	2	pKquM42Db: qnrB6
LVX	1
CST	2	None
Other drugsb	Not determined	Chromosome: fosA pKquM42Db: mphA, arr3, sul1c, tetA and dfrA27

ARG, antibiotic resistance genes; IPM, imipenem; MEM, meropenem; ETP, ertapenem; TZP, piperacillin-tazobactam; CAZ, ceftazidime; CTX, cefotaxime; CZA, ceftazidime-avibactam; AMK, amikacin; CIP, ciprofloxacin; LVX, levofloxacin; CST, colistin.

^aBroth microdilution method was used to determine MICs for IPM, MEM, ETP, CAZ, CTX, CIP, LEV, and CST. MICs for TZP, CZA, and AMK were examined using MIC test strips (LiofilChem, Italy). The CLSI breakpoints for resistance to IPM, MEM, ETP, TZP, CAZ, CTX, CZA, AMK, CIP, LVX and CST are ≥4, ≥4, ≥2, ≥32/4, ≥16, ≥4, ≥16/4, ≥16, ≥1, ≥2 and ≥4 μg mL⁻¹, respectively [21]. MIC values highlighted in bold were interpreted as resistance.

^bResistance phenotype related to other drugs including fosfomycin, azithromycin, rifamycin, sulfonamide, tetracycline, and trimethoprim were not determined.

^cTwo copies of sul1 were found.

Conjugation experiments were performed using the bla_NDM-1-positive strain M42D as a donor. Within 24 h of mating, strain M42D successfully transferred bla_NDM-1 to recipient E. coli J53 cells, with a conjugation frequency of 3.1 × 10⁻³ transconjugants per donor cell. The E. coli J53 transconjugant carrying bla_NDM-1 showed 16- and 32-folds increase in carbapenem MICs compared with E. coli J53 (4 vs 0.25 μg mL⁻¹and 8 vs 0.25 μg mL⁻¹for imipenem and meropenem, respectively). PCR analysis confirmed that bla_NDM-1 was located on an IncN2 plasmid in the transconjugant.

3.2. Basic genome features, subsystem analysis and acquired virulence traits of M42D

The hybrid Illumina-PacBio assembly approach yielded the complete genome of strain M42D. The genome consisted of a circular chromosome (5.3 Mbp) with a GC content of 57.9% and three plasmids with sizes of 41.2 kbp, 121.6 kbp and 194.8 kbp (Table 2). The GC contents of these three plasmids were 50.8–52.7%, lower than that of their host chromosome. The average GC content of the strain M42D was 53.1%. The ANIb analysis revealed that M42D shared the highest similarity with K. quasipneumoniae subsp. similipneumoniae 07A044ᵀ, with an ANIb value of 98.8% (86.6% aligned sequence). Furthermore, the ANIb values for other K. quasipneumoniae type strains, including DSM 28211 and FDAARGOS 1503, exceeded 96%, confirming its classification within this species. In contrast, the ANIb value for K. pneumoniae DSM 30104 was 93.2%, below the 95% species threshold, indicating that strain M42D is distinct from K. pneumoniae.

Table 2.

General genome data, virulence determinants and conjugal transfer systems of the bla_NDM-1-positive K. quasipneumoniae subsp. similipneumoniae M42D

Characteristics	M42D chromosome	pKquM42Da	pKquM42Db	pKquM42Dc
General genome features
Size in bp	5,314,524	41,190	121,583	194,781
% GC Content	57.9	50.8	52.7	51.1
No. of CDS (coding sequence)	5,122	59	170	228
No. of tRNA	90	0	0	0
No. of rRNA	25	0	0	0
plasmid Inc types	–	IncN2	IncFII	IncFIB(K)
Virulence factor profiling
Type 1 fimbriae	fimABCDEFGHIK	–	–	–
Type 3 fimbriae	mrkABCDFHIJ	–	–	–
Efflux pump	acrAB	–	–	–
Ent siderophore	entABCDEFS and fepABCDG	–	–	–
Salmochelin	iroEN	–	–	–
RcsAB regulators	rcsAB	–	–	–
Sti (Salmonella)	stiB	–	–	–
Pla (Yersinia)	–	–	–	pla
Conjugal transfer systems
Origin site of DNA transfer (oriT)	–	oriT (11,481–11,563)	oriT (94,219–94,275)	–
Relaxase	–	ORF_22 (MobF family relaxase)	ORF_73 (conjugative transfer relaxase/helicase TraI)	–
Type IV coupling protein (T4CP)	–	ORF_41 (coupling protein TrwD)	ORF_74 (coupling protein TraD)	–
Type IV secretion system (T4SS)	–	trwDEFG, trwIJKML, and trwN	traX, traD, traGH, traBQF, traNCU, traW, traC, traVBKELA, traM and orf169	–

K. quasipneumoniae subsp. similipneumoniae (hereafter referred to as Kqps) strain M42D was classified as sequence type (ST) 334 with a KL103 capsular type. Genome annotation predicted 5,122, 59, 170, and 228 coding sequences (CDSs) on the chromosome, pKquM42Da, pKquM42Db and pKquM42Dc, respectively (Table 2). The single circular chromosome also contained 90 putative tRNA genes and 25 rRNA genes, while no tRNA or rRNA genes were detected on the plasmids.

The total number of predicted genes was 5,694 (Table 2), of which 2,589 CDSs were assigned putative functions and were categorized into 11 subsystem categories (Fig. 1). The top five most abundant subsystem categories were metabolism (1,111 CDSs), energy (393 CDSs), protein processing (250 CDSs), stress responses (including defense and virulence, 225 CDSs), and membrane transport (170 CDSs).

Fig. 1.

Genome annotations and subsystem analysis of strain M42D. Circular genetic maps depicting the chromosome and extrachromosomal DNA, including pKquM42Da, pKquM42Db and pKquM42Dc (A). Functional gene analysis of the M42D genome categorized into 11 subsystems (B)

Virulence gene profiling revealed that many genes involved in adherence (Type 1 fimbria, Type 3 fimbria and Salmonella Sti), efflux pump (AcrAB), iron uptake (Ent siderophore and Salmochelin), and regulation of biofilm development (RcsAB) were identified on the chromosome as illustrated in Table 2. Three major virulence factors responsible for bacterial adherence were identified and predicted to be encoded by two groups of genes: fimABCDEFGHIK and mrkABCDFHIJ, and Salmonella stiB. Virulence determinants contributing to iron uptake and its transport system, such as aerobactin (iroEN), Ent siderophores (entABCDEFS and fepABCDG) and biofilm formation (rcsAB) were found in the studied isolate. A putative functional gene encoding for Yersinia Pla protease was detected on the largest plasmid pKquM42Dc.

3.3. Phenotypic and genotypic antibiotic resistance of strain M42D

The presence of antibiotic resistance genes (ARGs) was analyzed based on putative functional gene annotations using the open online resource ResFinder, with results presented in Table 1. The acquired bla_NDM-1, conferring resistance to carbapenems, was located on the smallest plasmid (pKquM42Da; 41.2 kb). No other ARGs were identified on this plasmid. Plasmid pKquM42Db carried bla_TEM-1, contributing to resistance to piperacillin/tazobactam, a β-lactam/β-lactamase inhibitor combination. Putative genes located on the chromosome (bla_OKP-B-17) and plasmid pKquM42Db (bla_CTX-M-15) were found to confer resistance to cephalosporins (ceftazidime, cefotaxime and ceftazidime-avibactam). Besides, mutations in chromosomal porin genes, contributing to cephalosporin and carbapenem resistance, were identified. Strain M42D exhibited eight and two amino acid substitutions in Omp36 and Omp37, respectively, while no mutations were found in OmpK35 (Table 3).

Table 3.

Mutations in chromosomal-mediated antibiotic resistance in K. quasipneumoniae strain M42D

Chromosomal gene	Strain M42D	Resistance
ompK35	No known mutation	–
ompK36	N49S, L59V, L191Q, Q227N, L229V, N304E	cephalosporin
	A217S, N218H	carbapenem
ompK37	I70M, I128M	carbapenem
gyrA, parC	No known mutation	–

Mutations in gyrA and parC, which are responsible for quinolone resistance, were not detected (Table 3). However, plasmid pKquM42Db carried qnrB6, a putative resistance determinant, that is predicted to confer resistance to ciprofloxacin. Additional resistance genes identified included those encoding for resistance to aminoglycoside (aac(6′)-Ib-cr and aadA16), fosfomycin (fosA), azithromycin (mphA), rifamycin, sulfonamide (arr-3), tetracycline (tetA), and trimethoprim (dfrA27) (Table 1).

3.4. Genetic characterization of M42D plasmids

Genomic analysis revealed that strain M42D carried two conjugative plasmids, pKquM42Da (IncN2 type) and pKquM42Db (IncFII type) (Table 2). These two conjugative plasmids carried genetic determinants involved in the origin site of DNA transfer (oriT), relaxase, type IV coupling protein (T4CP), and type IV secretion system (T4SS). The IncFIB(K) plasmid, pKquM42Dc harboring pla, lacked genes associated with conjugative transfer.

The bla_NDM-1-carrying pKquM42Da was a compact IncN2 plasmid with a size of 41.2 kb and contained only 59 predicted CDSs. A BLASTn-based search of the bla_NDM-1-carrying pKquM42Da revealed that it shared identical antibiotic resistance and conjugal transfer regions with bla_NDM-1 plasmids from various Enterobacterales species, such as E. coli, K. pneumoniae, Enterobacter spp. and Salmonella enterica, isolated from difference countries, with 100% similarity and 99.9–100% coverage (Table 4).

Table 4.

Genomic and meta data of pKquM42Da-like plasmids carried by various Enterobacterales species

Strain ID	Isolation year	Isolation place	Isolation source	Heath status	Plasmid name	Plasmid size (bp)	Similarity/Coverage	BioSample or GenBank accession no.
K. quasipneumoniae subsp. similipneumoniae M42D	2015	Phitsanulok, Thailand	rectal swab	hospitalized patient	pKquM42Da	41,190	100/100	This study
E. coli C31	NR	Singapore	urine	diseased human	pNDM1	41,190	100/100	SAMN30011701
E. coli ECS01	2012	Bangkok, Thailand	urine	diseased human	pNDM-ECS01	41,190	100/100	SAMN14226796
E. coli JN24	NR	Shangdong, China	NR	diseased human	pJN24NDM1	41,190	100/100	NZ_MK368725.1
K. pneumoniae TR3	NR	Singapore	NR	diseased human	pTR3	41,187	100/99.99	SAMN14226170
K. pneumoniae KPH3	2020	Bangkok, Thailand	NR	diseased human	p3	41,190	100/99.99	SAMN30176386
Enterobacter cloacae NH77	2018	Chiang Mai, Thailand	NR	diseased human	pEcloNH77	41,179	100/99.97	SAMN11866226
Enterobacter. hormaechei 189	2020	Kunming, China	NR	diseased human	pECL189-4	41,439	100/100	SAMN13832016
Salmonella enterica subsp. enterica AMA003584	2020	Denmark	human feces	diseased human	pAMA003584_NDM-1	42,517	100/100	NZ_MZ004973.1

NR, not reported.

3.5. Analysis of carbapenemase gene distribution among of K. quasipneumoniae subsp. similipneumoniae genomes

An in silico analysis of 361 Kqps genomes, including M42D, obtained via the NCBI database was conducted to assess the distribution of bla_NDM-1 and other carbapenemase genes. Carbapenemase genes were identified in a polyclonal population of Kqps isolates from different years, sources and regions. Of the genome analyzed, 9.1% (33/361) carried a single carbapenemase gene (Fig. 2 and Supplementary Table 1). Among detected carbapenemase producers, the predominant genes were bla_NDM-5 (10/33), bla_KPC-2 (6/33) and bla_NDM-1 (6/33). Among bla_NDM-carrying Kqps strains, bla_NDM-5 was primarily observed in migratory birds, whereas bla_NDM-1 was associated with human isolates (Fig. 2C). Other carbapenemase genes, including bla_IMP, bla_VIM, bla_KPC and bla_OXA, were detected at low frequencies.

Fig. 2.

Sankey diagram illustrating an indicative view of distribution of carbapenemase genes in K. quasipneumoniae subsp. similipneumoniae obtained from various years (A), country regions (B), sources (C) and MLST types (D). A total of 360 genomes of K. quasipneumoniae subsp. similipneumoniae retrieved from the publicly available database NCBI and M42D genome sequenced in this work were analyzed. Genome details and meta data are presented in Supplementary Table 1. The diameter of the lines is proportional to the number of isolates that are also labelled. Lines are colored based on different carbapenemase genes and their variants

Carbapenem-resistance genes were identified in isolates from eight geographical regions over a 19-year period (2001–2019) (Fig. 2A). Among the studied regions, high diversity of carbapenemase genes was found in Southeast Asia (Fig. 2B). Isolates harboring carbapenemase genes were predominantly from humans, followed by migratory birds (Fig. 2C). MLST-based strain typing revealed a polyclonal isolate carrying a carbapenemase gene. Among the detected STs, ST334 appeared to be a major ST compared to other STs (Fig. 2D and Supplementary Table 1). However, only the ST334 genome sequenced in this study, strain M42D, was found to harbor bla_NDM-1.

3.6. Core genome single-nucleotide polymorphisms and phylogenetic analysis of K. quasipneumoniae subsp. similipneumoniae ST334 isolates

Pangenome analysis of M42D and its closely related ST334 genomes identified 4,484 core genes together with 3,998 accessory genes within a total pangenome of 8,482 genes. These results revealed a relatively large accessory compartment and supported an open genome structure. The identified core genes were further subjected to core genome single-nucleotide polymorphisms (cgSNP) and phylogenetic study. Fourteen other Kqps ST334 isolates were selected, based on results obtained from the genome distance estimation tool, for these analyses. These isolates were samples during 2009–2019 from Pakistan (n = 6), Bangladesh, China, Hong Kong, Japan, United Kingdom, Malawi, Brazil and Thailand (one isolate each) (Fig. 3). Analysis of cgSNPs among the M42D-related ST334 genomes separated these genomes into three major clades (clades I–III). Clade I consisted of a single isolate from wastewater treatment plant effluent in Hong Kong (SWHEFF_72). Clade II comprised two human isolates from Japan (UTH00079) and Brazil (Kqp64R), showing 321 SNPs.

Fig. 3.

Phylogenomic tree inferred from concatenated core-genes identified among a collection of M42D-like strains. An all-versus-all SNPs distances among analyzed strain are shown as matrix heatmap. The metadata and other characteristics are included in the right-side panel of the tree

Strain M42D belonged to clade III and differed from genomes in this dataset by 142–3,253 SNP positions based on all-versus-all cgSNP comparison (Fig. 3). Kqps ST334 isolates from Pakistan (HE031 & HE034 and HE148, HE194 & HE196) demonstrated high genetic similarities, with only 1 SNP. The rest of the clade III isolates, obtaining from geographically distant locations over several years, were genetically diverse, with pairwise SNP differences ranged from 116 to 198 SNPs. Despite belonging to the same ST334 lineage, strain M42D differed from closely related genomes in clade III by 142–186 SNPs within core genes (Fig. 3). Notably, only strain M42D acquired the bla_NDM-1-carrying plasmid.

4. Discussion

4.1. Species identification and sequence type

In this study, the bla_NDM-1-positive carbapenem-resistant Klebsiella sp. strain M42D from a human carrier was phenotypically and genotypically characterized. Strain M42D exhibited high resistance to imipenem, ertapenem, and meropenem but remained susceptible to colistin, an antibiotic used for the treatment of carbapenem-resistant Enterobacterales infections. Initially identified as K. pneumoniae [20], strain M42D was reclassified, through whole genome sequencing, as Kqps ST334, recognized as the most prevalent Kqps ST globally (Fig. 2D). This finding aligns with previous reports demonstrating that Kqps ST334 is frequently detected among patients in Asian countries, including Singapore [19], Pakistan [36], Cambodia [37], Hong Kong [10], Bangladesh (BioSample: SAMN22108552), China (BioSample: SAMN23224935) and Japan (BioSample: SAMN29728679). In Cambodia, ST334 accounted for 30.5% of Kqps isolates tested [37]. In Thailand, multiple Kqps isolates have been reported from humans, both patients and carriers, animals and water environments. Most isolates were grouped into multiple STs, but not into a ST334 [14, 38]. To the best of our knowledge, only one isolate of Kqps ST334 has previously been documented in Thailand. This isolate, designated as KP5682, was obtained from a human clinical specimen in Southern Thailand [11]. Identification of carbapenem-resistant Kqps ST334 strain M42D in the present study raises concern since this ST has been described as an emerging antibiotic-resistant clone [39].

4.2. Carbapenemase genes and plasmid replicon types

Resistance to a broad spectrum of antibiotics, including ampicillin, ceftazidime, meropenem, fluoroquinolone and colistin has been observed in numerous Kqps isolates, irrespective of their origins. Production of various carbapenemases, including KPC-2, IMP, NDM-1, NDM-5, OXA-48, OXA-181, has been documented in Kqps isolates from human clinical samples from different countries, such as Saudi Arabia, China, Singapore, Nigeria, and Brazil [7–9, 19, 40, 41]. In addition, NDM-5-producing Kqps isolates of animal and environmental origin have been reported [17, 18]. Similarly, antibiotic-resistant K. quasipneumoniae isolates from humans and animals have been documented in Thailand [12–14]. Interestingly, resistance to both carbapenem and colistin was observed in a K. quasipneumoniae isolate from a Thai patient [38]. Reports from 2015 to 2016 revealed the presence of Kqps isolates carrying carbapenemase genes in Thai patients, including two bla_OXA-232-carrying Kqps isolates (ST3590 and ST3595) and two bla_IMP-14-positive Kqps isolates (ST1770 and ST476) (Supplementary Table 1, [38]). With respect to bla_NDM, a globally disseminated carbapenemase gene, two bla_NDM-1-positive Kqps isolates from Thai patients were identified. Both isolates belonged to ST1318 (Supplementary Table 1). Previous reports revealed that carbapenemase-producing Kqps isolates belonging to ST334 were detected in human clinical specimens from the USA, Brazil, and Singapore, harboring bla_KPC-3, bla_KPC-2 and bla_NDM-5 + bla_OXA-181, respectively [19, 40, 42]. Notably, strain M42D represents the first documented Kqps ST334 isolate harboring bla_NDM-1.

Previous report demonstrated that, in Thailand, bla_NDM-1 can be found either on chromosome or plasmid [43]. The present study revealed that bla_NDM-1 in Kqps M42D resided on a compact plasmid, pKquM42Da, with a size of 41.2 kb, carrying all the necessary genes for conjugation (Table 2). This finding is consistent with the observed transferability of bla_NDM-1, as confirmed by the broth mating method. PlasmidFinder identified pKquM42Da as an IncN2 replicon type, confirming the IncN2 detection by PCR. Similar plasmid sizes together with nearly identical genomic regions were observed among pKquM42Da-like plasmids, suggesting highly conserved plasmid backbone structures (Table 4). These plasmids are present in various Enterobacterales species isolated from human-associated specimens, including urine, feces, and rectal swab samples, from Singapore (BioSample: SAMN30011701; [44]), China [45, 46], Denmark [47] and many regions in Thailand (BioSample: SAMN11866226; [22, 48]) during 2012–2020. Although IncN2 is not a common replicon type for bla_NDM-1 plasmids, these results align with prior evidence suggesting that IncN2 plasmid is a primary vehicle for bla_NDM-1 dissemination in Thailand [43].

4.3. Antibiotic resistance

In addition to bla_NDM-1, Kqps strain M42D carried various ARGs on either plasmid or chromosome, encoding resistance to different antibiotic classes. The core β-lactamase gene, bla_OKP, specific to Kqps [39], was identified on the chromosome. With the exception of bla_NDM-1, all acquired ARGs were located on a 121.6 kb IncFII plasmid, pKquM42Db, which includes several conjugation genes, suggesting potential transferability (Table 2).

In Klebsiella spp., it is well documented that the synergy between porin alteration, including OmpK35, OmpK36 and OmpK37, and β-lactamase production contributes to high-level resistance to β-lactam [49]. OmpK36 and OmpK37 of strain M42D exhibited multiple amino acid substitutions (Table 3), which have been shown to be associated with cephalosporin and carbapenem resistance [50, 51]. Therefore, expression of bla_CTX-M and bla_NDM combined with alterations in OmpK36 and OmpK37 may lead to the high-level resistance to cefotaxime, ceftazidime and carbapenems in strain M42D (Table 1).

Quinolone resistance in K. pneumoniae is primarily driven by chromosomal mutations in the quinolone resistance-determining regions of gyrA and parC [49]. In this study, no mutations within the gyrA and parC were observed (Table 3). However, strain M42D carried qnrB6, a plasmid-mediated quinolone resistance gene, which may contribute to the low-level resistance to ciprofloxacin (MIC = 2 μg mL⁻¹, Table 1). The presence of aac(6′)-Ib-cr and aadA16 on pKquM42Db was linked to aminoglycoside resistance, although the isolate remained susceptible to amikacin. These results suggested that aac(6′)-Ib-cr and aadA16 were either not expressed or expressed at very low levels. However, these ARGs may be activated under certain environmental conditions leading to the expression of resistant phenotypes [52].

4.4. Virulence determinants

Diseases caused by hypervirulent Klebsiella spp., especially carbapenemase-producing strains, pose a significant threat to human health owing to their high morbidity and mortality rates. Carbapenemase-producing Kqps strains bearing virulence genes or exhibiting hypervirulent phenotypes have been reported [19, 41]. However, the virulence gene profile of strain M42D lacked biomarkers of hypervirulence (iucA, iroB, peg-344, rmpA, and rmpA2) [53], indicating that it is not hypervirulent. Nevertheless, multiple genes involved in bacterial adherence, biofilm formation regulation, iron uptake together with transport systems (Table 2) were identified on the M42D chromosome. Moreover, strain M42D possessed pla (plasminogen activator protease), which acts as an adhesin and a protease that limits neutrophil-mediated bacterial killing [54]. These findings suggest that, although not hypervirulent, strain M42D is capable of causing diseases and escaping host defenses.

4.5. Distribution of carbapenemase genes

An in silico analysis of 361 Kqps genomes from the NCBI database, including the sequenced M42D genome, was performed. From 2001 to 2019, 9.1% of Kqps isolates, predominantly from humans, carried a single carbapenemase gene (bla_NDM, bla_KPC, bla_IMP, bla_VIM or bla_OXA) (Fig. 2). Among these, bla_NDM was frequently detected (ten bla_NDM-5 and six bla_NDM-1) in different genetic backgrounds (various STs). Of the bla_NDM-1-positive Kqps isolates, five were previously recovered in Brazil (n = 2, ST138 and ST1859), China (n = 1, ST1859) and Thailand (n = 2, ST1318) (Supplementary Table 1). This study reports the other bla_NDM-1-positive Kqps strain, M42D, recovered from a human carrier. Notably, carbapenemase-producing Kqps isolates were predominantly found in Asia (Southeast, Southern, Western, and Eastern regions), particularly Southeast Asia (Fig. 2B), suggesting widespread dissemination of these strains in the region.

4.6. Phylogenetic relationship of K. quasipneumoniae subsp. similipneumoniae ST334 isolates

Genomic comparison of M42D with 14 closely related Kqps ST334 isolates from globally distant locations over multiple years revealed clustering within clade III, which included isolates from China, Pakistan, Bangladesh, the United Kingdom, Malawi and Thailand (Fig. 3). Based on a 21 SNPs cut-off for the genomic cluster of K. pneumoniae by the EuSCAPE Working Group and the ESGEM Study Group [55], isolates from Pakistan showed 1 SNP suggesting that a clonal expansion had occurred, although these isolates were obtained from different patients over a period of time. Strain M42D differed from other ST334 isolates in clade III by 142–186 SNPs, suggesting the distinct lineages rather than descending from a common ancestor. These results also suggest the independent adaptation of the isolates to different geographical locations.

Strain M42D and KP5682, another Kqps isolate from a urine sample of a Thai patient [11], possessed an identical capsular type (KL103), and shared similar ARG profiles (aac(6′)-Ib-cr, aadA16, qnrB6, mphA, arr-3, sul1;sul1, tetA, dfrA27, bla_TEM-1D and bla_CTX-M-15). However, they were not clonally related, as determined by 178 SNPs. Strain M42D carried an additional bla_NDM-1 gene, suggesting that acquisition of the carbapenem-resistance determinants likely occurred after lineage diversification during adaptation to different ecological niches and anatomical colonization sites. The intestinal origin of strain M42D suggests that it may have acquired the bla_NDM-1 plasmid from other gut bacteria. This hypothesis is supported by the evidence of in vivo interspecies transfer of bla_NDM-1 among Enterobacterales species in the human gut [56].

In conclusion, this study reports the first bla_NDM-1-harboring Kqps ST334 isolate, a major Kqps clone worldwide and provides baseline data on carbapenem-resistant Kqps for public health authorities not only in Thailand but also in other countries. Given the potential of Kqps to cause severe and life-threatening infections, accurate laboratory identification, by whole genome sequencing, is crucial. Furthermore, comprehensive genome analysis can provide valuable insights into the antibiotic resistance mechanisms, pathogenicity and spread of Kqps, which remain poorly understood. Our results emphasize the need for further genomic surveillance, including samples from humans, animals and environment, to track the dissemination dynamics of Kqps, which may be useful for implementing effective control measures to limit its transmission within the community.

Supplementary material

eujmi-16-256-s001.xlsx ^{(47.9KB, xlsx)}

Funding Statement

Funding sources: This study was supported by the Health Systems Research Institute, Thailand (HSRI 65-106).