From Turkey: First Report of KPC-3- and CTX-M-27-Producing Multidrug-Resistant Klebsiella pneumoniae ST147 Clone Carrying OmpK36 and Ompk37 Porin Mutations

Abstract

Aim:

The aim of this study was to identify antimicrobial resistance genes, virulence factor genes, and porin loss or mutations exhibited by the multidrug-resistant Klebsiella pneumoniae strain.

Materials and Methods:

Whole-genome sequencing was done via the Illumina NovaSeq 6000 platform. Strain identification and antibiotic susceptibility testing of strains were performed by the Vitek 2 automated system. Multilocus sequence typing analysis was carried out using seven conserved housekeeping genes.

Results:

The strain was resistant to penicillins, cephalosporins, carbapenems, aminoglycosides, fluoroquinolones, fosfomycin, and trimethoprim/sulfamethoxazole. The isolate was found to carry KPC-3, CTX-M-27, SHV-11, SHV-67, and TEM-1 β-lactamases. The clonal subtype of the isolate was ST147, and it possessed wzi64 and wzc38 alleles. Fifteen different point mutations (N49S, L59V, R146H, V178P, G189T, F198Y, V202L, F207Y, A217S, T222L, D223G, H235N, A280V, N304E, and S346N) were detected in the OmpK36 porin. A frame shift was observed in OmpK35 and two different point mutations (I70M and I128M) were found in the OmpK37 porin, in addition to seven mutations observed on the AcrR.

Conclusions:

This study demonstrated for the first time that the ST147 clone produced CTX-M-27 as well as KPC-3. In addition, new mutations were detected in the outer membrane proteins. These mutations together with the production of extended-spectrum β-lactamase and carbapenemase were found to contribute to the resistance of the ST147 clone to carbapenem and other antibiotics.

Introduction

Klebsiella pneumoniae is an opportunistic and nonmobile gram-negative rod bacterium that causes infections, especially in those who are immunocompromised.^1,2 K. pneumoniae is one of the bacterial pathogens with high-resistant clones responsible for nosocomial infections.² K. pneumoniae exhibits high resistance to various antibiotic groups, including β-lactam, fluoroquinolones, and aminoglycosides. This antibiotic-resistant feature has created a serious worldwide problem regarding the selection of effective antibiotics in the treatment of infections and consequently, is associated with high morbidity and mortality.¹ When treating infections, an increase in isolates of carbapenem-resistant K. pneumoniae, in particular, is a serious cause for concern.³ Therefore, carbapenem-resistant K. pneumoniae is included among the “priority/critical” pathogens in the list recently published by the World Health Organization.^3,4

β-Lactamase is classified into four Ambler classes: Class A (KPC, CTX-M, TEM, SHV, GES, IMI, NMC, and SME), Class B (metallo-β-lactamases NDM, VIM, IMP, GIM, SIM, and SPM), Class C (AmpC β-lactamase), and Class D (OXA-type β-lactamase).⁵ As one of the most important resistance mechanisms in clinical isolates, Klebsiella pneumoniae carbapenemase (KPC) was first identified in the United States in 1996 and then spread progressively throughout the world.⁶ The most commonly reported KPC-type enzymes in K. pneumoniae are KPC-2 and KPC-3.³ The CTX-M enzymes, which are Class A broad-spectrum β-lactamases, are rapidly proliferating among clinical isolates worldwide. In recent years, CTX-M enzymes have become the most common extended-spectrum β-lactamase (ESBL) enzymes found in clinical isolates. These CTX-Ms are divided into five main groups according to their amino acid sequences: CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9, and CTX-M-25.⁷

Porin loss is another resistance mechanism that enables the increase of carbapenem-resistant K. pneumoniae strains. OmpK35, OmpK36, and OmpK37 are three known porins in the outer membrane proteins of K. pneumoniae strains. The OmpK35 and OmpK36 porins play a critical role in the penetration of antibiotics into cells. Loss of OmpK35 or OmpK36 can counteract or reduce susceptibility to cephalosporins and carbapenems in K. pneumoniae strains.⁸ OmpK35, which increases resistance to ceftazidime, is not found in most clinical isolates of K. pneumoniae that are carrying ESBL. Loss of OmpK36 causes reduced susceptibility to cefotaxime, cefamycins, and carbapenems. Disruption of OmpK35 and OmpK36 by insertions or deletions to some isolates is associated with porin loss. Loss of OmpK35 or OmpK36 has been observed in some KPC-harboring isolates.⁹

More than 150 K. pneumoniae clonal groups (CGs) have been identified by whole-genome sequencing (WGS). Five commonly distributed CGs (CG258, CG14/15, CG17/20, CG43, and CG147) are known to be responsible for 72% of K. pneumoniae outbreaks.¹⁰ The aim of this study was to analyze the genome of the one K. pneumoniae clonal subtype ST147 to detect antimicrobial resistance markers, virulence factors, and porin loss or mutations.

Materials and Methods

Bacterial isolate

The K. pneumoniae strain was isolated from urine culture in 2017 from the Trabzon Fatih State Hospital in Turkey and evaluated for antibiotic susceptibility. AST-N327 (bioMérieux) card was used for antibiotics tested. The oligonucleotide primers of 27F (50-AGAGTTTGATCMTGGCTCAG-30) and 1492R (50-GGYTACCTTGTTACGACTT-30) were used to amplify 16S rRNA gene, and 16S rRNA sequencing was performed.¹¹ The sequence information of the 16S rRNA gene was analyzed by BLAST searches using the NCBI GenBank^® database. K. pneumoniae K8 was collected as part of a prior institutional review board (IRB)-approved study (IRB no. 2017/55).

Detection of antibiotic resistance genes

Polymerase chain reaction (PCR) tests were performed using primers of bla_VIM, bla_NDM, bla_KPC, and bla_OXA-48 genes.¹² The PCR mixture contained 5 μL of genomic DNA, 20 pM of each primer, 10 μL reaction buffer, 3 μL 25 mM MgCl₂, 200 mM of dNTPs, and 1.5 U of Taq DNA Polymerase (GeneON) in a final volume of 50 μL. All PCR results were analyzed on 1% agarose containing 0.5 μg/mL ethidium bromide and subsequently visualized under ultraviolet light.

DNA isolation

Bacterial genomic DNA was isolated using the Quick-DNA™ Fungal/Bacterial Miniprep Kit (Cat. No. D6005; Zymo Research) according to the manufacturer's instructions. Quality and quantity of the DNA were measured spectrophotometrically and fluorometrically.

Next-generation sequencing

The library for next-generation sequencing (NGS) was prepared using the Nextera XT DNA Library Preparation Kit and sequencing was performed via the Illumina NovaSeq 6000 platform as 2 × 150 paired-end bases reads. After sequencing, the quality of the raw reads was checked, and adapter sequences, contamination, and low-quality reads were removed. Putative genes in the consensus genome were extracted based on the reference genome annotation file using CLC genomics workbench tool. These genes were functionally annotated using OmicsBox software. Virulence factors were determined using the virulence factor database.

Multilocus sequence typing and capsular typing

Multilocus sequence typing (MLST) was performed using seven conserved housekeeping genes (gapA, infB, mdh, pgi, phoE, rpoB, and tonB). Capsular typing (wzi and wzc alleles) for K. pneumoniae strain was obtained by uploading NGS data to the Pasteur Institute website.

Accession numbers

Draft genome sequence data of the K. pneumoniae (K8) strain in this study have been submitted to the NCBI GenBank (Accession No. CP050819).

Results

Identification of the isolate was performed and the strain was determined to be K. pneumoniae. The antibiotic susceptibility test showed that it was resistant to penicillins (amoxicillin/clavulanic acid, and piperacillin/tazobactam), cephalosporins (cefuroxime, cefuroxime axetil, cefixime, ceftazidime, and ceftriaxone), carbapenems (ertapenem, imipenem, and meropenem), aminoglycosides (amikacin), fluoroquinolones (ciprofloxacin), fosfomycin, and trimethoprim/sulfamethoxazole (Table 1). The strain was identified as multidrug resistant (MDR). bla_VIM, bla_NDM, bla_KPC, and bla_OXA-48 genes were investigated by PCR, and it was determined that the isolate carried KPC.

Table 1.

Antibiotic Susceptibility Profile of Klebsiella pneumoniae

Antibiotics	MIC	R/S/I
Amoxicillin/clavulanic acid	≥32	R
Piperacillin/tazobactam	≥128	R
Cefuroxime	≥64	R
Cefuroxime axetil	≥64	R
Cefixime	≥4	R
Ceftazidime	≥64	R
Ceftriaxone	≥64	R
Ertapenem	≥8	R
Imipenem	≥16	R
Meropenem	≥16	R
Amikacin	≥64	R
Gentamicin	4	I
Ciprofloxacin	≥4	R
Fosfomycin	≥256	R
Trimethoprim/sulfamethoxazole	≥320	R

I, intermediate; MIC, minimum inhibitory concentration; R, resistant; S, sensitive.

According to WGS analyses, the strain was found to carry TEM-1, SHV-11, SHV-67, and CTX-M-27 β-lactamases and KPC-3 carbapenemase. The isolate also contained the aminoglycoside resistance genes aac(3)-IIb, aadA2, and aph(3′)-IIb. The MLST was performed for characterization of the clonal subtype of the strain. The clonal subtype of the isolate was found to be ST147. NGS data also were used to identify wzi and wzc variants. The ST147 isolate was determined to contain the capsular typing locus wzi-64 and wzc-38 alleles.

Virulence factor genes were evaluated via the VDBF. According to results, the virulence genes found in the strain included entA, entB, entF, entC, entD, entE, acrA, acrB, icmF, fimA, fimF, fimB, fimH, fimC, fimD, fimE, fimK, fimI, fimG, clpV/tssH, mrkC, vgrG/tssI, fepA, fepB, fepC, fepD, fepG, fyuA, ybtA, ybtE, ybtP, ybtS, ybtT, ybtU, ybtX, wbbM, wbbN, wbbO, tssF, vipA/tssB, vipB/tssC, vasE/tssK, yagX/ecpC, yagW/ecpD, yagY/ecpB, yagZ/ecpA, yagV/ecpE, gnd, ybdA, fes, glf, tssG, ugd, tle1, iroE, galF, wza, wzi, wzm, wzt, mrkA, mrkJ, mrkF, mrkH, mrkB, mrkD, mrkI, dotU/tssL, rcsB, rcsA, sciN/tssJ, hcp/tssD, tli1, impA/tssA, cpsACP, ompA, ykgK/ecpR, and KPHS_23120 (Table 2).

Table 2.

Klebsiella pneumoniae Virulence Factor Genes

Virulence factors	Related genes
Enterobactin	entA, entB, entF, entC, entD, entE, fepA, fepB, fepC, fepD, fepG, ybdA, fes
Salmochelin	iroE
Yersiniabactin	ybtA, ybtE, ybtP, ybtS, ybtT, ybtU, ybtX, fyuA
Efflux pump	acrA, acrB
Type 1 fimbriae	fimA, fimF, fimB, fimH, fimC, fimD, fimE, fimK, fimI, fimG
Serum resistance	wbbM, wbbN, wbbO, wzm, wzt, glf
Type VI secretion system	tssF, vipA/tssB, vipB/tssC, vasE/tssK, tssG, tle1, dotU/tssL, sciN/tssJ, hcp/tssD, tli1, impA/tssA, KPHS_23120, vgrG/tssI, clpV/tssH, icmf
Adherence	yagX/ecpC, yagW/ecpD, yagY/ecpB, yagZ/ecpA, yagV/ecpE, ykgK/ecpR, ompA
Type 3 fimbriae	mrkA, mrkJ, mrkF, mrkH, mrkB, mrkD mrkI, mrkC
Regulation of capsule synthesis	rcsB, rcsA
Immune evasion	cpsACP, galf, gnd, ugd, wza, wzi

Fifteen different point mutations were detected in the OmpK36 porin outer membrane. These mutations were identified as: N49S, L59V, R146H, V178P, G189T, F198Y, V202L, F207Y, A217S, T222L, D223G, H235N, A280V, N304E, and S346N. Two point mutations were found in the OmpK37 porin (I70M and I128M). In addition, a frame shift was observed in OmpK35. Seven different point mutations (P161R, G164A, F172S, R173G, L195V, F197I, and K201M) were detected on the AcrR, a repressor gene that directly controls the expression of the AcrAB efflux pump (Table 3). Furthermore, mobile genetic elements Ispa38, IS5, IS6, IS91, IS1182, and IS1661 were also found in K. pneumoniae.

Table 3.

Mutations Identified in Klebsiella pneumoniae ST147 Isolate

OmpK36	OmpK37	AcrR
N49S	I70M	P161R
L59V	I128M	G164A
R146H		F172S
V178P		R173G
G189T		L195V
F198Y		F197I
V202L		K201M
F207Y
A217S
T222L
D223G
H235N
A280V
N304E
S346N

Discussion

K. pneumoniae is among the most important pathogenic bacteria that cause many diseases such as urinary tract infections, bacteremia, and pneumonia as well as burn and wound infections and pyogenic liver abscesses. The pathogenicity of K. pneumoniae is caused by the presence of many virulence genes that encode virulence factors.¹³ Various virulence factors such as fimbriae, antifagocytic capsules, lipopolysaccharide, membrane carriers, and siderophores facilitate the survival of K. pneumoniae during infection. Its ability to form biofilms enables K. pneumoniae to protect itself from antibiotics and increases its persistence on medical device surfaces. Some of the virulence-related genes are also known to play a role in biofilm production.¹⁴

The relationship between the antibiotic resistance of K. pneumoniae and its biofilm formation ability has been demonstrated in many studies.¹⁴ To investigate the link between antibiotic resistance and biofilm formation, Hennequin et al. conducted a study showing that sub-MIC concentrations of cefotaxime caused increased biofilm formation.^14,15 One study also reported that MDR K. pneumoniae strains tend to form a richer biofilm than those of susceptible strains.¹⁶ In addition, a significant relationship was found between the biofilm-forming ability and ESBL production in K. pneumoniae strains isolated from sputum and urine.¹⁷ Aljanaby et al. found a positive relationship between the virulence genes present in K. pneumoniae and their antibiotic resistance ability.¹³ The present study showed that the K. pneumoniae isolate exhibited resistance against, amoxicillin/clavulanic acid, piperacillin/tazobactam, cefuroxime, cefuroxime axetil, cefixime, ceftazidime, ceftriaxone, ertapenem, imipenem, meropenem, amikacin, ciprofloxacin, and fosfomycin. Moreover, the WGS determined that the multidrug-resistant isolate carried virulence factor genes.

There are three basic mechanisms involved in the development of carbapenem resistance in K. pneumoniae isolates: carbapenemase production (KPC, OXA, and MBLs), expression or mutation of porin genes (OmpK35 and OmpK36), and upstream regulation of the efflux systems (AcrAB).^14,18,19

The spread of KPC-producing K. pneumoniae isolates worldwide is a serious health problem, which has been responsible for hospital outbreaks in the USA, Israel, and Greece, with mortality rates of about 35%.²⁰ The KPC-harboring K. pneumoniae isolates are resistant to all β-lactam, quinolone, and aminoglycoside antibiotics.²⁰ To date, 23 variants of the bla_KPC gene have been identified. The most common of these are KPC-2 in Colombia, Brazil, Argentina, Ecuador, and Venezuela, and KPC-3 in Europe.²¹ The bla_KPC gene has been found more frequently in the K. pneumoniae sequence type 258 clone (ST258) and this KPC-producing clone is the one most widely proliferating in various regions of the world.²¹ The KPC-2-producing K. pneumoniae ST-258 clone was identified for the first time in Turkey in 2014.²²

The KPC-2-producing K. pneumoniae ST147 is a globally successful clone and produces NDM, OXA-48, and CTX-M, as well as KPC. Many studies have shown that strain ST147 produces CTX-M-15, TEM1-b, and SHV-11, together with OXA-48.²³ In addition, the presence of CTX-M-27 ESBL in K. pneumoniae has been shown in previous studies.²⁴ The ST147 clone has been associated with VIMs and KPCs in Greece, with NDMs in Canada and the UK, and with VIMs in Scandinavia.²⁵ In a study conducted in Portugal, the ST147 clone was found to carry KPC-3 more frequently.²⁶ In a study conducted in Tunisia, the WGS analysis of K. pneumoniae ST147 strain was shown to carry NDM-1 and CTX-M-15 β-lactamase as well as virulence factor genes.²⁷ In 2020, it was determined by WGS analysis that ST147 strain has KPC-3 and GES-5 carbapenemase.²⁸ In another study conducted in 2020, it was shown by WGS characterization that five ST147 strains keep the bla_OXA-48 gene.²⁹ As a result of molecular characterization of two carbapenem-resistant ST147 strains, it has been shown that the strains carry bla_SHV-11, bla_CTX-M-15, bla_OXA-1, bla_NDM-7, and bla_TEM-1B.²

To the author's knowledge, the KPC-3-producing clone ST147 has been reported in this study for the first time in Turkey. In addition to producing KPC-3, this ST147 strain was found to produce CTX-M-27, SHV-11, SHV-67, and TEM-1. Therefore, the strain has a high resistance profile against antibiotics. Also, K. pneumoniae was found to carry the wzi64 gene, which is associated with ST147 and belongs to K-type 64. In several different studies, it was determined that the ST147 clone produced CTX-M-15, but this clone was not previously found to produce CTX-M-27. With this study, it was shown for the first time that the ST147 clone produced both CTX-M-27 and KPC-3.

There are three well-known pores in the K. pneumoniae outer membrane, OmpK35, OmpK36, and OmpK37. Since the loss of outer membrane proteins causes resistance to antibiotics, OmpK35 and OmpK36 play an important role in the transfer of antibiotics into the cell.³⁰ Alterations in OmpK35 and OmpK36 are among the contributing factors to the antibiotic resistance of ESBL-producing K. pneumoniae. It was also revealed that the ESBL or AmpC-type β-lactamase can play an important role in the carbapenem resistance of K. pneumoniae strains.³¹ Mutations or losses of OmpK35 and OmpK36 outer membrane porins have been shown to contribute to carbapenem resistance.³² Studies have reported up to 81% mortality caused by resistance from porins.^33–35 One study stated that resistance was associated with the OmpK35-OmpK37 deficiency linked to the OmpK36 variant in ceftazidime/avibactam-resistant KPC-producing K. pneumoniae strains.³⁵ A study conducted in 2019 confirmed that there has been a significant increase in MICs caused by mutations in the OmpK35 and OmpK36 porins in K. pneumoniae strains. The study also conclusively identified the primary role of OmpK36 in carbapenem resistance. In addition, they reported that a mutation that narrows the porin channel of OmpK36 (Gly134Asp135, OmpK36GD) created antibiotic resistance levels similar to those of the loss of this porin.³⁵ Recent studies have expressed similar implications for OmpK37, which has a narrower channel; however, information about these porins is limited. Antimicrobial resistance can only be indirectly inferred from expression levels in clinical isolates.³⁶ This study detected a frame shift mutation in the OmpK35 porin, mutations in 15 different amino acids in OmpK36, and a 2-point mutation in OmpK37. These mutations may be also related with resistance phenotype of strain.

Conclusions

This study found that the MDR K. pneumoniae ST147 isolate produced CTX-M-27, SHV, and TEM types of β-lactamase together with KPC-3 carbapenemase. In addition, new mutations were detected in the outer membrane proteins of this strain. To the author's knowledge, no study in the literature has previously demonstrated that the ST147 clone produces CTX-M-27. Therefore, this study reports for the first time that this K. pneumoniae clone may carry CTX-M-27 as well as KPC-3 carbapenemase.

Footnotes

Acknowledgments

The author wishes to thank the Trabzon Fatih State Hospital and Dr. Tuba Köse for their support.

Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

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