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Article

Detection of Delafloxacin Resistance Mechanisms in Multidrug-Resistant Klebsiella pneumoniae

by
András Kubicskó
1,
János Juhász
1,2,
Katalin Kamotsay
1,3,
Dora Szabo
1,4,5 and
Béla Kocsis
1,*
1
Institute of Medical Microbiology, Semmelweis University, 1089 Budapest, Hungary
2
Faculty of Information Technology and Bionics, Péter Pázmány Catholic University, 1083 Budapest, Hungary
3
Central Microbiology Laboratory, National Institute of Hematology and Infectious Disease, Central Hospital of Southern-Pest, 1097 Budapest, Hungary
4
HUN-REN-SU Human Microbiota Research Group, 1052 Budapest, Hungary
5
Department of Neurosurgery and Neurointervention, Semmelweis University, 1085 Budapest, Hungary
*
Author to whom correspondence should be addressed.
Antibiotics 2025, 14(1), 62; https://doi.org/10.3390/antibiotics14010062
Submission received: 29 November 2024 / Revised: 3 January 2025 / Accepted: 6 January 2025 / Published: 9 January 2025
(This article belongs to the Special Issue Antimicrobial Resistance Genes: Spread and Evolution)
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Abstract

Background: In this study, the mechanisms implicated in delafloxacin resistance in Klebsiella pneumoniae strains were investigated. Delafloxacin is a novel, broad-spectrum fluoroquinolone that has been approved for clinical application. Methods: In our study, 43 K. pneumoniae strains were assessed, antimicrobial susceptibility testing was performed via the broth microdilution method, and the minimum inhibitory concentration (MIC) values for ciprofloxacin, delafloxacin, levofloxacin, moxifloxacin, ceftazidime, cefotaxime, and imipenem were determined. Four delafloxacin-resistant K. pneumoniae strains were selected for whole-genome sequencing (WGS). Results: The MIC50 values for the 43 K. pneumoniae strains were as follows: ciprofloxacin 0.5 mg/L, levofloxacin 0.25 mg/L, moxifloxacin 0.5 mg/L, and delafloxacin 0.25 mg/L. All four selected delafloxacin-resistant K. pneumoniae strains showed extended-spectrum beta-lactamase production, and one strain exhibited carbapenem resistance. WGS enabled us to determine the sequence types (STs) of these strains, namely, ST307 (two strains), ST377, and ST147. Multiple mutations in quinolone-resistance-determining regions (QRDRs) were detected in all the delafloxacin-resistant K. pneumoniae strains; specifically, gyrA Ser83Ile and parC Ser80Ile were uniformly present in the strains of ST307 and ST147. However, in the ST377 strain, gyrA Ser83Tyr, Asp87Ala, and parC Ser80Ile, amino acid substitutions were detected. We also identified OqxAB and AcrAB efflux pumps in all delafloxacin-resistant K. pneumoniae strains. The association between beta-lactamase production and delafloxacin resistance was determined; specifically, CTX-M-15 production was detected in the ST147, ST307, and ST377 strains. Moreover, NDM-1 was detected in ST147. Conclusions: We conclude that multiple mutations in QRDRs, in combination with OqxAB and AcrAB efflux pumps, achieved delafloxacin resistance in K. pneumoniae. In our study, we report on NDM-1-producing K. pneumoniae ST147 in Hungary.

1. Introduction

Klebsiella pneumoniae is a member of the Enterobacteriaceae family; it is commonly found in the environment and can asymptomatically colonize the human nasopharynx and gastrointestinal tract [1]. However, K. pneumoniae is a major human pathogen, as it can cause a variety of diseases. Based on its pathogenic properties, K. pneumoniae is divided into classical and hypervirulent pathotypes. The classical K. pneumoniae pathotype is mainly acquired nosocomially among hospitalized patients and can cause bloodstream infections, ventilator-associated pneumonia, and catheter-associated urinary tract infections, among others. However, infections caused by hypervirulent K. pneumoniae show different clinical pictures, leading to severe community-acquired systemic and invasive infections and local infections (e.g., liver and extrahepatic abscesses) in otherwise healthy individuals [2,3,4,5,6,7,8].
Four major classes of virulence factors in K. pneumoniae have been characterized: capsule, including the production of a hypermucoviscosus capsule in hypervirulent K. pneumoniae strains; lipopolysaccharide (LPS); siderophores; and type 1 and 3 fimbriae [9]. The hypermucoviscous phenotype, in association with mucoviscosity-associated gene A (magA), is commonly seen in the K1, K2, K5, K20, K54, and K57 serotypes, with K1 and K2 being the most prevalent [5,10].
K. pneumoniae is included in the ESKAPEE pathogens (Enterococcus faecium, Staphylococcus aureus, K. pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species, and Escherichia coli), which are capable of causing a large number of difficult-to-treat infections. In addition, these pathogens all exhibit multidrug resistance, which enables them to survive and spread in different environments, such as in hospitals [11,12,13]. The antibiotic resistance of K. pneumoniae is of great concern worldwide, as different resistance mechanisms can be acquired, and multidrug-resistant (MDR) K. pneumoniae clones can develop and spread across the globe. The World Health Organization (WHO) has grouped both carbapenem-resistant and third-generation cefalosporin-resistant K. pneumoniae as top-priority pathogens in a critical group, indicating the urgent need for research into new antibiotics and the development of effective treatments for infections caused by these pathogens [14]. Resistance to beta-lactams in K. pneumoniae is defined through the production of different beta-lactamases. One of the major classification systems used for beta-lactamases is the Ambler molecular classification, which separates the enzymes into four classes (A, B, C, and D) based on their amino acid sequences. Ambler classes A, C, and D include serine beta-lactamases, while class B consists of metallo-beta-lactamases (MBLs) [15]. In the class A group, the TEM, SHV, and CTX-M types are the prominent beta-lactamases. Currently, CTX-M-15 is the most widespread extended-spectrum beta-lactamase (ESBL) among clinical isolates. Ambler class A also includes serine carbapenemases, e.g., KPC and SME. Ambler class C includes AmpC-type beta-lactamases, e.g., CMY and DHA. Class D, or oxacillin-hydrolyzing (OXA) beta-lactamases, includes a broad spectrum of enzymes with different activity profiles; namely, several OXA-type beta-lactamases hydrolyze cefalosporins, but OXA-48 exhibits carbapenemase activity [15]. The Ambler class B enzymes are MBLs, which are different from the previously mentioned beta-lactamases because, at their active sites, there is at least one catalytically functional divalent zinc atom. MBLs are capable of hydrolyzing most beta-lactams (e.g., cefalosporins and carbapenems) but not monobactams. The most important MBLs are IMP-, VIM-, and NDM-type beta-lactamases [15]. The acquisition of ESBLs and MBLs in K. pneumoniae can occur through the uptake of plasmids. These resistance plasmids can carry different resistance genes that encode ESBLs (e.g., blaTEM, blaSHV, and blaCTX-M-15) and carbapenemases (e.g., blaKPC, blaNDM, and blaVIM) simultaneously; furthermore, other resistance genes that confer resistance to other antibiotics, such as aminoglycosides, fluoroquinolones, and colistin, can be present on these plasmids [1,16]. Resistance plasmids can be taken up and passed on to other bacteria because these plasmids can be easily transferred among the Enterobacterales species. The acquistion of resistance plasmids plays an important role in the development of MDR bacteria [17]. The treatment options for infections caused by multidrug-resistant K. pneumoniae are often limited to certain antibiotics. The use of newly approved antibiotic agents such as delafloxacin can help reduce the selective pressure that has been caused by the widespread use of beta-lactams [18]. Delafloxacin is one of the fluoroquinolone agents that has been approved for clinical application in recent years [19,20,21,22]. Delafloxacin exhibits promising in vitro activity against a broad range of bacteria, including Gram-positive, Gram-negative, and anaerobe bacteria [23,24,25]. Delafloxacin exhibits enhanced bactericidal efficacy against Gram-positive and Gram-negative bacteria because delafloxacin targets both bacterial DNA gyrase and topoisomerase IV enzymes with equal affinity. Delafloxacin has an improved active site compared to that of earlier fluoroquinolones, which allows it to act with greater potency on the binding sites of target molecules. Delafloxacin also inhibits the ability of S. aureus to form a biofilm [26]. Delafloxacin has also been approved for the treatment of several infections, including acute bacterial skin and skin structure infections (ABSSSIs) and community-acquired bacterial pneumonia (CABP); additional indications for delafloxacin therapy include complicated urinary tract infections and intra-abdominal infections [22,27].
Fluoroquinolone resistance mechanisms are classified into two main groups. The first one is mutation in the quinolone-resistance-determining regions (QRDRs) of bacterial chromosomes, namely, the coding genes of gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE). Mutations in gyrA and parC tend to be more common than mutations of gyrB and parE [28]. AcrAB efflux pump has also been described as a chromosomally located fluoroquinolone resistance mechanism and is regulated by MarA, RamA, and SoxS determinants [29,30]. In addition to chromosomal mechanisms, plasmid-mediated quinolone resistance (PMQR) determinants have also been described; these confer reduced susceptibility to fluoroquinolones but enhance the development of chromosomal mutations, which lead to high-level fluoroquinolone resistance [31,32]. PMQRs include Qnr determinants, the aminoglycoside-acetyltransferase-(6′)-Ib-cr variant, and QepA and OqxAB efflux pumps. Qnr determinants are pentapeptide repeat proteins, which are capable of binding to gyrase and topoisomerase enzymes in order to protect them from fluoroquinolones. Qnr proteins are clustered into several groups based on their amino acid homology and include QnrA, QnrB, QnrC, QnrD, and QnrE. The aminoglycoside-acetyltransferase-(6′)-Ib-cr (aac(6′)-Ib-cr) variant leads to the enzymatic modification of certain fluoroquinolone agents (e.g., ciprofloxacin and norfloxacin); this reduces the antibacterial efficacy of these agents. QepA and OqxAB efflux pumps extrude fluoroquinolones, together with other bactericidal agents, from the bacterial cell [33,34,35,36,37,38].
This study aimed to investigate the antibacterial efficacy of delafloxacin against K. pneumoniae and analyze the mechanisms that confer resistance to delafloxacin in K. pneumoniae strains.

2. Results

2.1. Antimicrobial Susceptibility Testing

In total, 43 K. pneumoniae clinical isolates were included in our study. In addition, the minimum inhibitory concentration (MIC) values of fluoroquinolones and beta-lactams were determined. The distributions of the fluoroquinolone and beta-lactam MIC values are shown in Figure 1 and Figure 2, respectively. Among the strains included in this study, 21 were susceptible to ciprofloxacin, 26 were susceptible to levofloxacin, 21 were susceptible to moxifloxacin, and 23 were susceptible to delafloxacin. Altogether, 20 K. pneumoniae strains exhibited resistance to delafloxacin, and these strains were also resistant to the other fluoroquinolones tested.
In this collection of K. pneumoniae strains, an association between fluoroquinolone resistance and ESBL positivity was detected. Altogether, 47% of the K. pneumoniae strains were fluoroquinolone-resistant and showed an ESBL phenotype in this study. In this collection of strains, 16% of K. pneumoniae exhibited an ESBL phenotype but remained susceptible to fluoroquinolone. Two K. pneumoniae strains were resistant to imipenem in this study.
The MIC50 values of the 43 K. pneumoniae strains were as follows: ciprofloxacin 0.5 mg/L, levofloxacin 0.25 mg/L, moxifloxacin 0.5 mg/L, and delafloxacin 0.25 mg/L.
The MIC values were interpreted based on the EUCAST recommendations for ciprofloxacin, levofloxacin, and moxifloxacin. The MIC values of delafloxacin were interpreted based on the FDA recommendation [22].

2.2. Genome Sequencing

The whole-genome sequencing (WGS) analysis of four delafloxacin-resistant K. pneumoniae strains found that these strains belong to different sequence types (STs) according to the multi-locus sequence typing (MLST); these sequence types were ST307 (two strains), ST377, and ST147. Different beta-lactamase genes were detected in these strains; in ST307, these genes were blaSHV-28, blaOXA-1, blaTEM-1B, and blaCTX-M-15; in ST377, these genes were blaOXA-1, blaSHV-110, and blaCTX-M-15; and, in ST147, these genes were blaSHV-11, blaOXA-1, blaOXA-9, blaTEM-1A, blaCTX-M-15, and blaNDM-1 (Table 1).
The WGS analysis detected multiple QRDR mutations and PMQR determinants in delafloxacin-resistant K. pneumoniae strains. The following QRDR alteration was detected in the ST147 and ST307 strains: gyrA Ser83Ile. In contrast, double gyrA mutations were detected in ST377, namely, Ser83Tyr and Asp87Ala. In the case of the parC mutation, Ser80Ile was uniformly present in all delafloxacin-resistant K. pneumoniae strains of ST147, ST307, and ST377. Additionally, other mutations in ParC that may play a role in delafloxacin resistance were detected; specifically, a Pro402Ala amino acid substitution was present in all four delafloxacin-resistant K. pneumoniae strains. Another amino acid substitution in ParC Asn304Ser was present in the ST307 and ST147 strains.
Different PMQR determinants were detected in delafloxacin-resistant K. pneumoniae strains. The following determinants were detected: qnrS1 in the ST147, qnrB1 and aac(6′)-Ib-cr in both strains of ST307, and aac(6′)-Ib-cr in the ST377 strain. All tested K. pneumoniae strains had OqxAB and AcrAb/TolC efflux pumps (Table 2). According to an analysis of the plasmid-coded resistance genes of delafloxacin-resistant K. pneumoniae strains, the following resistance determinants were located on plasmids: qnrS1, qnrB1, aac(6′)-Ib-cr, blaOXA-1, blaOXA-9, blaTEM-1A, blaTEM-1B, blaCTX-M-15, and blaNDM-1. The WGS analysis detected virulence determinants in delafloxacin-resistant K. pneumoniae strains. These virulence determinants are shown in Table 3.
The genome structure of the NDM-1- and CTX-M-15-producing delafloxacin-resistant K. pneumoniae ST147 strain is shown in Figure 3. The whole-genome multi-locus sequence typing (wgMLST) data of four delafloxacin-resistant K. pneumoniae strains are analyzed in Figure 4.

3. Discussion

Antibiotic resistance poses a significant challenge worldwide; according to current estimations, 4.95 million patients die yearly due to antibiotic-resistant bacterial infections [39]. Multidrug-resistant K. pneumoniae is one of the major pathogens responsible for nosocomial infections; this is because it can cause various difficult-to-treat infections and a limited number of effective antibiotics are available for treatment. It is clear in clinical departments worldwide that new, effective antibiotics are needed. Therefore, in order to overcome the challenge of antibiotic resistance, new antibiotics are being introduced to treat such infections. Agents from different antibiotic groups are among the new antibiotics that have been approved for clinical use in recent years. Notably, beta-lactam and beta-lactamase inhibitor combinations (e.g., ceftazidime–avibactam, ceftolozane–tazobactam, and meropenem–vaborbactam), plazomicin from the aminoglycoside group, and new fluoroquinolones have also been approved for clinical application [40].
Delafloxacin is a new broad-spectrum fluoroquinolone agent that has a larger molecular surface compared to earlier fluoroquinolones, which enhances its potency. Delafloxacin has been approved for clinical application; however, the resistance of K. pneumoniae to delafloxacin has not yet been thoroughly investigated. In earlier studies, delafloxacin resistance was analyzed in Neisseria gonorrhoeae, S. aureus, and Helicobacter pylori [24,41,42,43]. In addition, we previously investigated the mechanisms implicated in delafloxacin resistance in high-risk clones of E. coli. In these earlier investigations, multiple QRDR mutations associated with PMQR determinants were detected in delafloxacin-resistant E. coli strains [44,45].
In our current study, we analyzed the resistance mechanisms of delafloxacin-resistant K. pneumoniae clinical isolates. Altogether, 43 K. pneumoniae isolates were included in this study and the distribution of the MIC values of fluoroquinolone was determined. The MIC50 values were 0.25 mg/L for delafloxacin, which is lower than that of ciprofloxacin and moxifloxacin (0.5 mg/L) but equal to levofloxacin (0.25 mg/L). However, considering that delafloxacin has a unique chemical structure compared to earlier fluoroquinolones, based on the MIC50 values, it can be assumed that delafloxacin has more potent effects on K. pneumoniae than ciprofloxacin or moxifloxacin.
Four delafloxacin-resistant K. pneumoniae strains were investigated via WGS in order to analyze their molecular mechanisms and genetic markers. Three out of the four strains belonged to internationally disseminated high-risk clones, namely, ST147 (one strain) and ST307 (two strains). However, one strain represented ST377, which has previously been reported to be a multidrug-resistant clone in certain countries [46,47,48].
K. pneumoniae ST147 clones are well-known international high-risk pathogens with MDR phenotypes that are associated with several carbapenemases; in some cases, the co-production of KPC-2, NDM-1, and OXA-48 has been established [49]. ST147 first appeared in Hungary in 2008 [50]; then, in the following years, it appeared in Italy, Greece, India, and North African countries [51,52]. ST147 strains were resistant to fluoroquinolones, with gyrA S83I and parC S80I QRDR mutations being associated with beta-lactamases such as VIMs, KPC-2, NDM-1, and OXAs. Nosocomial outbreaks of this clone were reported in several countries due to the appearance of acquired antimicrobial resistance determinants; these included SHV-36, CTX-M-15, KPCs, NDMs, VIMs, OXA-48, OXA-181, and OXA-204 [53,54].
K. pneumoniae ST307 is also an internationally disseminated high-risk clone. ST307 was reported in Hungary in 2019, with this strain found to be positive for CTX-M-15 and resistant to fluoroquinolone [55]. However, ST307 has disseminated worldwide, and it has been reported in Italy, Columbia, the United States of America, and South Africa [52]. ST307 has been found to exhibit different resistance mechanisms; in recent years, carbapenemases-producing strains of ST307 have been reported, namely, production of NDM-1, OXA-181, OXA-48, and KPC-2 has been detected [56,57,58,59,60].
In our study, an association between beta-lactamase genes and delafloxacin resistance was detected in all four K. pneumoniae strains via WGS. ST147 possessed blaNDM-1, blaSHV-11, blaOXA-1, blaOXA-9, blaTEM-1A, and blaCTX-M-15; both strains of ST307 possessed blaSHV-28, blaOXA-1, blaTEM-1B, and blaCTX-M-15; and ST377 possessed blaOXA-1, blaSHV-110, and blaCTX-M-15.
This study aimed to detect and analyze the genetic markers of delafloxacin resistance in K. pneumoniae strains. Among the fluoroquinolone resistance markers detected, all strains of internationally disseminated high-risk clones, namely, ST147 and ST307, uniformly presented gyrA Ser83Ile and parC Ser80Ile. However, ST377 possessed gyrA Ser83Tyr, Ser87Ala, and parC Ser80Ile. All delafloxacin-resistant K. pneumoniae strains carried oqxAB and acrAB efflux pumps.
An analysis of the chemical structure of the detected QRDR mutations revealed key insights into the development of a lower target-binding affinity of delafloxacin, which would ultimately lead to resistance. At position 83 in GyrA, a polar serine amino acid changes to an aromatic tyrosine molecule or to isoleucine; this indicates substitution to a hydrophobic amino acid. At position 87 in GyrA, the polar amino acid aspartate is substituted to alanine, which is a hydrophobic molecule. In ParC, the substitution of serine (polar) at position 80 to isoleucine (hydrophobic) increases the MIC values of delafloxacin (Figure 5).
Earlier studies found that a dissimilar fitness cost is observed among different clones of K. pneumoniae during the development of fluoroquinolone resistance [61]. Generally, the development of QRDR mutations results in an energy burden on strains of K. pneumoniae. However, high-risk internationally disseminated clones retain their fitness, which enables them to survive and disseminate. Specifically, double serine mutations in QRDRs were found to be a possible marker of major internationally disseminated clones that retain their fitness [62]. Interestingly, in our study, all delafloxacin-resistant K. pneumoniae strains harbored a double-serine mutation in QRDRs, which indicates that delafloxacin resistance can occur in major internationally disseminated K. pneumoniae clones.

4. Materials and Methods

4.1. Strains

In this study, 43 nonrepetitive K. pneumoniae strains were collected between September and December 2022 at South-Pest Central Hospital, National Institute of Hematology and Infectious Diseases, Budapest, Hungary. These K. pneumoniae strains were isolated from different clinical specimens, including blood culture and urine samples. All specimens were processed as part of routine laboratory procedures and the strains were selected based on inclusion criteria. The identification of isolates was performed using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI Biotyper, Bruker, Bremen, Germany). In this study, K. pneumoniae strains were included if they exhibited resistance to ciprofloxacin and/or resistance to third-generation cephalosporins or if their ESBL positivity had been confirmed via a double-disk synergy test.

4.2. Detection of the Minimum Inhibitory Concentration (MIC)

All K. pneumoniae strains were investigated via the broth microdilution method in order to determine the MIC values for delafloxacin, ciprofloxacin, levofloxacin, moxifloxacin, ceftazidime, cefotaxime, and imipenem. We performed antibiotic susceptibility testing using Muller–Hinton broth in 96-well microplates. The MIC results for ciprofloxacin, levofloxacin, moxifloxacin, ceftazidime, cefotaxime, and imipenem were interpreted according to the latest EUCAST protocol v14.0 (www.eucast.org (accessed on 1 January 2024)). However, the MIC values for delafloxacin were interpreted based on U.S. Food and Drug Administration (FDA) recommendations [22]. We used E. coli ATCC 25922 as a control strain in this study (https://www.atcc.org/products/25922).

4.3. Whole-Genome Sequencing (WGS)

In this study, WGS analysis was performed on four selected K. pneumoniae strains to detect resistance determinants. The selection criteria for WGS on K. pneumoniae strains were as follows: resistance to ciprofloxacin and delafloxacin, an ESBL phenotype, and the resistance of one K. pneumoniae strain to imipenem. The Illumina MiSeq platform was used to perform WGS at Eurofins BIOMI Kft (Gödöllő, Hungary). The genomic DNA of all four K. pneumoniae strains was extracted using the NucleoSpin Microbial DNA Mini kit (Macherey-Nagel, Düren, Germany). A qubit fluorometer was used to measure the isolated bacterial DNA, and then the quality of the DNA was checked via microcapillary electrophoresis (Tape Station 4150, Agilent, Waldbronn, Germany). The Illumina DNA Prep kit (San Diego, CA, USA) was used to prepare libraries. The Illumina Miseq system was used for sequencing, and the MiSeq Reagent Kit v2 was employed to generate 250 bp paired-end reads. SPAdes Genome assembler algorithm v3.15.3 was used for the genome assembly of K. pneumoniae strains. Antibiotic-resistant genes were detected in the assembled genomes using Bionumerics v8.1 software [44,45]. We also used the Comprehensive Antibiotic Resistance Database (CARD) and Resistance Gene Identifier program for the detection of resistance genes at https://card.mcmaster.ca/analyze/rgi (accessed on 10 September 2024).

4.4. Analysis of Genome Sequence

The assembled genomes of K. pneumoniae strains were analyzed using traditional seven-gene multi-locus sequence typing (MLST) [63]. Whole-genome MLST (wgMLST) was performed via Bionumerics v8.1 based on accessory schema with 19086 loci; this was complemented with the 634 core loci [64], 7 MLST loci [41], and 2 capsular typing loci (wzc [65] and wzi [66] sequencing) allele variants.
The genome characteristics of K. pneumoniae ST147 are shown in Figure 3. The plot was created using the Proksee server (https://proksee.ca/) (accessed on 10 September 2024) [67] based on the genome annotation generated by RAST (https://rast.nmpdr.org/rast.cgi) (accessed on 10 September 2024) [68,69] for K. pneumoniae, with default parameters.

5. Conclusions

Our study demonstrates that multiple mutations in QRDRs are present in delafloxacin-resistant K. pneumoniae, namely, gyrA Ser83Ile, parC Ser80Ile or gyrA Ser83Tyr, Ser87Ala, and parC Ser80Ile. It can be assumed that multiple specific amino acid substitutions mediate delafloxacin resistance; notably, at position 83 in GyrA, a polar serine is substituted with an apolar isoleucine or tyrosine; at position 87 in GyrA, an aspartate is substituted with alanine; and, at position 80 in ParC, a polar serine is substituted with an apolar isoleucine. We conclude that both gyrA and parC mutations in combination with OqxAB and AcrAB efflux pumps are needed to achieve delafloxacin resistance in K. pneumoniae. In our study, we report the detection of an NDM-1-producing K. pneumoniae ST147 strain in Hungary.

Author Contributions

A.K.: data curation, methodology, and writing—original draft, review, and editing; J.J.: software, visualization, and formal analysis; K.K.: data curation and methodology; D.S.: conceptualization, funding acquisition, and writing—original draft, review, and editing; B.K.: funding acquisition, validation, and writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by HUN-REN-SU, Human Microbiota Study Group No “0272”, and by the European Union’s Horizon 2020 research and innovation program (952491-AmReSu). B.K. was supported by the János Bolyai Scholarship (BO/00286/22/5) of the Hungarian Academy of Sciences.

Institutional Review Board Statement

This study was approved by Ethics Committee of Semmelweis University (SE RKEB: 218/2020). All procedures of this study were in accordance with the ethical standards of the Institutional National Research Committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.

Informed Consent Statement

Not applicable.

Data Availability Statement

Genomic data of four delafloxacin-resistant K. pneumoniae are submitted to NCBI Genbank at the following BioProject number: PRJNA1189473, and at the following accession numbers SRA: SAMN4497125 (Kpn 180); SAMN44971254 (Kpn 250/1); SAMN44971255 (Kpn 431); SAMN44971256 (Kpn 684).

Acknowledgments

We thank the sequencing service provided by Eurofins Biomi Kft, Gödöllő, Hungary.

Conflicts of Interest

The authors declare no conflicts of interest.

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Antibiotics 14 00062 g001
Figure 1. Distribution of MIC values for ciprofloxacin, levofloxacin, moxifloxacin, and delafloxacin among the 43 K. pneumoniae strains. Breakpoints are shown for ciprofloxacin (C), levofloxacin (L), moxifloxacin (M), and delafloxacin (D). EUCAST breakpoints were used for ciprofloxacin, levofloxacin, and moxifloxacin. In the case of delafloxacin, the FDA breakpoint was applied.
Figure 1. Distribution of MIC values for ciprofloxacin, levofloxacin, moxifloxacin, and delafloxacin among the 43 K. pneumoniae strains. Breakpoints are shown for ciprofloxacin (C), levofloxacin (L), moxifloxacin (M), and delafloxacin (D). EUCAST breakpoints were used for ciprofloxacin, levofloxacin, and moxifloxacin. In the case of delafloxacin, the FDA breakpoint was applied.
Antibiotics 14 00062 g001
Antibiotics 14 00062 g002
Figure 2. Distribution of MIC values for cefotaxime, ceftazidime, and imipenem among the 43 K. pneumoniae strains. Breakpoints are shown for cefotaxime (Ctx), ceftazidime (Caz), and imipenem (Imi). EUCAST breakpoints were used for cefotaxime, ceftazidime, and imipenem.
Figure 2. Distribution of MIC values for cefotaxime, ceftazidime, and imipenem among the 43 K. pneumoniae strains. Breakpoints are shown for cefotaxime (Ctx), ceftazidime (Caz), and imipenem (Imi). EUCAST breakpoints were used for cefotaxime, ceftazidime, and imipenem.
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Antibiotics 14 00062 g003
Figure 3. Genome characteristics of the NDM-1-producing K. pneumoniae ST147 strain. Major resistance genes are indicated in the figure. The circos plot shows the GC content skew in the inner circle histograms, the contigs (with grey arrows), and the positions of the detected resistance genes.
Figure 3. Genome characteristics of the NDM-1-producing K. pneumoniae ST147 strain. Major resistance genes are indicated in the figure. The circos plot shows the GC content skew in the inner circle histograms, the contigs (with grey arrows), and the positions of the detected resistance genes.
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Antibiotics 14 00062 g004
Figure 4. The wgMLST of four delafloxacin-resistant K. pneumoniae strains. Each circle indicates a K. pneumoniae strain. Lines between the circles denote differences in the allelic variant between the clones. Each clone is shown in a different color.
Figure 4. The wgMLST of four delafloxacin-resistant K. pneumoniae strains. Each circle indicates a K. pneumoniae strain. Lines between the circles denote differences in the allelic variant between the clones. Each clone is shown in a different color.
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Antibiotics 14 00062 g005aAntibiotics 14 00062 g005b
Figure 5. Role of amino acid substitutions in gyrase and topoisomerase enzymes in correlation with the action of delafloxacin. This figure illustrates the substitution of serine to isoleucine (a), the substitution of aspartate to alanine (b), and the substitution of serine to tyrosine (c) in correlation with the action of delafloxacin.
Figure 5. Role of amino acid substitutions in gyrase and topoisomerase enzymes in correlation with the action of delafloxacin. This figure illustrates the substitution of serine to isoleucine (a), the substitution of aspartate to alanine (b), and the substitution of serine to tyrosine (c) in correlation with the action of delafloxacin.
Antibiotics 14 00062 g005aAntibiotics 14 00062 g005b
Table 1. Genetic determinants of four delafloxacin-resistant K. pneumoniae strains. MLST: multi-locus sequence typing; PMQR: plasmid-mediated quinolone resistance determinants; beta-lactamases; MIC values of ciprofloxacin (Cip); levofloxacin (Lev); moxifloxacin (Mox); delafloxacin (Del); ceftazidime (Caz); cefotaxime (Ctx); and imipenem (Imi). All MIC values are shown in mg/L.
Table 1. Genetic determinants of four delafloxacin-resistant K. pneumoniae strains. MLST: multi-locus sequence typing; PMQR: plasmid-mediated quinolone resistance determinants; beta-lactamases; MIC values of ciprofloxacin (Cip); levofloxacin (Lev); moxifloxacin (Mox); delafloxacin (Del); ceftazidime (Caz); cefotaxime (Ctx); and imipenem (Imi). All MIC values are shown in mg/L.
MLSTPMQRBeta-LactamasesCipLevMoxDelCazCtxImi
K. pneumoniae 180ST147qnrS1,
oqxA, oqxB		blaNDM-1,
blaSHV-11,
blaOXA-1,
blaOXA-9,
blaTEM-1A,
blaCTX-M-15		12816128128128128128
K. pneumoniae 205/1ST307qnrB1,
aac(6′)-Ib-cr,
oqxA, oqxB		blaSHV-28,
blaOXA-1,
blaTEM-1B,
blaCTX-M-15		128832641281281
K. pneumoniae 431ST377aac(6′)-Ib-cr,
oqxA, oqxB		blaSHV-110,
blaOXA-1,
blaCTX-M-15		12883241281281
K. pneumoniae 684ST307qnrB1,
aac(6′)-Ib-cr,
oqxA, oqxB		blaSHV-28,
blaOXA-1,
blaTEM-1B,
blaCTX-M-15		128832321281280.5
Table 2. Four delafloxacin-resistant K. pneumoniae strains with fluoroquinolone resistance determinants. PMQR: plasmid-mediated quinolone resistance determinants, QRDR: quinolone-resistance-determining region, ST: sequence type.
Table 2. Four delafloxacin-resistant K. pneumoniae strains with fluoroquinolone resistance determinants. PMQR: plasmid-mediated quinolone resistance determinants, QRDR: quinolone-resistance-determining region, ST: sequence type.
STST377ST307ST307ST147
PMQRaac(6′)-Ib-cr,
oqxA, oqxB		qnrB1,
aac(6′)-Ib-cr,
oqxA, oqxB		qnrB1,
aac(6′)-Ib-cr, oqxA, oqxB		qnrS1,
oqxA, oqxB
QRDRgyrA: Ser83Tyr, Asp87Ala
parC:
Ser80Ile,
Pro402Ala		gyrA:
Ser83Ile
parC:
Ser80Ile,
Asn304Ser, Pro402Ala 		gyrA:
Ser83Ile
parC:
Ser80Ile,
Asn304Ser, Pro402Ala		gyrA:
Ser83Ile
parC:
Ser80Ile,
Asn304Ser, Pro402Ala
Efflux systemAcrAB/TolCAcrAB/TolCAcrAB/TolCAcrAB/TolC
delafloxacin MIC (mg/L)43264128
Table 3. Virulence determinants of four delafloxacin-resistant K. pneumoniae strains.
Table 3. Virulence determinants of four delafloxacin-resistant K. pneumoniae strains.
ST147ST307ST307ST377
Siderophores
fyu, iucB, ybtA,
ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX		 Siderophores
fyuA, irp1, irp2, ybtA, ybtE, ybtP, ybtQ, ybtS, ybtT, ybtU, ybtX
Type III fimbriae
mrkA, mrkC, mrkD, mrkF, mrkH, mrkI, mrkJ		Type III fimbriae
mrkA, mrkC, mrkD, mrkF, mrkH, mrkI, mrkJ		Type III fimbriae
mrkA, mrkC, mrkD, mrkF, mrkH, mrkI, mrkJ		Type III fimbriae
mrkA, mrkC, mrkD, mrkF, mrkH, mrkI
Mucus regulator
rmpA
rmpA2
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Kubicskó, A.; Juhász, J.; Kamotsay, K.; Szabo, D.; Kocsis, B. Detection of Delafloxacin Resistance Mechanisms in Multidrug-Resistant Klebsiella pneumoniae. Antibiotics 2025, 14, 62. https://doi.org/10.3390/antibiotics14010062

AMA Style

Kubicskó A, Juhász J, Kamotsay K, Szabo D, Kocsis B. Detection of Delafloxacin Resistance Mechanisms in Multidrug-Resistant Klebsiella pneumoniae. Antibiotics. 2025; 14(1):62. https://doi.org/10.3390/antibiotics14010062

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Kubicskó, András, János Juhász, Katalin Kamotsay, Dora Szabo, and Béla Kocsis. 2025. "Detection of Delafloxacin Resistance Mechanisms in Multidrug-Resistant Klebsiella pneumoniae" Antibiotics 14, no. 1: 62. https://doi.org/10.3390/antibiotics14010062

APA Style

Kubicskó, A., Juhász, J., Kamotsay, K., Szabo, D., & Kocsis, B. (2025). Detection of Delafloxacin Resistance Mechanisms in Multidrug-Resistant Klebsiella pneumoniae. Antibiotics, 14(1), 62. https://doi.org/10.3390/antibiotics14010062

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