RETRACTED: Drug Resistance and Molecular Characteristics of Carbapenem-Resistant OXA-48-Producing Klebsiella pneumoniae Strains in Hainan, China

: Background: The emergence and global spread of carbapenem-resistant hypervirulent


Introduction
Klebsiella pneumoniae (KP) is a common cause of respiratory tract, intestinal tract, blood, and abdominal organ infections in the world [1].Upon acquisition of a plasmid that harbors a carbapenemase gene, it becomes carbapenem-resistant K. pneumoniae (CRKP), a superbug that causes untreatable or hard-to-treat infections [1,2].CRKP strains usually lead to nosocomial infections and make therapeutic options very limited, which are related to high mortality [2].The main molecular mechanism in CRKP is the bearing of carbapenemaseencoding plasmids, and KPC are the most common types of carbapenemases [2].Apart from antimicrobial resistance, another worrisome development is related to the evolution of hypervirulent KP (hvKP).hvKP strains, which is usually thought to be attributed to the carriage of a virulence plasmid that harbors two CPS regulator genes (rmpA and rmpA2) and a number of siderophore gene clusters [1,3].The emergence of hvKP has also become a global concern because it poses a significant constraint on therapeutic strategies in clinics.
The study [4][5][6] revealed that the virulence traits of these strains have been related to the carriage of a typical virulence plasmid including multiple virulence-encoding genes (e.g., rmpA, regulator of the mucoid phenotype A) or specific KP capsular polysaccharide types (K1, K2, etc.).Studies have shown that virulence and carbapenem-resistance evolved separately in two distinct KP clonal groups [5,6].However, convergence of these two evolutionary paths has led to the emergence of KP strains that are hypervirulent and carbapenem-resistant at the same time; therefore, these strains are called carbapenemresistant and hypervirulent KP (CR-hvKP) [6,7].CR-hvKP is considered a real superbug that exhibits high transmissibility, hyper-resistance, and hypervirulence, posing a grave threat to human health.These CR-hvKP strains can be generated by at least three different evolution pathways.Firstly, CR-hvKP strains can arise from the transfer of a pLVPKlike virulence plasmid into a classic CRKP strain [5,6].Secondly, the transfer of a hybrid carbapenemase encoding and virulence plasmid in a KP strain can result in the emergence of CR-hvKP strains [8][9][10].Finally, CR-hvKP strains can acquire carbapenemase-encoding plasmids [6,7].In existing research reports, most CR-hvKP strains harboring the bla KPC-2 plasmid were identified in China [5][6][7].However, CR-hvKP strains bearing bla OXA-48 have never been documented in Hainan, China [11,12].In this research, we aimed to determine the clinical, drug-resistant, microbiological, and genomic characteristics of OXA-48-bearing CR-hvKP strains in the First Affiliated Hospital of Hainan Medical University, China.

Data Collection and Strains Identification
We collected OXA-48-producing KP strains from clinical samples (sputum, urine, blood, bile, pleural fluid, etc.) in the microbiological laboratories at the First Affiliated Hospital of Hainan Medical University from January 2022 to March 2023.The experimental study was approved by the institutional ethics board of the First Affiliated Hospital of Hainan Medical University (HYLL-2023-033).All experimental protocols were performed in accordance with the relevant ethical guidelines and regulations.Only the first culture was included for patients who had more than one culture that tested positive for OXA-48-producing strains of CR-hvKP to avoid duplication.Bacterial identification was determined using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (bioMérieux SA, Marcy l'Etoile, France).

Susceptibility Testing of Agents
All CRKP strains were cultured on Columbia blood agar (Oxoid, Hampshire, UK) containing 5% sheep blood (Luqiao, Beijing, China).Strain identity was confirmed by MALDI-TOF mass spectrometry (BrukerDaltonik GmbH, Bremen, Germany).Antimicrobial susceptibility was identified by the Vitek2 System, with the exception of tigecycline and colistin.The minimum inhibitory concentration (MIC) of tigecycline, ertapenem, and imipenem was determined by Etest, and colistin was determined by the broth microdilution method.The European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints were used for tigecycline and colistin susceptibility (http://www.eucast.org/clinical_breakpoints,accessed on 1 January 2020).All the tests were performed and interpreted as per Clinical and Laboratory Standard Institute (CLSI) 2020 guidelines [13].

Bacterial Conjugation
Plasmid conjugation was performed using the OXA-48-producing CR-hvKP strain as the donor strain (ertapenem-resistant) and Escherichia coli J53 (sodium azide-resistant) as the recipient strain.Standard bacterial strains used in the conjugation experiment were from urine and blood, respectively.The experiment was conducted using a previously described filter mating method [9].About 1 × 10 8 colony-forming units (cfu) of both the recipient strain and the donor strain were mixed and dotted on a sterilized filter, which was then incubated on a luria Bertani (LB) agar plate for 18 h at 37 • C. Transconjugants were selected using LB agar plates supplemented with 100 mg/L sodium azide and 0.125 mg/L ertapenem.Polymerase chain reaction (PCR) for rmpA/rmpA2 genes and bla OXA-48 and the MICs for carbapenem were performed on the transconjugants.

Stability of bla OXA-48 -Encoding Plasmid in CR-hvKP Strains
Bacterial cultures were grown at 37 • C in a shaking incubator at 200 revolutions per minute (RPM) and daily passaged by a 1:1000 dilution in LB broth without antibiotics for 28 days.From the plated culture, 100 colonies were randomly chosen and replica-plated onto LB agar plates, one containing ertapenem and one without antibiotics.Colonies growing on the antibiotic-free LB agar plate but not on the ertapenem-containing plate were then tested for the presence of bla OXA-48 using PCR.

Virulence Assessment
All animal care procedures and protocols were approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Hainan Medical University.Female C57BL/6 mice aged 6-8 weeks were given intraperitoneal injections of KP strains at various concentrations, according to previous studies [9,[14][15][16].Survival was determined by Kaplan-Meier analysis with a log-rank test using the Prism software package (GraphPad, Prism 9.0).The OXA-48-producing CR-hvKP strains and control strains were estimated in a mouse lethality study to calculate their 50% lethal dose (LD50).

Statistical Analysis
All the statistical analysis was conducted using MedCalc online software (https:// www.medcalc.org/calc/odds_ratio.php (accessed on 30 November 2023)) and GraphPad Prism Version 9 (San Diego, CA, USA: GraphPad Software; www.graphpad.com).Statistical significance for parametric and non-parametric variables between the two carbapenemase genes was tested by chi-square and Fisher's exact tests, and p-values < 0.05 were considered significant.Ninety-five percent confidence intervals (CIs) were calculated for various outcomes among the isolates producing the two genes to know the possible association between the presence of a gene and an outcome.

Bacterial Conjugation Experiment
A conjugation experiment was conducted to assess the transconjugation ability of the OXA-48-carrying plasmid.The results in Table 2 indicated that the carbapenem resistance phenotype of the three OXA-48-producing hvKP strains could be transferred to E. coli J53.The transconjugants all carried the bla OXA-48 gene, but the virulent determinant ituA or rmpA/rmpA2 were not detected in these transconjugants, suggesting the virulence plasmids were not co-transferred along with the OXA-48 plasmids.

Plasmid Stability
The three OXA-48-producing hvKP strains were passed daily for 28 days (~280 generations) to assess the stability of bla OXA-48 -bearing plasmids in CR-hvKP strains.At day 28, bla OXA-48 was not detected in these mutants by PCR; however, two plasmid-cured mutants of KP2185 were identified, and other β-lactamases and virulence genes such as rmpA and rmpA2 were retained.The MICs of imipenem and ertapenem decreased from 2 and 12 mg/L to 0.125 and 2 mg/L, respectively.Plasmid-cured KP2683-1 and NCRE-61 strains were not obtained in this experiment, suggesting that the bla OXA-48 -bearing plasmid is highly stable in CR-hvKP strains.

Antibiotic Sensitivity of OXA-48-Producing hvKP Strains
Table 3 presents the detailed MICs for the three OXA-48-producing hvKP strains.These strains exhibited varying antimicrobial resistance profiles.NCRE61 and KP2185 were resistant to the above-mentioned aminoglycosides, fluoroquinolones, and cephalosporins.Specifically, KP2683-1 was susceptible to almost all antibiotics tested, including aminoglycosides (amikacin and gentamycin), fluoroquinolones (ciprofloxacin and levofloxacin), cephalosporins (cefepime, ceftriaxone, cefuroxime, cefazolin, and ceftazidime), colistin, and tigecycline, with the exception of imipenem and ertapenem.The three strains had similar MICs against imipenem (2-3 mg/L) and were all susceptible to ceftazidime/avibactam.a The values were MIC-correlates determined by the Vitek2 System, except for ertapenem, imipenem, and tigecycline, determined by the E test, and colistin, determined by broth microdilution.
We conducted a core genome analysis and compared the two ST11 strains with an additional five non-hypervirulent ST11 KL64 strains collected from our hospital to increase resolution.The results showed that KP-2185 and NCRE-61 had 26 different core SNPs but differed from the other five ST11 strains by 78-81 core SNPs.The open reading frames were depicted as blocks.Black, yellow, red, green, and blue blocks represent replication genes, mobile elements, antimicrobial resistance genes, plasmid transfer genes, and plasmid backbone genes, respectively.Genes in the forward direction were located above the central line, while genes in the reverse direction were located below.The turkey red shading indicates regions of shared homology in the same direction among the different elements, while the blue shading signifies inverted shared homology.KP2683.The open reading frames were depicted as blocks.Black, yellow, red, orange, and blue blocks represent replication genes, mobile elements, heavy metal resistance genes, virulence genes, and plasmid backbone genes, respectively.The shading represents regions of shared homology among the different elements.Genes in the forward direction were located above the central line, while genes in the reverse direction were located below.

Discussion
K. pneumoniae, as a vital human pathogen, represents increasing multidrug-resistance, particularly to third-generation cephalosporins and carbapenems [11].Taking into account the mortality and morbidity of patients, medical burden, global drug resistance trends, and other relevant standards, CRKP was undoubtedly considered a key priority pathogen in the priority antibiotic-resistant bacteria list by the World Health Organization (WHO) in 2017 [3,4].Carbapenem resistance is primarily mediated by the production of carbapenemases (carbapenem hydrolyzing-lactamases), which have been found in KP isolates to fall into three categories: (1) metallo-lactamases or molecular class B lactamases (New Delhi metallo-lactamase/NDM-1, IMP, VIM) that hydrolyze all lactams except monobactams and are inhibited by ethylenediamine tetraacetic acid (EDTA) but not clavulanic acid; (2) serine-lactamases of molecular class A (NMC/IMI, SME, KPC, and GES) that hydrolyze even monobactams are inhibited by clavulanic acid and tazobactam but not by EDTA; (3) molecular class D serine β-lactamases (oxacillinases β-lactamases/OXA-48) that do not hydrolyze monobactams and are poorly inhibited by clavulanic acid and EDTA [4,5].These properties are used for differentiation in the laboratory.Most of these carbapenemases are acquired either by mutation or by horizontal gene transfer.
The recent emergence of hvKP strains has drawn global attention as they can cause severe infections that are challenging to treat with current antibiotics.Most CR-hvKP strains reported to date involve KPC or NDM-producing strains [5,6].OXA-48-producing hvKP strains have been reported in Europe, but the in vivo virulence of these strains has not been evaluated [12,22,24,25].
We discovered three CR-hvKP strains among 42 clinical OXA-48-producing KP strains.It should be noted that the rmpA2 gene in these plasmids is often structurally different from the WT rmpA2 gene by harboring various mutations or insertions/deletions; likewise, the rmpA gene in some plasmids also possessed mutations.Our findings revealed two evolutionary pathways for CR-hvKP.Firstly, the Prototypical ST23 hvKP strain KP2683-1 acquired an IncL/M type plasmid containing the OXA-48 gene; secondly, the ST11 CRKP strains KP2185 and NCRE61 acquired an IncFIB virulence plasmid The open reading frames were depicted as blocks.Black, yellow, red, orange, and blue blocks represent replication genes, mobile elements, heavy metal resistance genes, virulence genes, and plasmid backbone genes, respectively.The shading represents regions of shared homology among the different elements.Genes in the forward direction were located above the central line, while genes in the reverse direction were located below.
We conducted a core genome analysis and compared the two ST11 strains with an additional five non-hypervirulent ST11 KL64 strains collected from our hospital to increase resolution.The results showed that KP-2185 and NCRE-61 had 26 different core SNPs but differed from the other five ST11 strains by 78-81 core SNPs.

Discussion
K. pneumoniae, as a vital human pathogen, represents increasing multidrug-resistance, particularly to third-generation cephalosporins and carbapenems [11].Taking into account the mortality and morbidity of patients, medical burden, global drug resistance trends, and other relevant standards, CRKP was undoubtedly considered a key priority pathogen in the priority antibiotic-resistant bacteria list by the World Health Organization (WHO) in 2017 [3,4].Carbapenem resistance is primarily mediated by the production of carbapenemases (carbapenem hydrolyzing-lactamases), which have been found in KP isolates to fall into three categories: (1) metallo-lactamases or molecular class B lactamases (New Delhi metallo-lactamase/NDM-1, IMP, VIM) that hydrolyze all lactams except monobactams and are inhibited by ethylenediamine tetraacetic acid (EDTA) but not clavulanic acid; (2) serine-lactamases of molecular class A (NMC/IMI, SME, KPC, and GES) that hydrolyze even monobactams are inhibited by clavulanic acid and tazobactam but not by EDTA; (3) molecular class D serine β-lactamases (oxacillinases β-lactamases/OXA-48) that do not hydrolyze monobactams and are poorly inhibited by clavulanic acid and EDTA [4,5].These properties are used for differentiation in the laboratory.Most of these carbapenemases are acquired either by mutation or by horizontal gene transfer.
The recent emergence of hvKP strains has drawn global attention as they can cause severe infections that are challenging to treat with current antibiotics.Most CR-hvKP strains reported to date involve KPC or NDM-producing strains [5,6].OXA-48-producing hvKP strains have been reported in Europe, but the in vivo virulence of these strains has not been evaluated [12,22,24,25].
We discovered three CR-hvKP strains among 42 clinical OXA-48-producing KP strains.It should be noted that the rmpA2 gene in these plasmids is often structurally different from the WT rmpA2 gene by harboring various mutations or insertions/deletions; likewise, the rmpA gene in some plasmids also possessed mutations.Our findings revealed two evolutionary pathways for CR-hvKP.Firstly, the Prototypical ST23 hvKP strain KP2683-1 acquired an IncL/M type plasmid containing the OXA-48 gene; secondly, the ST11 CRKP strains KP2185 and NCRE61 acquired an IncFIB virulence plasmid carrying the genes rmpA2, iut, and iro gene clusters.In the above two evolutionary routes, the plasmids of the three CR-hvKP strains play a crucial role in the dissemination of resistance and virulence-associated genetic elements.
Our study first describes OXA-48-producing hvKP strains in the Hainan province of China and verifies their virulence through in vivo testing.The ST11 and capsular K64 strains (NCRE61 and KP2185) that acquired a virulence plasmid had an LD50 in the middle range compared to classic CRKP and KP2683-1, suggesting that the clonal backgrounds may have an impact on the virulence of CR-hvKP.The strain KP2683-1, with an ST23 and capsular K1 background, was resistant to carbapenems but retained susceptibility to most extended-generation cephalosporins, a profile consistent with the acquisition of the bla OXA-48 gene on an Inc.L plasmid.KP2683-1 also showed the highest virulence among the strains tested.Our study also found that the OXA-48-harboring plasmids are transmissible through conjugation, indicating that their transmissible nature will likely further contribute to the disease burden of CR-hvKP infection.
A recent study suggested that a hvKP ST23 strain was found to have acquired the OXA-181-bearing plasmid, but this strain lost the plasmid in subsequent subcultures without the selective pressure of carbapenem [26].It is widely accepted that antibiotic resistance often comes with a cost to the organism's fitness [27].The hypothesis is that hvKP strains may suffer a negative impact on fitness if they carry certain carbapenemase-encoding plasmids, and that these genes may be relatively unstable in the absence of selective pressure.
The stability of the OXA-48-harboring IncL-type plasmid in CR-hvKP strains has not been documented in previous literature.Interestingly, we were the first to discover that these bla OXA-48 -bearing plasmids were highly stable in CR-hvKP strains in our study, even without the selective pressure of carbapenem, which may be linked to the specific CR-hvKP strain and plasmid structure, posing an additional threat from this superbug.Our findings reveal that ceftazidime/avibactam, a novel β-lactam/β-lactamase inhibitor, can be effective against CR-hvKP, even when the strain is carbapenem-resistant.This is supported by previous studies indicating the efficacy of ceftazidime/avibactam against KPC-2-producing CRKP [28].Our results suggest that ceftazidime/avibactam may also be a viable option for treating OXA-48-producing hvKP.Our study emphasizes the need for new treatment options and highlights the difficulties in treating CR-hvKP strains.
Nevertheless, a few limitations exist in our study.First, to fully understand the virulence characteristics and strain backgrounds of these OXA-48-producing CR-hvKP strains, as well as their clinical impacts, a comprehensive molecular surveillance study on these strains is currently underway.This problem will be the focus of our future study.Second, this was a single-center study.We only collected strains from a tertiary hospital in China.Additionally, we did not perform the phenotypic disinfectant susceptibility testing of the CRKP strains in our study.Finally, despite only identifying three OXA-48-producing CR-hvKP strains, we were able to uncover two evolutionary routes in these strains.

Conclusions
In conclusion, we first reported three OXA-48-producing hvKP strains in Hainan Province, China, and demonstrated that bla OXA-48 -bearing plasmids are highly stable in two distinct clonal backgrounds.Our study highlighted the two evolutionary pathways of OXA-48-producing hvKP strains and confirmed their virulence through in vivo testing.The bla OXA-48 -encoding plasmid demonstrated high conjugation efficiency and stability in the CR-hvKP strains.Ceftazidime/avibactam may be a viable option for treating OXA-48-producing hvKP strains.Furthermore, our study emphasizes the need for further research on CR-hvKP strains worldwide to better comprehend the geographic variations and the relationship between virulence and resistance with the ongoing emergence of OXA-48-producing hvKP strains in Hainan Province, China.Informed Consent Statement: All authors have been personally and actively involved in the substantive work leading to the report and will hold themselves jointly and individually responsible for its content.Informed consent was obtained from all subjects involved in the study.

Figure 1 .
Figure 1.The plasmid structures of blaOXA-48 harbor plasmids.The open reading frames were depicted as blocks.Black, yellow, red, green, and blue blocks represent replication genes, mobile elements, antimicrobial resistance genes, plasmid transfer genes, and plasmid backbone genes, respectively.Genes in the forward direction were located above the central line, while genes in the reverse direction were located below.The turkey red shading indicates regions of shared homology in the same direction among the different elements, while the blue shading signifies inverted shared homology.

Figure 1 .
Figure 1.The plasmid structures of bla OXA-48 harbor plasmids.The open reading frames were depicted as blocks.Black, yellow, red, green, and blue blocks represent replication genes, mobile elements, antimicrobial resistance genes, plasmid transfer genes, and plasmid backbone genes, respectively.Genes in the forward direction were located above the central line, while genes in the reverse direction were located below.The turkey red shading indicates regions of shared homology in the same direction among the different elements, while the blue shading signifies inverted shared homology.

Figure 2 .
Figure 2. The plasmid structure of virulence plasmids from NCRE-61, KP2185, NTUH-K2044, and KP2683.The open reading frames were depicted as blocks.Black, yellow, red, orange, and blue blocks represent replication genes, mobile elements, heavy metal resistance genes, virulence genes, and plasmid backbone genes, respectively.The shading represents regions of shared homology among the different elements.Genes in the forward direction were located above the central line, while genes in the reverse direction were located below.

Figure 2 .
Figure 2. The plasmid structure of virulence plasmids from NCRE-61, KP2185, NTUH-K2044, and KP2683.The open reading frames were depicted as blocks.Black, yellow, red, orange, and blue blocks represent replication genes, mobile elements, heavy metal resistance genes, virulence genes, and plasmid backbone genes, respectively.The shading represents regions of shared homology among the different elements.Genes in the forward direction were located above the central line, while genes in the reverse direction were located below.

Author
Contributions: L.L., B.L. and M.Y. collected the epidemiological materials, clinical data, and samples.M.Y., B.L. and L.L. manipulated laboratory tests and statistical analysis of data.L.L. drafted the manuscript.X.Z. and Q.L. revised the final manuscript.All authors have read and agreed to the published version of the manuscript.Funding: The present study was supported by the National Natural Science Foundation of China (grant nos.82360003).The project is supported by the Education Department of Hainan Province, project number: Hnky2022ZD-11.The present study was also supported by the Hainan Provincial Natural Science Foundation of China, project number: 822MS176.The present study was supported by the Hainan Provincial Natural Science Foundation of China (grant nos.GHYF2022011, ZDKJ2021036, ZDYF2020223 and 820CXTD448) and the National Natural Science Foundation of China (grant nos.82160012, 82260001, 82311530776).The project is supported by the Hainan Province Clinical Medical Center.Institutional Review Board Statement:This study was approved by the institutional ethics board of the First Affiliated Hospital of Hainan Medical University (HYLL-2023-033), and informed consent was waived by the First Affiliated Hospital of Hainan Medical University.This study is reported in accordance with ARRIVE guidelines.