Analysis of the hypovirulent Klebsiella pneumoniae with the NDM-5 gene on IncN plasmids

ABSTRACT Our study aims to analyze the hypovirulent Klebsiella pneumoniae with the NDM-5 gene on IncN plasmids. This strain was isolated from a patient with a liver abscess, and the ST type was ST147. The characterization, antimicrobial drug resistance, and virulence of the strain with the NDM-5 gene on IncN plasmids were analyzed by whole-genome sequencing. Virulence testing was conducted using the Galleria mellonella infection model, serum killing, and biofilm formation assay. Growth measurement analysis, an in vitro competition experiment, and a plasmid stability assay were calculated using fitness costs. A conjugation assay was used to test NDM-5 transfer between bacterial strains. For statistical analysis and image visualization, GraphPad Prism 8.0 was employed. The result revealed that the hypovirulent strain with the NDM-5 gene was located on the IncN plasmid (92.284 kb), which is rare in Klebsiella pneumoniae. Whole-genome sequencing revealed the presence of 25 and 22 resistance and virulence genes, respectively. The strain KP4089 was resistant to carbapenems, β-lactam, ceftazidime-avibactam, and macrolides but remained susceptible to aminoglycosides, mucilage, and tigecycline in antimicrobial susceptibility tests. Furthermore, the strain KP4089 exhibited significantly lower serum killing and biofilm formation rates, demonstrating that the strain with the NDM-5 gene had low virulence. The strain with the NDM-5 gene on the IncN plasmid was genetically stable and had no fitness cost associated with identifying IncN plasmids. Future research should focus on clinical surveillance of hypovirulent multidrug-resistant Klebsiella pneumoniae to identify the potential strain and mechanistic studies to prevent its spread. IMPORTANCE It is crucial to strengthen the ongoing clinical surveillance of non-highly virulent, multi-resistant Klebsiella pneumoniae.

most common carbapenemases in clinically relevant Enterobacteriaceae strains.The ColE plasmid-located OXA-181 gene was initially discovered in India in 2007 (7).
Currently, clinical interest has been drawn to the New Delhi metallo-β-lactamase-5 due to its high treatment resistance and rapid transmission.Mei et al. (8) state that base replacements alter the sequence of the amino acids in NDM-5 and other antibi otics, conferring greater carbapenem resistance than other metallo-beta-lactamases.Ali et al. (9) demonstrated that NDM-5 has a higher hydrolytic activity than NDM-1, NDM-4, NDM-6, and NDM-7.The IncX3 plasmid (10,11) has been shown to diversify the dissemination of the blaNDM-5 gene to non-clonal E. coli hosts (ST12, ST167, ST354, ST361, ST410, ST617, ST746, ST6335, and ST6395) in China.However, it is inadequately understood that a strain with the NDM-5 gene is present in IncN plasmids.Consequently, we analyzed the strain with the NDM-5 gene on IncN plasmids, shedding light on its characteristics and fitness cost.

Isolation and antimicrobial susceptibility testing
Klebsiella pneumoniae (KP) was isolated from a patient with a liver abscess and then identified using a VITEK MS (Mérieux, France) automated rapid microbial mass spectrom etry detection system at a tertiary hospital in Jiangxi Province.
KP4089 and its transconjugant were tested for antimicrobial susceptibility using the K-B diffusion method (Table 1).The results were interpreted according to the Euro pean Committee on Antimicrobial Susceptibility Testing guidelines.CR-hvKP30457 and Escherichia coli ATCC25922 were used as quality control strains.

Plasmid analysis and conjugation assay
The plasmids of KP4089 were analyzed by S1-PFGE and southern blotting.In addition, a plasmid conjugation assay was performed between imipenem-resistant Klebsiella pneumoniae and rifampicin-resistant Escherichia coli EC600 to verify the transfer of plasmids containing resistance genes.Transconjugants (600 µg/mL rifampicin and 200 µg/mL imipenem) grown on the screening plate were transferred to a new screening plate, and successful plasmid transfer was verified by PCR and VITEK MS mass spectrome try.

Integrator analysis
The detection and identification of class 1, 2, and 3 integrons in the isolates were studied by amplifying the integrase genes intI1, intI2, and intI3 using the specific primers mentioned in Table S1.

Whole genome sequencing
The construction of a 10K SMRT Bell library was followed by the ligation, purification, and screening of DNA fragments using DNA adhesion enzymes, pure magnetic beads, and BluePipin fragments, respectively.Qualified DNA was divided into appropriate fragments using Covaris g-tubes.Additionally, AMPure PB magnetic beads were used to screen and purify the SMRT Bell library concentration.The size of the insert fragments was then quantified by measuring the constructed libraries with an Agilent 2100 bioanalyzer.HiSeq data were then used for sequencing, assembly, and further proofreading on the PacBio platform.

Comparative genomic analysis
Twenty-two strains of Klebsiella pneumoniae genomic sequences were analyzed by BLAST (https://blast.ncbi.nlm.nih.gov/).Whole-genome sequences for 21 strains were obtained from the NCBI website (https://www.ncbi.nlm.nih.gov/genome/).Twenty-one Klebsiella pneumoniae strains were included from different countries (Table 2).MEGE software was used to construct a maximum likelihood phylogenetic tree (Fig. 5).diluted bacterial solution and incubated, followed by applying the bacterial solution to the plates overnight.The bacterial survival rate was calculated by counting the number of colonies on both plates.

Biofilm assay
The biofilm formation assay was modified with reference to the method reported in the literature (12).The bacterial suspension was adjusted to 0.5 McFarland turbidity.This was followed by staining with crystalline violet, ethanol lysis, and transferring the ethanol lysate to a 96-well plate.The optical density (OD) values were recorded for each well, and the negative control values (Nc) were determined by the mean ± three times the standard deviation (x ¯ ± 3SD).The results were evaluated as follows: strongly positive (4 × Nc < OD); positive (2 × NC < OD ≤ 4 × Nc); weakly positive (Nc < OD ≤ 2 × Nc); and negative (OD ≤ Nc).

Galleria mellonella infection model
The Galleria mellonella infection model was used to assess strain virulence levels (8).A total of 10 larvae weighing around 250 mg (purchased from Tianjin Huiyude Biotechnol ogy Company, Tianjin, China) were used to test the virulence of each strain.Each was injected with 10 µl at a concentration of 1 × 10 6 CFU/mL injection, and the survival rate of the greater wax borer was recorded every 12 h for 3 days.All experiments were performed three times.Hypervirulent Klebsiella pneumoniae (HvKP) strains CRKP30457 and ATCC700603 were used as controls for hyper-and hypovirulence strains, respectively.

String test
Bacteria were inoculated onto sheep blood agar plates for overnight incubation and a string test was performed by stretching the colonies on the plates using an inoculation loop.The string test was positive if mucus filaments > 5 mm could be pulled out, which is highly suggestive of hvKP.

Growth measurement analysis
The CR-hvKP NUHL 30457, KP4089, the transformant, and EC600 were transferred to the Bioscreen C-Pro fully automated growth curve analyzer for continuous measurement of strain OD, as previously described (13).

In vitro competition assay
We adopted the method previously described by Chen et al. (14).The transformant and EC600 were inoculated into the LB broth and incubated for 24 h.The suspension was diluted, and 10 µL of the suspension was applied to a blank LB plate and incubated overnight.Every 24 h, 10 µL of the previous day's mixture was transferred to LB broth and incubated overnight for 4 days.Before each passaging of the strains, 10 μl of the diluted bacterial solution was taken and spread evenly on a blank LB plate; at the same time, the colonies on the blank plate were transferred to the plate containing imipenem (200 μg/ml), and the number of growing and non-growing colonies were counted separately.

Plasmid stability assay
The transconjugants were inoculated into LB broth and incubated for 21 days.Ten microliters of the diluted solution was evenly applied to a blank LB plate and incuba ted at 37°C.At the same time, the blank plate colonies were transferred to the mon oclonal antibody plate containing imipenem and incubated at 37°C.The number of grown and ungrown colonies was counted separately; plasmid stability = number of grown colonies/total number of colonies.The transformants were passaged under the screening plate of imipenem for 2 weeks.

Statistical analysis
GraphPad Prism 8.0 (windows) was used for statistical analysis and image visualization.P < 0.05 was considered to indicate a statistically significant difference.

Drug susceptibility testing
KP4089 exhibited resistance to carbapenems, β-lactam antibiotics, ceftazidime-avibac tam, macrolides, and aminoglutethimide; however, it remained susceptible to amino glycosides, mucilage, and tigecycline.In contrast, the transformant was resistant to many types of antibiotics, such as carbapenems, β-lactams, cephalosporins, macrolides, and aminoglutethimide.Nevertheless, the bacterium exhibited sensitivity to mucilage and tigecycline, mirroring the pre-transformant drug sensitivity.This finding provided additional evidence that the transfer of the resistant plasmid was likely to result in the development of multi-drug resistance, thus enhancing the bacteria's resistance to treatment, as depicted in Table 1.

Transferability of the blaNDM-5 gene
The conjugation assay result showed that the blaNDM-5 drug resistance gene and its resistance phenotype to imipenem were transferred to E. coli EC600, suggesting the blaNDM-5 resistance gene had a mobile genetic element.However, resistance genes such as OXA-181 and TEM-1 in the strain were non-transferable, suggesting that the transfer of resistance genes was influenced by multiple factors and was not absolute.This finding indirectly suggests that the presence of many plasmids in bacteria could be a potential source of transmission of hypervirulent resistant genes to other bacteria, which poses a risk to antibiotics commonly used to treat human infections.

Virulence assessment
The KP4089 survival rate after exposure to healthy human serum and inactivated serum was 1.37%.Control ATCC700603 and KP30457 survival rates were 28.8% and 1.88%, respectively.Statistical analysis showed that the two groups were statistically significant (P < 0.05) (Fig. 1A).Crystal violet staining exhibited that strain KP4089 had weaker biofilm formation than the reference group (Fig. 1B).Compared to the survival rate of KP30457, the KP4089 biofilm-forming capacity, serum killing level, and Galleria mellonella model were hypervirulent and close to that of ATCC700603 (Fig. 1C), but the string test was negative (Fig. 1D).This phenomenon indicates that the strain is a hypovirulent Klebsiella pneumoniae.
Whole-genome sequencing revealed that the strain with the NDM-5 gene was located on the IncN plasmid (92.284kb), which is rare in Klebsiella pneumoniae.Another OXA-181 gene was located on plasmid ColE (90.882 kb), which was highly homologous to the plasmid carrying the OXA-181 gene from Klebsiella pneumoniae (accession number: CP104800).This phenomenon suggested that horizontal transfer of the plasmid occurred between or within species.S1-PFGE and southern blotting analysis showed that the strains of NDM-5 contained three copies, each 812 bp in size, located on a plasmid 4.732 kb in size (Fig. 2).Whole-genome sequencing identified 25 resistance genes and 22 virulence genes.The strain of NDM-5 in ST147 typing contained several insertion elements, such as ISKpn26, IS52, IS5, and IS15, which contributed to the stability of the IncN plasmid.Pre-and post-junctional drug sensitivity results showed that the transformant had a resistance phenotype comparable to that of strain KP4089, and its growth was not significantly different from that of strain EC600 (Fig. 3A).The in vitro growth competition experiment showed that the relative fitness cost of the strain was 1.25, indicating the IncN plasmid did not impose a fitness cost on the host strain (Fig. 3C).

Comparative genomic analysis
The results of the evolutionary tree analysis showed that the 22 strains of Klebsiella pneumoniae were mainly located in three different branches (Fig. 4).Klebsiella pneumo niae strains that originated in the same country were more clustered in the second and third branches, while the first branch was more dispersed.Klebsiella pneumoniae strains originated in the United States were found in all three branches, but they were mainly clustered in the third branch.Klebsiella pneumoniae KP4089 was the only Klebsiella pneumoniae isolate among the 22 strains known to be isolated from patients with liver abscesses (Fig. 4).KP4089 was located in the first branch and formed a cluster with five other Klebsiella pneumoniae strains, thus inferring six strains that were more closely related.CP069206, LR890180, CP068607, AP021975, and CP040996 were the five strains that originated in India, Australia, Sweden, and America.KP4089 was located in the neighboring evolutionary branch, with a difference in isolation time (2019 and 2022), evolutionary branches, and a similarity identity of 99.8%, respectively.

FIG 3
The fitness cost of the strain 4089e.(A) Growth curve measurements of KP4089, 4089e, and Ec600.The growth of strain 4089e was not significantly different from that of strain EC600, while the growth capacity was significantly reduced compared to strain KP4089.(B) Plasmid stability for the strain 4089e in 0.5MIC-Imipenem and MIC-Imipenem medium.(C) The relative fitness of strain 4089e.The relative fitness cost of the strain was >1.structure contained the genes for a breakdown enzyme (mpkA), an efflux pump (oqxA6), and an ABC transporter protein (fepG).

DISCUSSION
KP is a prevalent pathogen that can infect the lungs, urinary tract, and genitalia (15).Carbapenem antibiotics are usually effective for treating KP infections.The prevalence of CRKP induced by NDM has been emerging, particularly for the NDM-5 gene, complicating clinical anti-infection treatment (16).Bacterial drug-resistant plasmids have been proven to mediate strain transmission and determine drug resistance both at home and abroad (17).The IncX3 plasmid (10,11) has been found to diversify the dissemination of the blaNDM-5 gene to non-clonal E. coli hosts (ST12, ST167, ST354, ST361, ST410, ST617, ST746, ST6335, and ST6395) in China.However, it is inadequately understood that a strain with the NDM-5 gene is present in IncN plasmids.Consequently, we analyzed the strain with the NDM-5 gene on IncN plasmids, shedding light on its characteristics and fitness cost.
The strain (KP4089) of the NDM-5 gene described in our study showed antimicrobial resistance to carbapenems, β-lactam, ceftazidime-avibactam, macrolides, and amino glutethimide but remained susceptible to aminoglycosides, mucilage, and tigecycline.Furthermore, the whole-genome sequencing revealed that the strain was located on a 92.284-kbIncN plasmid and contained the drug resistance genes (NDM-5, OXA-181, CTX-M-15, fosA6, and CatI) along with a class I integron.These results have similarities to the previous studies (18) demonstrating the presence of class I integrons in Klebsiella pneumoniae.However, additional studies are needed to explore the potential mecha nisms by which this bacterium is linked to multi-drug resistance.
The expression of various virulence genes in strain type KP4089 encompasses siderophore, bacteriophage hairs, and lipopolysaccharide (LPS).Previous research (19)(20)(21) has identified the genes ybtS, ybtX, and ybtQ as key determinants of external invasiveness.The bacteriophage function of this strain is facilitated by the yagV/ecpE gene, which also plays a role in promoting effective plasmid conjugation (22).The strain type KP4089 possesses certain virulence features that are associated with adhesion, such as the presence of the virulence genes yagW and ecpD.Virulence gene expression can be categorized into three classes: the iron uptake class (entA and entB), the invasive class (ompA), and the regulatory class (fepC, fepG).Formerly, infections caused by hvKP were commonly associated with the development of abscesses, specifically liver abscesses (23).We found that liver abscess patients' Klebsiella pneumoniae samples exhibited hypovirulence and numerous genes for carbapenemase resistance.This was due to the fact that they failed string tests, reduced biofilm formation, killed much serum, and possessed a plasmid with minimal virulence.As a result, the resistance genes could be shared between strains.In view of these findings, it is of utmost importance that clinicians understand that this potentially fatal condition may manifest itself even in the absence of an active hypervirulent Klebsiella pneumoniae infection.
The P4089-NDM5 plasmid is a mobile genetic element capable of disseminating genes associated with virulence and drug resistance.According to the results of our conjugation assay, the strains KP4089 and EC600 yielded a transconjugant with a drug resistance phenotype similar to strain KP4089.S1-PFGE confirmed that the transconju gant contained the NDM-5 plasmid, suggesting that the transconjugant was effective.In contrast, the transconjugation of the resistance genes OXA-181 and tetA failed, demon strating that the environment influences resistance plasmid transmission.This hints that NDM plasmids have a higher propensity for dissemination compared to other resistance plasmids.Notably, the growth pattern of the strain EC600 was comparable to that of the strain with inserted IncN plasmids, providing additional evidence supporting the notion that the presence of these plasmids did not impose any constraints on the adaptability of the strain.
Lastly, our in vitro growth competition experiments revealed that the acquisition of IncN plasmids had no effect on the host strain's fitness, confirming the strain's relative fitness cost of 1.25.Nevertheless, a treatment strategy is necessary due to the rapidity with which this could spread.In addition, we discovered that blaNDM-5 plasmids persisted for over 10 generations.This demonstrates that obtaining IncN plasmids enhances the ability of coliform bacteria to adapt to an environment with continuous transmission.The findings from our S1-PFGE and southern blot studies revealed the presence of three copies of P4089-NDM5.There are two probable reasons for this occurrence, namely the bacterium's endogenous regulatory mechanisms and the external selective pressures exerted by the environment.This indirectly suggests that the abundance of plasmids in bacteria may serve as a potential source for the dissemination of highly resistant genes to both other bacteria and humans, thus posing a threat to the efficacy of routinely employed antibiotics for infection treatment.

Conclusions
Whole-genome sequencing revealed that the NDM-5 gene strain was on the IncN plasmid (92.284kb), which is uncommon in Klebsiella pneumoniae.The drug resistance genes (NDM-5, OXA-181, CTX-M-15, fosA6, and CatI) were found in the strain KP4089, together with a class I integron.The relative fitness cost of the strain was greater than 1 in the in vitro growth competition experiment, indicating that the IncN plasmid did not impose a fitness cost on the host strain.To prevent the spread of hypovirulent multi drug-resistant KPs, continuing clinical surveillance of such strains must be increased, and future research should focus on determining the potential strain and mechanistic studies.

FIG 1
FIG 1 Analysis of virulence characteristics.(A) The serum resistance capability of KP4089.KP4089 has a weaker level of serum killing compared to hvKP30457 with ATCC700603.(B) Biofilm-forming ability of the strains is analyzed by the crystal violet method.(C) The Mann-Whitney test was used for statistical analyses.***P < 0.001.Virulence analysis of the strain KP4089 in a Galleria mellonella infection model.(D) The string test of KP4089 shows that the mucus filaments ＜ 5 mm.

FIG 2
FIG 2 PFGE, S1-PFGE, and southern hybridization analysis of KP4089.S1 nuclease digestion of plasmid bands is shown as linearized fragments on the gel.Southern blot hybridization of the marker gene (NDM-5) of the resistance plasmid, marked with a red arrow.Lane H9812, reference standard strain Salmonella serotype Braenderup H9812 restricted with Xbal.Note: the red box marks KP4089.

FIG 4 FIG 5
FIG 4 Maximum likelihood phylogeny based on the gene sequences of 22 strains of Klebsiella pneumoniae carrying the plasmid.

TABLE 1
Antibiotic resistance profile

TABLE 2
Comparative genomic analysis of the IncN plasmid of KP4089 with other IncN plasmids