Reduced virulence in tigecycline-resistant Klebsiella pneumoniae caused by overexpression of ompR and down-regulation of ompK35
Journal of Biomedical Science volume 30, Article number: 22 (2023)
The development of tigecycline resistance in hypervirulent Klebsiella pneumoniae strains has resulted in decreased virulence that is associated with reduced production of capsular polysaccharides (CPS). In this study, we investigated the mechanisms that link tigecycline susceptibility to decreased virulence.
We compared transcriptomes from tigecycline-susceptible wild-type strains and tigecycline-resistant mutants using mRNA sequencing. ompR-overexpressed and ompR-deleted mutants were constructed from wild-type strains and tigecycline-resistant mutants, respectively. Antibiotic susceptibility tests were performed, and string tests and precipitation assays were conducted to identify phenotypic changes related to tigecycline susceptibility and ompR expression. Bacterial virulence was assessed by serum resistance and Galleria mellonella infection assays.
Transcriptomic analyses demonstrated a significant decrease in the expression of ompK35 in the tigecycline-resistant mutants. We observed that tigecycline-resistant mutants overexpressed ompR, and that the expression of ompK35 was regulated negatively by ompR. While tigecycline-resistant mutants and ompR-overexpressed mutants exhibited reduced hypermucoviscosity and virulence, deletion of ompR from tigecycline-resistant mutants restored their hypermucoviscosity and virulence.
In hypervirulent K. pneumoniae strains, ompR expression, which is regulated by exposure to tigecycline, may affect the production of CPS, leading to bacterial virulence.
Klebsiella pneumoniae is one of the significant gram-negative pathogens that cause a variety of diseases including intra-abdominal infections, pneumonia, urinary tract infections, and pyogenic liver abscesses . Several virulence factors, capsules, lipopolysaccharides, siderophores, and fimbriae have been identified in K. pneumoniae . Particularly, the capsule is widely recognized as a major virulence factor that contributes to its defense against environmental pressures and host immune responses, as well as to antibiotic resistance [3, 4].
Due to the increased antimicrobial resistance in K. pneumoniae, tigecycline is often used as a last-resort antibiotic to combat multidrug-resistant K. pneumoniae . However, tigecycline resistance has been reported with increased frequency in K. pneumoniae during treatments with tigecycline, or even without exposure to tigecycline [6, 7]. It has been known that resistance to tigecycline is mainly attributed to the overproduction of efflux pumps such as AcrAB and OqxA, or to mutations in efflux pump regulator genes such as ramA, soxR, marR, and acrR .
In gram-negative bacteria, the outer membrane proteins play a crucial role in bacterial virulence and are also associated with antibiotic resistance . It is well-known that K. pneumoniae generates two major porins: OmpK35 and OmpK36, the levels of which are affected by a variety of environmental conditions such as osmolarity, temperature and pH . The tigecycline-resistant K. pneumoniae isolates exhibited significantly decreased expression of the porin OmpK35, compared to susceptible isolates . In addition, the development of tigecycline resistance in hypervirulent K. pneumoniae resulted in decreased virulence associated with reduced CPS . However, it is not known why CPS and virulence decrease in the tigecycline-resistant K. pneumoniae strain.
In this study, we investigated the mechanism for the association between OmpK35 and tigecycline resistance. We constructed mutants with deleted or overexpressed ompR, a negative regulator of ompK35, and compared the mucoviscosity, virulence, and gene expression between wild-type K. pneumoniae strains and their ompR mutants.
Materials and methods
Bacterial strains, plasmids, and culture conditions
In this study, two K. pneumoniae strains, 109 and 200, were used that were isolated from the blood of South Korean patients . Their capsular serotype was determined to be K1 and the 109 and 200 exhibited hypermucoviscous. Both strains belong to the multilocus sequence type 23 (ST23) and were susceptible to tigecycline. The tigecycline-resistant mutants 109-IR and 200-IR were derived from tigecycline-susceptible K. pneumoniae strains by methods previously described . Briefly, the susceptible strains were subcultured in Luria–Bertani (LB) broth with a serially increasing concentration of tigecycline (0.5 to 64 mg/L). All K. pneumoniae strains, mutants and plasmids that were used to construct mutants are presented in Table 1. All bacterial strains were grown in LB broth with shaking at 37 °C, and tigecycline-resistant mutants were cultured in media with 64 mg/L tigecycline. Where appropriate, gentamicin (30 mg/L) was added to the growth medium, and isopropyl-β-d-thiogalactopyranoside (IPTG) was added to 109/ompR and 200/ompR at a concentration of 0.25 mM, and to 109-IR∆ompR-C and 200-IR∆ompR-C at a concentration of 1 mM to induce OmpR.
Transcriptomic analysis by mRNA sequencing
Transcriptome profiling was performed to contrast the expression profiles between tigecycline-susceptible and resistant strains. For mRNA sequencing, all isolates were overnight cultured with vigorous shaking (220 rpm) at 37 °C and diluted into fresh LB broth (1:100). The RNA samples were extracted from mid-log phase bacterial cultures using a Qiagen RNeasy Mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s instruction. The TURBO DNA-free™ Kit (Invitrogen, MA, USA) was used to remove the contaminated DNA in RNA samples and we obtained mRNA from isolates. After isolation of RNA, cDNA was synthesized and sequencing libraries were generated in strand-specific manner according to the Illumine standard protocol for high-throughput sequencing. Library construction and sequencing were performed at Macrogen Inc. (Seoul, South Korea) using an HCS 3.3.52 Software for Illumina HiSeq 4000 sequencing system. The 101 bp paired-end raw reads were filtered and trimmed using Fase QC (version 0.11.7) and Trimmomatic (version 0.38). Expression levels of mRNA were measured as reads per kilobase per million sequence reads (RPKM), which considers the gene length for normalization. The complete genome sequence of K. pneumoniae NTUH-K2044 was used for aligning reads. The GenBank accession number of NTUH-K2044 is AP006725.
Construction of ompR deletion mutants
The ompR-deleted mutants were generated from 109-IR and 200-IR using the Lambda-Red recombinase method  and the pKD46 plasmid (Table 1). The pHK1014 plasmid was introduced into 109-IR and 200-IR by electroporation. Transformants were selected using 30 mg/L gentamicin, and then bacterial colonies were confirmed using the primer pairs pKD46-repA101-F/pKD46-repA101-R (Table 2). The kanamycin resistance gene (KmR) in pHK1009 was amplified using the primer pairs Del-ompR-F/Del-ompR-R that are upstream and downstream to the ompR gene (Table 2). The KmR amplicon was transformed into 109-IR and 200-IR which harbored pHK1014. Transformants were selected using 50 mg/L kanamycin, and bacterial colonies were confirmed using the primer pairs Checkpri-ompR-F/Checkpri-ompR-R (Table 2).
Cloning of ompR and complementation to mutants
For construction of the ompR expression vector, ompR was amplified from chromosomal DNA from the strain 109 using PCR and the primer pairs ompR-F/ompR-R (Table 2). The PCR product was purified using the extraction kit (iNtRON, Seongnam, Korea), digested with BamHI and ligated into a pHK251 precut using the same restriction enzyme. To confirm the mutation in ompR, Sanger DNA sequencing was performed. The pSY005 plasmid was electroporated into strains 109 and 200 for overexpression of ompR and it was also transformed into 109-IR∆ompR and 200-IR∆ompR for complementation by electroporation. The transformants were then plated on LB agar containing 30 mg/L gentamicin for selection.
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing was performed in accordance with the FDA guideline (https://www.accessdata.fda.gov/drugsatfda_docs/label/2013/021821s026s031lbl.pdf, susceptible, MIC ≤ 2 µg/mL; intermediate, MIC = 4 µg/mL; and resistant, MIC ≥ 8 µg/mL), since no tigecycline breakpoints exist in the Clinical Laboratory Standards Institute guideline for K. pneumoniae. The minimum inhibitory concentration (MIC) of tigecycline was determined by broth microdilution using Escherichia coli ATCC 25922 as the reference strain. All tests were performed in duplicate.
String tests and precipitation assays
To conduct string tests, all strains were inoculated overnight on LB agar plates at 37 °C and bacterial colonies on plates were extended using a loop. Hypermucoviscosity was determined to be positive when the strain produced a stretched string more than 5 mm in length using a loop . Precipitation assays were performed by centrifugation of the cultures. As the supernatant of hypermucoviscous strains remain dense after centrifugation, the supernatant density of centrifuged cultures can be a quantitative indicator of hypermucoviscosity . Prior to centrifugation, all bacterial strains were cultured in LB broth at 37 °C overnight with shaking. The samples were centrifuged at 2000×g for 10 min then the bacteria were suspended and diluted until the optical density at 600 nm (OD600) reached 4. The optical densities (OD) of the supernatants were measured at OD600. All tests were performed three biological replicates per strain.
Serum resistance assay
Serum resistance assays were performed to evaluate the resistance against killing by normal human serum (NHS) using a previously described method  with slight modifications. All strains were incubated overnight in LB broth at 37 °C with shaking and diluted 1:100 with fresh LB media and grown until the mid-log phase. Twenty-five μL of the bacterial solution were mixed with 75 μL of NHS in microtubes. Heat-inactivated human serum was used as a control to determine the ability to eliminate bacteria by NHS. The mixtures were incubated for 3 h with shaking and plated on LB agar after being serially diluted with phosphate buffered saline (PBS) for colony counting. For all strains, three independent tests were performed.
Galleria mellonella infection assays
Galleria mellonella larvae were purchased from the Sworm Corp. (Cheonan, South Korea). The G. mellonella larvae were kept at room temperature in the dark with food for ten days before use. Larvae with weights of approximately 150–200 mg were selected for further experiments.
Bacterial infections of G. mellonella were performed as previously described with minor modifications . Overnight bacterial cultures were harvested by centrifugation at 16,000 × g for 2 min then washed with 10 mM PBS. Bacterial cultures were adjusted with PBS to a McFarland standard of 0.5. The larvae were then infected with 10 μL of bacterial solutions by injection into the larvae’s last right proleg using an ultra-fine needle (BD Biosciences, San Jose, CA, USA). A PBS injection was used as a negative control, and K. pneumoniae strain ATCC 43816 as a positive control of hypermucoviscous strain. Ten larvae were infected with each bacterial strain and the viability of the larvae was examined until 72 h post infection. The experiments were performed three times independently.
RNA extraction and quantitative RT-PCR
To measure the relative fold changes of expression of ompK35 and ompR in the strains, quantitative real-time PCR (qRT-PCR) was performed. Overnight cultures of bacteria were inoculated into fresh LB broth and incubated at 37 °C with shaking until mid-log phase. Then, RNA was extracted using a Qiagen RNeasy Mini kit (Quiagen, Hilden, Germany) according to the manufacturer’s instructions. Contaminated DNA was eliminated from RNA samples using a TURBO DNA-free™ Kit (Invitrogen, MA, USA) and the reverse transcription reactions were conducted using a reverse transcription premix kit (iNtRON, Seongnam, South Korea). qRT-PCR was performed using TB Green Premix Ex Taq (TaKaRa, Shiga, Japan) with the QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems, CA, USA) using the primers listed in Table 2. The expression levels were determined by the 2−ΔΔCT method using the rpoB gene as a reference. The qRT-PCRs were performed three times and each sample was analyzed in duplicate.
Statistical analyses of all experiments were performed to assess the significance of the differences using Student’s t-test, a one-way ANOVA with Tukey’s multiple comparisons test, and a nonparametric Kruskal–Wallis test followed by Dunnett’s multiple comparison test with Prism v8.3 for windows (GraphPad Software, San Diego, CA, USA). P values of < 0.05 were considered to be statistically significant (*, p < 0.05; **, p < 0.001, ***, p < 0.0001).
Differentially expressed genes in the chromosome of Klebsiella pneumoniae
The chromosomal genes differently expressed in both tigecycline-resistant mutants (109-IR and 200-IR) were filtered with the criteria of fold-change ≥ 2. The filtered genes were categorized on the basis of the classification of clusters of orthologous groups (COGs) (Fig. 1). As a whole, 252 genes were up-regulated and 214 genes were down-regulated both in tigecycline-resistant mutants compared with the wild-type strains. Especially, 127 genes of metabolism pathway including carbohydrate transport and energy production were overexpressed.
Table 3 lists the top 10 genes with the highest differentially expressed genes in the tigecycline-resistant mutants compared with wild-type and tigecycline-susceptible K. pneumoniae strains. From the list we noted that ompK35 encodes a trimeric porin OmpK35, which is a homolog of OmpF in Escherichia coli. OmpK35 has been known to be associated with antibiotic resistance and virulence, along with another porin, OmpK36 .
mRNA expression of ompK35 and ompR
Using qRT-PCR, we measured mRNA expression of ompK35 and ompR, a repressor of ompK35 under high osmolar environments in E. coli  in each of two wild-type strains (109 and 200) and in the tigecycline-resistant mutants 109-IR and 200-IR. As indicated by transcriptomic analysis, ompK35 was down-regulated significantly in the tigecycline-resistant mutants 109-IR and 200-IR (p = 0.0108 and 0.0034, respectively) (Fig. 2A). ompR was up-regulated significantly in 109-IR and 200-IR, compared with their susceptible parental strains (p, 0.0005 and 0.0069, respectively) (Fig. 2A).
We then examined the change in ompR expression with increasing tigecycline concentrations in tigecycline-resistant mutants. As the concentration of tigecycline increased, the expression of ompR also increased gradually in both mutants (Fig. 2B). Figure 2C shows that ompR expression is well-regulated by the addition of IPTG.
The ompR-overexpressed mutants in the presence of IPTG exhibited significantly decreased expression of ompK35 (Fig. 2D). Expression of ompK35 increased slightly in the ompR-deleted mutants 109-IR∆ompR and 200-IR∆ompR, compared with the tigecycline-resistant mutants 109-IR and 200-IR (p, 0.0129 and 0.0011, respectively). The complementation of ompR and the induction of its expression in ompR-deleted mutants (109-IRΔompR-C and 200-IRΔompR-C) led to decreased expression of ompK35 (Fig. 2D). These results confirmed the role of OmpR as a repressor of ompK35.
The MIC for tigecycline increased eightfold in the ompR-overexpressed mutants 109/ompR/IPTG(+) and 200/ompR/IPTG(+), compared to those of tigecycline-susceptible, wile-type K. pneumoniae strains (Table 4). The tigecycline-resistant mutants exhibited very high levels of tigecycline resistance (MICs > 64 mg/L), and the MICs decreased dramatically in the ompR inactivated mutants 109-IR∆ompR and 200-IR∆ompR (4 and 8 mg/L, respectively). However, complementation of ompR did not increase the tigecycline susceptibility.
To evaluate the role of ompR on the production of mucoviscosity in K. pneumoniae, we evaluated the changes in colony phenotypes in the mutants. The tigecycline-susceptible strains 109, 200, 109/ompR/IPTG(‒), and 200/ompR/IPTG(‒) displayed large and glossy colony morphologies, while the tigecycline-resistant mutants 109-IR and 200-IR exhibited small and matt colony phenotypes (Fig. 3A). The ompR-overexpressed mutants 109/ompR/IPTG(+) and 200/ompR/IPTG(+) also exhibited small and matt colonies, as observed with 109-IR and 200-IR (Fig. 3A). In the ompR-deleted mutants from the tigecycline-resistant strains 109-IR∆ompR and 200-IR∆ompR, large and glossy colony shapes were observed, compared with 109-IR and 200-IR. The small and matt colony phenotypes of 109-IR and 200-IR were restored by complementation with ompR, 109-IR∆ompR-C, and 200-IR∆ompR-C (Fig. 3A).
The tigecycline-susceptible wild-type strains 109 and 200 exhibited a phenotype of hypermucoviscosity, ranging from 24 to 29 mm in the string test; while the ompR-overexpressed mutants 109/ompR/IPTG(+) and 200/ompR/IPTG(+) exhibited significantly reduced lengths of stretched strings, an average of 9 mm for both (P, < 0.0001 and 0.0005, respectively) (Fig. 3B). The tigecycline-resistant mutants 109-IR and 200-IR produced string lengths of 6–8 mm; however, the string lengths of the ompR-deleted mutants 109-IR∆ompR and 200-IR∆ompR increased dramatically to 23 mm and 21 mm, respectively (p, < 0.0001 for both). Complementation with ompR, 109-IR∆ompR-C and 200-IR∆ompR-C, reduced the length of stretched strings to those of tigecycline-resistant mutants (Fig. 3B).
Mucoviscosity in the K. pneumoniae strains was determined by measuring the OD at 600 nm of the supernatants after centrifugation. The tigecycline-susceptible wild-type strains 109 and 200 produced turbid and poor sediments due to hypermucoviscosity that causes extreme stickiness (Fig. 3C). The ompR-overexpressed mutants 109/ompR/IPTG(+) and 200/ompR/IPTG(+), and the tigecycline-resistant mutants 109-IR and 200-IR produced clear supernatants after centrifugation, unlike the dusty appearance observed with 109, 200, 109/ompR/IPTG(‒), and 200/ompR/IPTG(‒) (Fig. 3C). The 109-IR∆ompR and 200-IR∆ompR mutants produced a turbid appearance, which reverted to a clear appearance by complementation with ompR (Fig. 3C).
The measurement of optical density confirmed the visual observations (Fig. 2D). The overexpression of ompR or the development of tigecycline resistance lowered the turbidity of supernatants significantly, that is, the mucoviscosity.
To explore the relationship of ompR with virulence in K. pneumoniae, bacterial survival rates were evaluated against NHS (Fig. 4). The ompR-overexpressed mutants, 109/ompR/IPTG(+) and 200/ompR/IPTG(+), showed significantly decreased survival rates against serum, compared to the wild-type and the tigecycline-susceptible strains. The tigecycline-resistant mutants also exhibited very low survival rates against serum, which has been reported previously . Deletion of ompR in the tigecycline-resistant mutants 109-IR∆ompR and 200-IR∆ompR increased the serum resistance (p, 0.0012 and 0.0026, respectively), and the survival rates of ompR-complemented mutants were diminished.
Survival of G. mellonella larva
Most of the G. mellonella larva were killed within 72 h by the strains 109 and 200, as by hypervirulent K. pneumoniae strain ATCC 43816 (Fig. 5A and B). The G. mellonella larva survived significantly longer using the ompR-overexpressed mutants 109/ompR/IPTG(+) and 200/ompR/IPTG(+) compared to the wild-type strains (p, 0.0093 and 0.0009, respectively). The tigecycline-induced resistant mutants 109-IR and 200-IR exhibited dramatically increased survival rates with G. mellonella larva, compared to the wild-type and the tigecycline-susceptible strains (p, < 0.0001 and 0.0001, respectively) (Fig. 5A and B). Their survival rates were reduced by deletion of ompR in the tigecycline-resistant mutants and were restored by complementation with ompR (Fig. 5C and D).
Previously, we demonstrated that in vitro-induced tigecycline-resistant K. pneumoniae mutants exhibited a dramatic decrease in hypermucoviscosity associated with reduced capsular polysaccharide production, resulting in defects in virulence . Reduced virulence with respect to serum susceptibility and survivability of G. mellonella has previously been reported in tigecycline-resistant Acinetobacter baumannii . However, it is not yet known how the development of tigecycline resistance affects virulence in bacteria. In this study, we investigated which changes that occur during the development of tigecycline resistance in K. pneumoniae lower the virulence.
Transcriptomic analyses have confirmed that some genes that may be associated with tigecycline resistance are expressed differentially in two tigecycline-resistant mutants, for example, ramA . We detected decreased expression of a porin, OmpK35, in both tigecycline-resistant mutants. In K. pneumoniae, OmpK35 is a homolog of the E. coli OmpF porin . It is responsible for rapid influx of β-lactams including third generation cephalosporins and carbapenems in K. pneumoniae clinical isolates [20, 21]. Thus, a deficiency or a defect in the porin results in resistance to cephalosporins and carbapenems. Recently, decreased expression of OmpK35 was also identified in tigecycline-resistant K. pneumoniae strains . In addition to antibiotic resistance, decreased virulence has been reported in ompK35-deficient K. pneumoniae and ompF-deficient E. coli mutants [10, 22]. Since OmpK35 and OmpK36 are known to be regulated by a two-component regulatory system, OmpR-EnvZ-sensing osmotic signals [23, 24], we explored the effects of OmpR on the phenotypic changes that lead to virulence in the tigecycline-resistant mutants.
First, we confirmed that tigecycline-resistant mutants overexpressed ompR and that ompK35 expression is regulated negatively by ompR. In addition, the expression of ompR increased with increasing concentrations of tigecycline. That is, OmpR might sensor the tigecycline, which may act as an osmolarity factor.
Exposure to tigecycline produced a change in the phenotypes in hypervirulent K. pneumoniae strains. While tigecycline-susceptible and hypervirulent strains produced large, glossy, and mucoid colonies, the colonies of tigecycline-resistant mutants generated small, matt, and non-mucoid which may lead to the reduced virulence. Similar phenotypic changes were also observed in ompR-overexpressed mutants. In addition, after the deletion of ompR, tigecycline-resistant mutants were restored like phenotype of the tigecycline-susceptible K. pneumoniae strains. This implies that the overexpression of OmpR induced by tigecycline exposure can be responsible for the phenotypic changes in the hypervirulent K. pneumoniae stains.
The phenotype changes in tigecycline-induced resistance and ompR overexpression were clearly associated with decreased virulence judged from the survival of NHS and G. mellonella larvae. The altered susceptibility to tigecycline caused by the overexpression or deletion of ompR may be associated with OmpK35 expression, which is regulated by OmpR. The smaller change caused by complementation with ompR may indicate that the influence of OmpR on the tigecycline susceptibility is indirect.
Our study have some limitations. First, only a few strains were studied, limiting the generalization of the results. Second, it has not been revealed what features of tigecycline affect the expression of ompR and compK35. Nor have we found out why other antibiotics do not have these phenomena. CPS is known to be synthesized in different ways in K. pneumoniae K1 serotype, and thus would be further confirmed by various assays, for example, capsule stain and Periodic Acid Schiff stain. In addition, OmpK35 is a constituent of channel for extracellular polysaccharide (EPS) as well as CPS. As EPS may also be crucial for virulence, presence or amount of EPS should be compared between tigecycline-resistant and -susceptible strains.
Based on the findings in this study, we speculated that the cause of the increased resistance to tigecycline and reduced virulence in hypermucoviscosity and hypervirulent K. pneumoniae strains attributed to the overexpression ramA by exposure to tigecycline [7, 12]. Tigecycline may simultaneously stimulate OmpR. Overexpressed OmpR binds ompK35, and acts as a repressor. Down-regulated OmpK35 caused by OmpR resulted in reduced virulence in hypermucoviscous and hypervirulent K. pneumoniae strains.
In the present study, we demonstrated that ompR expression is regulated by exposure to tigecycline, thereby affecting the virulence associated phenotypes in K. pneumoniae strains. The reduced virulence in tigecycline-resistant mutants is probably an accompanying action of OmpK35, which is negatively regulated by OmpR.
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Reads per kilobase per million
Minimum inhibitory concentration
Normal human serum
Phosphate buffered saline
Quantitative real-time PCR
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Tigecycline was provided by Pfizer.
This research was supported in part by the Basic Science Research Program of the National Research Foundation (NRF) funded by the Korean government (MSIT) (NRF-2022R1A2B502001716).
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Park, S., Kim, H. & Ko, K.S. Reduced virulence in tigecycline-resistant Klebsiella pneumoniae caused by overexpression of ompR and down-regulation of ompK35. J Biomed Sci 30, 22 (2023). https://doi.org/10.1186/s12929-023-00910-w
- Klebsiella pneumoniae