Reduced virulence in tigecycline-resistant Klebsiella pneumoniae caused by overexpression of ompR and down-regulation of ompK35

Abstract

Background

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.

Methods

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.

Results

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.

Conclusions

In hypervirulent K. pneumoniae strains, ompR expression, which is regulated by exposure to tigecycline, may affect the production of CPS, leading to bacterial virulence.

Background

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 [1]. Several virulence factors, capsules, lipopolysaccharides, siderophores, and fimbriae have been identified in K. pneumoniae [2]. 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 [5]. 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 [8].

In gram-negative bacteria, the outer membrane proteins play a crucial role in bacterial virulence and are also associated with antibiotic resistance [9]. 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 [10]. The tigecycline-resistant K. pneumoniae isolates exhibited significantly decreased expression of the porin OmpK35, compared to susceptible isolates [11]. In addition, the development of tigecycline resistance in hypervirulent K. pneumoniae resulted in decreased virulence associated with reduced CPS [12]. 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 [12]. 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 [12]. 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.