Loss of hypermucoviscosity and increased fitness cost in colistin-resistant Klebsiella pneumoniae sequence type 23 strains

Abstract

In this study, we investigated the effects of colistin resistance on virulence and fitness in hypermucoviscous (HV) Klebsiella pneumoniae sequence type 23 (ST23) strains. Colistin-resistant mutants were developed from three colistin-susceptible HV K. pneumoniae ST23 strains. The lipid A structures of strains were analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. Changes in HV were investigated using the string test, and extracellular polysaccharide production was quantified. The expression levels of the phoQ, pmrD, pmrB, pbgP, magA, and p-rmpA2 genes, serum resistance, and biofilm-forming activity were determined. The fitness of colistin-resistant mutants compared to that of the parental strains was examined by determining the competitive index (CI). The colistin-resistant mutants exhibited reduced HV, which was accompanied by decreased formation of capsular polysaccharides (CPS) and reduced expression of genes (magA and p-rmpA2). While there was enhanced expression of pmrD and pbgP in all colistin-resistant derivatives, there were differences in the expression levels of phoQ and pmrB between strains. MALDI-TOF analysis detected the addition of aminoarabinose or palmitate to the lipid A moiety of lipopolysaccharide in the colistin-resistant derivatives. In addition, survival rates in the presence of normal human serum were decreased in the mutant strains, and CI values (0.01 to 0.19) indicated significant fitness defects in the colistin-resistant derivatives compared to the respective parental strains. In hypervirulent HV K. pneumoniae strains, the acquisition of colistin resistance was accompanied by reduced CPS production, impaired virulence, and a significant fitness cost.

FIG 1

Comparison of the expression levels of phoQ (A), pmrB (B), pmrD (C), and pbgP (D) in the three pairs of Klebsiella pneumoniae ST23 strains. Error bars indicate the standard deviations for three triplicate samples. *, P < 0.05 versus the result for a corresponding colistin-susceptible parental strain.

FIG 2

Lipid A structures of Klebsiella pneumoniae ST23 strains. Shown are the negative-ion matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry spectra of the lipid A moieties of lipopolysaccharide isolated from strains 08-B063 and 08-B063R (A), 07-B-060 and 07-B-060R (B), and 13703-3487 and 13703-3487R (C).

FIG 3

Mucoviscosity and capsular polysaccharide (CPS) production. (A and B) Mucoviscosity. The string test was used to assess the hypermucoviscosity of K. pneumoniae strains. A string of 5 mm or longer is defined as positive. (C) CPS production. CPS biosynthesis in the K. pneumoniae strains was determined by phenol-sulfuric acid assays. *, P < 0.05 versus the value for a corresponding colistin-susceptible parental strain; #, P < 0.05 versus the value for colistin-susceptible strain 08-B063. Error bars indicate the standard deviations for three triplicate samples.

FIG 4

Relative transcriptional levels of magA (A) and p-rmpA2 (B) in the three pairs of Klebsiella pneumoniae strains. Error bars indicate the standard deviations for three triplicate samples.

FIG 5

(A) Serum resistance assays of wild-type and colistin-resistant Klebsiella pneumoniae strains. Serum resistance is represented as percent viability (no. of colonies after 2 h of incubation/no. of colonies for the initial mixture). Error bars indicate standard deviations. Survival rates of each colistin-susceptible parental strain (white) and colistin-resistant mutant (gray) were evaluated and compared after incubation for 2 h with human serum. (B) Analysis of biofilm formation by colistin-susceptible and -resistant Klebsiella pneumoniae strains. Overnight cultures of K. pneumoniae strains were grown in fresh Luria-Bertani (LB) broth at a ratio of 1:100 in polystyrene plates at 37°C for 5 h. The bacteria were stained with crystal violet, washed to remove unbound cells, and eluted with 95% ethanol, and biofilm masses were detected by measuring the absorbance at 600 nm. *, P < 0.05 versus the value for a corresponding colistin-susceptible parental strain; #, P < 0.05 versus the value for colistin-susceptible strain 08-B063. Error bars indicate the standard deviations for three triplicate samples.

FIG 6

Growth rates and fitness costs. (A to C) Growth curves of colistin-susceptible (S) and -resistant (R) Klebsiella pneumoniae strains. y axis, optical densities (OD) of broth cultures at 600 nm; x axis, time of growth (hours). (D) Fitness costs of colistin-resistant mutants relative to those of their colistin-susceptible parental strains. The relative fitness of colistin-resistant clinical strains with 08-B063 was also measured. A CI value less than 1 indicates a fitness defect, and a value greater than 1 indicates a fitness benefit. Each of the three colistin-resistant mutants (CI values, 0.001 to 0.19) exhibited a marked fitness defect. Error bars indicate the standard deviations for three triplicate samples.

FIG 7

Mucoviscosity and CPS production of phoQ and pmrB deletion mutants. (A) Mucoviscosity of mutants. The string test was used to assess the hypermucoviscosity of mutants. A string length less than 5 mm was defined as negative. (B) CPS production of mutants. Error bars indicate the standard deviations for three triplicate samples. *, P < 0.05 versus value for colistin-susceptible strain 08-B063; #, P < 0.05 versus value for colistin-resistant strain 08-B063R.