Catalase Enhances Growth and Biofilm Production of Mycoplasma pneumoniae

Abstract

Mycoplasma pneumoniae causes chronic respiratory disease in humans. Factors thought to be important for colonization include the ability of the mycoplasma to form a biofilm on epithelial surfaces and the production of hydrogen peroxide to damage host tissue. Almost all of the mycoplasmas, including M. pneumoniae, lack superoxide dismutase and catalase and a balance should exist between peroxide production and growth. We show here that the addition of catalase to cultures enhanced the formation of biofilms and altered the structure. The incorporation of catalase in agar increased the number of colony-forming units detected and hence could improve the clinical diagnosis of mycoplasmal diseases.

Introduction

The human pathogen Mycoplasma pneumoniae causes diseases of the respiratory tract including lobar and bronchial pneumonia and tracheobronchitis and can exacerbate underlying diseases such as asthma [1,2]. Up to 40% of all community-acquired pneumonias are caused by M. pneumoniae, resulting in over 100,000 hospitalizations annually in the US. Macrolides are often prescribed to treat the infection but resistant strains are emerging [3]. The ability to culture these fastidious organisms is important to be able to test for antibiotic resistance and prescribe an appropriate treatment.

Hydrogen peroxide (H₂O₂) is produced by many species of mycoplasma and is considered a virulence factor causing oxidative damage to host cells [4,2,5,6]. Likewise, peroxide radicals produced from the respiratory burst of professional phagocytes serve to kill invading bacteria through oxidative damage to DNA [7]. Brennan and Feinstein showed that catalase activity in mice was essential to initially fight off infection caused by the murine respiratory pathogen Mycoplasma pulmonis but also enhanced the survival of the microorganisms later in the infection, suggesting a balance between the catalase-producing host and the peroxide-producing pathogen [8]. Like M. pulmonis, the human respiratory pathogen M. pneumoniae lacks catalase but produces H₂O₂ through pathways utilizing glycerol or glycerophosphocholine [4,9].

Growth of M. pneumoniae and many other mycoplasmas is generally slow and some species of mycoplasmas are uncultivable. We observed that cultures of M. pneumoniae grew to stationary phase more quickly in tissue culture flasks that were closed to the atmosphere as compared to vented flasks that permitted gas exchange. The possibility that growth was poor when oxygen was more abundant led us to investigate the effect of catalase on growth. We found that the number of colonies of M. pneumoniae increased when catalase was added to the agar. We propose that catalase be considered for use in clinical and research laboratories for cultivation of M. pneumoniae and possibly for other species of mycoplasma including those that are intractable. We also show that catalase affected the structure of the biofilms formed by M. pneumoniae.

Materials and Methods

CFU analysis on mycoplasma agar plates

M. pneumoniae strain UAB_PO1 was assayed as CFU on 60 mm plates containing 10 ml SP-4 agar as described [10]. Plates were either left unsealed or were sealed with several layers of parafilm to minimize gas exchange. To assess the effect of catalase on growth, bovine liver catalase (Sigma) was dissolved in either SP-4 broth or water at a concentration of 10 mg per ml or 3.7 mg mL⁻¹, respectively, and passed through a 0.2 µm polyethersulfone filter (VWR). One ml of catalase solution was allowed to absorb into the agar and dry in a laminar flow hood prior to CFU analysis. CFU were counted after growth for up to 3 weeks at 37°C. One-way analysis of variance and a Tukey post-hoc test was used to compare differences between the CFU detected on sealed or unsealed plates that were supplemented with catalase or not. The Student’s t-test was used to compare differences between the CFU detected in unsealed plates that were either supplemented with catalase or not. P values < 0.05 were considered significant.

Biofilm image analysis and quantification

Tissue culture flasks (Falcon, 25 cm²) containing 5 ml SP-4 broth alone or SP-4 broth supplemented with catalase to a final concentration of 1.5 mg mL⁻¹ were inoculated with 10⁵ CFU of M. pneumoniae. Some flasks had sealed caps while others had vented caps that allowed gas exchange with the external environment. The medium contained phenol red at 0.002 % as a pH indicator. The mycoplasmas were allowed to grow at 37°C for 7 days, 10 days, or until the culture reached stationary phase as determined by color change of the phenol red indicator.

For crystal violet analysis of the biofilms, the flasks were rocked gently in the growth medium to dislodge non-adherent mycoplasmas and the medium was discarded. Five ml of Gram’s Crystal Violet (0.3% crystal violet, 5.0% isopropanol, 5.0% methanol) diluted 1:1 in methanol was added to the flask and incubated overnight to fix and stain the biofilms. The stain was removed by suctioning, and the flasks washed gently twice with water to remove unincorporated stain.

The crystal violet-stained biofilms were imaged in the flasks using a Lieca HC brightfield microscopy at 50× magnification. ImageJ (National Institutes of Health, version 1.46r, http://imagej.nih.gov/ij) was used to correct the images for uneven illumination using the Contrast Limited Adaptive Histogram Equalization plugin. The images were thresholded and analyzed by the particle analyzer function of ImageJ. Particles with a surface area of greater than 170 µm² (50 pixels² in the original images) were accepted as towers. After imaging, the crystal violet was extracted from each of six replicate biofilms by washing the flasks twice with 1.0 ml methanol while rocking for 15 minutes for each wash. The 2 washes were combined and 200 µl were placed in triplicate into a 96-well plate. The relative amounts of biofilm were quantified by determining the OD₅₉₀. The results were analyzed by one-way analysis of variance. Pairwise comparisons were performed by the Tukey method. P values of less than 0.05 were considered significant.

For quantifying the protein content of the biofilm mass and of planktonic mycoplasmas, the mycoplasmas were grown as described above and the medium was removed from the flasks. The mycoplasmas were washed 3 times in 10 ml phosphate buffered saline (PBS) by centrifugation at 6900 × g to recover the planktonic cells. The biofilms were scraped from the flasks and washed 3 times in 10 ml PBS by centrifugation. The final pellets were suspended in known volumes of water. The total amount of protein locked into the biofilm or in the planktonic mycoplasmas was determined by the Pierce BCA Protein Assay (Thermo Scientific) reading the OD₅₆₀ for each sample in triplicate in 96-well plates with dilutions of bovine serum albumin used to establish a standard curve. The total amount of protein recovered from the flasks was calculated by adding the amounts of protein locked in the biofilms and the amount of protein in the planktonic cells. The percent of total protein adhered to the flasks was calculated by the formula: protein locked in the biofilm/ total protein in the flask × 100. The Student’s t-test was used to determine differences between the sealed flasks containing SP-4 alone or SP-4 supplemented with catalase. P values of less than 0.05 were considered significant.

Results

Catalase improves growth on agar

The addition of catalase had a positive effect on growth on agar. After 2 weeks of growth, about 6- to 10-fold more CFU were detected when catalase was added whether the plates were sealed or not (Fig. 1a; P < 0.01). In another experiment that studied colony growth on unsealed plates over a 3-week period, the effect of catalase was similar (Fig. 1b). The addition of catalase increased the number of colonies after 1 week (P < 0.01) and 2 weeks (P = 0.02). By 3 weeks, the number of CFU on plates without catalase approached the number of CFU on plates containing catalase, suggesting that catalase affected the rate of growth.

Fig. 1.

Effect of catalase on CFU. (a), CFU obtained with or without the addition of 10 mg catalase to the agar for plates that were either unsealed or sealed with parafilm. (b), growth of the mycoplasmas on unsealed agar plates with and without 3.7 mg catalase over a 3-week period. The black bars in (b) represent CFU on plates containing catalase and the gray bars represent CFU without catalase. Asterisks indicate significance between the number of CFU detected with or without catalase. N = 3 for each group except where noted in (a).

Poor biofilm formation in vented flasks

Crystal violet analysis of M. pneumoniae grown for 10 days in tissue culture flasks containing mycoplasmal broth indicated that biofilms formed more robustly in sealed flasks than in vented flasks whether catalase was added to the medium or not (Fig. 2a, P < 0.01). The overall poor growth in vented flasks, even in the presence of catalase, suggested that the level of H₂O₂ reached when oxygen is abundant could overwhelm the catalase.

Fig. 2.

Effect of catalase on biofilms grown in vented or sealed tissue culture flasks. In (a) the biofilms were stained with crystal violet. The OD₅₉₀ represents the amount of biofilm adhered to the flasks (n = 6 each group). Asterisks indicate significant differences in amounts of biofilms adhered between the groups. In (b) the protein content in mg per flask (left axis) of the biofilm adhered cells, the total protein content of the mycoplasmas in the flasks (adhered plus non-adhered), and the percentage of the protein content locked in the biofilm (right axis, % Adhered). The black bars represent protein from flasks containing catalase and the gray bars represent protein from flasks without additional catalase. The left graph shows these values for biofilms that were grown for 7 days (n = 3). The right graph shows the values for biofilms grown to stationary phase (n = 8). The asterisks indicate significance between protein recovered from flasks supplemented with catalase compared to flasks without catalase.

Effect of catalase on biofilm formation in sealed flasks

Crystal violet staining indicated that the addition of catalase to sealed flasks greatly enhanced the formation of the biofilms (Fig. 2a, P < 0.01). Strains of mycoplasma forming weak biofilms typically grow well but have a higher percentage of CFU that are planktonic than do strains of mycoplasmas that form more robust biofilms [11,10]. As the crystal violet assay was limited to assessing cells within the biofilm, we determined the amount of protein adhered to the sealed flasks as well as the protein from the unadhered cells in the medium. After 7 days growth, the addition of catalase to the medium resulted in a nearly 3-fold increase in protein adhered (P < 0.0001), a doubling of the percentage of protein adhered (P = 0.0025), and a substantial increase in the total protein recovered from the flasks (P = 0.0192) (Fig. 2b). When the cultures were allowed to grow to stationary phase as determined by color change of the medium (Fig. 2b, labeled “Stationary phase”), catalase enhanced the amount of adhered protein, the percentage of protein adhered and the total amount of mycoplasmal protein (P = 0.0001; P = 0.0178; P = 0.0072, respectively).

The addition of catalase to the medium affected the quality of the biofilms. Towers are prominent features of the biofilms formed by M. pneumoniae and M. pulmonis and protect mycoplasmas from lysis by complement and the pore-forming antimicrobial gramicidin [12,10]. Biofilms grown in medium without catalase contained many of these towers (see arrows in Fig. 3a and 3b and the outlines of the towers in Fig. 3c and 3d). When biofilms were grown in medium containing catalase, fewer and smaller towers formed and the biofilms were smoother and more homogenous.

Fig. 3.

Structure of the biofilms formed by M. pneumoniae. (a and c), brightfield images of biofilms grown in medium without or with supplementation with catalase, respectively, and stained with crystal violet. (b and d), outlines of the tower structures identified by the particle analysis function of ImageJ of (a and c). Arrows point out some of the towers from which the outlined towers were derived. Scale bars are 250 µm.

Discussion

These results indicate that the addition of catalase to cultures of M. pneumoniae has a beneficial impact on growth rate. Detection of mycoplasmas on agar is considered the gold standard for diagnosis. Although other methods such as PCR can be used for diagnostics, the ability to culture the organisms is essential for antibiotic susceptibility testing. The addition of catalase to medium may result in the more sensitive and rapid diagnosis of mycoplasma-induced diseases by culture and should be considered for use in diagnostic laboratories. The addition of catalase to the medium may improve the culturability and isolation of many other species of fastidious Mycoplasma.

Enhancement of biofilm formation by the addition of catalase is likely explained by the inactivation of H₂O₂ and its oxidative effects. The increased number of towers in the biofilms grown without catalase suggests that growth within towers may serve to protect the mycoplasmas from H₂O₂. Selective pressure for tower formation when oxygen is plentiful could be the result of inefficient penetration of H₂O₂ into the tower structures as reported for biofilms of Psuedomonas aeruginosa [13]. Changes in gene regulation may be a factor. It has been shown that brief treatment of M. pneumoniae biofilms with 0.1% H₂O₂ resulted in increased levels of transcripts for several genes including the MPN_131 and MPN_102 adhesin-like proteins [14], potentially contributing to the propensity of the mycoplasmas to form towers in the absence of catalase. The level of transcripts of the major adhesion protein P1 remained constant in that study, suggesting that proteins other than P1 may have a role in cell-to-cell interactions affecting tower formation. In addition to protecting mycoplasmas from complement and peptide antimicrobials, tower formation may be a mechanism for the mycoplasmas to cope with endogenous sources of H₂O₂.