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. 2023 Mar 2;12(3):499.
doi: 10.3390/antibiotics12030499.

Spiramycin Disarms Pseudomonas aeruginosa without Inhibiting Growth

Matteo Calcagnile  1 , Inès Jeguirim  2 , Salvatore Maurizio Tredici  1 , Fabrizio Damiano  1 , Pietro Alifano  1
Affiliations

Spiramycin Disarms Pseudomonas aeruginosa without Inhibiting Growth

Matteo Calcagnile et al. Antibiotics (Basel). .

Abstract

Spiramycin is a 16-membered macrolide antibiotic currently used in therapy to treat infections caused by Gram-positive bacteria responsible for respiratory tract infections, and it is also effective against some Gram-negative bacteria and against Toxoplasma spp. In contrast, Pseudomonas aeruginosa, which is one of the pathogens of most concern globally, is intrinsically resistant to spiramycin. In this study we show that spiramycin inhibits the expression of virulence determinants in P. aeruginosa in the absence of any significant effect on bacterial multiplication. In vitro experiments demonstrated that production of pyoverdine and pyocyanin by an environmental strain of P. aeruginosa was markedly reduced in the presence of spiramycin, as were biofilm formation, swarming motility, and rhamnolipid production. Moreover, treatment of P. aeruginosa with spiramycin sensitized the bacterium to H2O2 exposure. The ability of spiramycin to dampen the virulence of the P. aeruginosa strain was confirmed in a Galleria mellonella animal model. The results demonstrated that when G. mellonella larvae were infected with P. aeruginosa, the mortality after 24 h was >90%. In contrast, when the spiramycin was injected together with the bacterium, the mortality dropped to about 50%. Furthermore, marked reduction in transcript levels of the antimicrobial peptides gallerimycin, gloverin and moricin, and lysozyme was found in G. mellonella larvae infected with P. aeruginosa and treated with spiramycin, compared to the larvae infected without spiramycin treatment suggesting an immunomodulatory activity of spiramycin. These results lay the foundation for clinical studies to investigate the possibility of using the spiramycin as an anti-virulence and anti-inflammatory drug for a more effective treatment of P. aeruginosa infections, in combination with other antibiotics.

Keywords: Galleria mellonella; Pseudomonas aeruginosa; anti-virulence drugs; macrolide antibiotics; spiramycin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Biomass, PVD, and PYO production by P. aeruginosa GG-7R growing in LB for 24 h at 37 °C and 180 rpm. (A) Biomass was determined by absorbance at 600 nm measurement. (B) PVD in exhausted LB was assayed by fluorescence emission (excitation at 405 nm, emission at 450 nm). The value was normalized by the biomass (absorbance at 600 nm). (C) PYO in exhausted LB was assayed by optical absorbance at 520 nm. The value was normalized by the biomass (absorbance at 600 nm).
Figure 2
Figure 2
Absorption and fluorescence emission spectra of the exhausted LB broth of P. aeruginosa GG-7R grown either in the absence or in the presence of spiramycin (30 μg/mL). (A) Absorption spectra and (B) fluorescence emission spectra (excitation at 405 nm) of exhausted LB from P. aeruginosa cultures in multiwell plates. The samples were collected after the MIC experiment. The LB broth was diluted by 1 to 200 prior to measurement. The bacterium was grown at 37 °C and 180 rpm for 24 h.
Figure 3
Figure 3
Phenotype of P. aeruginosa GG-7R growing on LB agar in the absence or presence of spiramycin or erythromycin. (A) Effects of spiramycin at 24 h of incubation (37 °C). (B) Effects of spiramycin at 48 h of incubation (37 °C). (C) Effect of erythromycin at 24 h of incubation (37 °C).
Figure 4
Figure 4
Growth of P. aeruginosa (37 °C and 180 rpm) with and without spiramycin (30 or 120 μg/mL) and RT-qPCR on phzS, rhlC, and lasB genes. (A) Estimation of biomass by turbidity (optical absorbance at 600 nm), (B) pH of bacterial cultures, (C) estimation of the amount of PYO (optical absorbance at 520 nm). (D) Estimation of the amount of PYO (optical absorbance at 520 nm) normalized by the biomass (absorbance at 600 nm). This panel shows data from 24 h onwards when the optical absorbance at 520 is >0.15 in the control sample. (E,F) Results of transcript level analysis (RT-qPCR) at 24 h (E) and 48 h (F) of the phzS (pyocyanin synthesis), rhlC (rhamnolipid synthesis), and lasB (elastase) genes.
Figure 5
Figure 5
Growth of P. aeruginosa with hydrogen peroxide (H2O2) with or without spiramycin. The control value is indicated by a dotted line. Growth was carried out in LB at 37 °C and 180 rpm.
Figure 6
Figure 6
Effects of spiramycin on growth, biofilm, PYO, and PVD production by P. aeruginosa GG-7R growing on hydroxyapatite surfaces in multi-well plate filled with LB. (A) View of the plate after 72 h of incubation (37 °C, 150 rpm). (B) PYO quantification (optical absorbance at 520 nm) normalized by the biomass measured by the CV method. (C) PVD quantification (excitation at 405 nm, emission at 450 nm) normalized by the biomass measured by the CV method. (D, E) Estimation of the biomass using Crystal Violet method (D) or CFU counts (E). (F) Biomass of planktonic bacteria estimated by absorbance at 600 nm.
Figure 7
Figure 7
Effect of spiramycin on swarming motility and rhamnolipids production. The dishes were incubated at 37 °C for 48 h. (A,B) Growth and swarming motility of P. aeruginosa on BM2 solid medium in absence (A) or presence (B) of spiramycin (60 μg/mL). (C) Effects of spiramycin on rhamnolipid production.
Figure 8
Figure 8
Effect of spiramycin on G. mellonella larvae infected with P. aeruginosa. (A) Mortality of G. mellonella larvae infected with P. aeruginosa treated or not with spiramycin 24 h after infection. Control larvae: non-injected larvae; mock infection: larvae injected with resuspension solution without bacteria; toxicity control: larvae injected with resuspension solution and spiramycin. (B) Transcript levels of genes encoding three antimicrobial peptides (gallerimycin, gloverin, and moricin) and lysozyme in P. aeruginosa infected larvae treated or not with spiramycin.
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