Exploration of the feasibility of clinical application of phage treatment for multidrug-resistant Serratia marcescens-induced pulmonary infection

Abstract

Serratia marcescens (S. marcescens) commonly induces refractory infection due to its multidrug-resistant nature. To date, there have been no reports on the application of phage treatment for S. marcescens infection. This study was conducted to explore the feasibility of phage application in treating refractory S. marcescens infection by collaborating with a 59-year-old male patient with a pulmonary infection of multidrug-resistant S. marcescens. Our experiments included three domains: i) selection of the appropriate phage, ii) verification of the efficacy and safety of the selected phage, iii) confirmation of phage-bacteria interactions. Our results showed that phage Spe5P4 is appropriate for S. marcescens infection. Treatment with phage Spe5P4 showed good efficacy, manifested as amelioration of symptoms, hydrothorax examinations, and chest computed tomography findings. Phage treatment did not worsen hepatic and renal function, immunity-related indices, or indices of routine blood examination. It did not induce or deteriorate drug resistance of the involved antibiotics. Importantly, no adverse events were reported during the treatment or follow-up periods. Thus, phage treatment showed satisfactory safety. Finally, we found that phage treatment did not increase the bacterial load, cytotoxicity, virulence, or phage resistance of S. marcescens, indicating satisfactory phage-bacteria interactions between Spe5P4 and S. marcescens, which are useful for the future application of phage Spe5P4 against S. marcescens. This work provides evidence and a working basis for further application of phage Spe5P4 in treating refractory S. marcescens infections. We also provided a methodological basis for investigating clinical application of phage treatment against multidrug-resistant bacterial infections in the future.

Keywords: Phage treatment; Serratia marcescens (S. marcescens); efficacy and safety; multidrug-resistance; phage Spe5P4.

Figure 1.

Protocol of the treatments and examination of the microorganisms associated with phage treatment. This diagram presents the treatments and examination of microorganisms over time in this patient. Results of microorganism examinations are shown as different colours. This patient underwent three types of treatment: 1. Antifungal treatment: Voriconazole (150 mg, q12 h); 2. Antibiotics treatments: Clindamycin (0.6 g, q12 h), Cefuroxime (1.5 g, q12 h), Levofloxacin (0.5 g, q.d.), Amikacin/Tazocin (0.8 g/4.5 g, q.d./q8 h), Amikacin/Meropenem (0.4 g/1 g, q8 h); 3. Phage therapy (10⁹ PFU/mL, q12 h).

Figure 2.

Integrated phage treatment of S. marcescens infection and the related synergic effects. (A) Protocol of treatments and examinations. (B) Verification of the synergic effects of phage Spe5P4 + AMK. (C) Verification of the synergic effects of phage Spe5P4 + MEM. AMK: amikacin; MEM: meropenem.

Figure 3.

Observation of the efficacy of the phage treatment with time. (A) Representative chest CT images before (blue) and after phage treatment (green). Blue arrows present the lung tissue, red arrows present the pleural fluid. Before phage treatment, the lung tissue (black parts marked with a blue arrow) was markedly compressed indicating notable atelectasis. These black parts recovered slowly, indicating a slow recovery of atelectasis before phage administration. The pleural fluid (marked with a red arrow) was large and remarkable. The reduction in this part was slow, indicating slow absorption of the inflammatory hydrothorax. However, after phage treatment, the lung tussue achieved fast recovery, and the pleural fluid became smaller. At the end of follow-up, the lung tissue significantly recovered, and the pleural fluid was greatly aborted. Thus, phage treatment contributed to better amelioration of pulmonary conditions. (B) Appearance of the hydrothorax collected by thoracentesis. Obviously, the hydrothorax became clear with the phage treatment.

Figure 4.

Verification of the safety of phage treatment. (A) Variations of the hepatic and renal functions influenced by phage treatment. Although there were mild fluctuations during the treatments, they almost came within the range of normal value (gray zone). (B) Variations of the immunity-related indices. Levels of IL-8, and NEUT changed within the range of normal value (gray zone). Levels of IL-6 and CRP were higher than normal value, but significantly reduced after treatment at 25-day follow-up. (C) Variations of the indices of blood routine examination. Levels of WBC and ESR were unchanged at 25-day follow-up, although there were fluctuations during the treatment. ESR were higher than the normal range, but it presents a reducing trend (amelioration). ALT: alanine aminotransferase, AST: aspartate aminotransferase, Cr: creatinine, CRP: C-reactive protein, ESR: erythrocyte sedimentation rate, IL-6,8: interleukin-6,8, NEUT%: neutrophil percentage, WBC: white blood cell.

Figure 5.

Characteristics of microbiological interaction of phage Spe5P4 and bacteria during inpatient period. (A) Distribution of phage Spe5P4 in hydrothorax, serum, urine and faeces samples during phage therapy. Only phage Spe5P4 in hydrothorax could be detected, whereas phage in serum, urine and faeces samples were under the limitation of detection. (B) Detection of the bacteria using a 16S rRNA analysis during period of inpatient phage treatment. S. marcescens (Serratia, pink column) was well displayed. It presented a notable and progressive reduction with the course of phage treatment.

Figure 6.

Investigation of the potential phage-bacteria interactions between phage Spe5P4 and S. marcescens. (A) LDH release of the S. marcescens on A549 cells. The LDH release was unchanged with time of phage treatment. (B) Kaplan-Meier plots of the virulence in S. marcescens evaluated using a G. mellonella infection model. The Kaplan – Meier plots represent the survival of 20 larvae that were used for each strain. (C) Visualization of plaque forming units of the S. marcescens on the patient’s strains isolated before (D0) and during phage treatment (D7 – D15). (D) Value of biofilm formation capacity (OD₅₀) of the S. marcescens before and during phage treatment. (E) Examination of the swarming capacity of the S. marcescens before and during phage treatment. (F) Examination of the swimming capacity assay of the S. marcescens before and during phage treatment. * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001 (vs. the D0 isolate).