Case report: Personalized triple phage-antibiotic combination therapy to rescue necrotizing fasciitis caused by Panton-Valentine leukocidin-producing MRSA in a 12-year-old boy

Abstract

Maximal standard-of-care (SOC) management could not stop the life-threatening progression of a necrotizing fasciitis induced by Panton-Valentine Leukocidin-producing Methicillin-Resistant Staphylococcus aureus (MRSA) in a 12-year-old boy. Multi-route phage therapy was initiated along with antibiotics against Staphylococcus aureus, Pseudomonas aeruginosa and Stenotrophomonas maltophilia, eventually leading to full recovery with no reported adverse events.

Keywords: MRSA; Panton and Valentine leukocidin (PVL); bacteriophage; necrotizing fasciitis; pediatric intensive care unit; phage therapy.

Figure 1

Timeline of the clinical case's main events. Main medico-surgical events are represented as blue vertical arrows. Phage therapy-related events are represented in pink. Pink curve represents degree of Phage Immune Neutralization (PIN) against phage ISP in % of neutralized titer at 30 minutes. Error bars represent standard deviation of the means. Red droplets represent units (205 mL) of blood transfused. D , day; Sa, Staphylococcus aureus; Pa, Pseudomonas aeruginosa ; Sm, Stenotrophomonas maltophilia ; B, Blood culture ; R, Respiratory tract-originated sample culture (broncho-alveolar lavage or endotracheal aspirate) ; W, wound sample culture (swab or intraoperative biopsy); PT, phage therapy; IV, intravenous; PN, pulmonary nebulization; IP, intra-pleural; IA, intra-abdominal; CTX, Cefotaxime; FLX, Flucloxacillin; CLN, Clindamycin; VAN, Vancomycin; CPT, Ceftaroline; MEM, Meropenem; CIP, Ciprofloxacin; CST, Colistin.

Figure 2

Full body MRI, coronal view. Short-TI inversion recovery (STIR) mode. Image shows diffuse hyperintense inflammatory infiltration in subcutaneous soft tissues of both lower limbs, the upper left limb, the thorax and the abdomen, as well as disseminated inflammatory pulmonary lesions and two inflammatory fluid collections in the right thigh and left shoulder (red arrows).

Figure 3

Phage-antibiotic interaction testing. Activities of phage ISP (at multiplicity of infection [MOI] = 0.01 and 0.001) and ceftaroline (at 8mg/L) as well as the combinations of these phage titers with this ceftaroline concentration, were determined using an OmniLog® automated incubator (Biolog, Hayward, CA, USA). Data was analyzed with OmniLog data Analysis Software (v1.7). Experiments were done in 96-well plates in a final volume of 200 µl of LB supplemented with 100 times diluted tetrazolium dye mix, according to the manufacturer’s instructions. Bacterial cells were added at a concentration of 105 colony forming units(cfu)/well, calculated based on optical density (OD, at 600 nm) measurements (with an OD of 0.5 corresponding to 4 x 108 cfu/ml, on average), which were validated using a classical plate culture method. Plates were incubated at 37 ºC for 72 hours and a possible reduction (causing a color change) of the tetrazolium dye due to bacterial respiration (during growth) was monitored and recorded every 15 min by the Omnilog system. Bacterial proliferation is presented through relative units of cellular respiration : efficacious phages, antibiotics, and combinations thereof, suppress bacterial proliferation. "Control Sa" curve represent control growth of a triplicate containing only the aforementioned bacterial load (S. aureus), with no antibiotic or phage. Results are presented as mean values of biological triplicates. While ceftaroline alone at 8mg/L and phage ISP alone at two different MOIs appear unable to durably suppress bacterial growth over the 72 hours of growth analysis, combination of this ceftaroline concentration with either of these two sub-inhibitory phage titers leads to durable suppression of bacterial growth, suggesting in vitro synergistic properties.