Staphylococcus aureus α-Toxin Response Distinguishes Respiratory Virus-Methicillin-Resistant S. aureus Coinfection in Children

Abstract

Background: Development of methicillin-resistant Staphylococcus aureus (MRSA) pneumonia after a respiratory viral infection is frequently fatal in children. In mice, S. aureus α-toxin directly injures pneumocytes and increases mortality, whereas α-toxin blockade mitigates disease. The role of α-toxin in pediatric staphylococcal-viral coinfection is unclear.

Methods: We enrolled children across 34 North American pediatric intensive care units with acute respiratory failure and suspected influenza virus infection. Serial serum anti-α-toxin antibody titers and functional α-toxin neutralization capacity were compared across children coinfected with MRSA or methicillin-susceptible S. aureus (MSSA) and control children infected with influenza virus only. MRSA isolates were tested for α-toxin production and lethality in a murine pneumonia model.

Results: Influenza virus was identified in 22 of 25 children with MRSA coinfection (9 died) and 22 patients with MSSA coinfection (all survived). Initial α-toxin-specific antibody titers were similar, compared with those in the 13 controls. In patients with serial samples, only MRSA-coinfected patients showed time-dependent increases in anti-α-toxin titer and functional neutralization capacity. MRSA α-toxin production from patient isolates correlated with initial serologic titers and with mortality in murine pneumonia.

Conclusions: These data implicate α-toxin as a relevant antigen in severe pediatric MRSA pneumonia associated with respiratory viral infection, supporting a potential role for toxin-neutralizing therapy.

Keywords: bacteria; influenza; intensive care unit; pediatric; pneumonia; respiratory failure.

Figure 1.

α-Toxin–specific antibody measurements on a representative patient coinfected with methicillin-resistant Staphylococcus aureus (MRSA) and influenza virus. Four serial serum samples from 1 MRSA-coinfected patient, a 2.7-year-old male presenting with shock and acute lung injury, were subjected to the assays as described in “Methods” section; a healthy adult donor control serum is included. A, α-Toxin–binding antibody as measured by enzyme-linked immunosorbent assay (ELISA), using an immunoglobulin G (IgG)–specific secondary detector. The x-axis denotes the reciprocal dilution of patient serum. The dotted line denotes the threshold for computation of the end point titer, arbitrarily defined at 0.1 specific absorbance units. B, Residual hemolytic activity of 2 nM recombinant α-toxin incubated with serial dilutions of patient serum. Lines denote minimum, maximum, and half-maximum hemolytic activities. The computed titers and inhibitory concentration for half-inhibition (IC₅₀) levels are as listed.

Figure 2.

Methicillin-resistant Staphylococcus aureus (MRSA) coinfection elicited a prominent anti-α-toxin response relative to methicillin-susceptible S. aureus (MSSA) coinfection. A, α-Toxin–specific antibody level displayed as a function of study day and by coinfection with MRSA, MSSA, or neither. B, Ratio of highest convalescent titer vs acute titers for each patient when serial specimens were available, classified by coinfection status. C, α-Toxin–specific antibody assessed by functional neutralization of recombinant α-toxin in a rabbit erythrocyte lysis assay. “IC₅₀” indicates the inhibitory concentration of diluted serum that results in 50% inhibition of 2 nM recombinant α-toxin. D, Ratio of highest neutralization titer in convalescent sera vs acute sera for each patient when serial specimens were available, classified by coinfection status. Linear mixed-effects model analysis on log-transformed data revealed that the time profiles for MSRA are significantly different from those for MSSA and no S. aureus for α-toxin–binding immunoglobulin G (IgG) and α-toxin–neutralizing IC₅₀, as indicated above. Black symbols indicate patients who died during admission, while open symbols indicate patients with influenza-like illness who had an alternate viral diagnosis. Bars indicate geometric means. *P < .05, **P < .01, and ***P < .001.

Figure 3.

α-Toxin production by clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates correlated with admission α-toxin titer. MRSA isolates from 12 patients were analyzed for in vitro α-toxin production and normalized against S. aureus strain LAC, a known high producer of the toxin. α-Toxin–binding antibody titer (A) or toxin neutralization activity (B) of the corresponding patient's initial sera displayed as a function of in vitro α-toxin production. Mean toxin production from 3 independent determinations is shown. Black symbols indicate fatal cases, while open symbols indicate patients with influenza-like illness who had an alternate viral diagnosis. Abbreviations: IC₅₀, half-maximal inhibitory concentration; IgG, immunoglobulin G.

Figure 4.

α-Toxin production by clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates correlated with disease severity in experimental mouse S. aureus pneumonia. A, Kaplan–Meier survival curves of C57Bl/6 mice infected with various S. aureus strains (n = 19–20/group), including control strains LAC (MRSA USA300) and Newman (Nm; methicillin-susceptible S. aureus). Strains are arrayed in the legend as a function of increasing in vitro α-toxin production. B, Log_e-transformed hazard ratios of each strain against S. aureus LAC, graphed against strain in vitro production of α-toxin. The measures for strain LAC are 0 and 1, respectively (lines). Error bars indicate standard errors of the mean of each measure. Deming linear regression is shown.