A novel combination approach of human polyclonal IVIG and antibiotics against multidrug-resistant Gram-positive bacteria

Abstract

Background: Gram-positive bacteria, especially methicillin-resistant Staphylococcus aureus (MRSA) and enterococci, have shown a remarkable ability to develop resistance to antimicrobial agents.

Objective: We aimed to assess possible enhancement of the antimicrobial activity of vancomycin, amoxicillin, clarithromycin, and azithromycin by human polyclonal intravenous immunoglobulin G (IVIG) against 34 multidrug-resistant (MDR) bacterial isolates, including MRSA, Enterococcus faecium, and Enterococcus faecalis.

Materials and methods: Double combinations of the antibiotics with the IVIG were assessed by checkerboard assay, where the interaction was evaluated with respect to the minimum inhibitory concentration (MIC) of the antibiotics. The results of the checkerboard assay were verified in vitro using time-kill assay and in vivo using an invasive sepsis murine model.

Results: The checkerboard assay showed that IVIG enhanced the antimicrobial activity of amoxicillin and clarithromycin against isolates from the three groups of bacteria, which were resistant to the same antibiotics when tested in the absence of IVIG. The efficacy of vancomycin against 15% of the tested isolates was enhanced when it was combined with the antibodies. Antagonism was demonstrated in 47% of the E. faecalis isolates when clarithromycin was combined with the IVIG. Synergism was proved in the time-kill assay when amoxicillin was combined with the antibodies; meanwhile, antagonism was not demonstrated in all tested combinations, even in combinations that showed such response in checkerboard assay.

Conclusion: The suggested approach is promising and could be helpful to enhance the antimicrobial activity of not only effective antibiotics but also antibiotics that have been proven to be ineffective against MDR bacteria. To our knowledge, this combinatorial approach against MDR bacteria, such as MRSA and enterococci, has not been investigated before.

Keywords: Enterococcus faecalis; Enterococcus faecium; MRSA; amoxicillin; human polyclonal IVIG; multidrug resistance; nonconventional antimicrobials; vancomycin.

Figure 1

Double combination of the antibiotics with human polyclonal IVIG against the clinical isolates. Notes: Checkerboard assay was used to assess the combination of amoxicillin, vancomycin, azithromycin, or clarithromycin with polyclonal IVIG against 34 clinical isolates of MRSA (A), E. faecium (B), and E. faecalis (C). The interaction of the combined therapy was assessed with respect to the MICs of the antibiotics. The interaction is defined as synergistic if the MIC of the antibiotic decreased by twofold or more compared to its MIC alone. The interaction is indifferent if the MIC of the antibiotic remained unchanged, increased, or decreased by onefold concentration in combination. The interaction is antagonistic if the MIC of the antibiotic increased by twofold or more in combination with the polyclonal IVIG. Abbreviations: IVIG, intravenous immunoglobulin G; MRSA, methicillin-resistant Staphylococcus aureus; E. faecium, Enterococcus faecium; E. faecalis, Enterococcus faecalis; MIC, minimum inhibitory concentration.

Figure 2

Evaluation of double combination of amoxicillin with polyclonal IVIG against MRSA isolate C19 using time-kill assay. Notes: Isolate C19 was selected based on the synergistic response in checkerboard assay when IVIG was combined with amoxicillin (FIC = 0.125). The bacterial suspension at a density of 1 × 10⁵ CFU/mL was used to inoculate 50 mL MHB in 250 mL Erlenmeyer flasks, before being incubated at 37°C under shaking at 200 rpm for 8 hours. The samples were collected at 2-hour intervals, and the viable bacterial counts were determined. A given antibiotic and the polyclonal IVIG were tested individually and in combination using the following four sets of experiments: (A) 10 μg/mL of IVIG and amoxicillin at one-fourth of MIC, (B) 100 μg/mL of IVIG and amoxicillin at one-fourth of MIC, (C) 10 μg/mL of IVIG and amoxicillin at half of MIC, and (D) 100 μg/mL of IVIG and amoxicillin at half of MIC. Abbreviations: IVIG, intravenous immunoglobulin G; MRSA, methicillin-resistant Staphylococcus aureus; FIC, fraction inhibitory concentration; CFU, colony forming units; MHB, Müeller-Hinton broth; MIC, minimum inhibitory concentration; SD, standard deviation.

Figure 3

Evaluation of double combination of amoxicillin with polyclonal IVIG against E. faecalis isolate EF4 using time-kill assay. Notes: Isolate EF4 was selected based on the synergistic response in checkerboard assay when IVIG was combined with amoxicillin (FIC = 0.25). A given antibiotic and the polyclonal IVIG were tested individually and in combination using the following four sets of experiments: (A) 10 μg/mL of IVIG and amoxicillin at one-fourth of MIC, (B) 100 μg/mL of IVIG and amoxicillin at one-fourth of MIC, (C) 10 μg/mL of IVIG and amoxicillin at half of MIC, and (D) 100 μg/mL of IVIG and amoxicillin at half of MIC. Abbreviations: IVIG, intravenous immunoglobulin G; E. faecalis, Enterococcus faecalis; FIC, fraction inhibitory concentration; MIC, minimum inhibitory concentration; SD, standard deviation.

Figure 4

Evaluation of the antimicrobial activity of amoxicillin and vancomycin in combination with polyclonal IVIG against invasive MRSA infection in a murine model. Notes: Eight groups each of five mice were used to assess the combination therapy against invasive sepsis by MRSA isolate C19. The groups of the animal assigned for infection were injected IP with 1 mL of the bacterial suspension (1 × 10⁸ CFU/mL). Animals were treated with the antibiotics or the IVIG individually or in combination. On the fifth day post injection, mice were euthanized, the kidney, lung, heart, and liver were aseptically collected and homogenized, and viable bacterial counts were determined. Abbreviations: IVIG, intravenous immunoglobulin G; MRSA, methicillin-resistant Staphylococcus aureus; IP, intraperitoneally; CFU, colony forming units; SD, standard deviation.