Use of human polyclonal intravenous immunoglobulin (IVIG) as adjuvant therapy in combination with antibiotics against multi-drug resistant gram-positive bacteria M.H. Madkour, , K. Abou-Aisha, M. A. El Azizi Department of Microbiology and Immunology, Faculty of Pharmacy and Biotechnology Abstract Materials and Methods Results and Conclusions

Introduction: Gram-positive bacterial pathogens have shown a •Adjustment of bacterial inoculum size −Calibrated turbidity measurement at the wavelength 600nm showed correlation withcell remarkable ability to develop resistance to antimicrobial agents. 10 10 Infections caused by methicillin and glycopeptides- resistant density. The absorbance with the value of 1 is equivalent to a bacterial control control ½ MIC staphylococci and glycopeptides- resistant enterococci are getting cell density of 108 cells/ml. Then a tenfold serial dilution was done to 8 ¼ MIC 8 C 1 9 ) IVIG (100 µg/ml) obtain the final concentration 105 cells/ml. C 1 9 ) IVIG (100 µg/ml) 6 ½ MIC + IVIG more complicated by the ability of these bacteria to develop 6 ¼ MIC + IVIG (100 µg/ml) (100 µg/ml) MRSA resistance to antimicrobial agents. This study aims to study the MRSA 4 •Minimum inhibitory concentration (MIC) – MICs were determined in 4 effects of human polyclonal intravenous immunoglobulin (IVIG) Mueller-Hinton Broth (Oxoid Ltd, Hampshire, United Kingdom) 2 as adjuvant therapy against multi-drug resistant bacteria 2 log c fu ( containing antibiotics serially twofold diluted in order toobtainthe logc fu( including methicillin resistant -Staphylococcus aureus (MRSA), 0 following final concentrations 0.0625 to 32 µg/ml. MICs of a combination 0 0246810 0246810 Enterococcus faecium and Enterococcus faecalis. of the antibiotic and 10 µg/ml IVIG were also determined. Isolates were time (hours) Methods: The in vitro antimicrobial activity of the IVIG (5 µµµg/ml) sub-cultured on Nutrient agar (Oxoid Ltd, Hampshire, UnitedKingdom) time (hours) alone and in combination with amoxicillin, vancomycin, at 37 Covernighttheninoculumof105 cfu was applied. After incubation Fig. (1) Effect of the combination of 100µg/ml IVIG and Fig. (2)Effect of the combination of 100µg/ml IVIG and clarithromycin and azithromycin against MRSA (7 isolates), E. for 24h at 37 C. MICs were defined as the lowest concentration at which amoxicillin (¼ MIC) against the clinical isolate MRSA C19 amoxicillin (½ MIC) against the clinical isolate MRSA C19 faecium (8 isolates) and E. faecalis (19 isolates) was evaluated there was no visible growth. 10 10 using checkerboard and time kill assays. control control )

¼ MIC 4 •Minimum bactericidal concentration (MBC) – Bactericidal 8 8 ½ MIC

Results: IVIG showed synergistic effect when was combined with s i

IVIG (100 µg/ml) l

concentration of amoxicillin, vancomycin, azithromycin and a IVIG (100 µg/ml) 6 amoxicillin against, MRSA (14%) , E. faecium (12.5%)and E. 6 ¼ MIC + IVIG (100 c clarithromycin (Sigma, United States) were determined for all the isolates. e ½ MIC + IVIG (100 µg/ml) a

faecalis (16%) isolates. Using vancomycin with IVIG showed the f

(E.faecalis 4) 4 After 24h incubation at 37 C, MBCs were determined by the sub-culture 4 . µg/ml) E (

highest synergistic effect on MRSA isolates (29%) compared to

of all wells with no visible growth. u 2 2 f

E. faecium (12.5%) and E. faecalis (10.5%). Combination of IVIG c log c fu

g 0 o

with azithromycin was indifferent when tested against the 0 l •Fractional inhibitory concentration index (FIX) – The FIX was defined 0246810 246810 isolates. On the other hand the combination of clarithromycin as follows: FIX + . -2 and IVIG had an antagonistic effect on 47% of E. fecalis isolates. time (hours) time (hours) Conclusion: Use of IVIG as adjuvant therapy in combination The interaction was then defined as synergistic if the FIX was ≤ 0.5, with antibiotic in treatment of infections caused by multi-drug Fig. (3) Effect of the combination of 100µg/ml IVIG and Fig. (4)Effect of the combination of 100µg/ml IVIG and amoxicillin antagonistic if the FIX was ≥ 4andnointeractioniftheFIXwas>0.5–4. amoxicillin (¼ MIC) against the clinical isolate E.faecalis 4 (½ MIC) against the clinical isolate E.faecalis 4 resistant gram positive bacteria would improve the treatment

outcomes. This needs to be further investigated in in vivo model. •Death curve − For the most effective isolates a death curve experiment 10 10 control control

was performed. 500µlofthebacterialsolutionhavingtheconcentration )

6 ¼ MIC 8 ¼ MIC 8

5 N 10 cells/ml were added to a flask containing 50 ml Mueller-Hinton Broth. IVIG (100 µg/ml) Introduction IVIG (10 µg/ml) A 6 S 6 ¼ MIC + IVIG (100

Every 2 hours for a period of 8 hours a sample of 100 lwasdrawnand10 R µ ¼ MIC + IVIG (10 µg/ml)

M µg/ml) ( MRSA N6) MRSA fold serially diluted. Then sub-cultured on nutrient agar and incubated 4 4 u In 2014, 7 Millions patients died, suffering from infections caused f c overnight at 37 C. g

o 2 by multi-drug resistant pathogens. This number will increase 2 l dramatically by 2050 reaching 10 Millions patients, which is more log cfu ( •Analysis of the efficacy of IVIG in vivo − An over-night culture of MRSA 0 0 than patients dying from cancer nowadays. As for the economic 0246810 was washed and re-suspended in sterile saline (Nad EL Nasr 0246810 impact, it will reach 100 Trillion US Dollars in 2050. To understand time (hours) time (hours) Pharmaceutical Chemistry, Egypt) and used to prepare an inoculum of this number, the global GDP is 70 Trillion US dollars. 108 cfu/ml, which then injected as 1ml intra-peritoneal (IP). After that Fig. (5) Effect of the combination of 10µg/ml IVIG and Fig. (6) Effect of the combination of 100µg/ml IVIG and clarithromycin Resistance of pathogens is a major concern and the source of many the IVIG and the antibiotic were injected into the mice. 120 hours post clarithromycin (¼MIC) against the clinical isolate MRSA N6 (¼ MIC) against the clinical isolate MRSA N6 problems in hospitals and health care institutions. Over the last injection the mice were sacrificed and the number of remaining three decades the magnificent success of the pharmaceutical 1) When amoxicillin was used as antibacterial, IVIG provided a synergistic effect in 12 to 16% of strains of MRSA, E. pathogens were counted in several organs of the animals. industry in the United States, Japan, the United Kingdom, France faecium and E.faecalis. and Germany has caused and increased threat of bacterial 2) Using vancomycin with IVIG showed the highest synergistic effect on MRSA isolates (29%) compared to E.faecium resistance (5). Results and Conclusion (12.5%) and E.faecalis (10.5%). On of the often encountered classes of pathogens is Hospital- 3) On the other hand the combination of vancomycin and IVIG had an antagonistic effect on 14% of MRSA. associated MRSA (HA-MRSA). HA-MRSA are resistant to nearly all 4) IVIG alone was in no case able to kill the bacteria completely. However, when 100 µg/ml IVIG were administered in combinations of β-lactam inhibitors, and often show multiple MRSA E. faecium E. faecalis addition to 25 to 50% of the MIC of amoxcillin, the bacterial growth of MRSA strain C19 and E.faecalis strain 4 resistance also to other classes of antimicrobials (7). came to a complete stop after 8 hours. Enterococci, another example for the multi-drug resistant 100 86% 87.5% 84 % pathogens, are bacteria residing mostly in the gastrointestinal tract. 80 5) Adding 10 and 100µg/ml IVIG to 25% of the MIC of clarithromycin deacreased the bacterial growth from 8.985 Their activity as nosocomial pathogens has recently been a source of 60 0 .000 to 4.263 0.185 and 4.100 0.174. 40 severe infections not only of the urinary tract, but also of the blood 14% 16% Per cent age ( %) 20 12.5% stream. The choice of treatment for these infections has become 0 more and more difficult (2). Conclusion: Several reports have claimed IVIG ability to act as an antimicrobial agent by neutralizing the New compounds and alternative methods were investigated to find a pathogen toxins and super-antigen and preventing it to reach the target site. The combination therapy of the synergi sti c synergi st i c synergi st i c new treatment approach to overcome the multi-drug resistant no i nt er act i on no i nt er act i on no i nt er act i on antibiotic together with IVIG can be quite effective in certain cases and may help to overcome drug resistance. This nature of many pathogens, which made the treatment with needs to be further investigated in vivo model. conventional antibiotics does not assure desired clinical outcomes Addition of 10µg/ml IVIG to amoxicillin against MRSA, Enterococcus faecalis and Enterococcus faecium. (4). It was proven that many combination therapies increase the activity of the chosen antibiotic significantly (1). E. faecalis Commercially pooled polyclonal human Intravenous E. faecium References immunoglobulin (IVIG) is approved by the FDA as antimicrobial 100 MRSA agent. IVIG has shown benefits against numerous infections, 87.5% 89.5% 1) Aslangul, E., Ruimy, R., Garry, L., Andremont, A., & Fantin, B. (2005). Relationship between the Level of Acquired Resistance to Gentamicin and showing resistance to several antibiotic (3). Its mechanism of action 80 Synergism with Amoxicillin in Enterococcus faecalis. Antimicrobial Agents and Chemotherapy, 49(10), 4144–4148. doi:10.1128/AAC.49.10.4144. 57% 2) Gülhan, T., Aksakal, A., N, H. E. K., Savafian, S., & Boynukara, B. (2006). Virulence Factors of Enterococcus faecium and Enterococcus faecalis Strains was understood throughout the last few years. The mechanism of 60 40 Isolated from Humans and Pets. Turk. J. Vet. Anim. Sci. 30, 477–482. Retrieved from https://webvpn.uni- action depends mainly on the immunoglobulin dose and the 29% 14% ulm.de/+CSCO+ch756767633A2F2F77626865616E79662E67686F76676E782E7462692E6765+3753036114@5431296@1418493117@3ADF9EF6AFD pathogenesis of the disease. The administration of a low dose of Per cent age ( %) 20 12.5% 10.5% D3CE7984713CFA395AB76D9622A49+/veterinary/issues/vet-06-30-5/vet-30-5-8-0510-22.pdf IVIG has an antimicrobial activity, which could be explained by the 0 3) Jolles, S., Sewell, W. a C., & Misbah, S. a. (2005). Clinical uses of intravenous immunoglobulin. Clinical and Experimental Immunology, 142(1), 1–11. ability of the IVIG to neutralize the pathogen toxin and super- doi:10.1111/j.1365-2249.2005.02834.x. antigens and by preventing it to reach the target site. Here, we 4) Kyaw, B;Arora, S; Lim, C. (2012). Bactericidal antibiotic-phytochemical combinations against methicillin resistant. Brazilian Journal of Microbiology, 4062, 938–945. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3768864/pdf/bjm-43-938.pdf.

propose the addition of IVIG to the antimicrobial agent, which synergistic synergistic synergistic antagonistic

no i nt er act i on no i nt er act i on no i nt er act i on 5) Neu, H. C. (1993). The Crisis in Antibiotic Resistance. Siecence, 21. DOI: 10.1126/science.257.5073.1064. could help in the treatment of multi-drug resistant strains due to its 6) Nimmerjahn, F., & Ravetch, J. V. (2008). Anti-Inflammatory Actions of Intravenous Immunoglobulin. Annual review of Immunology, 26, 513-533. antimicrobial mechanism (6). doi:10.1146/annurev.immunol.26.021607.090232.

Addition of 10µg/ml IVIG to vancomycin against MRSA, Enterococcus faecalis and Enterococcus faecium 7) Palavecino, E. (2007). Clinical, epidemiological, and laboratory aspects of methicillin-resistant Staphylococcus aureus (MRSA) infections. Methods in molecular biology (Clifton, N.J.) (Vol. 391, pp. 1–19). doi:10.1007/978-1-59745-468-1_1.