Screening of Pakistani honeys for antibacterial potential against multi-drug resistant Salmonella typhi

Abstract

The development of resistance to conventional anti-typhoid drugs and recent emergence of fluoroquinolone resistance had made it very difficult and expensive to treat typhoid fever. More recently, the appearance of extended-spectrum beta-lactamases (ESBLs) in Salmonella typhi is leading to a situation where, in some conditions, the disease may well be untreatable without extremely expensive injectables or combination therapies. As the therapeutic strategies are likely to dwindle, it becomes imperative to look for non- conventional modalities to meet any future possible challenges. Recently medical profession has rediscovered the unique and repertoire of antibacterial properties (acidity, osmolarity, hydrogen peroxide and plant derived non-peroxide activity) of honey. On the basis of these properties, Food and Drug Administration (FDA) of USA and European countries approved manuka honey (New Zealand) and medihoney (Australian) as therapeutic agents for the treatment of chronic skin infections and burns. However, its role in systemic infections like typhoid fever is not determined. In addition, striking variation in the potency of antibacterial activity of honey between different floral sources and even within the same floral source does exist. Hence, it is imperative to assay antibacterial activity of honey before consideration for therapeutic use. In Pakistan, there is great diversity in honey varieties because of the richness in bee flora; therefore, it is believed that there are several unrecognized honeys of high therapeutic value in this part of the world. Twenty four blood culture isolates of typhoid salmonellae including MDR-S. typhi (n=16) were investigated for their susceptibility to manuka honey, black seed honey, shain honey and simulated honey. An agar dilution assay was adopted for the determination of minimum inhibitory concentration. Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853) Enterococcus faecalis (ATCC 29212) and Acinetobacter baumannii (ATCC 29212) were included as the standard control strains. Additionally, one hundred samples of Pakistani unifloral honey from nineteen common bee flora, produced by bees (Apis mellifera) were collected from different geographical areas of Pakistan. They were screened for both total (acidity, osmolarity, hydrogen peroxide and non-peroxide activity) and plant derived non-peroxide antibacterial activity against one blood culture isolate of multi-drug resistant (MDR) S. typhi by agar well diffusion assay. Manuka (Unique Manuka Factor-21) honey was used as standard. The total antibacterial activity of these honey samples were standardized in accordance with phenol equivalence % (w/v). Regarding MICs, manuka honey inhibited all clinical isolates of typhoidal salmonellae at concentration median 7.67+1.0% (v/v), black seed honey median 9.0±1.0% (v/v) and shain honey at median 12.0±1.0% (v/v). Simulated honey inhibited these isolates at concentration median 30±0.0% (v/v). Whereas, manuka honey inhibited all ATCC reference strains between the MICs range of 5.0 to 9.0% (v/v), black seed honey at 6.0% to 10.0% (v/v), shain honey at 10 to 14 % (v/v) and simulated honey at 30 to 35% (v/v). This demonstrates that antibacterial effect of natural honey is not linked with high osmolarity alone and there are other factors contributing to antibacterial activity. Screening of antibacterial activity of one hundred unifloral honey samples against blood culture isolate of MDR-S. typhi revealed that nineteen honey samples (19%) displayed higher antibacterial activity (16-20 % of phenol), which is more than that of manuka honey (21-UMF). Thirty percent honey samples demonstrated antibacterial activity between the ranges of 11-15% of phenol, similar to that of manuka honey and about 51% of honey samples did not exhibit any zone of inhibition against MDR-S. typhi at 50% (w/v) dilution. Since manuka honey used in this study is standardized product with 21- UMF and approved by FDA as therapeutic agent. Therefore, those honey samples which displayed antibacterial activity equal to or greater than manuka honey, may be utilized in those clinical conditions, where higher hydrogen per-oxide related antibacterial activity is needed like leg ulcers, diabetic foot ulcers, burns, skin graft donor sites and surgical wounds. These newly identified honeys may add value to the existing honey resource. Keeping in view the tremendous variation in the potency of antibacterial activity of honey, it is essential that research may continue beyond commercially available honey to those, locally produced, as yet untested. However, none of Pakistani honey samples displayed non-peroxide activity. Only manuka honey showed non-peroxide activity when catalase enzyme was added to neutralize the hydrogen peroxide related antibacterial activity at 25% dilution (w/v). Nevertheless, MICs of black seed honey and manuka by agar dilution assay are comparable. The agar dilution assay provides more reliable, reproducible and quantitative results as compared to agar well diffusion assay which is less sensitive and only provides qualitative results. Therefore, both manuka honey and black seed honey warrants further evaluation in suitable typhoid animal model for future prevention and treatment of typhoid fever.