title: Determination of antibacterial activity and minimum inhibitory concentration of larval extract of fly via resazurin-based turbidometric assay.
abstract: BACKGROUND
  Antimicrobial resistance is currently a major global issue. As the rate of emergence of antimicrobial resistance has superseded the rate of discovery and introduction of new effective drugs, the medical arsenal now is experiencing shortage of effective drugs to combat diseases, particularly against diseases caused by the dreadful multidrug-resistant strains, such as the methicillin-resistant Staphylococcus aureus (MRSA). The ability of fly larvae to thrive in septic habitats has prompted us to determine the antibacterial activity and minimum inhibitory concentrations (MICs) of larval extract of flies, namely Lucilia cuprina, Sarcophaga peregrina and Musca domestica against 4 pathogenic bacteria [Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Pseudomonas aeruginosa and Escherichia coli] via a simple and sensitive antibacterial assay, resazurin-based turbidometric (TB) assay as well as to demonstrate the preliminary chemical profile of larval extracts using gas chromatography-mass spectrophotometry (GC-MS).
  
  
  RESULTS
  The resazurin-based TB assay demonstrated that the L. cuprina larval extract was inhibitory against all tested bacteria, whilst the larval extract of S. peregrina and M. domestica were only inhibitory against the MRSA, with a MIC of 100 mg ml-1. Subsequent sub-culture of aliquots revealed that the larval extract of L. cuprina was bactericidal against MRSA whilst the larval extracts of S. peregrina and M. domestica were bacteriostatic against MRSA. The GC-MS analysis had quantitatively identified 20 organic compounds (fatty acids or their derivatives, aromatic acid esters, glycosides and phenol) from the larval extract of L. cuprina; and 5 fatty acid derivatives with known antimicrobial activities from S. peregrina and M. domestica.
  
  
  CONCLUSIONS
  The resazurin-based turbidometric assay is a simple, reliable and feasible screening assay which evidently demonstrated the antibacterial activity of all fly larval extracts, primarily against the MRSA. The larval extract of L. cuprina exerted a broad spectrum antibacterial activity against all tested bacteria. The present study revealed probable development and use of novel and effective natural disinfectant(s) and antibacterial agent(s) from flies and efforts to screen more fly species for antibacterial activity using resazurin-based TB assay should be undertaken for initial screening for subsequent discovery and isolation of potential novel antimicrobial substances, particularly against the multi-drug resistant strains.
authors.affiliation: Medical Entomology Unit, Infectious Disease Research Centre, Institute for Medical Research, Jalan Pahang, Kuala Lumpur, 50588, Malaysia. chienhuey@imr.gov.my.
authors.forename: Chien Huey
authors.identifier: http://orcid.org/0000-0001-8978-9965
authors.initials: CH
authors.lastname: Teh
authors.affiliation: Medical Entomology Unit, Infectious Disease Research Centre, Institute for Medical Research, Jalan Pahang, Kuala Lumpur, 50588, Malaysia.
authors.forename: Wasi Ahmad
authors.initials: WA
authors.lastname: Nazni
authors.affiliation: Medical Entomology Unit, Infectious Disease Research Centre, Institute for Medical Research, Jalan Pahang, Kuala Lumpur, 50588, Malaysia.
authors.forename: Ab Hamid
authors.initials: AH
authors.lastname: Nurulhusna
authors.affiliation: Medical Entomology Unit, Infectious Disease Research Centre, Institute for Medical Research, Jalan Pahang, Kuala Lumpur, 50588, Malaysia.
authors.forename: Ahmad
authors.initials: A
authors.lastname: Norazah
authors.affiliation: Medical Entomology Unit, Infectious Disease Research Centre, Institute for Medical Research, Jalan Pahang, Kuala Lumpur, 50588, Malaysia.
authors.forename: Han Lim
authors.initials: HL
authors.lastname: Lee
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D001419:Bacteria / Q000187:drug effects; D004175:Diptera / Q000737:chemistry; D055808:Drug Discovery; D004353:Drug Evaluation, Preclinical; D004926:Escherichia coli / Q000187:drug effects; D004952:Esters / Q000737:chemistry / Q000494:pharmacology; D005227:Fatty Acids / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods; D006027:Glycosides / Q000737:chemistry; D006793:Houseflies / Q000737:chemistry; D007814:Larva / Q000737:chemistry*; D055624:Methicillin-Resistant Staphylococcus aureus / Q000187:drug effects; D008826:Microbial Sensitivity Tests; D010078:Oxazines / Q000737:chemistry*; D019800:Phenol; D011550:Pseudomonas aeruginosa / Q000187:drug effects; D058493:Sarcophagidae / Q000737:chemistry; D013211:Staphylococcus aureus / Q000187:drug effects; D014020:Tissue Extracts / Q000494:pharmacology*; D014966:Xanthenes / Q000737:chemistry*
chemical_list: D000900:Anti-Bacterial Agents; D004952:Esters; D005227:Fatty Acids; D006027:Glycosides; D010078:Oxazines; D014020:Tissue Extracts; D014966:Xanthenes; C005843:resazurin; D019800:Phenol
keywords: Antibacterial activity; Lucilia cuprina; MRSA; Maggot therapy; Resazurin