Determination of antibacterial activity and minimum inhibitory concentration of larval extract of fly via resazurin-based turbidometric assay

The advent of antibiotics had improved mankind’s health status and quality of life tremendously. However, overuse and misuse of antibiotics had resulted in increased development and occurrence of bacterial resistance against commercially available antibiotics [21]. Therefore, the search for novel antibiotics of natural origins, particularly from marine resources [5, 15] and plants [7, 11] has been undertaken persistently. Nonetheless, studies which looked into the possibility of discovery of antimicrobial agent(s) from insects are scarce.

Insects, particularly the immature of flies, by nature of their biology and the ability to breed and thrive in septic habitats such as cadavers, carcasses, wounds and decaying organic matters, are infested with a great variety of microorganisms, some of which are pathogens [2]. In order to protect themselves from the undesirable effects of these microbes, insect immatures are known to produce and secrete potent anti-microbial substances [1, 4, 10, 16, 17] which may be the potential sources of novel antibiotics, especially for treatment of diseases caused by the multi-drug resistance strains such as the methicillin-resistant Staphylococcus aureus (MRSA).

The promising antibacterial activity of the local blowfly larvae, Lucilia cuprina against a wide range of pathogenic bacteria [19] which had in turn made it an effective medicinal larvae in maggot debridement therapy [13] as well as the ability of fly to thrive in septic environment have prompted our interest to screen the larval extract of other fly species against pathogenic bacteria. For screening purposes, it is essential to employ an in vitro antibacterial assay that is simple, reliable, sensitive, and most importantly, require a minimal amount of crude extract. The inability of turbidometric assay to differentiate dead and alive bacteria as well as the insensitivity of well-diffusion assay [9] had hindered the screening process.

The resazurin-based turbidometric (TB) assay was first used to quantify bacterial content in milk by Pesch and Simmert in 1929 [14]. Resazurin (7-Hydroxy-3H-phenoxazin-3-one 10-oxide) is a blue dye which can be irreversibly reduced to a pink and highly red fluorescent substance, resorufin by oxidoreductase within viable cells. The resorufin can be further reduced to a colorless and non-fluorescent molecule, hydroresorufin. In light of the simplicity and high-throughput of the resazurin-based TB assay, it has been employed in many studies as an antibacterial screening assay (Sarker et al., 2007; [18]; Hussain et al., 2011 [6]; Gahlaut & Chhillar, 2013; [3]), however, these studies only involved screening of antimicrobial activity of phytochemicals.

Therefore, the present study aimed to determine the antibacterial activity and properties (bactericidal or bacteriostatic) as well as minimum inhibitory concentrations (MICs) of larval extracts of Lucilia cuprina, Sarcophaga peregrina, and Musca domestica against Staphylococcus aureus, MRSA, Pseudomonas aeruginosa and Escherichia coli via resazurin-based TB assay, as well as to demonstrate the chemical profiles of these larval extracts using gas chromatography-mass spectrophotometry (GC-MS).

In the resazurin-based turbidometric (TB) assay, all sterility control wells for all tested bacteria remained as blue colour after an overnight incubation and followed by a 4-h incubation with resazurin. In contrast, all wells in the growth control column (contained growth medium and bacteria) of all tested bacteria had changed from blue to pink colour or from blue to pale pink (Additional files 1, 2, 3 and 4).

In contrast, the MIC of chloramphenicol against MRSA was as high as 25 mg ml^-1 and all larval extracts (L. cuprina, S. peregrina and M. domestica) were inhibitory against MRSA at MICs of 100 mg ml^-1. On the other hand, for the gram-negative bacteria, P. aeruginosa and E. coli, the MICs of gentamicin were 1.56 mg ml^-1 and 0.78 mg ml^-1, respectively. The larval extract of L. cuprina was active against both P. aeruginosa and E. coli at MIC of 100 mg ml^-1, however, similar to those observed in S. aureus, larval extracts of S. peregrina and M. domestica did not exhibit any antibacterial activity against these gram-negative bacteria. The MICs of standard antibiotics and larval extracts against all tested bacteria were summarised in Table 1.

When aliquots were removed from the corresponding MIC wells of standard antibiotics (gentamicin or chloramphenicol) or L. cuprina larval extract, no bacterial growth was observed for all BHIA plates because bacteria cells were unable to resume growth (Additional file 5). However, aliquots of MRSA resumed growth on BHIA plates after it was removed from the MIC wells of S. peregrina and M. domestica larval extract (Additional file 6) though the resazurin-based TB assay demonstrating that the growth of MRSA was inhibited.

In resazurin-based TB assay, viable and metabolically active bacteria cells irreversibly reduced the blue dye, resazurin to a pink and highly red fluorescent compound, resorufin and finally to a colourless and non-fluorescent molecule, hydroresorufin by oxidoreductase [8]. Such change of colour can be observed visually and therefore spectrophotometer is not needed in this assay as compared to the conventional TB assay.

Generally, the larval extract of L. cuprina was inhibitory against all tested bacteria, whilst the larval extract of S. peregrina and M. domestica were only active against MRSA. The broad spectrum inhibitory effects of the larval extract of L. cuprina against all tested bacteria in the resazurin-based TB assay were in agreement with those reported by Teh et al. [19] who employed conventional TB assay, and this again substantiated that the resazurin-based TB assay generated comparable results with the conventional TB assay and therefore can be considered as a simple, reliable and feasible antibacterial assay since it does not require a spectrophotometer to determine the bacterial growth. On the other hand, it was noteworthy that the MICs of L. cuprina larval extract as determined in the present study (100 mg ml^-1) was relatively higher than those reported by Teh et al. [19]. The inconsistency in the MICs value though using the same fly species (L. cuprina) could be due to different definitions of MIC. Teh et al. [19] defined the MIC endpoints of larval extract against bacteria as the lowest concentration of larval extract (mg ml^-1) resulting in at least 50% bacterial growth inhibition relative to that of the corresponding controls. In contrast, the present study defined MIC as the lowest concentration of larval extract resulting in color change (from blue to pink) or resazurin reduction. Since reduction of resazurin could only performed by viable bacterial cells, therefore, the MICs determined by the resazurin-based TB assay were relatively higher as compared to those determined via conventional TB assay [19] because more larval extract was required to inhibit bacterial growth to less than 80 bacterial cells ([12] had demonstrated that visible change of color from blue to pink can be detected in as few as 80 cells). Besides, the use of heavier bacterial inoculum in the present study i.e. 1.5 x 10⁶ colony-forming unit/ ml (CFU/ml) compared to only 1.0 x 10² CFU/ml in the previous study by Teh et al. [19] may also attribute to the discrepancy in MICs.

To the best of the author’s knowledge, the inhibitory effect as well as the MICs of the S. peregrina and M. domestica larval extract against MRSA had never been determined. The apparent potency of the L. cuprina, S. peregrina and M. domestica larval extract against MRSA provided promising input for probable identification, isolation and purification of novel effective antibacterial compound(s) of natural origin, particularly to combat the dreadful bacterial strain, MRSA.

On the other hand, the inactivity of M. domestica larval extract against S. aureus, P. aeruginosa and E. coli was not in agreement with those reported by other investigators. Golebiowski et al. [4] who had successfully identified 7 compounds from the larvae of M. domestica had revealed that 2,4-decadienal exhibited the strongest antibacterial activity among the other 6 compounds, with MIC of 64 mg ml^-1 against S. aureus and 512 mg ml^-1 against P. aeruginosa and E. coli. The discrepancies between the present study and those reported by Golebiowski et al. [4] could be partly due to a lower bacterial inoculum tested (5 x 10⁵ CFU/ml) and also the employment of different antibacterial assay [turbidometric assay in the study by Golebiowski et al. [4]. In the study by Golebiowski et al. [4], the MICs of active compounds were defined as the lowest concentration of active compound at which growth inhibition was clearly visible (absence of turbidity and a pellet at the bottom of the well). Such MIC determination method could result in overestimating the inhibitory effects of the active compounds since visual absence of turbidity and a pellet at the bottom of the well may not guarantee bacterial inhibition as bacterial growth can still occur microscopically. On the other hand, for resazurin-based TB assay, the reduction of resazurin to resorufin by viable cells which in turn lead to visible change of color from blue to pink can be detected in as few as 80 cells [12] and therefore reduced the likelihood of overestimating the inhibitory effect of larval extract.

In terms of antibacterial properties, the inability of bacterial cells to resume growth on the brain heart infusion agar (BHIA) after being transferred from the MIC wells indicated that the standard antibiotics (chloramphenicol for S. aureus and MRSA; gentamicin for P. aeruginosa and E.coli) and L. cuprina larval extract were indeed bactericidal against all tested bacteria at the corresponding MICs. On the other hand, larval extracts of S. peregrina and M. domestica larval extract were unable to suppress the growth of MRSA on BHIA and this signposted that the larval extracts of S. peregrina and M. domestica exerted bacteriostatic effect against MRSA. Although larval extracts exhibited different properties of antibacterial activity (bactericidal or bacteriostatic) against bacteria, the clinical importance of bacteriostatic versus bactericidal effect on microorganisms is under dispute. Therefore, when screening potential substances for antibacterial activity, the antibacterial properties (bactericidal or bacteriostatic) of that particular substance should never be used to rule out its potential value as an efficient antibacterial drug. It should also be noted that although the MICs of larval extract were higher than the MICs of standard antibiotics, the standard antibiotics were composed of purified active ingredients as compared to the crude extracts of fly larvae. Therefore, a smaller amount of larval extract is expected to exhibit the antibacterial activity if the purified form of larval extract could be produced which would serve as the leads for synthesis of novel antimicrobial products of natural origin.

The chemical analysis of larval extracts revealed that fatty acids were the dominant compounds. Fatty acids had been reported to inhibit bacterial growth by disruption of bacterial membranes or inhibition of fatty acid synthesis [20]. Zheng et al. [22] reported that long chain unsaturated fatty acids such as oleic acid, linoleic acid, palmitoleic acid and arachidonic acid inhibited bacterial growth (S. aureus) by inhibiting the bacterial enoyl-acyl carrier protein reductase (FabI), which is an essential components of bacterial fatty acid synthesis. Therefore, it is not surprising that the antibacterial activities of larval extracts were contributed by the combination of fatty acids. This may partly explains the apparent potency of L. cuprina larval extract as compared to the larval extracts of S. peregrina and M. domestica since it contained more fatty acids. Nonetheless, one should underscore that these preliminary GC-MS analysis only demonstrated the chemical profile of non-volatile compounds from the larval extracts, in order not to overlook the other volatile compounds which may have antibacterial activities, future work to derivatise the larval extract using silylating reagent such as N,O-bis(trimethylsilyl) trifluoroacetamide (BSTFA) should be undertaken for potential identification and isolation of novel antimicrobial substance(s). Once these compounds have been identified, their antibacterial activity will be tested, both singly and in combinations in the future studies. Subsequently, the cytotoxicity of these compounds on mammalian cell lines will also be assessed to warrant safe use of these compounds in human.

In short, the resazurin-based turbidometric assay is a simple, reliable and feasible screening assay in assessing the antibacterial activity of larval extract of flies. This assay evidently demonstrated the antibacterial activity of L. cuprina, S. peregrina and M. domestica larval extracts against MRSA, with L. cuprina exerted the broadest antibacterial activity against both gram-positive (S. aureus and MRSA) and gram-negative bacteria (P. aeruginosa and E. coli). The present study also revealed a potential room for the development of novel and effective natural disinfectant(s) and antibacterial agent(s) from flies. Further work of derivatisation and characterization of the larval extract samples to retrieve other non-volatile compounds is greatly warranted to produce a detailed chemical profile as an informative guidance for subsequent identification of antibacterial compound(s). In addition, additional work to screen more fly species using resazurin-based TB assay for screening of antimicrobial activity for probable identification and isolation of potential antimicrobial substances should also be undertaken. If successful, these isolated and purified active substances may then be used as an alternative for maggot debridement therapy for entomo-phobia patients as well as in combating the increasing threat of emergence of multidrug resistance bacterial strains, particularly the MRSA.