Expanding the microbiological testing parameters

The extract has only been shown to definitively inhibit E. coli with no evidence to support gram positive nor antifungal activity. In order to gauge the spectrum and effectiveness of the antimicrobial, tests will be conducted parallel to the biochemical analysis.

Various gram-negative bacteria will be tested as well as testing the extract against a multi drug resistant strain of E. coli. Testing the samples against resistant strains will inform us of the potential uses of the compound and if it could be used as a replacement for

conventional antibiotics (Jan et al. 1998; Tankhiwale et al. 2004).

As well as testing the range of effectiveness of the crude extracts with expanded

susceptibility testing; we will also be testing each of the sub-fractionations from fraction 19 to try and identify which of the LC/MS peaks was the antimicrobial.

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10.1. Testing if the extract is bacteriostatic or bactericidal

It would be of interest to determine if the extract is a bacteriostatic or a bactericidal compound. This can be achieved by using the protocol set out by Vanhoof (1980). The antimicrobial would be introduced to a bacteria-inoculated plate, the plate allowed to incubate ensuring the antimicrobial can take effect. Colonies once exposed to the

antimicrobial are then transferred to a fresh NA plate with no antimicrobials present. If the colonies are able grow on the new plate the antimicrobial is bacteriostatic, if not then the compound is bactericidal (Vanhoof et al. 1980; Mayaud et al. 2008).

10.2. Could the antimicrobials be produced by Drosophila gut bacteria?

It is clear there is a high likelihood of the compounds listed in table 1 being produced directly through the Drosophila is extremely low. Halogenated antimicrobial compounds have been shown to be produced by bacteria therefore there is a likelihood that within the gut of the Drosophila, a species is producing the antimicrobial. We will discuss the possibility of some of the most common Drosophila gut bacterial species being responsible and if possible find methods to both increase the yield and purity of the product for further testing.

10.3. Common gut bacteria within Drosophila

There is a probability that the necessary biological pathways and mechanisms required to bind a halogenated atom to an organic structure can be found in the gut microbiota of the flies rather than produced directly by Drosophila. As the origin of the compound is unknown we must look to see if there is a possibility of the Drosophila harbouring a microbe that can provide it with protection.

There are examples of insects using a bacterium to actively produce antimicrobials. The beewolf wasp has a close symbiotic relationship with Streptomyces, which it houses on its head. The bacterium is used by the female wasps as a source of antibiotics used to protect their offspring from pathogens. This is yet another example of the measures taken by an insect to provide protection to its offspring (Kroiss et al. 2010).

Compared to mammals, flies have realtively simple gut microbiota. Some of the most abundant species belong to Lactobacillaceae, Enterocococcaceae, Acetobacteraceae and Enterobacteriaceae families. We will explore the possibility of these families being responsible for the antimicrobial activity by searching potential candidates which may produce smiliar analogues to those found during the analysis of fraction 19 (Erkosar et al.

No literature was found to support the notion that Lactobacillacae species produce

antimicrobial products. They are used as probiotics in livestock rearing as an alternative to antibiotic probiotics, therefore helping to reduce the burden of antibiotic resistance. There is no evidence Enterococcaceae producing haogenated antimicrobials nor do the

Acetobacteraceae, however Acetobacteraceae species have been found to have a key role within honey bee hives; they are critical for larval nutrition. Unfortunately, none of the common families shown above produce any analoguesthat could be used to infer a potential pathway nor do any of these species prove to be potential candidates of further study. A more in-depth look will be needed once the nature and structure of the

antimicrobial is identified (Ocaña et al. 2006; Zhou et al. 2005; Corby-Harris et al. 2014).

10.4. Bacteria x a potential source or antimicrobial?

Whilst testing if there was a disparity in the density of growth of bacteria on mated male and female food a common bacterium was identified and isolated. The gram-negative, rod bacterium was found in differing levels between the two mated fly groups. The unknown bacteria would later become known as Bacteria X

A pure isolation of the bacteria was isolated and tested against the extracts; it was found to be susceptible to the extract. The susceptibility to the extract would suggest that the bacterium does not produce the antimicrobial in question; the deep-sea bacterium Altermonas produce an antimicrobial to regulate their rate of growth dependant on the available resources. The marine violet antimicrobial produced by Altermonas is a

brominated compound (Gauthier and Flatau 1976).

The identity of bacteria X is unknown however the API test results produced the most likely genus in which the bacteria belongs; these being either Pseudomonas, Burkholderia or Sphingomonas. Each of these species have been found on the egg surface of drosophila; there is no supporting literature that these species could be responsible for the compounds isolated in fraction 19 (Wong et al. 2015).

As discussed earlier Streptomyces have been shown to exhibit mutualistic relationships with host insects which can then be used to protect offspring. Streptomyces belongs to the Actinobacteria phyla, Wong et al (2015) showed the presence of Actinobacteria on the surface of eggs however the study states that more than 99% are Actinomycetes, it does not elaborate on the other possibilities. Actinomycetes too are known for producing antibiotic compounds, these are mainly beta-lactam and macrolides however 166 compounds are known to be produced. Unlike the colony isolated from the fly food these are gram positive bacterium (Wong et al. 2015: Mahajan and Balachandran 2012).

Not all species of bacteria can be cultured Wong et al (2015) used a liquid medium to culture some bacteria as well as pyrosequencing qPCR to obtain a broader picture of the bacterial species present. If the antimicrobial within fraction 19 is indeed produced by a gut bacterium it may be difficult to isolate and grow the bacterium outside the fly gut