Lucilia sericata Larvalarının Tüm Vücut Ekstrakt Metabolitlerinin Araştırılması ve Potensiyel Antibakteriyel Etkileri

Amac: Yakin donemde, tamamlayici tip uygulamalari modern tibbin ilgi alanina girmistir. Maggot Debritman Tedavisi dunya capinda yaygin olarak kullanilan bir yontemdir. Bu yontem, ozellikle kronik yaralarin tedavisinde tavsiye edilmektedir ve dusuk maliyet, kolay uygulanabilirlik ve nadir yan etkiler gibi avantajlari bulunmaktadir, ancak yontemin etki mekanizmasi henuz tam olarak ortaya konulamamistir. Bu calismanin amaci, Lucilia sericata larvalarinin tum vucut ekstraktinin metabolitlerini ortaya koymak ve bunlarin potensiyel antibakteriyel niteligini arastirmaktir. Gerec ve Yontemler: Antibakteriyel etkinligi arastirmak icin, onceden ozel iklim odalarinda uretilmis larvalarinin tum vucut ekstraktlari, Staphylococcus aureus , Pseudomonas aeruginosa , Escherichia coli ve Enterococcus faecalis bakterileri icin agar difuzyon ve akan hucreolcer ile test edilmistir. Antibakteriyel incelemeyi takiben, iki boyutlu elektroforez ile protein arastirilmasi yapilmistir. Bulgular: S.aureus (16mm), E.coli (22mm) ve E.faecalis (14mm) icin inhibisyon alani gozlenmis ancak P.aeruginosa icinalan olusmamistir. Hucreolcer ile farkli dilusyonlarda farkli oldurme oranlari gozlenmis ve en dusuk dilusyonda (1/64), P.aeruginosa, E.faecalis, E.coli ve S.aureus icin sirasiyla%51,9, %75, %80ve %98,7 oranlari alinmistir. Iki boyutlu elektroforezde farkli molekuler agirlik (<10-260kDa) ve izoelektrik noktada (3-9) proteinler tespit edilmistir. Sonuc: Maggot ekstraklarinin test edilen suslar uzerine antibakteriyel etkisi net olarak gozlenmistir. Farkli molekuler agirlik ve izoelektrik noktada proteinler tespit edilmistir. Antibakteriyel etkinin bu proteinler tarafindan saglanmasi muhtemel olsa da, proteinlerin kutle spektrometrisi ve protein veri bankalari ile ayrica arastirilmasi gerekir.


Introduction
Professionals have put a distance between complementary medicinal techniques and current medicine, but recently, scientific researches indicate that these methods may actually have utilities in medical care [1][2][3]. Among these techniques, maggot debridement therapy (MDT) or larval therapy or biosurgery, by far, is one of the most studied and accepted application, and is routinely performed in many country [4].
The main area for application of MDT is chronic wound care.
Chronic wounds has become more frequent and cheap, effective, easily-applicable methods are actually needed, especially when patient comorbidities are also under consideration [5][6][7][8][9]. Venous stasis ulcers, pressure wounds, neuropathic ulcers (diabetic foot ulcer), traumatic and post surgical non-healing wounds were major indications. Many studies were published that focus on effect mechanisms, but it seems there is no "one" action to define, and there is a serious mesh consisting of serial activities working simultaneously. Although the modes of action have not been entirely enlightened yet, but it seems the result of the therapy is affected by maggot itself, patient immunity, wound type, infective microorganisms. Lucilia sericata larvae is by far the most investigated and applied maggots worldwide [4,10,11].
Excretions/secretions (ES) and whole body extracts (WBE) of Lucilia sericata larvae have become topics of many investigations. Researchers found various components that may have impact on chronic wounds towards healing. They have different molecular mass, isoelectric points and structure, which indicate that the components may have different and mutiple duties on wound debridement, antimicrobial effect, biofilm degradation and wound healing. Some studies stated potential homologies with "known" proteins and enzymes in databases, but unfortunately these studies actually focused on very limited components [12][13][14][15][16][17][18][19][20].
The aim of this study is to detect components and to investigate potential antibacterial effects of WBE metabolites of maggots. Flow-cytometry is recently used in antimicrobial susceptibility analysis, and this method was not previously used for maggot ES and WBEs. Since previous studies were generally performed on sterilized and/or pure maggots, we have focused on "provoked (encountered to pathogen)" maggots to see potential differences from previous studies to observe changes on components. livers and sawdust at the bottom, and the cage was covered air-permeable clothing. When adult flies were observed in the cage, the same feeding process was applied and new eggs were obtained via liver again. This time the eggs were fed with additional fresh liver since instar 2 and 3 larvae were observed.

Material and Methods
These larvae were further collected and after cleaning with sterile saline, they were ready to use. McFarland turbidity, and these solutions were poured onto fresh livers as in two seperate groups. Instar 2 and 3 maggots were inoculated onto livers and they were caged with airpermeable clothing. These boxes were incubated at 5% CO 2 atmosphere and 37 o C for 48 hours.
Obtaining Whole Body Extract: The maggots were collected and after cleaning with sterile saline, the E.coli and S.aureus subsp.
aureus maggot groups were seperately smashed in mortar. The collected body fluid were centrifuged in 13.000 rpm for 10 min, and supernatant fluid were used for further tests immediately without any delay to prevent protein destruction.
Agar Well Diffusion Method: The test was performed according to the same procedures in Kirby-Bauer disc diffusion method regarding Clinical and Laboratory Standards Institute (CLSI) [22] and European Committee on Antimicrobial Susceptibility Testing (EUCAST) [23] guides and Dogandemir's study [24]. cfu/ml) is provided [26]. According to Nuding et al [27] and manufacturer application notes [28], dilutions, mixtures and incubations were applied. Dilutions of WBEs were decided from 1/2 to 1/64, based on MIC levels in Dogandemir'study [24].
The analysis were done with BD Accuri C6 flow-cytometry device (BD, Maryland, USA) and rates of living/dead cells were defined according to data from detectors and software applications. Thus, by comparing fluorescence of TO and PI, rates of bacterial cells killed by WBEs were detected.
2D-PAGE: Two dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the major step of protein analysis, which gives important structural data (molecular mass -kDa, isoelectric point -pI), but not functional information. Preperations, solutions and staining were done according to manufacturer's instructions and previous studies [29][30][31]. The analysis were done on 12% seperating gel with Protean IEF-Cell and Criterion SDS-PAGE elektrophoresis cell devices (BioRad, CA, USA) and silver staining were performed to evaluate in optimal sensitivity.
The spots were compared according to immobilized pH gradient (IPG) strip (BioRad, CA, USA) (pI values) and size marker.

Discussion
Although several studies focused on MDT, a very limited data have been obtained until now. These researches are mainly based on ES and WBE of sterile and/or patient-applied maggots [24,[32][33][34][35][36][37][38]. In this study, maggots are collected from laboratory conditions and differently from "liver culture" to create a counterfeit environment that maggots suppose like they are on an infected wound. This method has never been performed before, but as we know from entomological studies, L.sericata larvae particularly chooses infected and dead tissue [4,10]. Previous researchers stated that obtaining ES and/ or WBE is a thorny procedure, because too many maggots are necessary to gain enough material to analyse. With this method, we aim to create a standardized maggot pool, which is consisted of maggots grown in and encountered the same conditions and microorganisms, and also, we were able to get and Masiero et al [47] showed strong activity of maggot ES and body fluids against P.aeruginosa and its biofilms. Pöppel et al [20], found various peptides and genetic arrangements of L.sericata, particularly against this species. They also stated synergistic effects of these peptides. However, clinical efficiency of MDT on Pseudomonas or Acinetobacter-infected wounds is contraversial [48]. Dogandemir [24] used patientapplied maggots and activity against P.aeruginosa was seriously limited. Among all these arguements, researchers have a concensus that antibacterial activity of maggots is strongly related with so called "provocation". This issue is about maggots showing spesific and specialized activity against the encountered pathogen. Huberman et al [36] and Kerridge et al [38] claimed that following the first encountering, maggots secrete low-molecular weight proteins immediately, but after a while, high-molecular weight complex proteins are secreted in greater amounts than sterile larvae. Data of Pöppel et al [20] also supported this information, which indicates that maggots do somehow adapt and fight in a particular way against what the "enemy" is. So, this in vitro undetectable antipseudomonal effect may be a result of facing with P.aeruginosa and/or synergistic activities of secreted peptides. it is important to observe the "Time-Kill Analysis", which gives minimum bactericidal concentration, can be very beneficial to understand the actual antibacterial activity alterations [51].
In our study, the activity against other species (S.aureus subsp. aureus, E.faecalis, E.coli) was very promising. Kruglikova et al [13] and Chernysh et al [14] reported that L.sericata larvae ES had bacteriostatic act on E.coli, bactericidal act on many gram negative and positive bacteria and finally fungicidal activity.
Similar results were stated by Cazander et al [43,44] and Van der Plas et al [49]. In overall, there is an opinion that maggot fluids are more effective against gram positives [12,52].
Despite of this, we found a strong activity against E.coli, which indicates that bacterial cell wall is not the only target and multilple mechanisms are on the move.  [20] reported 47 different genes encoding antimicrobial peptides and they recombinantly produced 23 of them such as "cecropin", "cecropin like", "proline rich", "stomoxyn", "dephencin". Additionally, they detected proteins called "elevated during infection -edin" that are coded in case of infection. As previously stated, they also showed synergistic and additive effects of these proteins. As understood, the studies on maggot ES and WBE is just on a preliminary phase that there is a huge black hole to explore.
In our study, SDS-PAGE (1D-PAGE) showed many protein bands (12-260 kDa). Since the band intensities were different from each other, it can be noted that protein concentrations may have varied. This interpretation might also be valid for SDS-PAGE, but this kind of quantitation can be stated by automized analysis devices, which we did not used. In 2-D PAGE analysis, many protein spots with various pI were observed. Table 2 gives detected bands and spots. In previous studies, proteins were mainly isolated seperately, thus lowmolecular weight peptides could be purified [33][34][35][36]. As previously stated, Huberman et al [36] and Kerridge et al [38] reported that high-molecular weight proteins were secreted following an "enemy-encountering". As seen, our results indicated high-molecular weight proteins. It should be noted that we investigated on WBE and used "provoked" maggots.
Since we incubated maggot on a virtual infected wound, this was actually expected. However, WBE may have contained structural proteins, so it is a major limitation that functional analysis was not applied.
In this study, "liver culture" was infected by S.aureus subsp.
aureus and E.coli. As noticed, we found the highest antibacterial activities against these agents. We believe that protein analysis should be performed to sterile and S.aureus-, E.coli-, K.pneumonaie-, P.aeruginosa-, Proteus spp.-, Enterococcus spp.-, Acinetobacter spp.-provoked maggots, and finally comparison should be made. This analysis will uncover main differences between sterile and provoked maggots, and it may also prove "spesific and specialized antibacterial action". In addition, WBEs and ES of these maggots should also be tested with functional analysis with mass spectrometry and Protein ID. But, synergistic and additive effects should not be forgotten.
In conclusion, MDT is a very effective method as a part of multidiciplinary approches in treatment of chronic wounds.
For a chronic wound treatment, the main attempts are debridement, antimicrobial action, provoking wound healing and biofilm distruction. In our study, it was obvious that there is a certain antibacterial effect, and various proteins may have a role on this. Furthermore, these proteins may also act in other attempts, which is in need of further studies. Dilutional antimicrobial tests, time-kill analysis and advanced functional protein identifications should be performed to clarify actual effect mechanisms of MDT.

Declaration of conflict of interest
The authors received no financial support for the research and/or authorship of this article. There is no conflict of interest.