IN VITRO EFFECT OF Chloroprocta SP . MAGGOTS SECRETION ON S taphylococcus epidermidis BIOFILM AND THE EXPRESSION LEVEL ica A OF GENE

Biofilm formation and the expression of ica A gene can be induced by environment conditions that are potentially toxic for bacterial cells. The effect of green flies maggots secretion to biofilm was studied some years ago to investigate in vitro effect of secretion of Chloroprocta sp . maggots on the formation of Staphylococcus epidermidis biofilm (phenotype) and the expression level of ica A gene (genotype) for indicating its mechanism on bacterial biofilm eradication. Microtiter plate biofilm assay was used to measure the effect of Chloroprocta sp . maggots secretion at various concentration on S. epidermidis biofilm. The expression level of ica A gene was performed by Real TimePCR using lightcycler method. The biofilm susceptibility test was done against maggots excretion/secretion using MTT assay. Whereas planktonic susceptibility testing was carried out using Kirby Bauer method. In the presence of maggots secretion at low concentration (5%), biofilm formation of S. epidermidis 734 was induced. In contrast, the expression level of icaA gene in production of maggots excretion/ secretion at concentration of 5% was lower than that of without maggots secretion (1/2 Fold). Eradication of bacterial biofilm was demonstrated after 48h incubation (MD=-0,011;P<0,05), but planktonic cell. In vitro difference effect of the Chloroprocta sp . maggots secretion at low concentration to phenotype and genotype of S. epidermidis biofilm showed that the possibility of maggots secretion ability to eradicate bacterial biofilm was not mainly due to the expression level of ica A gene.


INTRODUCTION
S. epidermidis is a major pathogen of medical device-associated infections. (Rupp and Archer, 1994). Polysaccharide intercellular adhesin (PIA) is the main virulence factor of S. epidermidis biofilm. Polysaccharide intercellular adhesin play a significant role in biofilm formation, especially in initial bacterial adherence and intracelullar adhesion (Mack et al., 1996;O'Gara and Humphreys, 2001). Polysaccharide intercellular adhesin is synthesized by enzymes encoded genes icaA, icaD, icaB, icaC genes. icaD was always expressed in biofilm-production strains and non-biofilm production strains, meanwhile icaA is only expressed on biofilm-production strains (Cafiso et al., 2004).
Biofilm-producing bacteria show 10-1,000-fold less susceptible to many antimicrobial agents than planktonic cells (Davies, 2003). The emergence of antibiotic resistant of biofilm-producing bacterial has caused a resurgence in the research of component to enhance the effect of conventional antibiotics. Subinhibitory concentrations of some antibiotics can influence the expression of significant bacterial virulence factors. The expression of ica operon can be highly enhanced by combination of streptogramin and quinupristin-dalfopristin and by tetracycline .
Previous study has evaluated the effect of green flies secretion , Lucilia sericata, maggots on nascent biofilm formation and the disruption of pre-formed biofilms of S. epidermidis strains in accordance to the polysaccharide intercellular adhesin or the accumulation of associated protein mechanisms for biofilm accumulation (Harris et al., 2009). Chloroprocta sp. is one of green flies of the family Calliphoridae found predominantly in Semarang, Central Java. Therefore, the present study was aim to evaluate in vitro effect of Chloroprocta sp. maggots secretion at low concentration on the formation of S. epidermidis biofilm (phenotype) and the expression level of icaA gene (genotype). The data obtained were used for evaluating the mechanisms of maggots excretion/ secretion in eradicating bacterials biofilm at phenotype and genotype levels.

Larvae and collection of maggots excretion/ secretion
Late second or early third instar larva of Chloroprocta sp. maggots were obtained from the Laboratory of Nutritional Biochemistry of Agriculture and Husbandry, Faculty of Diponegoro University, Semarang. Excretion/ secretion were collected from these maggots. Briefly, maggots were transferred to sterile tubes to provide a density of 100 maggots in 200µL of phosphate buffer saline and incubated in the dark at room temperature (25°C) for one hour. The obtained liquid was transferred to another tube and sterilized (Arora et al., 2010).

Bacterial strains
The bacterial strains used in this study were biofilm-positive and icaA-positive S. epidermidis 294, 734, 169; while the clinically isolated negative control of biofilm is S. epidermidis ATCC12228. S. epidermidis 294 is a clinical isolate from the Department of Microbiology, Faculty of Medical Diponegoro University-Kariadi Hospital Semarang. Meanwhile, the other strains were the collection of the Department of Microbiology Faculty of Medicine, Gadjah Mada University. S. epidermidis strains were cultured in Trypticase Soy Broth (TSB) at 37°C for 24h (overnight culture).

Biofilm assay
The biofilm phenotype isolate of S. epidermidis was determined with a microtiter plate biofilm assay using 96-well tissue culture plates to measure the attachment and the accumulation on the plastic surface, as described previously with modification (Merrit et al., 2011). Briefly, overnight cultures of the strains in Trypticase Soy Broth were diluted 1:100 in fresh Trypticase Soy Broth and 20μL cultures were inoculated into 200μL Trypticase Soy Broth in each well. After 24h of incubation at 37°C, the plates were washed with phospat buffer saline and the adherent bacteria were stained with 1% crystal violet. The absorbance at 595nm (A595) of the stained adherent bacterial films was quantified with microplate reader (Biorad). To quantify the effect of maggots secretion concentration on S. epidermidis biofilm formation, the biofilm assays were performed using secrete dilutions: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% and no excretion/secretion was added to the control wells. Briefly, 96 wells of tissue culture plates were filled with 100µL Trypticase Soy Broth containing maggots secretion from 0% to 100% for each well as mentioned above. All experiments were performed three times. S. epidermidis ATCC12228 was used for negative biofilm control.
To determine the lowest concentration of maggots secretion enhancing S. epidermidis biofilm formation, the biofilm assays were performed using a series of maggots secretion dilutions ½ fold from 5% to 0.0045% and no maggots secretion was added to the control wells. Experiments were tested in duplo. Strain with higher optical density than the negative control after administration of maggots secretion (0 to 100%) was examined (734). S. epidermidis ATCC12228 was used for negative control.

The expression level of icaA gene
Total RNA was isolated from 24h biofilm-producing bacteria cultures with and without 5% maggots secretion in 12-well tissue culture plate as described previously with some modification (Nuryastuti et al., 2009). Briefly, after suspending, the biofilm-producing bacteria were pelleted by centrifugation and frozen at -56°C. The samples were thawed slowly on ice and resuspended in 100µL diethylpyrocarbonate (DEPC)-treated water. The total RNA was isolated using the ambion purelink RNA mini kit according to the manufacture's instructions. Approximately, 11 µL of RNA sample was used for cDNA synthesis according to the manufacture's instructions (Transcriptor First Strand cDNA synthesis kit). Real TimePCR was performed. The expression level of icaA gene was analyzed by Real TimePCR in a LightCycler method with untreated biofilms as controls and gyrB as the reference. Primer used for LightCycler metode were: icaA-1 forward (sequence GAAGTT CTGATAATACTGCTG; icaA-1 reverse (GATG CTTGTTTGATTCCCTC), 129 bp in size. Reference primers were gyrB-3 forward (GGAGGT AAATTCGGAGGT) and gyrB-3 reverse CTTGATGATAAATCGTGCCA), 129 bp in size.
Meanwhile, the planktonic susceptibility testing was done againts maggots secretion on agar plate using Kirby Bauer method (Cappuccino and Sherman, 2001), with some modifications. Briefly, blank disc was filled with the maggots secretion (10, 20, 30µL). Cefoxitin disk was served as a control. The disk were placed in a space equidistant from each other on Mueller Hinton agar plate and gently touched with a steril forceps. One well in the same plate was made and filled with 100µL maggots secretion. Following the incubation, diameter of each zone of inhibition was measured.

Effect of maggots secretion on biofilm formation
Descriptively, optical density (OD) measurements at 595nm (OD595) was observed using microtiter plate assay. It indicated that biofilm formation of S. epidermidis 294 and 169 were lower than that of S. epidermidis ATCC12228 (mean were 0.0637; 0.0665; 0.0839 respectively) for all maggots secretion concentration tested. Meanwhile, S. epidermidis 734 biofilm formation enhanced relatively by low concentration (<50%) of maggots secretion (mean was 0.0861) in comparison to S. epidermidis ATCC12228 (mean was 0.0839). Relative inhibition of biofilm formation showed in S. epidermidis 734 strain by higher concentration (>50%) of maggots secretion ( Figure 1). Therefore, to determine the lowest concentration which enhancing biofilm formation, measurement of OD of S. epidermidis 734 strain was continued in concentration lower than 10%. In the presence of low concentration (5%) of maggots secretion, S. epidermidis 734 biofilm formation was extremely induced compared to that of S. epidermidis ATCC12228 (Figure 2).
The different effect of maggots excretion/secretion concentration to the different strains descriptively indicated variability between strains. These results was in contrast to the previous study which showed a significant correlation between concentration of secretion of L.sericata maggots with the decrease of S. epidermidis biofilm formation.
In the other side, the same study showed that the inhibitory activity on biofilm formation not only depends on the concentration, but also on the incubation time (Harris et al., 2009). Meanwhile, the measurement of the effect of maggots secretion to biofilm formation in the present study was based on concentration only.
Naturally the dose response is biphasic, high-dose inhibition and low-dose induction of biofilm formation. Some antimicrobials can be antagonists of biofilm formation at low doses, in the other side they can be agonists at higher doses (Kaplan, 2011). Other possible explanations of this discrepancy were caused by different type of flies used in this study and the amount and type of maggots secretion' components.

IcaA gene expression level
The expression level of icaA gene in 5% concentration of maggots secretion was about ½ fold than that of without. The result  demonstrated a significant decrease of the expression level of icaA gene after the addition of 5% excretion/ secretion compared to that of without ( Figure 3) Some previous studies showed that enhanced biofilm formation and icaA gene expression in the addition of low concentrations of antibiotics are based on varied ways of action. The biofilm formation can be induced by some conditions that are potentially toxic for the bacterial cell such as high osmolarity, detergents, urea, ethanol, and oxidative stress Kaplan, 2011). A global response to cell stress plays an important role in the mechanism of conditioninduced biofilm formation of many bacteria (Kaplan, 2011).  The results of the present study showed that the response of bacteria to low concentrations of maggots secretion was different in phenotype and genotype level. Low concentration of maggots secretion enhanced biofilm formation, contrarily, the same concentration decreased the expression level of icaA gene.
Some possible explanation for the present study were ica genes migth be suppressed in S. epidermidis 734 as a weak biofilm producer and different mechanisms leading to ica expression between different strains in the same species of staphylococci (Kaplan 2011). In addition, the regulation of icaA, icaD, icaB, icaC expression is mediated by complex regulatory factors such as global regulatory proteins (SarA and σB), IcaR and TcaR directly or indirectly. It was also possible that different laboratories (hence different condition) induced biofilm formation and ica expression differently (Cue et al., 2012).

Biofilm susceptibility to maggots secretion
In order to determine biofilm-bacteria susceptibility to maggots secretion, secretion was added to growing biofilm-bacteria. The culture were then incubated for various durations (2, 24, 48h). Biofilm-bacteria viability was meassured by MTT assay. The results showed that after incubated for 48h, S. epidermidis 734 curve (mean=0.049) was under S. epidermidis ATCC12228 curve (mean=0.153) consistently, in comparison with incubation time of 2h and 24h (Figure 4). F test of the multivariat effect of time showed F=72.084, P<0.05. Mean difference (MD) of bacterial viability of S. epidermidis 734 compared with S. epidermidis ATCC12228 was -0.011, P<0.05. This indicated that bacterial viability of S. epidermidis 734 strain was lower than the negative control. It was in accordance to the standard of biofilm minimal bactericidal concentration (MBC). Minimal bactericidal concentration MBC was the concentration of maggots secretion showing A595 values equal to or lower than that of the negative control biofilm S. epidermidis ATCC12228 (Nuryastuti et al., 2009). As described previously, MTT assay was used to analyse the effect of secretion of maggots on viability of biofilm-bacteria. Therefore, the result indicated that the control of bacterial biofilm occured after 48h in vitro.
In the present study, the eradication of bacterial biofilm was dependent upon the incubation periods but independent upon the concentration of maggots secretion. Meanwhile, the previous study showed that the total number of bacteria in the wells was not significantly changed; demonstrating that secretion of L. sericata maggots did not interrupt biofilms by killing bacteria. In vitro killing study discovered that only the highest concentration of maggots secretion (400μg) which reduced the number of viable Staphylococcus aureus after 3h. Conversely, the maggots secretion did not reduce the number of viable Pseudomonas aeruginosa up to the concentration of 800μg (van-der-Plas et al., 2008).
In the other side, treatment of excretion /secretion of Chloroprocta sp. maggots by Kirby-Bauer method determined no zone of inhibition formed from all of excretion/ secretion doses (data is not shown). The results revealed that the current doses of secretion did not kill S. epidermidis as a planktonic form. Previous study by using RDA assay, the most sensitive in vitro killing assay, determined that S. aureus was not killed at the biofilm-effective doses of maggots secretion while P. aeruginosa was not killed at all (van-der-Plas et al. 2008).
This in vitro study was a preclinical test for investigating of Chloroprocta sp. maggots secretion effects on biofilm bacteria. Recently, the aim of antibiotics/antimicrobials discovery have concerned on those proteins or processes important for bacterial cell viability (Alksne and Projan, 2000). Therefore, study of antimicrobials that have ability to either improve suppression of biofilm or conventional antibiotics efficacy and their underlying mechanisms lead to the development of novel co-therapeutic agents that can decrease biofilm infections.

CONCLUSSION
In vitro difference effect of low concentration of secretion of Chloroprocta sp. maggots to phenotype (biofilm formation) and genotype (the expression level of icaA gene) of S. epidermidis biofilm showed a possibility that maggots secretion ability to control bacterial biofilm not only with regard to the expression level of icaA gene.