EFFECTIVENESS OF THE FERMENTATIVE EXTRACT OF Lactobacillus acidophilus AS ANTIMICROBIALS AGAINST Aeromonas hydrophila

The purpose of this study was to evaluate the in vitro and in vivo antimicrobial activity of commercial Lactobacillus acidophilus (L. acidophilus) cells and cell free extract against Aeromonas hydrophila (A. hydrophila). The in vitro method was carried out using well diffusion method. For in vivo evaluation, the effect of L. acidophilus on the survival rate of Pangasianodon hypophthalmus (P. hypophthalmus) infected with A. hydrophila was evaluated. The well diffusion method showed a significant inhibition ability of L. acidophilus cells against A. hydrophila compared to the cell free extract. The inhibition diameters obtained with cells and cell free extract were 17.23 mm and 15.17 mm, respectively. P. hypophthalmus injected with L. acidophilus cells and cell free extract following challenged with A. hydrophila cells showed survival rate of 70% and 60% respectively, at 2-week post challenged. The gas chromatography-mass spectrophotometry (GC-MS) result revealed that a diverse of compounds was detected in both the L. acidophilus cells and cell free extract, among them the most abundant component was pyrrolo[1,2a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl), which showed a promising anticancerous activity and might be played a significant role in the recovery of the infectious P. hypophthalmus. The current study revealed that both cells and cell free extract of L. acidophilus have antimicrobial activity against A. hydrophila. ____________________________________________________________________________________________________________________


INTRODUCTION
Aeromonas hydrophila (A.hydrophila) is an opportunistic and worldwide available freshwater bacterial species exist in the intestine of various aquatic mammals including fish (Austin and Adams, 1996;Popović et al., 2000).A. hydrophila is the aetiological agent of ulcer disease, haemorrhagic septicemia, motile aeromonas septicemia (MAS), or red sore disease in a variety of freshwater fish species (Newman, 1993).It is frequently linked with stressed or immunocompromised to hosts (Roberts, 1993).It also has been recognized as the principal infectious agent of fish bacterial septicaemia in freshwater striped catfish.
Diseases of striped catfish are being considered as a prime constraint and may ultimately turn into a limiting factor in the economics of a booming as well as sustainable aquaculture industry (Crumlish et al., 2010).Losses of Vietnamese P. hypophthalmus production systems due to disease outbreak caused by A. hydrophila have been described previously by Subagja et al. (1999).Although, disease caused by Aeromonas species showed very high resistance capability to antibiotics (Harikrishnan and Balasundaram, 2005), still worldwide many aquaculture industry completely depend on the use of antibiotics and various chemicals for the prevention and control of bacterial diseases (Villamil et al., 2014).On the other hand, presently probiotics have been used as an alternative method of controlling ulcerative dermatitis disease caused by A. hydrophila by adding them in the feed.
Currently, lactic acid bacteria has been recognized as a most effective probiotic in aquaculture (Al-Dohail, 2010;El-Ezabi et al., 2011;Talpur et al., 2014) as well as in the dairy farms (Karska-Wysocki et al., 2010), due of its antagonistic effect against a wide variety of bacteria (Savadogo et al., 2004;Al-Dohail, 2010).Lactobacillus acidophilus (L.acidophilus) is one of the most significant probiotic candidates among all of the lactic acid bacteria, which has revealed a strong antagonistic effect against a variety of bacteria including A. hydrophila (Aly et al., 2008;Al-Dohail, 2010); Staphylococcus xylosus and S. agalactiae (Al-Dohail, 2010); meticillin-resistant S. aureus (Karska-Wysocki et al., 2010), and Chlostridium difficile (Mkrtchyan et al., 2010).Therefore, a preliminary study was design to find out the most abundant bioactive compound that are responsible for the antimicrobial activity of L. acidophilus against A. hydrophila in order to confirm their potential effect in striped catfish culture.

Isolation and Identification of A. hydrophila
Fresh water pathogenic species of A. hydrophila collected from the National Fish Health Research Center Penang, Malaysia was applied in this current study.A. hydrophila was re-isolated from the experimentally infected kidney of striped catfish and incubated on tryptic soy agar (TSA, Himedia, India) for about 24 hours at 30° C (Figure 1A).Morphologically different and well-shaped colonies were separately selected and streaked to a new TSA plates until pure colonies were gained (Figure 1B).

Gram Staining Method
A Gram staining technique was conducted in order to confirm whether the bacterium is Gram negative or positive.A pure single colony of newly cultured A. hydrophila was inoculated into 10 ml of triptic soy broth (TSB) for 24 hours in a constant shaking incubator (INFORS HT electron, 180 rpm) at 30° C. A loop full of the broth culture was taken and placed on a sterile slide in order to prepare a thin smear.Then the inoculating loop was spread by means of rounded motion to create 1 cm in diameter.After air dried, the slide including sample was fixed by moving quickly the entire slide over the flame of a Bunsen burner two to three times.The heat-fixed smear slide was then Gram stained following the method described by Bhattacharyya et al. (2015).

Molecular Identification Method
For molecular identification and confirmation of A. hydrophila, a single colony from a 24 hours culture of isolated bacterium in TSA was transferred to 10 ml of sterile TSB and incubated for 24 hours in a constant shaking incubator (INFORS HT electron, 180 rpm) at 30° C. The bacterial deoxyribonucleic acid (DNA) was isolated according to the protocol of genomic DNA isolation from Gram positive and Gram negative bacteria as described in the Wizard Genomic DNA Purification kit (Promega, USA) and stored the isolated DNA at 2-8° C until used.
Amplifications of A. hydrophila DNA were performed with the MyCycler TM Thermal Cycler (BioRad, USA).The universal primers (68F 5'TNANACATGCAAGTCGAKCG'3, Tm 52.7° C and 1392R5'ACGGGCGGTGTGTRC'3, Tm 51.4° C) were used in this study (Mashayekhan, 2002).A total reaction volume of 50 µL was used.Each reaction contained 1x reaction buffer, 1 µL of 0.2 mM dNTP, 4 µL of 2.0 mM MgCl2, 2.5 µL of 0.5 µM each primers, 0.25 µL of Taq DNA polymerase, 2 µL of extracted DNA as template and 27.75 µL ddH2O.PCR was performed according to a simplified hot start protocol.Briefly, the reaction tubes were directly heated to 95° C and the temperature kept at 95° C for 5 min.Cycles were: for initial denaturation 2 min at 95° C, 30 sec at 95° C for denaturation, 30 sec at 47° C for annealing, 90 sec at 72° C for extension, and 5 min at 72° C for final extension.
The amplified DNA was detected through agarose gel electrophoresis.Briefly, the gel was prepared by adding 1 g agarose powder in 100 mL of Tris-acetate-EDTA (TAE) buffer (0.5x).The solution was heated in a small conical flask for 3-4 minutes until dissolved completely in microwave.Then, the gel was poured into a gel-tray which contained a comb in one end and left to solidify at room temperature.The gel was placed in the electrophoresis tank containing 0.5x TAE buffer.Then a ratio of 1:5 loading dye to sample were gently mixed by pipetting and loaded into the well.About 2 µL of the 1kb DNA ladder (Fermentas, USA) was also loaded to determine the sizes of the DNA fragments.The gel electrophoresis was then run at 60 volts for 40 minutes.The gel was then stained in ethidium bromide (EtBr) solution in order to check the appearance of bands.Gel doc imaging system (VersaDoc TM Imaging System Bio-Rad, USA) was used to take the image of the band in the gel.
The purification of the DNA was done by following the protocol described in the QIAquick gel extraction kit (Qiagen, USA).The gel containing the target DNA fragments were carefully removed using a sharp and clean scalpel.The gel slices were transferred into eppendorf tubes and weighed.The QG buffer were then added to the tube at a ratio of 1:3 gel to QC buffer and Figure 1.Cultures of Aeromonas hydrophila.A= Aeromonas hydrophila isolated from infected striped catfish, Pangasianodon hypophthalmus kidney and liver; B= Pure colonies of Aeromonas hydrophila re-isolated from infected kidney of striped catfish incubated at 50° C for 10 minutes and mixed by vortex the tube every 2-3 minutes until the gel completely dissolved.One mL isopropanol was added to the sample and mixed well.The samples were then transferred into QIAquick columns (with collection tubes at bottom) and centrifuged at 10,000 g for 1 minute.The solution in collection tubes was discarded and about 500 µL of QG buffer was added to QIAquick column and centrifuged with same speed.Then, 750 µL of PE buffer was added to each QIAquick column and centrifuged for 1 minute.Centrifugation was once again conducted to remove residual ethanol from PE buffer.In the final step, QIAquick column was placed into sterile 1.5 mL microcentrifuge tube and 50 µL of EB buffer (10 mM Tris-CI, pH 8.5) was added to QIAquick column, and then centrifuged for 1 minute.The tubes were then kept at -20° C. The purified sample was sent to the service provider, First Base, Malaysia for sequencing.

Antimicrobial Activity of L. acidophilus Against A. hydrophila
Aeromonas hydrophila was grown-up in a similar approach as mentioned previously.Bacterial cell free extract was then removed from the cells by centrifuging at 3000 g for 10 minutes at 4° C and the cells were then washed two times with phosphate buffered saline (PBS) (pH 7.4) and re-suspended in the same buffer (Zheng et al., 2011).The turbidity of the washed samples was measured to achieve an OD 600 nm value of 1, which corresponded to 1x10 8 CFU mL -1 of bacterial suspension resulted from the plate counting.
Commercial L. acidophilus (International Food Grade, Laboratory of USA) was used as a probiotic bacterial strain in this current study.For seed culture, 1 g of commercial L. acidophilus (LAB) was incubated into 50 mL of MRS broth (2% w/v) (De Mann et al., 1960;Wang, 2011) for 12 hours at 37° C in a shaking incubator (INFORS HT electron, 180 rpm).Thereafter, 1 mL of those cultured bacterial suspension was transferred to 99 mL of MRS broth for mass culture and incubated for 36 hours (Al-Dohail, 2010).The bacterial cells were then harvested by centrifugation at 3000 g for 10 minutes and washed twice with PBS (pH 7.4) and re-suspended in the same buffer (Villamil et al., 2014).The cell free extract was transferred and filtered through a filter (ministart, 0.20 µm) and kept in a sterile tube for further use.Colony-forming units (CFU) were determined by a decimal dilution method in the same buffered saline.100 µL of the diluted bacterial cells were plated in petri dishes containing MRS agar (Himedia, India) and after 48 h incubation at 37° C (Drago et al., 1997;Andani et al., 2012) counted manually.Concurrently, one mL of bacterial suspension contains 1x10 9 CFU, which also corresponded to 1.0 OD at 600 nm wavelength.

In vitro Antimicrobial Activity of L. acidophilus Against A. hydrophila
The well diffusion method was conducted to assess the antimicrobial activity of LAB against A. hydrophila.About 0.2 mL of cultured A. hydrophila cells in PBS prepared following the method as described earlier was mixed to 15 mL of cool molten TSA (0.2%).Well mixed samples were then transferred to a new sterile petri dish and left to solidify for about 3 hours at room temperature.Then five holes (6 mm) were made using a sterilized cork tool on each petri dish.Among five holes, two holes filled with 20 µL of the cells of LAB (A), another two holes were filled with 20 µL of the cell free extract of LAB (B) and the last hole filled with 20 µL sterile PBS to serve as control (C).Three replicate petri dishes were prepared in the similar way and incubated for 24 hours at 37° C (Das et al., 2006).The inhibition zone around the hole was measured for the determination of antimicrobial activity.

Effect of L. acidophilus on Survival of P. hypophthalmus infected to A. hydrophila
In order to verify the in vitro antimicrobial activity of LAB against A. hydrophila a second in vivo experiment was conducted.Ninety P. hypophthalmus (50.23±1.34g) were selected randomly and distributed into 9 aquariums (each of 10 fish).A. hydrophila and LAB were prepared in the similar way as mentioned before.The first group of fish was intraperitoneally (IP) injected with 0.2 mL of A. hydrophila cells (AM) suspension containing 1x10 8 CFU mL -1 in PBS (considered as a lethal dose as observed in the pathogenicity test), which served as a control.While, a second group of fish was injected with 0.2 mL of LAB cells (LABP) suspension containing 1x10 9 CFU mL -1 and the third group was injected with 0.2 mL of LAB cell free extract (LABS).The next day, all fish from the second and third groups were injected with the similar concentration of A. hydrophila cells (that injected in the first group) and the mortality was recorded for 2-week.

Identification of Bioactive Compounds from L. acidophilus Cells and Cell Free Extract
The extraction of bacterial metabolized was performed by following the method of Mithun and Rao (2012) with some modification.Twenty mL of the cultured bacterial samples (OD600= 1.0) were harvested by centrifuging at 3000 g for 10 minutes.Then the cell free extract was separated and filtered using Ministart (20 µm mesh size) to remove almost all the cells.Bacterial cells re-suspended in 20 mL of broth and 20 mL of cell free extract were used for the extraction of metabolized after mixing with another 20 mL of mixture (methanol:chloroform:distilled water= 2:2:1) separately and transferred to a separating funnel and vertically left for the two phases.The lower phase was then collected after 1 hour and the upper phase was resuspended with the similar volume of the mixture (methanol:chloroform:distilled water= 2:2:1).Similar steps were followed twice in order to extract almost all the metabolized.The extract was then evaporated using a rotary vacuum evaporator.The bacterial extract was dissolved in 1 mL of 100% methanol and mixed well by pipetting.After filtering, the bacterial extract was transferred to a sterile 2 mL capacity vial and stored at -20° C until GC-MS analysis.
The derivative extracts separated from the cultured bacterial samples were analyzed using GCMS-QP2010 Ultra, SHIMADZU.Approximately, 1 µL of the aliquot of the extracts were injected into a BPX5 capillary column (L.30m; I.D. 0.25 mm; film thickness 0.25 µm; max.temp.360/370° C) using auto injector (AOC-20i, SHIMADZU).The initial gas chromatography oven temperature was 70° C, 5 min after injection the GC oven temperature was increased from 5° C/min to 320° C and held for 5 min at 320° C. Helium gas was used as a carrier gas, and pressure programmed such that the helium flow was kept constant at a flow rate of 1.7 mL/min.Detection was achieved using MS detection in electron-ionization mode and full scan monitoring mode (m/z 35-500).The ion source temperature was set at 200° C and interface temperature was set at 320° C.

Data Analysis
The results were analysed statistically using oneway analysis of variance (ANOVA) and the mean differences among the three different treatments were tested with a significance level of P<0.05 using a Duncan's multiple range test (Duncan, 1955).The data were presented as mean ± SD.An independent-samples T test was also performed in order to determine the mean differences within the two different groups.

Gram Staining
After Gram staining, the slide was examined under a light microscope and the result revealed that the isolated bacteria showed pink colour colonies which indicated that the bacterium was Gram negative (Figure 2).

Molecular Identification
Based on morphological and biochemical characteristics, a number of colonies representing all recovered aeromonads in this study were amplified using PCR.The agarose gel (1%) stained with ethidium bromide (EtBr) of PCR product is shown in Figure 3.The obtained sequences were then aligned using the Basic Local Alignment Search Tool (BLAST) for the identification of bacteria species.The 16S rRNA gene sequences showed 100% similarity with A. hydrophila in the existing NCBI database (Accesion no KR067615.1)(Table 1)

In vitro Antimicrobial Activity of L. acidophilus against A. hydrophila
The inhibition ability of the commercial Lactobacillus acidophilus (International Food Grade, Laboratory of USA) cells and cell free extracts against the growth of A. hydrophila is presented in Table 2 and Figure 4.In the well diffusion method, the significantly higher inhibition zone (mm) was observed, in the cells of LAB (17.23±0.25) to A. hydrophila compared to the cell free extract (15.17±0.15).The evidence of these clear zones produced by the inhibition of pathogens revealed that LAB successfully inhibited the growth of   the A. hydrophila.However, the actual mechanisms involved in inhibiting growth are not completely understood yet.This inhibition zone afforded by the cells of LAB might be due to antagonism to the pathogen and/or the competition between the probiotic and the pathogenic bacteria for their adhesion sites in agar (Al-Dohail, 2010), or in the mucous membrane (Olsson et al., 1992) or for their nutrition (Enany et al., 2012).It has been well recommended that the LAB may exert their antimicrobial activities through the production of bioactive compounds, such as bactericins, organic acids and hydrogen peroxide during their metabolism (Drago et al., 1997).Similar to the present study, Ajitha et al. (2004) also reported the positive influence of four strains of lactic acid bacteria (LAB) such as Lactobacillus acidophilus, Streptococcus cremoris, Lactobacillus bulgaricus-56 and Lactobacillus bulgaricus-57 in vitro against some bacterial pathogens and observed cell free extracts of those probiotics are effective in inhibiting the growth of Vibrio alginolyticus.In contrast with the present study, no inhibition was observed in the well diffusion method in the case of cell free extract of lactobacilli cultures incubated for 48 hours in MRS broth (Drago et al., 1997).

Effect of L. acidophilus on Survival of P. hypophthalmus infected to A. hydrophila
The fish in all the three groups started to be evidence for clinical signs 1 day after injection with A. hydrophila.Typical clinical sings of this haemorrhagic septicaemia disease caused by this pathogenic bacterium includes lesions of haemorrhages at the base of the pelvic fins, on the ventral surface of the body and abdominal distension (Figure 5B, 5C) when the bacteria and their toxins are exist within various organs of the fish, and severe ulcers of the fish abdominal skin (Figure 5A).Interestingly, the fish injected with either LABP or LABS following challenge using A. hydrophila showed recovery from severe infection (Figure 6).
The cumulative survival of striped catfish after being infected with A. hydrophila is presented in Figure 7 and Table 3.The result revealed that A. hydrophila cells (AM) injected fish started to give evidence of its infection sign from the first day after injection and all the fish were died within the first four days.Whereas, the fish injected with LABP and LABS following challenged with A. hydrophila cells showed 70% and 60% survival rate respectively, at 2-week post challenged, but did not differ significantly (P>0.05) when compared between these two groups.
The GC-MS result revealed that a diverse of compounds was detected in both the LAB cells and cell free extract.The most abundant component is pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2methylpropyl).This component was also detected in the metabolites of Micrococcus luteas previously, which showed a promising anti-cancerous activity (Mithun and Rao, 2012).The recovery from severe infection of fish previously injected with LAB cells and cell free extract might be due to the presence of this anti-cancerous component.Beside this component, a large number of components includes antimicrobials, organic acids and alcohols were also detected in both the LAB cells and cell free extract, which might be a reason of inhibition the growth of A. hydrophila as well as higher survival of previously injected striped catfish using LABP and LABS.Several studies also reported the ability of lactic acid bacteria in producing antimicrobial compounds, organic acid and alcohols (De Keersmaecker et al., 2010;Rattanachaikunsopon and Phumkhachorn 2010).

Figure 2 .Figure 3 .
Figure 2. Gram staining of the isolated bacteria (pink to red stain indicate Gram-negative bacteria)

Figure 4 .Figure 5 .
Figure 4. Zone of antimicrobial inhibition of Lactobacillus acidophilus against Aeromonas hydrophila used well diffusion method.A= Indicates cells of Lactobacillus acidophilus; B= Cell free extract of Lactobacillus acidophilus, C= Control

Figure 6 .Figure 7 .
Figure 6.P. hypophthalmus followed challenged with Aeromonas hydrophila cells showed a healing from severe infection.A= Injected with Lactobacillus acidophilus cells (LABP), B= Injected with Lactobacillus acidophilus cell free extract (LABS)

Table 1 .
Identification of strain using BLAST analysis

Table 2 .
Inhibition zone diameter (mm) of cells and cell free extract of Lactobacillus acidophilus against Aeromonas hydrophila

Table 3 .
Survival of P. hypophthalmus injected with A. hydrophila cells (AM), Lactobacillus acidophilus cell (LABP), and Lactobacillus acidophilus cell free extract (LABS) following challenged with A. hydrophila

Table 4 .
Bioactive compounds identified from the Lactobacillus acidophilus cells (LABP) and cell free extract (LABS) by GC-MS