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Aminnezhad S, Rahimifard N, Kermanshahi R. K, Ranjbar R, Baghery O, Bagheri F. Synergistic Effects of Supernatant from Lactobacillus plantarum Cultured Medium and Conventional Antibiotics Against Pseudomonas Aeruginosa. Biosci Biotech Res Asia 2012;9(2)
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Synergistic Effects of Supernatant from Lactobacillus plantarum Cultured Medium and Conventional Antibiotics Against Pseudomonas Aeruginosa

S. Aminnezhad1, N. Rahimifard2, R.K. Kermanshahi3*, R. Ranjbar 4,Ozra Baghery4 and F. Bagheri5*

1Department of microbiology,Damghan branch,Islamic Azad University, Damghan, Iran.

2Department of Microbiology, Food and Drug Laboratory Research Center (FDLRC), Tehran, Iran 3Department of Microbiology, Food and Drug Control Laboratories (FDCLs), Ministry of Health (MOH), Tehran, Iran

3Department of Biology, School of Science, Alzahra University, Tehran, Iran.

4Molecular Biology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran.

5Department of Microbiology, Pharmaceutical Sciences Branches, Islamic Azad University, Tehran, Iran.

DOI : http://dx.doi.org/http://dx.doi.org/10.13005/bbra/1024

ABSTRACT: Antimicrobial resistance development resulted by using of antibiotics in treatment of infectious diseases. Therefore, there is a big demand for new sources of treatment again such as using of drug. In The other hand reduce antibiotic dose required to decrease the associated side effect. In this study the synergistic action of Conventional Antibiotics and cell free supernatant (CFS) of probiotic (L. plantarum ATCC:8014) against P. aeruginosa ATCC: 27853was evaluated. Cultured medium of probiotic bacteria were separated by centrifuging at 15000 rpm. The antimicrobial effects CFS of L. plantarum was evaluated by well diffusion method. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by micro dilution method according to CLSI 2006. Finally interaction between CFS and Amikacin, Gentamicin, Ciprofloxacin and Azithromycin against P. aeruginosa ATCC: 27853 were examined through checkerboard method and fractional inhibitory concentration (FIC) was determined. The results showed a significant effect by CFS on the P. aeruginosa. The MIC and MBC of CFS from L. plantarum were 62.5 ìlD ml and 125µlD ml. Using the FIC indices, synergistic interactions were observed in combination of CFS and antibiotics. FIC indices of CFS from L. plantarum and Gentamicin, and Azithromycin were 0.124 and 0.312 respectively showing synergism effect, while FIC indices of CFS and Amikacin and Ciprofloxacin were 1.6 and demonstrated indifference action. Our finding indicated that L. plantarum as probiotic bacteria have a significant inhibitory effect on the growth of Pseudomonas aeruginosa ATCC: 27853. The antimicrobial potency of this combination can be useful for designing and developing alternative therapeutic strategies again P. aeruginosa infections.

KEYWORDS: Pseudomonas aeruginosa; Minimal inhibitory concentration (MIC)

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Aminnezhad S, Rahimifard N, Kermanshahi R. K, Ranjbar R, Baghery O, Bagheri F. Synergistic Effects of Supernatant from Lactobacillus plantarum Cultured Medium and Conventional Antibiotics Against Pseudomonas Aeruginosa. Biosci Biotech Res Asia 2012;9(2)

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Aminnezhad S, Rahimifard N, Kermanshahi R. K, Ranjbar R, Baghery O, Bagheri F. Synergistic Effects of Supernatant from Lactobacillus plantarum Cultured Medium and Conventional Antibiotics Against Pseudomonas Aeruginosa. Biosci Biotech Res Asia 2012;9(2). Available from: https://www.biotech-asia.org/?p=9887

Introduction

Nowadays many attentions have been paid to in the other hand Pseudomonas aeruginosa as a top three opportunistic pathogen in hospitalized, immune compromised, and cystic fibrosis patients (1).

Infection caused by P. aeruginosa are often life_ threatening and difficult to treat because of its primary limited susceptibility to commonly use antimicrobial agents (2).

It is necessary to the utilization of alternative antibacterial therapies against Pseudomonas aeruginosa infections. Synergistic combinations of antibiotics and other antimicrobials maybe effective against infections where the development of resistance and/or subsequent failure to monotherapy is prevalent associate with prevention of emergence of bacterial resistance (3,4). One such preference is the possible therapeutic use of probiotics as an adjunct to chemotherapy (5). Probiotics are dietary supplements containing potentially useful yeasts or bacteria. According to the currently adopted description by FAO/WHO in 2001, probiotics are ‘live microorganisms, which when administered in requisite amounts confer a health benefit on the host’ (6, 4).supernatant  of Most lactobacillus bacteria(LAB) such L. plantarum contains several antimicrobials such as organic acids, hydrogen peroxide, aroma components, fatty acid  and low-molecular-mass compounds which kill pathogens(7).

LAB strains can produce organic acid through hetero-fermentative pathways, and these compounds may interact with cell membranes, induce intracellular acidification and diffuse passively across the membrane and protein denaturation (8). H2O2  by peroxidation of membrane lipids thus the  altering the cell membrane permeability and can act as a precursor of the production of bactericidal free radicals such as superoxide (O2ˉ ) and hydroxyl (OHˉ  ) radicals that can damage DNA (9).

Therapeutic role of probiotics in the P. aeruginosa (10,11,12), Staphylococcus aureus (13) and salmonella (6,14, 5) infections have been reported. Treatment with combination of probiotics and antibiotic have been reported to be successful in the management of Helicobacter pylori infection (15).

Combinatorial therapy by probiotic and antibiotic may provide higher antimicrobial activity and decrease the dose of antibiotic required in addition to replenishing the intestinal flora thereby providing benefit to the host and also decrease other antibiotics side effects (16). In the present study the possible effect of combinations between the cell-free lactobacillus supernatant (CFS) and Conventional Antibiotics such Amikacin or Gentamicin Ciprofloxacin and Azithromycin on their antibacterial potencies against Pseudomonas aeruginosa were evaluated.

 Materials and Methods

Bacterial strains and growth conditions

Pseudomonas aeruginosa  ATCC: 27853 and Lactobacillus plantarum ATCC:8014  was procured from Persian Type Culture Collection (PTCC).

aeruginosa sub cultured in nutrient broth (Merck, Germany) for 24 h at 37°C. L. plantarum were grown in De Mann Rogosa Sharpe (MRS) broth  (Merck, Germany) for 48 h at 37°C under anaerobic conditions in a Coy Laboratory anaerobic chamber. (17).

Antibiotic susceptibility tests

Qualitative antibacterial susceptibility of the P. aeruginosa were determined according to the standard disk diffusion (Kirby-Bauer) method (18) using paper disk  including (μg /disc): Azithromycin (15); Ceftriaxone (30); Imipenem (10); Amikacin (30); Chloramphenicol (30); Ceftazidim (30); Tobramycin (10); Gentamycin (10); Ciprofloxacin (5) purchased from Mast Co(Liverpool, UK)

Microbial suspension with 106 colony forming units (CFU/ml) of P. aeruginosa have in NB were prepares specific on to Muller Hilton agar medium. The plates were incubated at 37°C for 24 h in aerobic condition and examined for the inhibition zones diameter appearing around each antibiotic disc. test were carried out thrice for each antibiotics  .Inhibitory zone diameters were compared with the standards provided by the National Committee for Clinical Laboratory Standards (NCCLS) (17).

Preparation of cell free supernatant (CFS) from Lactobacilli strains

CFS was prepared according to the method Ogunbanwo (19).Lactobacillus was grown in 1L deMan, Rogosa and Sharpe (MRS) broth (pH 5.7) for 48 h at 37°C in anaerobic condition. Cell free supernatant obtained by centrifuging the culture at 15000 rpm for 15 min at 4°C and then filtered through 0.45 μm filters (Millipore, Bedford,MA).

In-vitro inhibitory effect of CFS and Antibiotics

Well diffusion method

Antimicrobial activity of CFS was determined by the well diffusion method. In this method, bacterial inoculum colonies from overnight nutrient agar were used to make suspension of the test organisms to be equivalent to the 0.5 McFarland standard. Wells with 6 mm diameter were punched in the agar plates and they were filled with 100 µl of different concentrations of CFS including 10 µl/ml, 50 µl/ml and 100 µl/ml of CFS. The plates were then incubated at 37° C for 24 h and the diameter  zones of inhibition were assessed (20)

The experiments were repeated three times and the mean values of the diameter of inhibition zone with ± standard deviation were calculated.

Determination of minimum inhibitory concentrations (MICs)

The MICs were determined by broth micro dilution assay  according  to the procedures recommended by the Clinical and Laboratory Standards Institute (formerly the National Committee for Clinical Laboratory Standards 2006).

Dilutions of the antibiotics (Amikacin , Gentamicin, Ciprofloxacin and Azithromycin ), ranging from 0.125-256 µg/ml in Muller Hinton Broth were prepared by incorporating the antibiotic stock solution into Muller Hinton Broth. Dilutions of the CFS in the range of 0.12-250 µl/ml were also prepared by incorporation of the CFS into Muller Hinton Broth. Each plate includes positive and negative controls (17).

Briefly, a bacterial inoculum (100 μl), corresponding to 5  105 CFU/ml, was added to 100 μl of serial twofold dilutions of the antibiotics in the wells of microtiter plates. The final volume of each well was 200 μl. The plates were incubated at 37°C for 24 h. The MIC was defined as “the lowest concentration of antibiotic which can inhibited visible growth of microorganism”.

100 μl of liquid from each well without visible growth on to MHA for determination of MBC was used and incubated at 37C for 48-72 hrs.

Finally the lowest concentration of antimicrobial agent being able to reduced  99.9% of the bacteria was assessed as MBC (21). Experiments were done in triplicate.

Fractional inhibitory concentration (FIC) determination and interaction effect of two antimicrobial agents (antibiotics + CFS) on test bacteria

Drug interactions were assessed by using a checkerboard microdilution method, the concentrations of antimicrobial agent were typically ranged from four or five below the expected MIC to twice the anticipated MIC as in the 45 degree line in figure 1 (each square represents one plate), using two fold dilutions of each antimicrobial agent, also concentration of MIC point and dilution lower that it for each antimicrobial agent alone.

Inocula were prepared spectrophotometrically and further diluted in order to procure final concentrations ranging from 0.5  106 CFU/ml. Each microdilution well included 100 μl of the diluted (two times) drug concentrations of both antimicrobials (antibiotics and CFS) was inoculated with 100 μl of the diluted (two times) inoculum suspension (final volume of each well, 200 μl). The trays were incubated at 37°C, and the results were read at 24 h visually with a ELISA reader system (statfax-2100, Awareness Technology Inc., USA).

The FIC index was then calculated by using the observing equation with summing the separate FICs for each drugs present in that well:

FICindex = FICA+ FICB =  +

Where A is the concentration of drug A in well that is the lowest inhibitory concentration in its row, MIC A is the MIC of the organism to drug A alone, and FIC A  is the fractional inhibitory concentration of drug A. Also B is the concentration of drug B in a well that is the lowest inhibitory concentration in its column; MIC B and FICB are defined in the same fashion for drug A.

According to this method, synergism has traditionally been defined as an FIC index of 0.5 or less and additivity as a FIC index of 1.0; antagonism has been defined as a FIC index of 2.0. Synergy was further sub-classified as marked (FIC ≤0.50) and weak (FIC index, between 0.50 and 1.0) (22,23).

Analysis of cell free supernatant (CFS) from L. plantarum for antimicrobial compounds

CFS of each strain of L. plantarum were prepared according to the method of Ogunbanwo(19). The supernatant was filtered using 0.45 µm Millipore filters and 2 aliquot were stored at –20°C until analysed for antimicrobial compounds contain lacticAacid,acetic acids and H2O2 (Sigma) by using reversed-phase fast performance liquid chromatography(RP-FPLC) (24).

Standard stock solutions of lactic acid pKa=3.086  (5.2 mg/mL), acetic acid (5.4  mg/mL) and Hydrogen peroxide (35%) were prepared in ultrapure water and stored at 4 °C (25) .

Standard solutions of organic acids and H2O2 were determined by RP-FPLC, using an AKTA Purifier system (GE Healthcare) equipped with YMC-Triart C18 (250 ˟ 4.6 mmI.D, S-5um, 12nm).

The degassed mobile phase of 0.009 M KH2PO4 adjusted by phosphoric acid to PH 2.06.filtered through a 0.45 µm membrane filter was used at a flow rate of 1ml/min. The wavelength of detection was optimized at 210 nm and the sample injection was 50 µL (24, 26) .CFS from Lactobacilli strains were analyzed by RP-HPLC under the same conditions (24, 27) .

Statistical analysis

The data were analyzed using Graph Pad Prism version 5 (Graphpad Software In, San Deigo, USA). All data were expressed as mean ± S.D. Statistical analyses were evaluated by one-way analysis of variance (ANOVA). Significant level for all tests was considered (p<0. 05)

 

 

Results

Antibiotic susceptibility pattern of P. aeruginosa were shown that was sensitive to Amikacin and Gentamicin, Ciprofloxacin while was resistance to Azithromycin (Table1).

The antimicrobial activities assayed against P. aeruginosa by agar well diffusion assay.

The antimicrobial activity of CFS from L.plantarum in different concentration were determined using the agar well diffusion method summarized in  Table 2. The activity was quantitatively assessed on the basis of the inhibition zone, and their activity index was also calculated along with the minimum inhibitory concentration (MIC), In Table 3, we shown the result of the and MIC and minimum bactericidal concentration (MBC) of the CFS and antibiotics were determined by microdilution method. Also, the FIC value for CFS and antibiotics were shown in Table 4. antimicrobial combination  between CFS and Gentamicin or Azithromycin demonstrated synergistic actions against P. aeruginosa. the combination of CFS and Ciprofloxacin or Amikacin demonstrated indifference action.

Table 1: Antibiotic susceptibility pattern of standard strain P.aeruginosa as determined by disc-diffusion technique

Microorganism              

 

                        Antibiotic

                Conc.) ug.disc(

 

Azithromycin(15)

 

Ciprofloxacin(5)

 

Gentamicin(10)

 

Ceftazidim(30)

 

Tobramycin(10)

 

Amikacin(30)

 

Tobramycin(10)

 

Chloramphenicol(30)

 

Imipenem(10)

 

 

P.aeruginosa

PTCC:1430

 

 

R

 

S

 

S

 

S

 

S

 

S

 

S

 

R

 

S

S-sensitive R-resistant

Table 2: Inhibition zone diameter of CFS from lactobacillus strains  against P.aeruginosa  PTCC:1430 a

Conc.(

 

CFS

10

 

50 100 P value
L.plantarum

ATCC:8014

6a 14.5±0.70 18±00 0.0003
Blank(MRS medium) 6

 

6 6 _

zone of inhibition,including the diameter of the well(6mm);mean value of three independen experiments.

Table 3: Minnimum inhibitory concentration (MIC)and Minnimum bactericidal concentration (MBC)  of antimicrobial agents on  the  P.aeruginosa  PTCC:1430 a

       Antimicrobial

agent

 

MIC & MBC

 

 

CFS ( )b

 

Antibiotics( )

 

L.plantarum

ATCC:8014

 

Amikacin

 

Gentamicin

 

 

Ciprofloxacin

 

 

 

 

 

 

Azithromycin

 

MIC

62.5 8       1 0.25

 

128

 

 

MBC

125 16 4 0.5

 

_

 

aAll determinations were done in  triplicate.

bCFS dissolved in Cation Adjusted Muller Hinton Broth

antimicrobial compound in the CFS of lactobacillus were identified by comparison of retention times and the UV absorption spectra with those obtained from the corresponding standards.

Peaks  of standard solution  were  observed at (6.00±0.13) min for lactic acid, (6.02±0.06) min for acetic acid, and (3.06±0.0.6) min  for  H2O2, for an average of 5 injections. As a control, the antimicrobial compound profile of sterile MRS medium was analysed (Fig. 2A). Compared with this control chromatogram, the CFS of  L. plantarum (Fig. 2B) contained same peaks that corresponding  to acetic  acid(AA) (6. 23 min), lactic acid(LA) (5.73) and H2O2 (3.02 min).

Table  4: Effect of CFS from  lactobacillus  of  different antibiotics against P.aeruginosa

combination of two compounds  

MIC A(

 

MICB

 

MIC A+B

 

Checkerboard FIC index

 

Checkerboard effect

A

(CFS of lactobacillus)

B

(antibiotic)

 

 

 

 

 

 

 

L.plantarum

ATCC:8014

 

Azithromycin

 

 

 

62.5

 

128

 

7.8+32

 

0.312

 

marked synergy

 

Gentamicin

 

62.5

 

1

 

3.9+0.06

 

0.124

 

marked synergy

 

Ciprofloxacin

 

62.5

 

0.25

 

3.9 + 25

 

 

1.6

 

indifference

Amikacin

 

 

62.5

 

8

 

3.9+8

 

1.6

 

indifference

Table 5: Determination of antimicrobial compounds in CFS from Lactobacilli strains by using HPLC method

 

CFS from  lactobacillus

 

antimicrobial compound

 

Retention time(ml)

 

Peak start

(ml)

 

Peak end

(ml)

 

Area/ Total  area

()volume(%)

 

 

 

Blank

(MRS medium)

 

 

H2 o2

 

3.02

 

2.65

 

3.19

 

13.09

 

Lactic acid pKa=3.086

 

5.79

 

5.57

 

6.06

 

 

3.68

 

Acetic acid

pKa=4.76

 

6.24

 

6.06

 

 

6.66

 

3.80

 

 

L.plantarum

ATCC:8014

 

H2 o2

 

3/03

 

2/67

 

3/22

 

13/50

Lactic acid pKa=3.086  

5/73

 

5/50

 

6/08

 

13/50

Acetic acid

pKa=4.76

 

6/22

 

6/08

 

6/68

 

4/23

 

However, this  study  showed that  antimicrobial compound is already  present in sterile MRS medium,  but significantly change were abserved in the amount of compound  during growth of L. plantarum (p<0. 05) (Fig 2Aand 2B).

Figure 1: Simplified checher-board method (MIC A and B drugs is considered as 1 µg/ml. Figure 1: Simplified checher-board method (MIC A and B drugs is considered as 1 µg/ml.

 

Click here to View figure

 

Figure 2: HPLC profile of the cell free supernatant of sterile De Man-Rogosa Sharpe(MRS) medium(A), Lactobacillus plantarum ATCC:8014 (B). Figure 2: HPLC profile of the cell free supernatant of sterile De Man-Rogosa Sharpe(MRS) medium(A), Lactobacillus  plantarum ATCC:8014 (B).

 

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Discussion

aeruginosa infection is on of the most difficult or impossible to eradicate infections and therefore this bacterial infection needs new therapeutic protocol and strategy (10, 28).

Antimicrobial combinations has been utilized as an effective therapeutic strategy with the using of benefit various mechanisms of action (16).

A synergestic combination of aminoglycosides (Gentamicin, Tobramycin and Amikacin), fluoroquinolone (Ciprofloxacin) and Penicillins (Carbenicillin) has been used to treat Pseudomonas aeruginosa infections (29). Also, some P.aeruginosa strains have been reported to have resistance to many of  antibiotics (30, 31).

In the present study, the possible effect of CFS on the bactericidal activities of Conventional Antibiotics such  Amikacin or Gentamicin Ciprofloxacin and Azithromycin were evalute Consequently, the study of Conventional antibiotics in combination with probiotics might prove the benefit as to the using of combination the lower the dose of antibiotic alone.

In the present study, we put forward the hypothesis that whether treatment with combination of cell free supernatant from L.plantarum with Conventional antibiotics have higher antimicrobial activity against P. aeruginosa or not.

Keeping in view the combination of cell free supernatant from L.plantarum  and Conventional Antibiotics was further tested to evaluate the possible synergistic effect against P.aeruginosa.

Aminoglycosides and Azithromycin interfere and affect bacterial protein synthesis through binding to the 30s and 50s ribosomal subunits  of the bacterial cell respectively(32,33). The quinolones such ciprofloxacin exhibit their bactericidal action by blocking DNA replication through gyrase inhibition(34).

Previously, studies reported that aminoglicosids  caused increases  reactive oxygen species (ROS) levels in rats (35). In addition, an increase of reactive oxygen species in the bacterial cells in response to Ciprofloxacin has been shown (36).

In previous studies,  separation, purification and identification of antimicrobial agents produced by  LAB, were conducted by several techniques (15, 37, 38,39) and in this study presence of lactic acid ,acetic acid and H2O2 in CFS of L.plantarum  was confirmed by HPLC analysis.

The organic acids acts by collapsing the electrochemical proton gradient, and H2O2  by per oxidation of membrane lipids thus the  altering the cell membrane permeability which results in disruption of substrate transport systems (40, 41, 42).

Alakomi and colleagues (2000) also found that  lactic acid, in addition to its antimicrobial property  due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiate of the effects of other antimicrobial substances (43).

In the present study a significant increase in antibiotics performance in the presence of CFS was observed in compared ,when P.aeruginosa is treated with antimicrobial agent for each of only.

These antibacterial mechanisms of H2O2  and Gentamicin to production of reactive oxygen species (ROS) might have act cooperatively with each other, leads to a higher bactericidal effect of the combination. in support of our finding , Hoffmann have also reported the sub-MICs of Azithromycin suppress the QS-controlled lasB expression  in parallel  with the  inhibition  of QS- controlled  virulence  factors, alginate and increased sensitivity to hydrogen peroxide in P. aeruginosa(44). Our results  showed that  the synergism abserved between CFS and   Azithromycin Can be made by the influence of Azithromycin increasing sensivity of  P. aeruginosa  on H2O2   produced by L.plantarum.

FIC index also further substantiated the synergism effect between the two compounds. We have previously demonstrated that CFS also reduced the MICs of Gentamicin and Azithromycin. This synergistic effect was also confirmed by checkerboard testing (Fig. 4). Gentamicin and Azithromycin had FIC indices of ˂0.5, indicating synergy between CFS and the antibiotic.

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