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Determination and Partial Characterization of Antimicrobial Material of Pseudomonas aeruginosa Isolated from Milk
Jayant Pawar1*, N. K. Bipinraj2, E. A. Singh3, Nikhil Nikam4 Department of SoST (Nanobiotechnology), International Institute of Information Technology, Pune, India 2 Department of Microbial Biotechnology, BVPs RGI Biotechnology, Pune, India 3 Department of Plant Biotechnology, BVPs RGI Biotechnology, Pune, India 4 Department of SoST (Nanobiotechnology), International Institute of Information Technology, Pune, India
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ABSTRACT Since the broad-spectrum antimicrobials inhibit almost all bacteria in food including the probiotic bacterial strains, this method is not a good choice for food protection. This study presents the partial characterization of bacteriocin-like (inhibiting molecule showing properties of bacteriocin) material produced by Pseudomonas aeruginosa, having narrow-spectrum activity against specific food pathogens. Bacteriocin-like substance was found to be active against Staphylococcus aureus. The maximum antibacterial activity of Pseudomonas aeruginosa was recorded at 16th hour incubation in LB broth at 37 C. The antibacterial activity was found to be extracellular since cell-free supernatant showed inhibition. The inhibitor was inactivated by the addition of proteases, thus confirming the proteinaceous nature of the inhibiting molecule. The inhibitor was stable after extended refrigerated storage and freezing-thawing cycles and also at high temperature incubation (60 C, 100 C, 121 C for 15 min). This fact suggests that bacteriocin-like material produced by Pseudomonas aeruginosa may find application in food protection as a biopreservative in raw and minimally processed food. Keywords: Pseudomonas aeruginosa, Staphylococcus aureus, antimicrobial activity, bacteriocin-like material, narrow-spectrum activity.
poisoning and are often resistant to a wide 1. INTRODUCTION range of traditional antimicrobials [6]. Thus, there is a pressing necessity to develop The food safety issues acquire a great attention since the pathogenic narrow-spectrum antimicrobials for specific food pathogens.
microorganisms are found in the food stuffs. In addition, there is a rapid increase of resistance in pathogenic bacteria for traditional antibiotics [14]. Since the broadspectrum antimicrobials inhibit almost all bacteria in food including the good bacterial strains [5], this method is not a good choice for food protection. bacteria like Sometimes, the Although some traditional antimicrobials and other techniques like refrigeration,
pasteurization, salting, irradiation, artificial food additives addition are available to protect the food from pathogenic
microorganisms but, because of broadspectrum activity of such techniques and antimicrobials, the good bacteria and nutritive value of the food get damaged [7]. Excessive
pathogenic
Staphylococcus
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use of artificial food additives in food is found to have adverse effect on community health. The authors work approach was determining and characterizing bacteriocin which has narrow-spectrum activity against specific food pathogens [8]. This approach makes food safe and free from food pathogens. The bacteriocin not only kills selective pathogenic bacteria but it also preserves the nutritive quality and probiotic microorganisms of food [9]. Bacteriocins can also be used as the next generation of veterinary [1012]. and human some 2.1. Strains and Culture Conditions The Pseudomonas aeruginosa was 2. MATERIALS AND METHODS materials and methodology used in this work is described in the second section. Third section describes the result and discussion and the objective of this work is concluded in the fourth section. Finally, references are mentioned in the last section.
subcultured on LB agar slant (24 h, 37 C) and stored in a refrigerator at 4 C. Standard bacterial species used as indicator
antimicrobials
Recently,
microorganisms, Staphylococcus aureus, was subcultured on LB agar slant (24 h, 37 C) and stored in the refrigerator at 4 C.
bacteriocins generally recognized as safe (GRAS) have received particular attention for their potential applications in food
2.2. Screening for Antagonistic Activity An agar well diffusion assay (AWDA) was
This implies that more attention should be focused on narrow-spectrum antimicrobials. In this paper, authors report on the screening and partial characterization of bacteriocinlike material produced by Pseudomonas aeruginosa against Staphylococcus aureus both of which were found in milk. The Pseudomonas species having antimicrobial properties against plant pathogens [14] and some strains can produce antibacterial active compounds against Gram-positive bacteria [15].
used for detection of antagonistic activity [16]. LB agar plates (1.8% agar), in which wells were formed, were spread inoculated with an overnight culture of the
Staphylococcus aureus. Wells, of 5 mm in diameter and of 18 L in capacity, were formed by using sterile gel borer on the surface of agar plate. The 15 L of an overnight culture of the Pseudomonas
aeruginosa were placed in each well. The plates were then incubated aerobically for 24 h at 37 C and were subsequently examined for zones of inhibition. Inhibition
This paper further proceeds in four different sections to describe the materials,
was recorded as negative if no zone was observed around the agar well. Each
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antagonistic activity was related to the area (mm ) of the inhibition zone displayed [17].
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60 C, 100 C and also in autoclave (121 C) for 15 min. The effect of extended storage at low refrigerated temperature on
2.3. Detection of Inhibitory Activity The Pseudomonas aeruginosa were grown overnight at 37 C in 20 mL LB broth. Cellfree supernatants were bacterial obtained culture by broth
supernatants stability was evaluated by placing supernatants at 4 C up to 15 days [18, 19]. The activity was checked against Staphylococcus aureus by AWDA. In all cases, a positive control, consisting of freshly prepared cell-free supernatants was tested on parallel.
centrifuging the
(10,000 rpm for 10 min at 4 C) (sample 1) and culture pellets were dissolved in sterile distilled water (sample 2) [18]. The
(ii) To test pH influence, cell-free culture supernatants were adjusted to pH 5.0, 6.0, 7.0, 8.0, 9.0 with dilute HCl or NaOH, mixed, and allowed standing at room temperature for 2 h and their activity was checked against
2.4. Kinetics of Growth and Bacteriocin Biosynthesis To elucidate the time of incubation at which Pseudomonas aeruginosa exhibited the
(iii) The effects of trypsin, proteinase K, and 0.2N NaOH on AMS activity were
maximum bacteriocin production, sterile flasks containing LB broth inoculated with 1% of an overnight culture were incubated at 37 C. At intervals of 0, 4, 8, 12, 16, 20, 24, and 28 h samples were removed for
determined by the method described by [20, 21]. The absence of inhibition zone in presence of the protease confirmed
measurement of biomass by absorbance at 600 nm [18], for protein concentration by Lowerys method and for antimicrobial activity of supernatants by the AWDA method as described in 2.2. 3.1. Results 3.1.1. Screening for Antagonistic Activity Pseudomonas aeruginosa were shown to 2.5. Characterization of the Antibacterial Compound (i) Sensitivity of antibacterial compounds to heat was investigated by treating the culture supernatant of P, aeruginosa in water bath at
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produce
inhibition
zone
against
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3.1.2. Detection of Inhibitor Site of production of Pseudomonas aeruginosa
Synthesis Both sample 1 (cell-free supernatants) and sample 2 (culture pellets dissolved in sterile distilled water) showed antagonistic activities against Staphylococcus aureus by the AWDA as described above. It was proved that the inhibitor synthesis site was extracellular since cell-free supernatants of P, aeruginosa showed inhibition. (Figure 1)
Graph 2: Protein Concentration. Fig. 1: Cell-free supernatants of Pseudomonas aeruginosa showed inhibition.
3.1.3. Kinetics of Growth and Bacteriocin Biosynthesis Graphs 1, 2 and 3 show the profiles of the growth kinetics, antimicrobial activity under optimal growth conditions and protein
Graph 3: Activity of Inhibitor.
concentration for Pseudomonas aeruginosa. The Pseudomonas aeruginosa has shown maximum growth at 20th hour and maximum activity of inhibitor and protein concentration at 16th hours incubation time. Therefore, the optimum incubation time for inhibitor
3.1.4. Characterization of the Inhibitor Effect of Thermal Treatments: Supernatant of Pseudomonas aeruginosa isolate showed stronger resistance to heat treatment,
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60 C, 100 C, 121 C (Figure 2) and also at low refrigerated temperature (4 C) up to 13 days. (Table I)
Table I: Effect of Thermal Treatments on CellFree Supernatant of Pseudomonas aeruginosa against Staphylococcus aureus.
Producer Strains Indicator Microorganisms pH of Producer Strain AMC Pseudomo nas aeruginosa pH 5.0 pH 6.0 pH 7.0 pH 8.0 pH 9.0 + + + + Staphylococcus aureus
aeruginosa grown at 37 C during 16th hour, since this led to maximum antimicrobial activity. The supernatant of Pseudomonas aeruginosa showed maximum antimicrobial activity (zone of inhibition) at pH 6.0 and 7.0 (Table II).
Sensitivity
to
Proteolytic
Enzymes:
Application of Trypsin and Proteinase K at final concentration of 1 mg/mL and 0.2N NaOH led to inactivation of the antagonistic activities of culture supernatants which was confirmed by AWDA. (Table III; Figure 3)
Table II: Influence of Different pH on Activity of Cell-Free Supernatant of Pseudomonas Table III: Effect of Proteolytic Enzymes on Activity of Cell-Free Supernatant of Pseudomonas
Staphylococcus aureus
Fig. 2: Supernatant of Pseudomonas aeruginosa Showed Stronger Resistance to Heat Treatment, Retaining Antibacterial Activity after15 min at 60 C, 100 C, 121 C.
Fig. 3: Inactivation of the Antagonistic Activities of Culture Supernatants after Treatment with Protease.
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3.2. Discussion As the final goal was to apply a hurdle to reduce the prevalence of Staphylococcus aureus, thus to enhance the safety and keeping quality of food, we restricted our investigations in the following sections to the study of only Pseudomonas aeruginosa exhibiting antagonistic aureus. activity against aeruginosa bacteriocin was produced during the exponential growth phase, with the greatest production occurring during the beginning of the stationary phase [26, 27]. In our study, Pseudomonas aeruginosa were sensitive to Ampicillin and other antibiotics and showed hemolytic activity. Therefore, our strain may not be suitable to be used as a starter culture or probiotic culture in food or feed directly. Nevertheless, additional
Staphylococcus
Pseudomonas
aeruginosa is able to produce proteinaceous antimicrobial compound that inhibits the growth of Staphylococcus aureus. It may also be due to the production of bacteriocin or bacteriocin-like compounds [19, 23]. Since bacterial strains need to withstand the competition of other microorganisms to survive in their hostile natural environment, so they often produce antimicrobials [24]. The outer membrane of Gram-negative bacteria membrane antimicrobial may protect the the cytoplasmic of the
experiments need to be designed to test the allergenicity bacteriocin-like and pathogenicity produced of by
material
Pseudomonas aeruginosa for further use in food or feed or anywhere in medications. However, Pseudomonas aeruginosa is an opportunistic human pathogen that can cause a wide range of clinical symptoms and infections [28]. Thus, its use as a biopreservative is not recommendable. But bacteriocin-like material of Pseudomonas aeruginosa can be used as a bio-preservative if it does not show any allergenic and pathogenic effects on human and other animals. displayed However, by antibacterial activity
from
action [25].
compound
Hence,
Pseudomonas aeruginosa cannot be killed by Staphylococcus aureus since it is Gramnegative bacteria. In the antagonistic activity, Pseudomonas aeruginosa was sensitive to proteolytic enzymes, indicating that it was due to proteinaceous nature of the inhibitor. The bacteriocin produced by producer strains was thermostable and thus would be a very useful characteristic if it was to be used as a food preservative, because many foodprocessing procedures involve a heating step. It is also stable over a wide range of pH and may be useful in acid as well as nonacid foods. Like most bacteriocins, Pseudomonas
Pseudomonas
aeruginosa
4. CONCLUSIONS
The Pseudomonas aeruginosa inhibits the growth of Staphylococcus bacteriocin-like antimicrobial aureus material. activity by The of
producing maximum
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showed inhibition. The antimicrobial activity of pseudomonas aeruginosa was inactivated by the addition of proteases, thus confirming the proteinaceous nature of the inhibitor. The bacteriocin activity was stable after extended refrigerated storage and freezing-thawing cycles and also at high temperature (60 C, 100 C, 121 C for 15 min). Thus the bacteriocin like- material of Pseudomonas aeruginosa may be used as barrier flora against the settlement of undesirable
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