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Effect of calcium on Pseudomonas aeruginasa and Bacillus cereus metabolites

Efeito do cálcio nos metabólitos de Pseudomonas aeruginasa e Bacillus cereus

Abstract

The effects of Calcium (Ca+2) on virulence and some parameters should be analyzed in this study. Pseudomonas aeruginosa Gram (-) and Bacillus cereus Gram (+) were used. Both bacteria are soil bacteria. In this study; the effect of Ca+2 on protease, amylase, LasB elastolytic assay, H2O2, pyorubin and biofilm on metabolites of these bacteria were investigated during 24 hour time. In this study, the effect of Ca+2 on the production of some secondary metabolites on P. aeruginosa and B. cereus was investigated and presented for the first time by us.

Keywords:
calcium; Bacillus cereus; Pseudomonas aeruginosa; seconder metabolite

Resumo

Os efeitos do cálcio (Ca+2) na virulência e alguns parâmetros devem ser analisados neste estudo. Pseudomonas aeruginosa Gram (-) e Bacillus cereus Gram (+) foram usados. Ambas as bactérias são bactérias do solo. Neste estudo, o efeito do Ca+2 sobre a protease, amilase, ensaio elastolítico LasB, H2O2, piorubina e biofilme nos metabólitos dessas bactérias foram investigados durante 24 horas. Neste estudo, o efeito do Ca+2 na produção de alguns metabólitos secundários em P. aeruginosa e B. cereus foi investigado e apresentado pela primeira vez por nós.

Palavras-chave:
cálcio; Bacillus cereus; Pseudomonas aeruginosa; metabólito seconder

1. Introduction

α-amylase (E.C.3.2.1.1; 1,4-α-D-glucan-glucanohydrolase) is a hydrolytic enzyme that hydrolyzes α-1,4-glycosidic linkages in starch and forms products such as glucose or maltose. This enzyme is one of the most important biotechnological products used in various industrial processes such as food, paper, textile and detergent (Liu and Xu, 2008LIU, X.D. and XU, Y., 2008. A novel raw starch digesting α-amylase from a newly isolated Bacillus sp. YX-1: purification and characterization. Bioresource Technology, vol. 99, no. 10, pp. 4315-4320. PMid:17920264.).

α-amylase can be isolated from plants, animals or microorganisms. It is found in many bacteria (Bacillus spp. (B. amyloliquefaciens, B. subtilis, B. cereus, B. amyloloquefaciens, B. amloloquefaciens, B. megaterium, B. licheniformis, Lactobacillus sp, Escherichia sp, Proteus sp, Clostridium sp and Pseudomonas sp.) and fungi (Aspergillus, Penicillum, Sefalosporium, Neurospora and Rhizopus). Microorganisms in particular, are used more in the production of this enzyme. Bacillus species are more widely used in commercial protease production (Parmar and Pandya, 2012PARMAR, D. and PANDYA, A., 2012. Characterization of amylase producing bacterial isolates. Bulletin of Environment, Pharmacology. Life Sciences, vol. 1, no. 6, pp. 42-47.; Venkata and Divakar, 2013VENKATA, N.R.E. and DIVAKAR, G., 2013. Production of amylase by using Pseudomonas aeruginosa ısolated from garden soil. International Journal of Advance Pharmaceutical and Biological Chemistry, vol. 2, no. 1, pp. 50-56.; Khannous et al., 2014KHANNOUS, L., JRAD, M., DAMMAK, M., MILADI, R., CHAABEN, N., KHEMAKHEM, B., GHARSALLAH, N. and FENDRI, I., 2014. Isolation of a novel amylase and lipase-producing Pseudomonas luteola strain: study of amylase production conditions. Lipids in Health and Disease, vol. 13, no. 9, pp. 1-9. PMid:24405763.; Sundarram and Murthy, 2014SUNDARRAM, A. and MURTHY, T.P.K., 2014. α-Amylase production and applications: a review. Journal of Applied and Environmental Microbiology, vol. 2, no. 4, pp. 166-175.; Keharom et al., 2016KEHAROM, S., MAHACHAI, R. and CHANTHAI, S., 2016. The optimization study of α-amylase activity based on central composite design-response surface methodology by dinitrosalicylic acid method. International Food Research Journal, vol. 23, no. 1, pp. 10-17.; Özcan and Çorbaci, 2017ÖZCAN, K. and ÇORBACI, C., 2017. Streptomyces sp. K22 ve K30 suşlarından lipaz ve proteaz enzim üretimi. Karadeniz Fen Bilimleri Dergisi, vol. 7, no. 2, pp. 128-135.).

P. aeruginosa is a metabolically versatile Gram (-) pathogenic bacterium that has adapted to many environments associated with terrestrial, aquatic, animal, human and plants. In addition, Pseudomonas species are bacterial groups with many scientific and technological importances. It is a metabolically versatile and powerful organism that can use many simple or complex organic compounds. Pseudomonas has a fairly large genome in its genes that contains many different virulence factors. In this way, it has the ability to adapt to almost any environment. As a result of processes such as phase variation or adaptive mutations to changing environmental conditions, rapid change of the P. aeruginosa genotype is possible in producing morphologically different phenotypic variants. Pseudomonas sp. is capable of producing many extracellular enzymes such as lipase and amylase. Proteases secreted, play an important role in pathogenesis during acute infections. Apart from this, P. aeruginosa can produce LasB elastase and LasA staphylolytic protease secretion (Kessler et al., 1993KESSLER, E., SAFRIN, M., OLSON, J.C. and OHMAN, D.E., 1993. Secreted LasA of Pseudomonas aeruginosa is a staphylolytic protease. The Journal of Biological Chemistry, vol. 268, no. 10, pp. 7503-7508. PMid:8463280.; Kong et al., 2005KONG, K.F., JAYAWARDENA, S.R., INDULKAR, S.D., DEL PUERTO, A., KOH, C.L., HØIBY, N. and MATHEE, K., 2005. Pseudomonas aeruginosa Amp R is a global transcriptional factor that regulates expression of Amp C and Pox B β- lactamases, proteases, quorum sensing and other virulence factors. Antimicrobial Agents and Chemotherapy, vol. 49, no. 11, pp. 4567-4575. PMid:16251297.; Hosseinidoust et al., 2013HOSSEINIDOUST, Z., TUFENKJI, N. and VAN DE VEN, T.G., 2013. Predation in homogeneous and heterogeneous phage environments affects virulence determinants of Pseudomonas aeruginosa. Applied and Environmental Microbiology, vol. 79, no. 9, pp. 2862-2871. PMid:23435883.; Jiménez et al., 2015JIMÉNEZ, T., NOYOLA, A., ROMERO, R., BARRERA, R., ROMÁN, R., RAMOS, G., CARLOS, R.L.J., SANCHEZ, A. and ARADILLAS, V., 2015. Pseudomonas aeruginosa strains resistant to antibiotics and heavy metals, producing biosurfactant, pyocyanin and biofilm from surfaces hospital environment. Bothalia Journal, vol. 45, no. 4, pp. 36-45.; Georgescu et al., 2016GEORGESCU, M., GHEORGHE, I., CURUTIU, C., LAZAR, V., BLEOTU, C. and CHIFIRIUC, M.C., 2016. Virulence and resistance features of Pseudomonas aeruginosa strains isolated from chronic leg ulcers. BMC Infectious Diseases, vol. 16, no. 92, suppl. 1, pp. 1-7. PMid:27169367.; Kalaiarasan and Narasimha, 2016KALAIARASAN, E. and NARASIMHA, H.B., 2016. Antimicrobial resistance patterns and prevalence of virulence factors among biofilm producing strains of Pseudomonas aeruginosa. European Journal of Biotechnology and Bioscience, vol. 4, no. 11, pp. 26-28.; Marou et al., 2016MAROU, I., ABOULKACEM, A., TIMINOUNI, M. and BELHAJ, A., 2016. Virulence profiles of clinical and environmental Pseudomonas aeruginosa isolates from Central Morocco. African Journal of Microbiology, vol. 10, no. 14, pp. 473-480.).

Metalloproteinase elastase A, which belongs to Pseudomonas, has been reported to break down the elastin and increase the substrate range of elastase B. Elastase B and alkaline protease specifically destroy host defense proteins. This is very important in virulence (Caballero et al., 2001CABALLERO, A.R., MOREAU, J.M., ENGEL, L.S., MARQUART, M.E., HILL, J.M. and O’CALLAGHAN, R.J., 2001. Pseudomonas aeruginosa protease IV enzyme assays and comparison to other Pseudomonas proteases. Analytical Biochemistry, vol. 290, no. 2, pp. 330-337. PMid:11237336.). Elastase has three active amino acids. These are the catalytic triads that work together; aspartate, histidine and serine. Elastase, E. coli and other Gram (-) bacteria outer membrane elastase, also have the property of breaking down Shigella virulence factors, which can be done by carboxy of small and hydrophobic amino acids such as glycine, alanine and valine (Vijay et al., 2014VIJAY, A., BABU, S. and GUDLAPALLY, J., 2014. Isolation and extraction of elastase producing bacteria from natural habitat (water-soil sample) and targeting it to APP, a cause of alzheimer’s disease. Helix, vol. 2, pp. 516-520.).

The structures we call biofilms are actually a group of microorganisms attached to a surface and covered with an exopolysaccharide matrix. It is most commonly created by P. aeruginos. The presence of chemotaxis, motility, surface adhesions and surfactants towards the surface are factors affecting biofilm formation (Bose et al., 2009BOSE, S., KHODKE, M., BASAK, S. and MALLICK, S.K., 2009. Detection of biofilm producing Staphylococci: need of the hour. Journal of Clinical and Diagnostic Research, vol. 3, pp. 1915-1920.). Biofilm, in phytopathogenic microorganisms and animal pathogens, adaptation promotes survival. Cells in biofilm are said to be more resistant to oxidative stress than free cells (Zhang et al., 2018ZHANG, Y., KONG, J., XIE, Y., GUO, Y., CHENG, Y., QIAN, H. and YAO, W., 2018. Essential oil components inhibit biofilm formation in Erwinia carotovora and Pseudomonas fluorescens via anti-quorum sensing activity. Lebensmittel-Wissenschaft + Technologie, vol. 92, pp. 133-139.).

Bacillus cereus can be found widely in soil and plants. Bacteria possessing psychrotrophic, spore, Gram (+), flagella, aerobic, and peritric flagels are aerobic. Optimum growing is usually 30 °C. B. cereus has lecithinase, gelatinase, amylase and protease activity. It can reduce nitrate and is resistant to polymyxin. Many strains can also breed in 7.5% salt. Cereus takes its name from cereal, which means grain (Kalkan and Halkman, 2006KALKAN, S. and HALKMAN, K., 2006. Bacillus cereus ve içme sütünde oluşturduğu sorunlar. Orlab On-Line Mikrobiyoloji Dergisi, vol. 4, no. 1, pp. 1-11.).

Piyorubin is a bright red color phenazine pigment produced by some P. aeruginosa strains, insoluble in chloroform and soluble in water. It is irreversibly discolored at low oxygen concentration. It is red in all pH grades (Ogunnariwo and Hamilton-Miller, 1975OGUNNARIWO, J. and HAMILTON-MILLER, T., 1975. Brown- and red-pıgmented Pseudomonas aerugınosa: differentiation between melanin and pyorubrin. Journal of Medical Microbiology, vol. 8, no. 1, pp. 199-203. PMid:805242.; Hosseinidoust et al., 2013HOSSEINIDOUST, Z., TUFENKJI, N. and VAN DE VEN, T.G., 2013. Predation in homogeneous and heterogeneous phage environments affects virulence determinants of Pseudomonas aeruginosa. Applied and Environmental Microbiology, vol. 79, no. 9, pp. 2862-2871. PMid:23435883.; Kalaiarasan and Narasimha, 2016KALAIARASAN, E. and NARASIMHA, H.B., 2016. Antimicrobial resistance patterns and prevalence of virulence factors among biofilm producing strains of Pseudomonas aeruginosa. European Journal of Biotechnology and Bioscience, vol. 4, no. 11, pp. 26-28.; Lo et al., 2016LO, Y.L., SHEN, L., CHANG, C.H., BHUWAN, M., CHIU, C.H. and CHANG, H.Y., 2016. Regulation of motility and phenazine pigment production by fliA is cyclic-di-GMP dependent in Pseudomonas aeruginosa PAO1. PLoS One, vol. 11, no. 5, e0155397. PMid:27175902.; Marou et al., 2016MAROU, I., ABOULKACEM, A., TIMINOUNI, M. and BELHAJ, A., 2016. Virulence profiles of clinical and environmental Pseudomonas aeruginosa isolates from Central Morocco. African Journal of Microbiology, vol. 10, no. 14, pp. 473-480.).

Studies suggest that cellular Ca+2 in a host can be an environmental clue for opportunistic bacteria and can trigger their virulence. As already known, Ca+2 in prokaryotes has roles in many physiological events such as spore formation, motility, cell differentiation, transport and virulence (Guragain et al., 2013GURAGAIN, M., LENABURG, D.L., MOORE, F.S., REUTLINGER, I. and PATRAUCHAN, M.A., 2013. Calcium homeostasis in Pseudomonas aeruginosa requires multiple transporters and modulates swarming motility. Cell Calcium, vol. 54, no. 5, pp. 350-361. PMid:24074964.).

In this study, it was aimed to observe the effect of calcium on secondary metabolite production in two different bacteria and investigated for the first time.

2. Materials and Methods

2.1. Reagents

All chemicals were of the highest purity available commercially.

2.2. Microorganism

P. aeruginosa (ATCC 27853) and B. cereus (ATCC 10876) obtained from the ATCC and used this study.

2.3. Growth conditions

Microorganisms were grown and cultivated as follows: 3 mL of 24 h bacterial inoculum (OD600 = 0.3-0.4) was inoculated into 5 mL growth Nutrient Broth media and agitated at the rate of static for 24 h at 37 °C. Crude extracellular enzyme solutions were prepared by removing the cells by centrifugation at 13.500 rpm and room temperature for 5 min. Supernatant harvested was assayed. The concentrations of calcium based in preliminary experiments; in NB was 0,05 M, 0,15 M and 0,15 M were used in Nutrient Broth.

2.4. Amylase activity assays

The amount of the reducing sugars released by the action of amylases on starch was currently performed at 37 °C and pH 7 phosphate buffer for 15 min and the increase in the glucose was determined by antron method. The reaction mixture contained 0.5 mL starch (2% in 0.01 M mM phosphate buffer) and the 0.5 mL enzyme solution in a final volume of 1 mL. One unit of amylase was defined as the amount of enzyme, required to produce reducing sugars equivalent to 1 μmol glucose/min at 37 °C and at pH 7.0 (Geok et al., 2003GEOK, L.P., RAZAK, C.N.A., ABD RAHMAN, R.N.Z., BASRI, M. and SALLEH, A.B., 2003. Isolation and screening of an extracellular organic solvent-tolerant protease producer. Biochemical Engineering Journal, vol. 13, pp. 73-77.; Venkata and Divakar, 2013VENKATA, N.R.E. and DIVAKAR, G., 2013. Production of amylase by using Pseudomonas aeruginosa ısolated from garden soil. International Journal of Advance Pharmaceutical and Biological Chemistry, vol. 2, no. 1, pp. 50-56.).

2.5. Pyorubin

Bacteria were incubated at 37 °C with static for 24 h. The pigments in the bacterial culture were extracted with chloroform. After centrifugation, the reaction mixture contained 1 mL supernatan and 1.5 mL chloroform added. The chloroform layer was mixed with 0.5 mL of 0.2 N HCl, which yielded a pink solution and the absorbance, was measured at OD525. Similarly, the OD525 of the aqueous phase was alsomeasured and normalized to indicate pyorubin production per cell (Hosseinidoust et al., 2013HOSSEINIDOUST, Z., TUFENKJI, N. and VAN DE VEN, T.G., 2013. Predation in homogeneous and heterogeneous phage environments affects virulence determinants of Pseudomonas aeruginosa. Applied and Environmental Microbiology, vol. 79, no. 9, pp. 2862-2871. PMid:23435883.; Kalaiarasan and Narasimha, 2016KALAIARASAN, E. and NARASIMHA, H.B., 2016. Antimicrobial resistance patterns and prevalence of virulence factors among biofilm producing strains of Pseudomonas aeruginosa. European Journal of Biotechnology and Bioscience, vol. 4, no. 11, pp. 26-28.; Lo et al., 2016LO, Y.L., SHEN, L., CHANG, C.H., BHUWAN, M., CHIU, C.H. and CHANG, H.Y., 2016. Regulation of motility and phenazine pigment production by fliA is cyclic-di-GMP dependent in Pseudomonas aeruginosa PAO1. PLoS One, vol. 11, no. 5, e0155397. PMid:27175902.; Marou et al., 2016MAROU, I., ABOULKACEM, A., TIMINOUNI, M. and BELHAJ, A., 2016. Virulence profiles of clinical and environmental Pseudomonas aeruginosa isolates from Central Morocco. African Journal of Microbiology, vol. 10, no. 14, pp. 473-480.).

2.6. Assay of protease activity

The overnight culture of P. aeruginosa and B. cereus isolates was inoculated in Nutrient Broth. Protease activity was measured by some modification of the reaction mixture consisted of 1.0 mL enzyme solution preincubated at 37 °C for 10 min. The reaction was started by the addition of 1.0 mL casein 6.5 mg/mL (in 0.05 M phosphate buffer pH 7.0). The reaction mixture was then incubated in the incubator at 37 °C for 10 min shaker and terminated by the addition of 1 mL 10% trichloroacetic acid (TCA). A vortex mixer was used. This mixture was further incubated at 37 °C for 20 min, followed by centrifugation at 13,500 rpm for 10 min. The supernatant was harvested. To 300 μL supernatant, 750 μL of 0.5 M Na2CO3 and 150 μL folin ciocalteau reagent: water (1:3 v/v) was added to yield a blue color. The colored mixture was incubated in an incubator at 37 °C for 20 min. Absorbance was read at OD660 nm. The amount of tyrosine was determined from the tyrosine standard curve (Geok et al., 2003GEOK, L.P., RAZAK, C.N.A., ABD RAHMAN, R.N.Z., BASRI, M. and SALLEH, A.B., 2003. Isolation and screening of an extracellular organic solvent-tolerant protease producer. Biochemical Engineering Journal, vol. 13, pp. 73-77.; Özcan and Çorbaci, 2017ÖZCAN, K. and ÇORBACI, C., 2017. Streptomyces sp. K22 ve K30 suşlarından lipaz ve proteaz enzim üretimi. Karadeniz Fen Bilimleri Dergisi, vol. 7, no. 2, pp. 128-135.; Fitriani and Güven, 2018FITRIANI, S. and GÜVEN, K., 2018. Isolation, screening, partial purification and characterization of protease from halophilic bacteria isolated from Indonesian fermented food. Anadolu Üniversitesi Bilim ve Teknoloji Dergisi C - Yaşam Bilimleri ve Biyoteknoloji, vol. 7, no. 2, pp. 130-142.).

2.7. Biofilm assay

Biofilm generated by P. aeruginosa was evaluated using the following crystal violet assay (Jiménez et al., 2015JIMÉNEZ, T., NOYOLA, A., ROMERO, R., BARRERA, R., ROMÁN, R., RAMOS, G., CARLOS, R.L.J., SANCHEZ, A. and ARADILLAS, V., 2015. Pseudomonas aeruginosa strains resistant to antibiotics and heavy metals, producing biosurfactant, pyocyanin and biofilm from surfaces hospital environment. Bothalia Journal, vol. 45, no. 4, pp. 36-45.). The biofilm forming ability isolates were tested using glass tube with little modifications. The overnight culture of P. aeruginosa isolates were inoculated in Nutrient Broth supplemented with Ca+2 (0; 0.05; 0.1 and 0.15 M) for 24 hours at 37 °C. After incubation, removing the planktonic bacteria, the wells were carefully rinsed three times-distilled water and then stained with 0.1% of crystal violet (10 minutes, at room temperature) and washed with distilled water (three-times). After air drying, ethanol (95%) was added and incubated for 15 min to remove the bound crystal violet. The absorbance was measured spectrophotometrically at OD570 nm, for quantification of biofilm biomass (Jiménez et al., 2015JIMÉNEZ, T., NOYOLA, A., ROMERO, R., BARRERA, R., ROMÁN, R., RAMOS, G., CARLOS, R.L.J., SANCHEZ, A. and ARADILLAS, V., 2015. Pseudomonas aeruginosa strains resistant to antibiotics and heavy metals, producing biosurfactant, pyocyanin and biofilm from surfaces hospital environment. Bothalia Journal, vol. 45, no. 4, pp. 36-45.; Kalaiarasan and Narasimha, 2016KALAIARASAN, E. and NARASIMHA, H.B., 2016. Antimicrobial resistance patterns and prevalence of virulence factors among biofilm producing strains of Pseudomonas aeruginosa. European Journal of Biotechnology and Bioscience, vol. 4, no. 11, pp. 26-28.; Mirani et al., 2018MIRANI, Z.A., FATIMA, A., UROOJ, S., AZIZ, M., KHAN, M.N. and ABBAS, T., 2018. Relationship of cell surface hydrophobicity with biofilm formation and growth rate: a study on Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli. Iranian Journal of Basic Medical Sciences., vol. 21, no. 7, pp. 760-769. PMid:30140417.; Zhang et al., 2018ZHANG, Y., KONG, J., XIE, Y., GUO, Y., CHENG, Y., QIAN, H. and YAO, W., 2018. Essential oil components inhibit biofilm formation in Erwinia carotovora and Pseudomonas fluorescens via anti-quorum sensing activity. Lebensmittel-Wissenschaft + Technologie, vol. 92, pp. 133-139.).

2.8. LasB elastolytic assay

Liquid cultures were inoculated on Starch Agar plates. The test was performed with some modifications. 1 mL supernatan, 1 mL reaction mixture [1 mg/mL ECR in 200 mM Trizma-base buffrer (pH 8.8)] and then incubated with shaking at 37 °C for 30 min. Then 0.5 mL 100 mM EDTA added and reaction stopped. Insoluble ECR was removed by centrifugation (6000 rpm and room temperature 5 min), and the absorption of the supernatant was measured at OD495 nm. Activity was expressed as change in OD495 g/protein (Kong et al., 2005KONG, K.F., JAYAWARDENA, S.R., INDULKAR, S.D., DEL PUERTO, A., KOH, C.L., HØIBY, N. and MATHEE, K., 2005. Pseudomonas aeruginosa Amp R is a global transcriptional factor that regulates expression of Amp C and Pox B β- lactamases, proteases, quorum sensing and other virulence factors. Antimicrobial Agents and Chemotherapy, vol. 49, no. 11, pp. 4567-4575. PMid:16251297.; Vijay et al., 2014VIJAY, A., BABU, S. and GUDLAPALLY, J., 2014. Isolation and extraction of elastase producing bacteria from natural habitat (water-soil sample) and targeting it to APP, a cause of alzheimer’s disease. Helix, vol. 2, pp. 516-520.; Jose et al., 2017JOSE, D., JOSE, S. and SINGH, B., 2017. Prufication and characterization of higly active LasB protease from Pseudomonas aeruginosa MCCB123. Indian Journal of Experimental Biology, vol. 55, pp. 303-310.).

2.9. H2O2 sensitivity assay

The test was performed with some modifications. Liquid cultures were inoculated on Müller Hinton agar plates. Empty antibiogram discs were placed on which 4 ul H2O2 (30%) was added. Plates incubated for at 37 °C and 24 hours. Then, diameter measurements were recorded (Zheng et al., 2018ZHENG, Y., LI, Y., LONG, H., ZHAO, X., JIA, K., LI, J., WANG, L., WANG, R., LU, X. and ZHANG, D., 2018. bifA regulates biofilm development of Pseudomonas putida MnB1 as a primary response to H2O2. Frontiers in Microbiology, vol. 9, pp. 1490. PMid:30042743.).

All experiments were carried out in triplicates.

2.10. Statistics

Data were presented as mean ± SD. Student’s t-test was used for comparing and significant difference was claimed when P < 0.05.

3. Results

The determination of reducing sugars was generally carried out by the antrone method. The concentration of the glucose was determined at OD620 nm spectrophotometrically. It is mainly used in the assay of α-amylase activity (Keharom et al., 2016KEHAROM, S., MAHACHAI, R. and CHANTHAI, S., 2016. The optimization study of α-amylase activity based on central composite design-response surface methodology by dinitrosalicylic acid method. International Food Research Journal, vol. 23, no. 1, pp. 10-17.). In our study, various seconder parameters of B. cereus and P. aeruginosa were investigated in solid media, and 24-hour incubation at 37 °C was evaluated. The effects of Ca+2 on virulence and some parameters were tried to be analyzed in this study. P. aeruginosa and B. cereus were used as Gram (-) bacteria as a control and Gram (+) bacteria as a control. Both bacteria are soil bacteria. The concentration was left at 0.15M because the bacteria lost its ability to reproduce in the above concentrations. A severe inhibitory effect was observed. For this reason, as they were found to be the most suitable concentrations as a result of optimization studies, 0.05, 0.1 and 0.15 M Ca+2 were used.

Why did P. aeruginosa compare with B. cereus? a) Both are soil bacteria; b) P. aeruginosa Gram (-) and B. cereus Gram (+). Therefore, it gives the opportunity to compare; c) P. aeruginosa sporeless and B. cereus sporulated bacteria; d) Pathogenicity levels are different. e) We used our work in this preference.

3.1. Amylase activity

The determination of reducing sugars was generally carried out by the antrone method. The concentration of the glucose was determined at OD620 nm spectrophotometrically. It is mainly used in the assay of α-amylase activity (Keharom et al., 2016KEHAROM, S., MAHACHAI, R. and CHANTHAI, S., 2016. The optimization study of α-amylase activity based on central composite design-response surface methodology by dinitrosalicylic acid method. International Food Research Journal, vol. 23, no. 1, pp. 10-17.). Amylase activity, in the presence of Ca+2, B. cereus, achieved a maximum increase of up to 1.2 times. The highest increase was in the presence of 0.15 M Ca+2 with 25.32 U/mL. For P. aeruginosa, an increase of 1.6 times more amylase activity was observed on average compared to B. ceres. When P. aeruginosa was evaluated in itself, no significant increase was observed in the presence of Ca+2. The highest P. aeruginosa amylase activity was observed in the presence of 0.1 M Ca with 32.13 U/mL. Although P. aeruginosa is more advantageous on the graph, in its production it caused a 102% increase in 0.1 M Ca+2 concentrations. However, this ratio in B. cereus caused an increase in 123% amylase production in the presence of 0.15 M Ca+2. All of these values are calculated according to the controls (Figure 1).

Figure 1
Amylase unit activity of B. cereus (○) and P. aeruginosa (●), grown in NB medium under static conditions at 37 °C.

3.2. Biofilm activity

As was expected, biofilm formation in B. cereus was low. The highest biofilm formation occurred in OD570 with 0.284 in the presence of 0.1 M Ca+2 for B. cereus. According to the control, an increase of up to 2.5 times was observed in the presence of Ca+2. In P. aeruginosa, a higher biofilm formation occurred, as was expected. Unlike B. cereus, in the presence of 0.1 M Ca+2, a higher biofilm formation was observed in OD570 with 0.735. It appears to be the most suitable concentration for biofilm formation for P. aeruginosa. The difference was two times more than in the control and there was approximately three times more biofilm formation than in B. cereus. Although P. aeruginosa is advantageous, a 200% increase in 0.1 M Ca+2 concentrations was observed compared to the control. However, this ratio in B. cereus caused a 252% increase in the presence of 0.15 M Ca+2 compared to the control (Figure 2).

Figure 2
Biofilm levels of B. cereus (○) and P. aeruginosa (●), grown in NB medium under static conditions at 37 °C.

3.3. LasB activity (ECR)

The addition of Ca+2 in both bacteria decreased ECR activity. There was a decrease of 7.14 times in B. cereus and 4.8 times in P. aeruginosa. It occurred mostly in B. cereus. The greatest decrease in both bacteria occurred in the presence of 0.15 M Ca+2. In accordance with B. cereus, ECR activity decreased 1.7 times more than with P. aeruginosa. The graphic here shows a harmonious decrease (Figure 3).

Figure 3
Las B activity of B. cereus (○) and P. aeruginosa (●), grown in NB medium under static conditions at 37 °C.

3.4. H2O2

In B. cereus, the highest sensitivity was observed in the presence of 0.15 M Ca+2 with 33 mm. H2O2 resistance only increased compared to the control in the presence of 0.05 M Ca+2. This was observed with a 7 mm zone decrease. While P. aeruginosa was 35.3 mm in the highest zone control, the highest resistance was realized in the presence of 0.1 M Ca+2, just like in B. cereus. This was observed with a zone diameter of 14 mm. Resistance increase in both bacteria was observed in the presence of 0.05 M Ca+2. An increase in sensitivity started to occur above these concentrations. While Ca+2 increase in B. cereus causes a sensitivity increase compared to the control, in P. aeruginosa, resistance increased continuously compared to the control. H2O2 resistance in P. aeruginosa caused an increase in resistance in all three concentrations compared to the control (Figure 4). P. aeruginosa increased by 60% resistance at a concentration of 0.05 M Ca+2. However, this ratio in B. cereus caused only a 3% increase in the presence of 0.05 M Ca+2. These values are calculated according to the controls.

Figure 4
H2O2 diameter of B. cereus (○) and P. aeruginosa (●), grown in NB medium under static conditions at 37 °C.

3.5. Protease activity

The presence of Ca+2 in both bacteria caused an increase in protease activity. This increase caused enzyme activity in B. cereus in the presence of 0.05 M Ca+2 up to 0.746 U/mL and up to 5.6 times according to the control. Therefore, this appears to be the most appropriate concentration in protease activity for B. cereus. In P. aeruginosa, only a 2.3-fold increase in the presence of Ca+2 was 1.635 U/mL in the presence of 0.1 M Ca+2. Consequently, this appears to be the most appropriate concentration in protease activity for P. aeruginosa. However, the concentration of 0.15 M Ca+2 caused a decrease in both bacteria. In general, the presence of Ca+2 increased protease activity. Although P. aeruginosa is advantageous when the graph is examined, it caused an increase of 0.1% Ca+2 concentrations by 220%. However, this ratio in B. cereus caused a 548% increase in the presence of 0.05 M Ca+2. These values are calculated according to the controls (Figure 5).

Figure 5
Protease activity of B. cereus (○) and P. aeruginosa (●), grown in NB medium under static conditions at 37 °C.

3.6. Pyorubin activity

Pyorubin, a pigment, caused an increase in the presence of Ca+2. However, these increases are not seen as significant increases. Subsequent concentrations, the highest in the presence of 0.1 M Ca+2, also began to decline. The value of 88.21 was obtained. As a result, Ca+2 had no effect on the increase or decrease of pyorubin. As a result, it increased the production of 0.1 M Ca+2 pyorubin, which has the highest effect, by only 102% (Figure 6). Why are there no B. cereus values in this figure? Because of pyorubin is a metabolite specific to P. aeruginosa. Therefore, it is not shown on the figure.

Figure 6
Pyorubin production of P. aeruginosa (●), grown in NB medium under static conditions at 37 °C.

4. Discussion

The determination of reducing sugars was generally carried out by the antrone method. The concentration of the glucose was determined at OD620 nm spectrophotometrically. It is mainly used in the assay of α-amylase activity (Keharom et al., 2016KEHAROM, S., MAHACHAI, R. and CHANTHAI, S., 2016. The optimization study of α-amylase activity based on central composite design-response surface methodology by dinitrosalicylic acid method. International Food Research Journal, vol. 23, no. 1, pp. 10-17.). In some studies, it has been observed that calcium ion influences the biofilm structure in P. aeruginosa cultures. They stated that by adding calcium it was 10 to 20 times thicker than the formation of non-added calcium biofilm (Sarkisova et al., 2005SARKISOVA, S., PATRAUCHAN, M.A., BERGLUND, D., NIVENS, D.E. and FRANKLIN, M.J., 2005. Calcium-induced virulence factors associated with the extracellular matrix of mucoid Pseudomonas aeruginosa biofilms. Journal of Bacteriology, vol. 187, no. 13, pp. 4327-4337. PMid:15968041.; Cruz et al., 2012CRUZ, L.F., COBINE, P.A. and DE LA FUENTE, L., 2012. Calcium increases Xylella fastidiosa surface attachment, biofilm formation and twitching motility. Applied and Environmental Microbiology, vol. 78, no. 5, pp. 1321-1331. PMid:22194297.). The extracellular elastase (LasB) and LasA amounts, which are the products of the Type II secretory pathway, increase in the presence of additional calcium. In addition, the amount of extracellular protease increased with the addition of Ca+2 (Sarkisova et al., 2005SARKISOVA, S., PATRAUCHAN, M.A., BERGLUND, D., NIVENS, D.E. and FRANKLIN, M.J., 2005. Calcium-induced virulence factors associated with the extracellular matrix of mucoid Pseudomonas aeruginosa biofilms. Journal of Bacteriology, vol. 187, no. 13, pp. 4327-4337. PMid:15968041.). Another study showed a 20-fold increase in biofilm thickness after 24 hours in the presence of Ca+2 (1.0 and 10.0 mM CaCl2). In this study, by growing the culture with 10 mM CaCl2, approximately two to three times more biofilm structures were observed compared to the non-Ca+2 medium (Sarkisova et al., 2005SARKISOVA, S., PATRAUCHAN, M.A., BERGLUND, D., NIVENS, D.E. and FRANKLIN, M.J., 2005. Calcium-induced virulence factors associated with the extracellular matrix of mucoid Pseudomonas aeruginosa biofilms. Journal of Bacteriology, vol. 187, no. 13, pp. 4327-4337. PMid:15968041.). Some studies have stated that the amount of protease and pyocyanin increase with the addition of Ca+2. The addition of calcium has been shown to increase the biofilm structure in Vibrio cholerae. Although, in Pectobacterium carotovorum, it has been found to increase the activity of the type III secretion system and the expression of effector proteins, as well as the modulation of hydrolytic enzymes (such as polygalacturonase and pectate lyase), which are considered to be important in virulence. Calcium also plays a role in X. fastidiosa infection. In addition, the presence of Ca+2 is said to increase biofilm formation, cell binding and motility in vitro. These results show that the role of Ca+2 in biofilm formation is important (Sarkisova et al., 2005SARKISOVA, S., PATRAUCHAN, M.A., BERGLUND, D., NIVENS, D.E. and FRANKLIN, M.J., 2005. Calcium-induced virulence factors associated with the extracellular matrix of mucoid Pseudomonas aeruginosa biofilms. Journal of Bacteriology, vol. 187, no. 13, pp. 4327-4337. PMid:15968041.; Cruz et al., 2012CRUZ, L.F., COBINE, P.A. and DE LA FUENTE, L., 2012. Calcium increases Xylella fastidiosa surface attachment, biofilm formation and twitching motility. Applied and Environmental Microbiology, vol. 78, no. 5, pp. 1321-1331. PMid:22194297.). It is also stated in Prokaryotes that the presence of Ca+2 regulates and increases bacterial gene expression. Many prokaryotes, including Escherichia coli, Propionibacterium acnes, Streptococcus pneumoniae, Bacillus subtilis and Cyanobacteria, have also been shown to maintain intracellular Ca+2 levels at micromolar levels, producing in response to environmental and physiological conditions. This suggests that Ca+2 plays an important role in prokaryotic physiology and virulence. Apart from this, it is stated that Ca+2 increases biofilm formation in P. aeruginosa and protease, and pyocyanin virulence factors induce biosynthesis (Guragain et al., 2013GURAGAIN, M., LENABURG, D.L., MOORE, F.S., REUTLINGER, I. and PATRAUCHAN, M.A., 2013. Calcium homeostasis in Pseudomonas aeruginosa requires multiple transporters and modulates swarming motility. Cell Calcium, vol. 54, no. 5, pp. 350-361. PMid:24074964.; Domínguez et al., 2015DOMÍNGUEZ, D.C., GURAGAIN, M. and PATRAUCHAN, M., 2015. Calcium binding proteins and calcium signaling in prokaryotes. Cell Calcium, vol. 57, no. 3, pp. 151-165. PMid:25555683.). In addition, it has been stated in a study that the presence of Ca+2 in the environment increases the expression of genes that cause proteolysis and stress response (Guragain et al., 2013GURAGAIN, M., LENABURG, D.L., MOORE, F.S., REUTLINGER, I. and PATRAUCHAN, M.A., 2013. Calcium homeostasis in Pseudomonas aeruginosa requires multiple transporters and modulates swarming motility. Cell Calcium, vol. 54, no. 5, pp. 350-361. PMid:24074964.). In another study, the presence of Ca+2 has been shown to increase the virulence of P. aeruginos and the thickness of the biofilm structure. It also shows that Ca+2, X. fastidiosa's biofilm formation, has the ability to cling to the cell surface and play a role in the regulation of the movement of twitches (Sarkisova et al., 2005SARKISOVA, S., PATRAUCHAN, M.A., BERGLUND, D., NIVENS, D.E. and FRANKLIN, M.J., 2005. Calcium-induced virulence factors associated with the extracellular matrix of mucoid Pseudomonas aeruginosa biofilms. Journal of Bacteriology, vol. 187, no. 13, pp. 4327-4337. PMid:15968041.; Cruz et al., 2012CRUZ, L.F., COBINE, P.A. and DE LA FUENTE, L., 2012. Calcium increases Xylella fastidiosa surface attachment, biofilm formation and twitching motility. Applied and Environmental Microbiology, vol. 78, no. 5, pp. 1321-1331. PMid:22194297.). The chemical analysis showed an increased production of pyocyanin with Ca+2 additions in P. aeruginosa FRD1 (Sarkisova et al., 2005SARKISOVA, S., PATRAUCHAN, M.A., BERGLUND, D., NIVENS, D.E. and FRANKLIN, M.J., 2005. Calcium-induced virulence factors associated with the extracellular matrix of mucoid Pseudomonas aeruginosa biofilms. Journal of Bacteriology, vol. 187, no. 13, pp. 4327-4337. PMid:15968041.). Chemical analysis shows that pyocyanin production increases with the addition of Ca+2 (Sarkisova et al., 2005SARKISOVA, S., PATRAUCHAN, M.A., BERGLUND, D., NIVENS, D.E. and FRANKLIN, M.J., 2005. Calcium-induced virulence factors associated with the extracellular matrix of mucoid Pseudomonas aeruginosa biofilms. Journal of Bacteriology, vol. 187, no. 13, pp. 4327-4337. PMid:15968041.). We did not find any study on the effect of Ca+2 on pyoverdin. It is stated that protease enzymes need some metal ions in order to maintain their stability and maintain their active form. According to this study; it has been stated that Ca+2, Mg+2 and Mn+2 ions increase the protease activity (Guragain et al., 2013GURAGAIN, M., LENABURG, D.L., MOORE, F.S., REUTLINGER, I. and PATRAUCHAN, M.A., 2013. Calcium homeostasis in Pseudomonas aeruginosa requires multiple transporters and modulates swarming motility. Cell Calcium, vol. 54, no. 5, pp. 350-361. PMid:24074964.).

Acknowledgements

This work was supported by a Grant (APYB 2019/1811) from Research Fund Unit of Inonu University, Turkey.

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Publication Dates

  • Publication in this collection
    11 June 2021
  • Date of issue
    2022

History

  • Received
    04 Sept 2020
  • Accepted
    23 Nov 2020
Instituto Internacional de Ecologia R. Bento Carlos, 750, 13560-660 São Carlos SP - Brasil, Tel. e Fax: (55 16) 3362-5400 - São Carlos - SP - Brazil
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