Effect of Ca+2 on P. aeruginasa and B. cereus metabolites

Background: 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. Results: Amylase; P. aeruginosa is 102% increase in 0.1 M Ca +2 concentrations and B. cereus caused an increase in 123%. Biolm P. aeruginosa; 200% increase in 0.1 M Ca +2 concentrations. B. cereus caused a 252% increase in the presence of 0.15 M Ca +2 . Las B; in accordance with B. cereus, Las B activity decreased 1.7 times more than with P. aeruginosa. H 2 O 2 ; P. aeruginosa increased by 60% resistance. B. cereus caused only a 3% increase. Protease in P. aeruginosa increase of 220%. B. cereus caused a 548% increase. Pyorubin, a pigment, caused an increase in the presence of Ca +2 . However, these increases are not seen as signicant increases. Conclusion: In this study; the effect of Ca +2 on protease, amylase, LasB elastolytic assay, H 2 O 2 , pyorubin and biolm was investigated in a 24 hour time. In the study we have conducted here, the effect of Ca +2 meat on the production of some secondary metabolites on Gram (-) P. aeruginosa and Gram (+) B. cereus was investigated for the rst time. As a result, although there are many different related studies, they are not comparative as in ours. Therefore, our study has progressed in a different direction and it is a rst in its eld.

and bio lm was investigated in a 24 hour time. In the study we have conducted here, the effect of Ca +2 meat on the production of some secondary metabolites on Gram (-) P. aeruginosa and Gram (+) B. cereus was investigated for the rst time. As a result, although there are many different related studies, they are not comparative as in ours. Therefore, our study has progressed in a different direction and it is a rst in its eld. Introductıon α-amylase (E.C.3.2.1.1; 1,4-α-D-glucan-glucanohydrolase) is a hydrolytic enzyme that hydrolyzes α-1,4glycosidic 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 [1].
α-amylase can be isolated from plants, animals or microorganisms. It is found in many bacteria (Bacillus spp. B. amyloliquefaciens, Bacillus subtilis, B. cereus, B. amyloloquefaciens, B. amloloquefaciens, B. megaterium, B. licheniformis, Lactobacillus, Escherichia, Proteus, Clostridium and Pseudomonas sp.) and fungi (A. niger, Penicillum, Sefalosporium, Neurospora and Rhizopus). For this reason, microorganisms in particular, are used more in the production of this enzyme. However, Bacillus species are more widely used in commercial protease production [2][3][4][5][6][7]. 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 scienti c 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 [8][9][10][11][12][13][14] 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 speci cally destroy host defense proteins. This is very important in virulence [15]. 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 [16].
The structures we call bio lms 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 bio lm formation [17]. Bio lm, in phytopathogenic microorganisms and animal pathogens, adaptation promotes survival. Cells in bio lm are said to be more resistant to oxidative stress than free cells [18].
Bacillus cereus can be found widely in soil and plants. Bacteria possessing psychrotrophic, spore, Gram (+), agella, aerobic, and peritric agels are aerobic. Optimum breeding 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 [19].
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 [20].
In the study we have conducted here, the effect of Ca + 2 's meat on the production of some secondary metabolites on Gram (-) P. aeruginosa and Gram (+) B. cereus was investigated for the rst time.

Reagents
All chemicals were of the highest purity available commercially. Microorganism P. aeruginosa (ATCC 27853) and B. cereus (ATCC 10876) obtained from the ATCC and used this study.

Enzyme production
Microorganisms were grown and cultivated as follows: 3 ml of 24 h bacterial inoculum (OD 600 = 0.3-0.4) was inoculated into 5 ml growth 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 for proteolytic activity [21].

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 phosphate buffer) and the 0.5 ml enzyme solution in a nal volume of 1 ml. One unit of amylase was de ned as the amount of enzyme, required to produce reducing sugars equivalent to 1 µmol glucose/min at 37 °C and at pH 7.0 [7,21].

Pyorubin
Pyorubin, a pigment, caused an increase in the presence of Ca + 2 . However, these increases are not seen as signi cant 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% (Fig. 6). Why are there no B. cereus values in this chart? Because of pyorubin is a metabolite speci c to Pseudomonas aeruginosa. Therefore it is not shown on the chart.

Assay of protease activity
Protease activity was measured by some modi cation 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 Na 2 CO 3 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 OD 660 nm. The amount of tyrosine was determined from the tyrosine standard curve [4,21,22].

Bio lm assay
Bio lm generated by each strain of P. aeruginosa were evaluated using the following crystal violet assay [10]. The bio lm forming ability isolates were tested using glass tube with little modi cations. The overnight cutlers 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 OD 570 nm, for quanti cation of bio lm biomass [10][11][23][24][25].

LasB elastolytic assay
The test was performed with some modi cations. 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 reaction stopped 0.5 ml 100 mM EDTA added. Insoluble ECR was removed by centrifugation (6000 rpm and room temperature 5 min), and the absorption of the supernatant was measured at OD 495 nm. Activity was expressed as change in OD 495 g/protein [13,16,26].

Results
In our study, various physiological 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 0.15 M 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 we work with a high level of Ca + 2 ? We preferred it because it has the highest values we can reach with optimization studies.
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.

Amilaz
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. cereus. When P. aeruginosa was evaluated in itself, no signi cant 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 (Fig. 1).

Bio lm
As was expected, bio lm formation in B. cereus was low. The highest bio lm formation occurred in OD 570 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 bio lm formation occurred, as was expected. Unlike B. cereus, in the presence of 0.1 M Ca + 2 , a higher bio lm formation was observed in OD 570 with 0.735. It appears to be the most suitable concentration for bio lm formation for P. aeruginosa.
The difference was two times more than in the control and there was approximately three times more bio lm 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 (Fig. 2).

LasB elastolytic assay (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 (Fig. 3).

| Protease
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 (Fig. 5).

Pyorubin
Pyorubin, a pigment, caused an increase in the presence of Ca +2 . However, these increases are not seen as signi cant 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% (Fig. 6). Why are there no B. cereus values in this chart? Because of pyorubin is a metabolite speci c to Pseudomonas aeruginosa. Therefore it is not shown on the chart.

Conclusion
The determination of reducing sugars was generally carried out by the antrone method. The concentration of the glucose was determined at OD 620 nm spectrophotometrically. It is mainly used in the assay of α-amylase activity [2]. In some studies, it has been observed that calcium ion in uences the bio lm 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 bio lm [27,28]. 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 [28]. Another study showed a 20-fold increase in bio lm thickness after 24 hours in the presence of Ca + 2 (1.0 and 10.0 mM CaCl 2 ). In this study, by growing the culture with 10 mM CaCl 2 , approximately two to three times more bio lm structures were observed compared to the non-Ca + 2 medium [28]. 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 bio lm 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 bio lm formation, cell binding and motility in vitro. These results show that the role of Ca + 2 in bio lm formation is important [27,28]. 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 play an important role in prokaryotic physiology and virulence. Apart from this, it is stated that Ca + 2 increases bio lm formation in P. aeruginosa and protease, and pyocyanin virulence factors induce biosynthesis [20,29]. 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 [20]. In another study, the presence of Ca + 2 has been shown to increase the virulence of Pseudomonas aeruginos and the thickness of the bio lm structure. It also shows that Ca + 2 , X. fastidiosa's bio lm formation, has the ability to cling to the cell surface and play a role in the regulation of the movement of twitches [27,28]. However, when Ca + 2 were added, intense protein bands of AprA, LasB, and PrpL were observed. The chemical analysis showed an increased production of pyocyanin with Ca + 2 additions in P. aeruginosa FRD1 [28]. Chemical analysis shows that pyocyanin production increases with the addition of Ca + 2 [28]. We did not nd 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 [20]. In our study, it is a rare study because it is studied with high molarity Ca + 2 , which is not found in the literature. As a result, although there are many different related studies, they are not comparative as in ours. Therefore, our study has progressed in a different direction and it is a rst in its eld.

Declarations
Authors' contributions HK and CCK designed this study. HK and ZBK performed most experiments. Authors analyzed the data, wrote the manuscript, read and approved the nal manuscript.

Availability of data and materials
All data generated or analyzed during this study are included in this published article.
Ethics approval and consent to participate     Pyorubin production of P. aeruginosa (•), grown in NB medium under static conditions at 37 °C.