Mechanism of calcium hydroxide inhibiting methicillin resistant Staphylococcus aureus

Zheng-Dong Guo, Yang-Yang Bian, Xiao-Qian Liu, Dong Wang, Si-Yuan Zhang, Jian Yang, Lei Peng✉

1. Trauma medical center of the First Affiliated Hospital of Hainan Medical College , Haikou, Hainan 570216, China

2. Key Laboratory of Emergency and Trauma Ministry of Education, Hainan Medical University , Haikou, Hainan 570216, China

3. Key Laboratory of Hainan Trauma and Disaster Rescue, The First Affiliated Hospital of Hainan Medical University , Haikou, Hainan 570216,China

Keywords:Calcium hydroxide Methicillin-resistant staphylococcus MRSA Bacteriostasis Antibacterial mechanism Cytomembrane

ABSTRACT Objective:To investigate the inhibitory effect of calcium hydroxide on methicillin-resistant Staphylococcus aureus and the related inhibition mechanism. Methods: To determine the minimum inhibitory concentration of calcium hydroxide using microplate dilution method;to compare the effects of calcium hydroxide at 8MIC, MIC, 1/4MIC and 0 concentrations on MRSA using growth curve method; to determine the effects of calcium hydroxide on the cell membrane of methicillin-resistant Staphylococcus aureus using calcium xanthophyll and propidium iodide fluorescence staining The effect of calcium hydroxide on the morphology of methicillin-resistant Staphylococcus aureus was observed by scanning electron microscopy; the inhibition mechanism of calcium hydroxide on MRSA was investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis of protein bands. Results: The MIC of calcium hydroxide on MRSA was 3.125 mg/mL; the fluorescence intensity showed significant changes after co-culture of calcium hydroxide with bacteria; MRSA appeared to be significantly crumpled and broken in the presence of calcium hydroxide; the SDS-PAGE experimental bands indicated that the protein inside the bacteria decreased accordingly with the increase of calcium hydroxide concentration. Conclusion: Calcium hydroxide has a significant inhibitory effect on the growth of MRSA, and its bactericidal mechanism may be related to the destruction of bacterial body structure.

1. Introduction

Traumatic osteomyelitis is an inflammatory disease secondary to bone and its surrounding soft tissues, and is a relatively common and difficult problem for clinical orthopedic surgeons, often referred to as the "undead cancer"[1]. In recent years, with the rapid improvement of our national economy, the mechanization level of the primary and secondary industries has also increased significantly,resulting in an increasing accident rate and an increasing number of trauma patients [2]. With the irrational use and even abuse of broadspectrum antimicrobials and glucocorticoids, the number of patients infected with drug-resistant strains of bacteria is on the rise, and the antimicrobial efficacy of using antibiotics against clinically resistant strains of bacteria is significantly reduced, and currently infections with drug-resistant strains of bacteria are a difficult and difficult clinical problem to deal with[3]. Staphylococcus aureus is one of the common organisms in traumatic osteomyelitis, and MRSA is caused by various reasons such as failure of timely debridement and irregular use of antimicrobial drugs[4, 5]. At present, for the problem of drug resistance of the main pathogenic bacteria of traumatic osteomyelitis, such as the treatment of MRSA, the choice of available antibiotics is rather narrow, so it is necessary to explore a drug that plays an efficient antibacterial and non-resistant role against MRSA, and it is also very meaningful for the cure of traumatic osteomyelitis.

Calcium hydroxide is a white powdered inorganic compound that, in the field of clinical dentistry, calcium hydroxide [6, 7] has become a common drug for the treatment of pulpitis. It can slowly release calcium ions and hydroxide ions with alkaline nature in water to form an alkaline environment, and its Ph can be as high as 12. Hydroxide ions can effectively kill bacteria in the pulp cavity with no drug resistance and wide antibacterial spectrum, and can play a good antibacterial effect on the root canal area of diseased teeth[8, 9]. There are relatively few studies on the mechanism of MRSA inhibition by calcium hydroxide. Our group used the strong alkalinity of calcium hydroxide to kill MRSA, in order to provide a basis for the development of new safe, efficient and non-resistant antibacterial drugs for the treatment of traumatic osteomyelitis.

2. Materials and Methods

2.1 Experimental materials

MRSA, calcium hydroxide (provided by College of Pharmacy,Wenzhou Medical University); nutrient broth medium 11B01,nutrient agar medium 11A10 (Beijing SanPharma Technology Development Company); wheat turbidimetric tube (Wenzhou Kangtai Biotechnology Co., Ltd.).

Preparation of bacterial broth: Inoculate the bacterial broth on solid agar broth medium for 24h, pick a colony and inoculate it in nutrient broth medium, incubate it in constant temperature shaker at 37℃ for 24 h. Before use, use 0.9% saline to dilute the bacterial broth to 0.5 McFarland (the amount of bacteria is equivalent to 1.5×108cfu/mL)according to the McFarland method.and then take the appropriate amount of diluted bacterial solution and dilute it to 106 cfu/mL with sterile broth medium, and set aside. [10]

2.2 Determination of minimum bacterial inhibition concentration[11]

The minimum inhibitory concentration of MRSA was determined using the microplate dilution method. A 96-well plate was taken,and 100 μL of calcium hydroxide solution and 100 μL of MRSA solution at 106 cfu/mL were added to each well from 1 to 8, so that the final concentrations of calcium hydroxide per well were 100, 50,25, 12.5, 6.25, 3.125, 1.5625, and 0.78125 in mg/mL. Well 9 was added with nutrient broth medium 100 μL and 100 μL of MRSA solution at a concentration of 106 cfu/mL to observe if bacterial growth was favored. The 10th well was added with only 200 μL of nutrient broth medium as a blank test control group. Each well was mixed thoroughly and incubated in a constant temperature incubator for 24 h. The supernatant turbidity of each group of bacteria was observed by removing the well plate, and the minimum inhibitory concentration was determined as no turbidity in the supernatant.

2.3 Determination of bacterial growth curve [12]

Calcium hydroxide was weighed and dissolved in the bacterial solution with a concentration of 106 cfu/mL MRSA, so that the final concentration of calcium hydroxide in the mixed bacterial solution was 8MIC, MIC, 1/4MIC, and the one without calcium hydroxide was used as the control group, and the OD600 values of each group were measured by enzyme standard at 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, and 24 h, respectively.

2.4 Fluorescence microscopy analysis of cell membrane damage

The logarithmic growth period of MRSA bacterial solution was taken, and the concentration of bacterial solution was adjusted to 107 cfu/mL using the Mackenzie turbidimetric method, and the concentration was adjusted to 4 MIC by adding calcium hydroxide,and no calcium hydroxide was added as the control group. After co-cultivation in a constant temperature shaker at 37℃ for 1 h, the bacterial broth was collected separately, and the bacterial bodies were collected after centrifugation, washed by PBS centrifugation three times, and resuspended by PBS solution. Staining was performed using Calcein-AM/PI double-staining kit, incubated for 15 min at room temperature and protected from light, and 10 μL of samples were fixed on slides by aspiration, and photographed and observed by fluorescence microscopy.

2.5 Scanning electron microscope observation of the morphology of MRSA [13, 14]

Two 10 ml EP tubes were taken separately, the first EP tube with calcium hydroxide prepared with nutrient broth medium to the minimum inhibitory concentration MIC, and the second EP tube with nutrient broth medium without calcium hydroxide as a blank control group, and incubated with MRSA in a constant temperature incubator at 37℃ for 1 h, respectively, and centrifuged with a centrifuge (5000 r/min) for 5 min, the supernatant was discarded, and the precipitates was washed twice with PBS at Ph 7.2, respectively,and fixed overnight with 2.5% glutaraldehyde at 4℃. Afterwards,the precipitates were dehydrated with gradient ethanol, dried and sprayed with gold, and the morphology of MRSA was photographed by scanning electron microscopy. [15]

2.6 SDS-PAGE analysis of bacteriophage proteins [16-18]

The MRSA in logarithmic growth phase was centrifuged to prepare the bacterial broth with OD600=1.9, and calcium hydroxide was added to the broth to make the final concentrations of 0, MIC,and 4MIC, respectively, and incubated in a constant temperature incubator for a total of 1.5 h. After centrifugation, the bacteria were collected, rinsed with PBS buffer for 3 times, and the bacterial broth was crushed with an ultrasonic crusher, and the crushed broth was incubated with The crushed fluid was centrifuged at high speed for 10 min with a refrigerated centrifuge at 4℃, and the supernatant was taken and mixed with 5x of the loading buffer and boiled for 5 min,followed by loading for SDS-PAGE experiments and photographed and analyzed with a gel imaging system.

2.7 Statistical analysis

Data statistics and significance of differences analysis were performed on the experimental data using GraphPad Prism 8.0 data processing software. p≤0.05 was considered significant (marked in the text*: p＜0.05;**: p＜0.01;***: p＜0.001;****: p＜0.0001).

3. Results

3.1 Measurement of MIC

The supernatant of well 6 of the 96-well plate (calcium hydroxide concentration of 3.125 mg/mL) was the clarified sample, thus determining this concentration as the MIC of MRSA.

3.2 Determination of bacterial growth curve

The growth trend of MRSA to different concentrations of calcium hydroxide solution is shown in Fig. The blank control group without calcium hydroxide increased the number of bacteria after 2 h with time; while the logarithmic growth period of bacteria in the experimental group was delayed backward as the amount of calcium hydroxide increased. When the bacterial solution contained 8MIC of calcium hydroxide, the OD value of bacteria did not increase and maintained a flat trend, indicating that calcium hydroxide had a significant inhibitory effect on MRSA (see Fig 1).

Figure 1 Effect of calcium hydroxide on proliferation of MRSA

3.3 Fluorescence microscopy analysis of cell membrane breakage

Most of the plots in the control group showed green fluorescence and relatively little red fluorescence, indicating that MRSA did not undergo obvious damage to the cell membrane; whereas most of the bacterial group co-cultured with calcium hydroxide showed red fluorescence and almost no green fluorescence could be seen,indicating that the cell membrane of the bacterium of MRSA was completely destroyed and almost all of it died. According to the analysis of the fluorescence images, the red fluorescence was close to 100% after the action of calcium hydroxide. This data indicates that calcium hydroxide has a strong destructive effect on MRSA bacteriophage(see Fig 2).

Figure 2 Fluorescence micrograph of MRSA

3.4 Scanning electron microscope observation of the morphology of MRSA

The effect of calcium hydroxide on MRSA was observed by SEM,as shown in the figure, the bacteria in the control group without calcium hydroxide treatment had a clear and smooth surface; in the experimental group treated with calcium hydroxide, the bacterial surface adsorbed calcium hydroxide and showed wrinkling,deformation, and even surface lysis, and the integrity of the bacteria was severely damaged. This shows that calcium hydroxide can effectively destroy the bacterial structure of MRSA (see Fig3).

Figure 3 Scanning electron micrograph of MRSA

3.5 SDS-PAGE analysis of bacteriophage proteins

Protein is the main bearer of life activities, and interfering with protein synthesis can achieve the purpose of bacterial inhibition.Therefore, the effect of calcium hydroxide on the proteins inside the MRSA bacterium can be studied by SDS-PAGE, and the results are shown in Fig. The color of the protein bands in the MIC and 4MIC groups became relatively lighter compared with the 0MIC group. Combined with the results of scanning electron microscopy,it was speculated that calcium hydroxide severely damaged the bacteriophage structure of MRSA, resulting in the leakage of proteins inside the bacteria.(see Fig 4).

Figure 4 Effect of calcium hydroxide on MRSA protein

4. Discussion

The presence of infection of bones and their soft tissues in traumatic osteomyelitis has led to the difficulty of removing its pathogenic bacteria, which has been a difficult problem for clinical orthopedic surgeons to overcome. Staphylococcus aureus is one of the common bacteria in traumatic osteomyelitis, and the failure of timely debridement and irregular use of antibacterial drugs have led to the emergence of drug-resistant strains (such as MRSA) for various reasons, making the treatment of traumatic osteomyelitis more difficult. [19] Calcium hydroxide is an antimicrobial agent with broad-spectrum antimicrobial effects and is easily sourced and inexpensive, so related in-depth research on antimicrobial agents has a high social and economic value.

Bacteria are mainly composed of cell walls, cell membranes, and cytoplasm. [20] The antibacterial mechanism of most antibiotics is achieved by destroying the cell wall and cell membrane of the bacterium and inhibiting the synthesis of proteins by ribosomes.β-lactam antibiotics work by inhibiting the cell wall mucopeptide synthetase, thus preventing the bacteria from synthesizing cell wall mucopeptides, which destroys the integrity of the bacterial cell wall and the bacterium loses the protection of the cell wall and thus lyses and dies. [21] The mechanism of action of tetracycline is mainly to restrict the binding of amino acids to ribonucleoprotein bodies in bacteria, preventing the synthesis of proteins, thus achieving the effect of bacterial inhibition [22]. The long-term irrational use of antibacterial drugs in the clinical process has led to a corresponding increase in bacterial resistance. [23, 24]Calcium hydroxide is a common drug for the treatment of pulpitis and is clinically very effective. [25, 26].According to the scanning electron microscopy, it can be seen that after co-culture of calcium hydroxide solution with MRSA for 1 h, most of the bacteria showed crumpling and lysis,and the integrity of the cell membrane was severely damaged. The fluorescence staining results of the bacteria indicated that calcium hydroxide had a destructive effect on the MRSA cell membrane, thus exerting an inhibitory effect, which was consistent with the scanning electron microscopy observations.

In summary, calcium hydroxide can inhibit the proliferation of MRSA at a minimum inhibitory concentration of 3.125 mg/mL,and the results of the inhibition mechanism showed that calcium hydroxide can disrupt the morphology of MRSA, causing its surface to crumple or even break, thus achieving the inhibition effect. However, whether there are other mechanisms involved in the effect of calcium hydroxide on bacteria, such as the destruction of bacterial DNA, needs to be further verified. This experiment can provide a basis for the later development of new safe, efficient and non-resistant antibacterial drugs for the treatment of traumatic osteomyelitis.

Author contribution First author: Zhengdong Guo, involved in the experiment, collecting and organizing data and writing the paper; corresponding author:Lei Peng, project design and paper proofreading; remaining authors:Yangyang Bian, Xiaoqian Liu, Siyuan Zhang, Dong Wang and Jian Yang, involved in the experiment and managing the accounts.