MRSA Eradication Using Chlorine Dioxide
Antimicrobial resistant infections (AMRs) currently claim at least 50.000 lives each year in Europe and the US alone, and many hundreds of thousands more die in other areas of the world. In 15 European countries, more than 10% of Staphylococcus aureus infections in the bloodstream are caused by methicillin-resistant strains (MRSA),
and several of these countries register resistance rates close to 50%. 1 Furthermore, while the number of antibiotic-resistant infections is increasing, the number of new antibiotics is decreasing. 1,2 Therefore, it is imperative that new and novel treatments are sought for ADRs, and this is the premise of this research: to use natural substances to eradicate MRSA, which do not create further resistance. Chlorine dioxide used in vitro, has been our main focus of this research, since it was the most effective, compared to other natural substances tested.
Keywords: antimicrobial resistant strains, methicillin resistant, Staphylococcus aureu,
toxic shock syndrome, erythromycin, chlorine dioxide
Abbreviations: MRSA, methicillin-resistant Staphylococcus aureus; AMR. antimicrobial resistant; TSST-1, toxin-1 toxic shock syndrome; ClO 2, Chlorine Dioxide, PVL, Panton-Valentine Leukocidin; MSSA, methicillin-sensitive saphylococcus aureus
Introduction
Nosocomial infections acquired in hospitals or ICUs are often caused by antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus Aureus (MRSA). This resistance to antibiotics is accompanied by high rates of morbidity, mortality and the high cost of health facilities.
What is MRSA?
Staphylococcus aureus is a gram-positive coconut that is both catalase and coagulase +. Staphylococcus aureus has evolved to develop numerous immune evasion strategies to combat neutrophil-mediated death, including neutrophil activation, migration to the site of infection, bacterial opsonization, phagocytosis, and subsequent neutrophil-mediated destruction. Up to 40 S. aureus immune-evading molecules are known, and new functions for these evading proteins are being identified.
They produce a variety of toxins, including alpha-toxin, beta-toxin, gamma-toxin, delta-toxin, exfoliatin, enterotoxins, Panton-Valentine leukocidin (PVL), and Toxic Shock Syndrome Toxin 1 (TSST-1); enterotoxins and TSST-1 are associated with toxic shock syndrome; Progressive multifocal leukoencephalopathy is associated with necrotic infections of the skin and lungs and is an important virulence factor for pneumonia and osteomyelitis. 3
S. aureus expresses a wide range of virulence factors, including
toxins (hemolysins and leukocidins), immunoevasive surface factors (eg, capsule and protein A), and enzymes that promote tissue invasion (eg, hyaluronidase). 3
Colonization by MRSA increases the risk of infection, and infecting strains coincide with colonizing strains in up to 50–80% of cases. 4,5
Almost anything in contact with the skin can serve as a fomite in the transmission of MRSA, from coats and white ties to pens and cell phones.
Colonization can persist for long periods. MRSA can also persist in the home environment, complicating eradication attempts. 6
At the same time, colonization is not static, as strains have been found to evolve and even replace within the same host. 7
Drug resistance
MRSA has acquired MGE carrying antibiotic resistance genes on multiple independent occasions. Resistance to penicillin (blaZ), trimethoprim (dfrA and dfrK), erythromycin (ermC), clindamycin (constitutively expressed ermC), and tetracyclines (tetK and tetL) have been identified in insertion sequences, transposons, and sometimes plasmids in both MRSA as in methicillin. Susceptible Staphylococcus aureus (MSSA). 8 Probably reflecting the strong selective pressures within the hospital setting, antibiotic resistance is often genetically related to resistance to disinfectants or heavy metals (eg, quaternary ammonia compounds, mercury, or cadmium) among HA-MRSA strains. 9
What is chlorine dioxide?
The now commercially important compound chlorine dioxide (ClO 2) is not a recent discovery. The gas was first produced by Humphrey Davy in 1811 by reacting hydrochloric acid with potassium chlorate. This produced "euchlorine", as it was called then. Watt and Burgess, who invented alkaline pulp bleaching in 1834, mentioned euchlorine as a bleaching agent in their first patent. 10,11
Chlorine dioxide later became known as bleach and later a disinfectant. The production of ClO 2 from the mineral chlorate is complicated, however, and the gas is explosive so it could not
be easily used practically until the production of sodium chlorite powder by Olin Corporation in 1940.
Chlorine dioxide could now be released when needed from the chlorite salt. In municipal water supplies, this is usually done by adding chlorine to the chlorite solution and in the laboratory by adding an acid to the chlorite solution. Alliger showed in 1978, 10,11 that
ClO 2 could be applied topically by the individual user.
ClO 2 is a small molecule with a molecular weight of 67,46 and forms a stable radical. 12 ClO 2 is an oxidant, which is reduced to a chlorite ion (ClO 2 -) by capturing an electron (ClO 2 + e- → ClO 2 -). The redox potential (Eº) is relatively high as 0,95 V, so it does not harm the human microbiome. 13,14
Chlorine dioxide (ClO2) solution
Chlorine dioxide is: bactericidal, virucidal, sporicidal, cysticidal, algaecidal, and fungicidal. 15 Chlorine dioxide, a strong oxidant, has been reported to inhibit or destroy microorganisms in concentrations ranging from 1 to 100 ppm that produce potent antiviral activity, inactivating> or = 99,9% of viruses with a treatment of 15 second sensitization.
15-19
Furthermore, ClO 2 can remove biofilms quickly 20 because it is highly soluble in water and, unlike ozone, does not react with
extracellular polysaccharides from biofilm. In this way ClO 2 can penetrate the biofilms quickly to reach and kill the microbes that live within the film: a great advantage that is different from addressing both for Natural
and Allopathic Medicine. There are many reports that ClO 2 Solution has virucidal activity. 21-25 The inactivation concentration against
various viruses is 1-2 ppm in poliovirus. 21,22 2,19 ppm in the coronavirus that causes SARS. 23 7.5ppm in hepatitis A virus, 24 and 0,2 ppm in rotavirus. 25
Chlorine dioxide safety
Many evaluations have shown ClO 2 non-toxic compounds. Five decades of use have not indicated any adverse health effects.
The main areas of use have been disinfection of water supplies, removal of unwanted tastes and odors, and bleaching in the pulp and paper and textile industries.
Toxicology tests include ingestion of ClO 2 in drinking water, additions to tissue culture, injections into the blood, seeds
disinfection, 26,27 disinfection of insect eggs, injections under the skin of animals and into the brain of mice, burns administered to more than 1500 rats, and injections into plant stems. Standard tests include Ames mutation, Chinese hamster, rabbit eye, skin abrasion, pharmacodynamics, and teratology. 28
In one study, human volunteers drank ClO 2 or ClO 2 ¯ in solution up to 24 ppm and showed no adverse effects. 28
Several studies examined the effects on reproductive toxicity or teratology. There is no evidence of fetal malformation or birth.
defects in ClO 2 concentrations, both in the drink and in the skin route, up to 100 ppm. 29-31
With prolonged feeding, toxicity occurs mainly in red blood cells. Rats fed 1000 mg / l chronically for 6 months did not show significant hematological changes. However, after 9 months, red blood cell counts, hematocrit, and hemoglobin decreased in all treatment groups.
Lack of long-term toxicity, but low-level baseline is dramatically illustrated in two separate studies in which 32 rats and 33 bees were fed high-dose ClO2 for two years. No harmful effects were observed with up to 100 ppm added to the water supply.
Materials and methods
Methicillin-resistant Staphylococcus aureus (MRSA), grown on blood agar plates, which were provided by a local certified clinical laboratory, was used in this research study.
MRSA culture
In a Safety Class 2 cabinet, from the blood agar plates (Columbian Agar), a MRSA sample was taken from isolated cultures using a sterilized loop and placed in sterile tubes with 5 ml of Tryptic Soy Broth (TSB) . These culture tubes were incubated at 37 degrees centigrade for 48 hours. These culture tubes could be stored in the refrigerator at 4 degrees Celsius for up to 10 days, again for which samples would be made.
Counting bacteria
One of the most common methods for quantifying bacteria is by counting colony forming units (CFU). This widely used method is simple, gives a good general idea of cell viability, and is sensitive even to low concentrations of bacteria.
One big downside is that it takes days to get results that are estimates at best. A colony can arise from one or a thousand cells and sample preparation can vary from technology to technology, as well as each time, depending on sample conditions. For the sake of precision, the QUANTOMTx microbial cell counter from Logos Biosystems (logosbio.com) was used in this investigation. It is an automated image-based cell counter that can identify and count individual bacterial cells in minutes.
The QUANTOM Tx automatically focuses, captures, and analyzes multiple fluorescence-stained cell images to detect bacterial cells with high sensitivity and precision. It contains a sophisticated cell detection and removal algorithm that can accurately identify individual bacterial cells even in the smallest groups. In these experiments, we use the Viable Cell Staining Kit to detect live or viable cells.
The Quantom Microbial Cell Counter has been compared and found to be as accurate as flow cytometry and hemocytometer measurements, but greatly reduces time as each count takes no more than 30 seconds and can distinguish between groups . Stained cells are mixed with QUANTOM I Cell Loading Buffer, loaded onto QUANTOM M50 Cell Counting Slides, and centrifuged in the QUANTOM Centrifuge to immobilize and evenly distribute cells along a single focal plane to ensure consistent precise cell detection. Counting results and images can be viewed and saved immediately after counting.
To prepare the sample for the Quantom, 10 microliters (ul) of the culture medium were taken using a previously calibrated DLAB electronic pipet and placed in a sterile 1,5 ml Eppendorf tube. To this was added 2 ul of viable cell staining dye and incubated in a Heraeus incubator at 37 degrees centigrade for 30 minutes. 8 ul of buffer was added to this sample to enhance the fluorescence signal. To save on Quantom consumable slides, we recycle slides by washing them in Imrali Inventions iWash® Slide Cleaning Systems (www.imraliinventions.com).
To these tubes, was chlorine dioxide added in different concentrations, for different durations? The chlorine dioxide concentration ranged from 0,5 µl (0,5 ppm) to 5 µl (5 ppm), and the duration of exposure to the sample ranged from 30 minutes to 30 seconds.
For each experiment based on time and duration, two sample tubes were set up to keep the dilution factor constant. Depending on the amount of chlorine dioxide added to the experimental tube, the same amount of water was added to the control tube.
From these control and experimental tubes, 6 µl of the sample was taken using an electronic pipet and placed on the M50 cell count slides. The slides were placed in the QUANTOM centrifuge for 8 minutes at 300 RCF (relative centrifugal force) and then placed in the Quantom microbial cell counter to take a reference measurement (control) and another measurement from the experimental tube.
The optimal configuration of the Quantom Microbial Cell Counter for the MRSA protocol that we found during the test was established in Dilution Factor 2, Minimum size of fluorescence object 0.4um, Maximum size of fluorescent object 15μm Roundness 50%, Decluster level 7 and Detection sensitivity 7.
Chlorine dioxide preparation
Traditional chlorine dioxide, called MMS, was prepared as a Traditional chlorine dioxide, called MMS, was prepared as a solution using two components, sodium chlorite solution (25% solution in water) and 4% hydrochloric acid solution. One drop from each of these solutions was placed in a sterile 1,5 ml Eppendorf tube and allowed to activate for 30 seconds. In addition, more experiments were conducted using a new generation of chlorine dioxide called CDSplus, a proprietary product manufactured by Aquarius Pro-Life as a water treatment product. This is a buffered form of chlorine dioxide at a standard pH of 7 and a concentration of 3000 ppm when active (250 ml). From the activated CDSplus (250 ml), 83 µl = 1 ppm, 166 µl = 2 ppm were extracted; 0,25 ml = 3 ppm.
Experimental protocols
Various concentrations of chlorine dioxide
MMS and CDSplus were used. The range was 1 ppm to 5 ppm. The time of MMS and CDSplus were used. The range was 1 ppm to 5 ppm. The exposure time to chlorine dioxide ranged from 30 minutes to 30 seconds. In initial experiments it was not clear what time would be required for inhibition, but it was quickly shown to be less than a minute of exposure. Most of the experiments, therefore, had an exposure time of 1 minute.
Results Initial experiments
We started taking different concentrations of chlorobased dioxide in traditional MMS and tested these concentrations with MRSA in solution for different times ranging from 30 minutes to 30 seconds. 1μl of chlorine dioxide is equivalent to a concentration of 1 ppm. The lowest concentration of chlorine dioxide used to completely eradicate MRSA in these experiments was 0,5 ppm, with an exposure time of 30 seconds.
Table 1 below shows the different concentrations as a function of time, with the MRSA cell concentration measured by the Quantom cell counter. As you can see, for all chlorine dioxide concentrations ranging from 1 to 5 ppm, and time of exposure from 30 minutes to 30 seconds, MRSA growth inhibition was 99,99% throughout all these experiments.
Table 1 Comparison of bacterial counts before and after exposure to chlorine dioxide.
Experiment 1
Table 2 shows the cell count numbers for the 6 concentrations of chlorine dioxide used, namely: 0,5, 1, 2, 3, 4 and 5 ppm were used and a baseline count was measured for each concentration. Experiment number 0 is the baseline (control) count for each experiment group using different concentrations of chlorine dioxide. For each concentration, the experiment was repeated 5 times, with mean concentrations given.
From the initial experiments, since chlorine dioxide was found to kill 99,99% of MRSA bacteria at concentrations of 5 ppm for only 30 seconds, all other experiments used an exposure time of one minute as the standard, while they tried different concentrations.
In this experiment, ClO2 concentrations ranging from 0,5
- 5ppm were taken, using the Traditional MMS. At each of the 5 concentrations, the inhibition rate was 100%; refer to Table 2 and Figure
- Figure 1 shows the repeatability of the MRSA bacteria count using different concentrations ranging from 1 to 5 ppm. A baseline count was taken for each concentration; this was repeated 5 times. In all 5 replicates, MRSA growth inhibition was 100%.
Figure 2 compares MRSA cell count to MMS concentration over 1 minute. The area covered is equal to the number of cell count. The initial counts for each concentration are displayed on the left side of the graph and the final counts are displayed on the right side of the graph. The inhibition rate was 100% for all chlorine dioxide concentrations, with an exposure time of 1 minute.
Figure 1 Chlorine dioxide at different concentrations using MMS
Figure 2 Different concentrations of traditional MMS for a duration of 1 min.
Table 2 Chlorine dioxide (traditional MMS) at different concentrations repeated 5 times
Table 3 compares the concentrations of 1, 2, 3, 4 and 5 ppm for a Table 3 compares the concentrations of 1, 2, 3, 4 and 5 ppm for a 1 minute exposure to chlorine dioxide. The control was compared with the experimental one for the different concentrations. For all these concentrations of chlorine dioxide, the inhibition rate was 100%.
Experiment 2: using CDSplus
The same previous experiment was repeated using the CDS plus generation, using concentrations of 1-3 ppm. At each of the 3 concentrations, the inhibition rate was again 100%; See Table 4 and Figure 3. Figure 3 shows MRSA cell eradication using different concentrations of CDSplus, namely 1, 2 and 3 ppm. A baseline count was measured for the control group, and then each concentration of CDS plus was added and repeated twice.
For all concentrations, the inhibition rate was 100%.
Table 4 compares the concentrations of 1, 2 and 3 pp for a 60 second exposure to chlorine dioxide, using the new generation CDS plus.
The control was compared with the experimental one for the different concentrations.
Figure 3 MRSA-CDSPlus with different concentrations.
Figure 4 compares MRSA cell count with chlorine dioxide concentration (CDS plus) for 1 minute. The top line shows the reference cell count for the control group. The bottom line shows the MRSA cell counts after exposing the cells for 1 minute to different concentrations of chlorine dioxide; the inhibition rate was 100%.
Figure 4 Different concentration of CDSPlus for 60 seconds.
Table 4 Chlorine dioxide (CDSplus) at different concentrations for a 1 minute exposure Table 4 Chlorine dioxide (CDSplus) at different concentrations for a 1 minute exposure
Conclusions
MRSA is versatile and unpredictable. Their genetic adaptability and MRSA is versatile and unpredictable. Its genetic adaptability and the serial appearance of successful epidemic strains mean that it remains a major threat to human health.
The persistently high mortality associated with invasive MRSA infection, despite the fact that the FDA has approved multiple antibiotics effective against MRSA since 2014, highlights the need for high-quality trials to determine the optimal management for these patients. In these in vitro experiments, the efficacy of chlorine dioxide against MRSA has been consistently demonstrated, with growth inhibition of 99,99% -100% even at the smallest concentrations of 0,5 ppm.
Given the proven safety of chlorine dioxide in animal and human experiments to date, there is an urgent need for high-quality clinical trials to determine the efficacy of chlorine dioxide in individuals infected with MRSA today.
These studies will be carried out by the clinical community, starting with individual clinical trials in different countries of the world, with the creation of a network of clinical trials to collect all the data and develop safe and effective clinical protocols. Regarding safety, in a carefully designed experiment, the characteristic time required to kill a microbe was found to be only a few milliseconds. As ClO 2 is a fairly volatile compound, its contact time (its permanence on the treated surface) is limited to a few minutes.
While this stay is safe enough (being at least 3 orders of magnitude longer than the death time) to inactivate all bacteria in
the surface of the body is too short for ClO 2 to penetrate deeper than a few tenths of a millimeter; therefore, it cannot cause any real harm to an organism that is much larger than a bacterium.
There are also many testimonies of the use of chlorine dioxide by human volunteers for the eradication of many infectious diseases, including malaria and HIV, but one of the pioneers in Africa, Jim Humble. There is much controversy over this anecdotal evidence, but the number of witnesses who testify cannot be ignored: politics and personal interests must be set aside and science must examine the evidence for the benefit of humanity! 34,35
References and original document from the following link:
