MRSA Eradication Using Chlorine Dioxide
MRSA Eradication Using Chlorine Dioxide
Abbreviations: MRSA, methicillin-resistant staphylococcus At the same time, colonization is not static, as strains have been found
aureus; AMR. antimicrobial-resistant; TSST-1, toxic shock syndrome to evolve and even to be replaced within the same host.7
toxin–1; ClO2, chlorine dioxide, PVL, panton-valentine leucocidin;
Drug resistance
MSSA, methicillin-susceptible saphylococcus aureus
MGEs carrying antibiotic resistance genes have been acquired by
Introduction MRSA on multiple independent occasions. Resistance to penicillin
Frequently, Nosocomial Infections acquired in hospitals or (blaZ), trimethoprim (dfrA and dfrK), erythromycin (ermC),
ICUs, are caused by antibiotic-resistant bacteria such as Methicillin- clindamycin (constitutively expressed ermC) and tetracyclines (tetK
Resistant Staphylococcus Aureus (MRSA). This antibiotic resistance and tetL) have all been identified on insertion sequences, transposons
is accompanied by high rates of morbidity, mortality, and the high cost and sometimes plasmids in both MRSA and methicillin-susceptible
of health care facilities. Staphylococcus aureus (MSSA).8 Likely reflecting the strong selective
pressures within the hospital environment, antibiotic resistance is
What is MRSA? often genetically linked to disinfectant or heavy metal resistance (for
example, quaternary ammonia compounds, mercury or cadmium)
Staphylococcus aureus is a gram-positive coccus that is both among HA-MRSA strains.9
catalase- and coagulase+. Staphylococcus aureus has evolved to
develop numerous immune evasion strategies to combat neutrophil- What is chlorine dioxide
mediated killing, such as neutrophil activation, migration to the site
of infection, bacterial opsonization, phagocytosis, and subsequent The compound chlorine dioxide (ClO2), now commercially
neutrophil-mediated killing. As many as 40 immune-evasion important, is not a recent discovery. The gas was first produced by
molecules of S. aureus are known, and new functions are being Humphrey Davy in 1811 when reacting hydrochloric acid with
identified for these evasion proteins. potassium chlorate. This yielded “euchlorine”, as it was then termed.
Watt and Burgess, who invented alkaline pulp bleaching in 1834,
They produce a range of toxins, including alpha-toxin, beta-toxin, mentioned euchlorine as a bleaching agent in their first patent.10,11
gamma-toxin, delta-toxin, exfoliatin, enterotoxins, Panton-Valentine
leukocidin (PVL), and toxic shock syndrome toxin–1 (TSST-1); Chlorine dioxide then became well known as bleach and later
enterotoxins and TSST-1 are associated with toxic shock syndrome; a disinfectant. The production of ClO2 from the mineral chlorate is
PVL is associated with necrotic skin and lung infections and is a complicated, however, and the gas is explosive so that it could not
major virulence factor for pneumonia and osteomyelitis.3 be easily utilized practically until the production of sodium chlorite
powder by Olin Corporation in 1940.
S. aureus expresses a wide range of virulence factors, including
toxins (haemolysins and leukocidins), immune-evasive surface factors Chlorine dioxide could now be released when necessary from
(for example, capsule and protein A), and enzymes that promote tissue the chlorite salt. In municipal water supplies this is usually done
invasion (for example, hyaluronidase).3 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
MRSA colonization increases the risk of infection, and infecting ClO2 could be applied topically by the individual user.
strains match colonizing strains in as many as 50–80% of cases.4,5
Nearly any item in contact with skin can serve as a fomite in MRSA ClO2 is a small molecule with a molecular weight of 67.46, and
transmission, from white coats and ties to pens and mobile telephones. it forms a stable radical.12 ClO2 is an oxidizer, which is reduced to
chlorite ion (ClO2 -) by capturing an electron (ClO2 + e- → ClO2 -).
Colonization can persist for long periods. MRSA may also persist The redox potential (Eº) is relatively high as 0.95 V, therefore does not
within the home environment, complicating attempts of eradication.6 harm the human microbiome.13,14
Chlorine dioxide (ClO2) solution centigrade for 48 hours. These culture tubes could be stored in a
refrigerator at 4 degrees centigrade for up to 10 days, whereby new
Chlorine dioxide is: bactericidal, virucidal, sporicidal, cysticidal, samples would be made.
algicidal, and fungicidal.15 It has been reported that chlorine dioxide, a
strong oxidant, can inhibit or destroy microorganisms at concentrations Counting bacteria
ranging from 1 to 100 ppm which produced potent antiviral activity,
inactivating >or= 99.9% of the viruses with a 15-sec treatment for One of the most common methods to quantify bacteria is counting
sensitization.15-19 colony-forming units (CFUs). This widely used method is simple,
gives a good general idea of cell viability, and is sensitive even to low
Moreover, ClO2 can remove biofilms swiftly20 because it is concentrations of bacteria.
highly soluble in water and unlike ozone, it does not react with the
extracellular polysaccharides of the biofilm. This way ClO2 can A major disadvantage is that it takes days to get results that are
penetrate biofilms rapidly to reach and kill the microbes living within estimations at best. One colony may arise from one or a thousand
the film – a huge advantage that is different to tackle for both Natural cells and sample preparation can vary from tech to tech, as well as
and Allopathic Medicine. There are many reports that ClO2 solution each time, depending on sample conditions. For the sake of increased
has a virucidal activity.21-25 The inactivation concentration against accuracy, in this research, the QUANTOM Tx Microbial Cell Counter
various viruses is 1-2ppm in poliovirus.21,22 2.19ppm in coronavirus was used from Logos Biosystems (logosbio.com). It is an image-
which causes SARS.23 7.5ppm in hepatitis A virus,24 and 0.2ppm in based, automated cell counter that can identify and count individual
rotavirus.25 bacterial cells in minutes.
The QUANTOM Tx automatically focuses on, captures, and
Safety of chlorine dioxide
analyzes multiple images of fluorescence-stained cells to detect
Many evaluations have shown ClO2 compounds to be non-toxic. bacterial cells with high sensitivity and accuracy. It contains a
Five decades of use have not indicated any adverse effects on health. sophisticated cell detection and declustering algorithm that can
The main areas of use have been disinfecting water supplies, the accurately identify individual bacterial cells in even the tightest
elimination of unwanted tastes and odours, and bleaching in the pulp clusters. In these experiments, we used the Viable Cell Staining Kit to
and paper, and textile industries. detect live or viable cells.
Toxicology tests include ingestion of ClO2 in drinking The Quantom Microbial Cell Counter has been compared and
water, additions to tissue culture, injections into the blood, seed found to be as accurate as Flow Cytometry and Haemocytometer
disinfection,26,27 insect egg disinfection, injections under the skin of measurements, but greatly reducing the time as each count takes
animals and into the brains of mice, burns administered to over 1500 no longer than 30 seconds, and it can distinguish between clusters.
rats, and injections into the stalks of plants. Standard tests include Stained cells are mixed with QUANTOM Cell Loading Buffer I,
Ames Mutation, Chinese Hamster, Rabbits Eye, Skin Abrasion, loaded into QUANTOM M50 Cell Counting Slides, and spun in the
Pharmacodynamics and Teratology.28 QUANTOM Centrifuge to immobilize and evenly distribute the cells
along a single focal plane to ensure accurate cell detection. Counting
In one study, human volunteers drank ClO2 or ClO2¯ in solution up results and images can be viewed and saved immediately after the
to 24 ppm and showed no adverse effects.28 count.
Several studies examined the effects on reproductive toxicity To prepare the sample for the Quantom, 10 microlitres (ul) of the
or teratology. There is no evidence of fetal malformation or birth culture medium was taken using a DLAB electronic pipette that had
defects at ClO2 concentrations, in drinking as well as skin route, up been previously calibrated and placed in a 1.5ml sterilized Eppendorf
to 100ppm.29-31 tube. To this was added 2ul of Viable Cell Staining Dye and this
With prolonged feeding toxicity is produced mainly in the red was incubated in a Heraeus incubator at 37 degrees centigrade for
blood cell. Rats fed up to 1000mg/l chronically for 6 months showed 30 minutes. To this sample was added 8ul of Buffer to enhance the
no significant hematological changes. After 9 months, however, red fluorescence signal. To save on the consumable Quantom slides, we
blood cell counts, hematocrit, and hemoglobin were decreased in all recycled the slides by washing them in the iWash®️ Slide Cleaner
treatment groups. Systems from Imrali Inventions (www.imraliinventions.com).
Lack of toxicity in the long term, but the low-level basis is To these tubes, was added chlorine dioxide in different
dramatically illustrated by two separate studies where rats32 and concentrations, for differing durations? The concentration of chlorine
honeybees33 were fed ClO2 in high doses over two years. No ill effects dioxide ranged from 0.5μl (0.5 ppm) to 5μl (5ppm), and the duration
were noted with up to 100 ppm added to the water supply. of exposure to the sample ranged from 30minutes to 30 seconds.
Materials and methods For each experiment based on time and duration, two tubes of the
sample were prepared to keep the dilution factor constant. According
Methicillin-resistant Staphylococcus Aureus (MRSA) was used in to the amount of chlorine dioxide added to the experimental tube, the
this research study, grown on blood, agar plates, which were provided same quantity of water was added to the control tube.
by a local clinical laboratory with certification.
From these Control and Experimental tubes, 6 μl of the sample was
Culturing MRSA taken using an electronic pipette and placed on the M50 Cell Counting
Slides. The slides were placed into the QUANTOM Centrifuge for
In a Safety Class 2 cabinet, from the Blood agar plates (Columbian
8mins at 300 RCF (Relative Centrifugal Force) and then placed into
Agar), a sample of MRSA from isolated cultures was taken using a
the Quantom Microbial Cell Counter to take a baseline measure
sterilized loop and placed in sterile tubes with 5 ml of Tryptic Soy
(Control) and another measurement from the Experimental tube.
Broth (TSB). These culture tubes were incubated at 37 degrees
Citation: Georgiou G. MRSA eradication using chlorine dioxide. J Bacteriol Mycol Open Access. 2021;9(3):115‒120. DOI: 10.15406/jbmoa.2021.09.00306
Copyright:
MRSA eradication using chlorine dioxide ©2021 Georgiou 117
The optimum Quantom Microbial Cell Counter setting for the MMS and CDSplus were used. The range was from 1ppm - 5ppm. The
MRSA protocol that we found during testing was set to Dilution time of exposure of the chlorine dioxide ranged from 30minutes down
Factor 2, Minimum Fluorescence Object Size 0.4um, Maximum to 30 seconds. It was not clear in the initial experiments what time
Fluorescent Object Size 15μm Roundness 50%, Declustering Level 7, would be required for inhibition, but it was quickly demonstrated that
and Detection Sensitivity 7. it was less than one minute exposure. Most experiments, therefore,
had an exposure time of 1minute.
Preparing Chlorine Dioxide
The traditional chlorine dioxide, called MMS, was prepared as Results
a solution using two components, Sodium chlorite solution (25% Initial Experiments
solution in water) and Hydrochloric Acid 4% solution. One drop of
each of these solutions was placed in a 1.5ml sterile Eppendorf tube We began taking different chlorine dioxide concentrations based
and left for 30 seconds to activate. In addition, more experiments on the Traditional MMS and tested these concentrations with MRSA
were performed using a new generation of chlorine dioxide called in solution for different times spanning from 30minutes to 30 seconds.
CDSplus, a patented product manufactured by Aquarius Pro-Life as 1μl of Chlorine dioxide is the equivalent of 1ppm concentration. The
a water treatment product. This is a buffered form of chlorine dioxide lowest concentration of Chlorine dioxide used to completely eradicate
at a standard pH of 7 and a concentration of 3,000ppm when activated MRSA in these experiments was 0.5ppm, with an exposure time of
(250ml). From the activated CDSplus (250ml), was taken 83μl = 30 seconds. Table 1 below shows the different concentrations against
1ppm; 166 μl= 2ppm; 0.25ml = 3ppm. time, with the MRSA cell concentration as measured by the Quantom
Cell Counter. As can be seen, for all concentrations of chlorine dioxide
Experimental Protocols ranging from 1 to 5ppm, and time of exposure from 30minutes down
Several concentrations of chlorine dioxide – both the Traditional to 30 seconds, the growth inhibition of the MRSA was 99.99%
throughout all these experiments.
Table 1 Comparison of Bacterial Counts Before and After Chlorine Dioxide Exposure
C, Control; E, Experimental
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
From the initial experiments, given that Chlorine dioxide was used and a baseline line count was measured for each concentration.
found to eliminate 99.99% of the MRSA bacteria at concentrations of Experiment number 0 is the baseline count (control) for each
0.5 ppm for only 30 seconds, all other experiments used a one-minute experiment group using different concentrations of chlorine dioxide.
exposure time as standard, while testing different concentrations. For each concentration, the experiment was repeated 5 times, with
In this experiment, concentrations of ClO2 ranging from 0.5 average concentrations given.
– 5ppm were taken, using the Traditional MMS. In each of the 5 Figure 2 compares the cell count of MRSA cells against MMS
concentrations, the inhibition rate was 100% - see Table 2 and Figure concentration for 1 minute. The covered area is equal to the cell count
1. Figure 1 shows the repeatability of counting MRSA bacteria using number. The initial counts for each concentration are shown on the
different concentrations ranging from 1 - 5ppm. A baseline count left side of the graph and the final is shown on the right side of the
was taken for each concentration – this was repeated 5 times. In all 5 graph. The inhibition rate was 100% for all concentrations of chlorine
repeats, the growth inhibition of the MRSA was 100%. dioxide, with an exposure time of 1 minute.
Citation: Georgiou G. MRSA eradication using chlorine dioxide. J Bacteriol Mycol Open Access. 2021;9(3):115‒120. DOI: 10.15406/jbmoa.2021.09.00306
Copyright:
MRSA eradication using chlorine dioxide ©2021 Georgiou 118
Concentration
of Chlorine 0.5ppm 1ppm 2ppm 3ppm 4ppm 5ppm
dioxide
Experiment no.
1 0 0 0 0 0 0
2 0 0 0 0 0 0
3 0 0 0 0 0 0
4 0 0 0 0 0 0
Table 3 compares the concentrations of 1, 2, 3, 4, and 5 ppm for Table 4 Chlorine Dioxide (CDSplus) at different concentrations for 1 minute
exposure
1-minute exposure to chlorine dioxide. The control was compared
to the experimental for the different concentrations. For all these Concentration of Chlorine dioxide 1ppm 2ppm 3ppm
concentrations of chlorine dioxide, the inhibition rate was 100%.
Experiment no.
Table 3 Chlorine Dioxide (Traditional MMS) at different concentrations for 0 - Control 3256 2750 2565
1minute exposure 1 1 0 0
2 1 0 0
1 PPM 2 PPM 3 PPM 4 PPM 5 PPM
Citation: Georgiou G. MRSA eradication using chlorine dioxide. J Bacteriol Mycol Open Access. 2021;9(3):115‒120. DOI: 10.15406/jbmoa.2021.09.00306
Copyright:
MRSA eradication using chlorine dioxide ©2021 Georgiou 119
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Citation: Georgiou G. MRSA eradication using chlorine dioxide. J Bacteriol Mycol Open Access. 2021;9(3):115‒120. DOI: 10.15406/jbmoa.2021.09.00306