Action of Polyhexamethylene Guanidine Hydrochloride on Micro- and Macroorganisms: two perspectives of the same issue

Several thousand compounds described in research literature possess biocide properties. However, safety considerations leave only hundreds of them for practical application. Tens of biocide types are taken out of production every year due to either low antimicrobial activity or high toxicity.

Some of the microorganisms have a natural resistance towards biocide chemicals. Besides that, microorganisms have an ability to adapt themselves to the adverse conditions including the action of antimicrobial chemicals.

This phenomenon is due to the survival of the most resistant germ cultures of the bacterial colony after the contact with biocides. Therefore due to mutation only cells with aberrant genes survive and multiply. Cases of pathogen multiplying in solutions prepared for disinfecting are described in literature. Adaptation of microorganisms to the therapeutic doses of antibiotics and their polyresistance to tens antimicrobial chemicals have been reported.

The number of the resistant strains grows constantly due to the misuse, inaccurate, or inexpert application of antibiotics and disinfectants. Polyresistant pathogens tend to spread in nature. So, for example, a lot of criticism has been published recently concerning the quaternary ammonium compounds (QACs).

Researchers have found that several types of microorganisms have natural stability towards QACs, others acquire the resistance through forming a biological film that neutralises the active chemicals. Furthermore, QACs were found to be inactive towards tuberculosis pathogens, picornoviruses, pseudomonades, and mucoid strains of staphylococci.

The search for essentially novel environment-friendly and efficient biocides never stops, because of fast adaptation of microorganisms, growing requirements concerning environmental safety of the biocide production, toxicity and sensitising effect of biocides.

Enhancement of antimicrobial activity is not the most urgent issue in the development of new biocides, because such enhancement is usually accompanied by the growth of their toxicity.

The efforts are rather concentrated on the increasing of environmental safety and of durability of antimicrobial protection of the surfaces treated with biocides, and on decreasing of toxicity and allergenicity. Wide spectrum of biocide activity against the pathogens, raw materials availability for industrial production, technological feasibility, large storage term, acceptable physical and chemical properties, hygienic and consumer properties are also important issues.

Comparison of various groups of chemical compounds (chlorine-containing, oxygen-containing, aldehydes, phenols and their derivatives, QACs, surfactants), according to the criteria listed above, shows that ionogenic and ampholytic surfactants are the most perspective group of compounds.

The Institute for Ecotechnologies has developed unique non-toxic polymers with a wide range of biocide activity, namely, polyalkylene guanidines (PHMG). PHMG are high-molecular cationic surfactants. PHMG are used in new disinfectants and original chemicals for protection of various materials from biodestruction.

High-molecular salts (chloride and phosphate) of polyhexamethylene guanidine (PHMG) are the most perspective and the best studied compounds of the PHMG series.

PHMG have a wide range of antimicrobial, antiviral, fungicide, pesticide, insecticide, and algaecide activity, low toxicity and corrosive activity. They can be stored for a long time without loss of bactericide properties. Still more important is the durability of bactericidal action, because PHMG form a thin polymeric film on the treated surface. This ensures a durable protection of the surfaces from microorganism attacks.

The polar guanidine groups that destroy microorganisms ensure high biocidal activity of the polymers. On the other hand, these groups are compatible with the macroorganisms that metabolise the guanidine-containing polymers using fermentative systems. Polymeric structure of the PHMG ensures their higher biocide activity and lower toxicity for the humans and animals than that of chlorhexidine bigluconate and other low-molecular cationic surfactants.

The antimicrobial action of biocides is known to depend primarily on their chemical structure and on the cell structure of the microorganisms. The first obstacle in the way of microorganism interaction with the antimicrobial chemicals is the cell membrane and cytoplasmic membrane.

These membranes provide an osmotic barrier and selective penetration of the substances into the cell.

The cell membrane structure is different in various microorganisms. This is responsible for selective interaction of the microorganisms with different antimicrobials and their different stability with the respect to the action of chemical compounds of different types. So, cationic surfactants are targeted at the carboxyl groups of aminoacids and acidic polysaccharides of the bacteria.

Anionic surfactants are targeted at the protein ketone groups, aminogroups of the corresponding hydrocarbons and lipids, and also at the phosphate groups of the teichoic acids.

Microbe cells have a negative overall charge in natural conditions. Therefore cationic surfactants are widely applied, because they kill gram-positive and gram-negative bacteria, yeast and filamentous fungi. There are positive-charged molecules inside the cell, so anionic surfactants kill the microorganisms, too. However, greater concentration of anionic surfactant is necessary in this case.

The mechanisms of surfactant action on the gram-positive and gram-negative microorganisms as well as on prokaryotic and eukaryotic cells differ. This is due to the differences in chemical structural organisation of the cell membranes, as well as in the cytoplasmic contents of the cells.

Note that microorganisms have very effective protection mechanisms against the antimicrobial chemicals, and the level of protection of different microorganisms can differ by a factor of several hundreds or even several thousands.

Efficiency of various antimicrobial chemicals against micro-organisms depends first of all on their ability to change the cell membrane permeability and to penetrate the cell.

The efficiency of antimicrobial action depends first of all on their ability to penetrate the cell membrane. Therefore the chemicals that stimulate the passive transport and membrane penetrability are of great interest. Low-molecular cationic surfactants (cetyl pyridine chloride, chlorhexidine, alkyl dimethylbenzylammonium chloride) destabilise strongly the cell membrane.

However, these chemicals have too high toxicity. This property and the absence of the action selectivity limit the application of low-molecular cationic surfactants. High-molecular chemicals that interact with cell membranes have lower toxicity and are of a great practical interest.

Once inside the cell, the biocide chemicals interact with different functional groups of the cell contents, depending on the chemical properties of the biocides. This disrupts the metabolism process and the formation of cell structures. Below we discuss several examples of the biocide action types. Sodium fluoride, phenylhydrazine, etc. inhibit the ferment activity.

Inhibition even of a single ferment that participates in an important metabolic process interrupts the corresponding process and can be fatal for the whole organism. The toxicity of heavy metal cations is due to their interaction with various protein functional groups.

This leads to the structure disturbance and disfunction of the proteins. Organotin compounds mainly inhibit the electron transport in the process of oxidative phosphorylation. QACs chiefly cause protein denaturation and disrupt the cell membranes.

Organo-chlorine compounds, phenols and quinones act on the respiratory processes. Phenols can also form insoluble co-ordination compounds with cell membrane polysaccharides, thus affecting the membrane function. Quinones interact with the cell metabolites, cause their blocking and exclusion from the exchange processes.

According to the recent research results, the mechanism of PHMG interaction with microorganisms is as following:

  • guanidine polycations are adsorbed by the negative-charged bacterial membrane, thus blocking the respiratory and nutrition processes, and metabolite transport through the bacteria cell membrane (this effect depends on the ionic charge of the polycation);
  • PHMG hydrochloride macromolecules penetrate the cell membrane, cause irreversible structural damage of the cytoplasmic membrane, nucleotide, and cytoplasm (this process depends on the surfactant activity, lipophility, solubility in water, molecular volume of the diffusing particle);
  • PHMGH react with the acidic phospholipids and cytoplasmic cell proteins; this causes the membrane disruption (this effect depends on the antiseptic concentration and on its molecular mass);
  • this results in blocking of the glycolytic ferments of the respiratory system, loss of the pathogenic properties, and death of the microbe cell.

The perspectives of PHMG practical application required a thorough toxicological and sanitary evaluation of these chemicals. This was necessary to determine the maximum concentration limits for chemical residual on the treated surfaces, in water and food.

The experiments have shown that the intoxication intensity depends on the chemical structure of the biocide, exposure type, and dose, and on the exposure time (acute, subacute, and chronic action).

PHMG are insoluble in fats, but they are well soluble in water. Experiments have shown that PHMG can be absorbed through undamaged skin coverlets. However, due to the low oil / water distribution ratio and high molecular mass of the PHMG, the transepidermic, transfollicular, and transglandular penetration rates is as low as 1.5 mkg / (cm2 hour).

For comparison, penetration rate of low-molecular chlorhexidine it is as great as 10.3 mkg / (cm2 hour). Penetration rate for PHMG grows with the increase of the chemical’s dose in contact with skin.

On the other hand, transcutaneous resorbtion of PHMG occurs for the most part during the first 5-30 minutes of the contact, because PHMG form a polymer film on the skin surface after drying. This film prevents further transdermal penetration of the antiseptic into organism. All the PHMG studied were proven harmless in chronic experiments for doses below 50 mg/kg. This is important from the hygienic point of view. Under these conditions PHMG do not endanger the development and functioning of the gametes, testicles, and ovaries, they do not impact the oestrous cycle, do not harm embryogenesis, no not cause mutations in somatic or gametic cells, or tumour formation.

All the studied PHMGH in dose below 50 mg/kg in case of skin exposure and chronic exposure are safe for human organism.

Oral exposure to PHMG causes the same pathogenic reactions including biological membrane destruction and thence the disruption of metabolic process regulation in macroorganisms, as well as in microorganisms. However, blocking of respiratory system ferments in microorganisms leads to the loss of their pathogenic properties, and finally, to their death.

PHMG action on macroorganisms is limited to the derangement of metabolism that can result in anaemia development.

Haemolytic effect caused by the membranotoxic action of PHMG is the most dangerous implication. This effect has been found only for oral exposure to high doses of the chemical in chronic experiments.

In real PHMG application conditions the haemolytic effect is not dangerous for the macroorganisms, because doses lethal for microorganisms are considerably below the level of PHMG doses that can cause anaemia.

The calculated certain safety factor (CSF = LD50 / ED100) for PHMG equals 3667 on the average. This factor shows the ratio of toxicity of PHMG towards the pathogenic microflora and towards the humans and animals.

Antimicrobic activity and toxic action of PHMG depends, apart from the dose and exposure duration, on the polymer structure and, first of all, on the chemical properties of the anion. So, e.g., it was established that the action of different PHMG salts studied on the colibacillus differs by a factor of 120.

Note that PHMG are normally biodegradable compounds. Live organisms produce fermentative systems that cause destruction of these chemicals, thus preventing their accumulation in the organism.

Experiments have shown that the first stage of PHMG chloride and PHMG phosphate in a live organism is the substitution of the chlorine or phosphate ion with the gluconate anion. Thus one of the least toxic PHMG chemicals is formed. Later the guanidine groups are hydrolysed and converted into ureal groups, and the polymer chain falls into fragments.

Action of different PHMGH salts on various animal types has been studied. The experiments have shown that the average lethal dose for skin exposure (LD50 per cut) equals 8 900 – 15 500 mg / kg, and for oral exposure (LD50 per os) it equals 815 – 3 200 mg / kg.

Other parameters of PHMG hydrochloride necessary to establish the hygienic norms have been measured. According to the Russian state standard ГОСТ 12.1.007-76, all the known chemicals of the PHMG series are rated as low-hazard compounds for skin exposure (4th class), and as medium-hazard compounds for oral exposure (3rd class).

Macroorganism exposure to PHMG hydrochloride is possible in case of their application in disinfecting solutions and also for drinking water and swimming pool water decontamination.

Various water-polluting chemicals (suspended substances, colloid particles, oil products, anionic surfactants, dye molecules, etc.), that have, as a rule, anionic properties, effectively absorb PHMG.

This is essential for PHMG behaviour in natural water bodies. This feature causes rapid precipitation of PHMG, and activated sludge increases its biodestruction rate. Experiments using a standard technique for estimating flocculant biodestruction rate have shown that 80 % of the chemical decomposes after a single pass through an activated sludge layer.

All the described features of PHMG hydrochlorideallow concluding that these chemicals are very perspective for application as biocides. They can meet the demand for modern disinfectant chemicals and biocidal materials.

Nikolay Polykarpov,
Ph.D. (biology)

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