Bactericidal properties of inorganic acids against mycobacteria
A. Paliy*, O. Pavlichenko, A. Berezovskyi, A. Fotin, D. Kisil and O. Panasenko
Abstract
Despite current successes in fighting animal tuberculosis, sporadic cases of infection with both the disease’s causative agents and atypical mycobacteria still occur in animals.
Since animal mycobacteriosis cannot be treated with medication, priority must be given to their prevention and elimination as non-specific measures, including disinfection. The unique structure of the mycobacteria cell determines their high resistance to antimicrobial agents, therefore the constant search for effective disinfectants is an urgent task of veterinary science. This study aimed to determine the bactericidal properties of several inorganic acids against mycobacteria. Experiments were carried out following modern methodological aspects using the atypical mycobacteria M. fortuitum, pathogens of tuberculosis M. bovis and M. avium. Orthophosphoric (phosphoric) acid (H3PO4) and nitric acid (HNO3) were used in the experiments. Using the suspension method, orthophosphoric acid was found to have a bactericidal effect against atypical mycobacteria M. fortuitum and the pathogen of tuberculosis M. avium when tested at a concentration of 1.5% (24 hours) and 2.0% (1–24 hours), against the causative agent of tuberculosis M. bovis at a concentration of 1.5% (5–24 hours) and 2.0% (1–24 hours). Nitric acid showed a bactericidal effect when tested in solution against mycobacteria M. fortuitum and M. avium at a concentration of 2.0% (5–24 hours), and against M. bovis at a concentration of 2.0% after 1 hour exposure. A different level of mycobacterial growth intensity in test tubes was observed after the action of inorganic acids in sub-bactericidal and bacteriostatic concentrations, which was directly dependent on the concentration and exposure of the acid, and on the type of test culture of microorganisms. It has been shown that inorganic acids can disinfect surfaces contaminated with the tuberculosis-causing agent. This refers to various surfaces like wood, tile, fabric, glass, and metal. It has been found that orthophosphoric acid (1.5% for 24 hours) and nitric acid (2.0% for 5 hours) can be used to disinfect in cases of tuberculosis infection. These findings have been confirmed through bioassays on laboratory animals. Future research will focus on discovering and developing new medications with strong bactericidal properties against mycobacteria.Key words: orthophosphoric acid; nitric acid; mycobacteria; concentration; exposure; test objects; bactericidal effect
Introduction
Existing measures to combat infectious animal diseases can be effective and efficient only if it is possible to break the epizootic chain or its links. To this end, it is necessary to increase the resistance of susceptible animals and to ensure the destruction of pathogens at all phases of their life cycle in the environment by available and effective means of mechanical, chemical, physical, and biological effects (Sander et al., 2020; Matsuzaki et al., 2021; Ghanbari et al., 2022).
Today, tuberculosis still holds a special place among zoo anthroponotic diseases. According to the world’s leading tuberculosis experts, this disease remains a particularly dangerous infection for humans today (Boisson-Dupuis, 2020; Migliori et al., 2021).
Bovine tuberculosis is a serious infectious disease affecting a wide range of domesticated and wild animals, representing a global economic and public health burden (Turgenbayev et al., 2021; Borham et al., 2022). M. bovis is a pathogen at the human-livestock-wildlife interface. Diseases transmitted between humans, livestock, and wildlife are increasingly challenging public and veterinary health systems (Mohamed, 2019; Byrne et al., 2022).
Cattle can contract tuberculosis due to various reasons, such as feeding young animals with non-disinfected skim milk, bringing in cattle from TB positive farms and exposing them to the general herd, contact with infected cattle while grazing or drinking water, and most importantly, failure to follow proper health and anti-tuberculosis prevention measures.
These measures include maintaining good sanitation on holdings, disinfecting facilities and farm territories, and using disinfected manure (Bouchez-Zacria et al., 2018; Sedighi and Varga, 2021).
Many experiments have shown that mycobacteria are highly resistant to environmental pressures, chemicals, and physical factors (Saxena et al., 2021).
While sunlight can be effective in killing mycobacteria in the external environment, this outcome can be influenced by various factors. Therefore, it is essential to use disinfectants to sanitise contaminated objects (Zhang et al., 2022; Balani et al., 2023).
Various disinfectants from different groups of chemical compounds are used to neutralise mycobacteria in the environment (Bocian et al., 2014; Shinoda et al., 2016). However, when studying the properties of different disinfectants, we found that most of them do not cause the devitalisation of mycobacteria or have only a bacteriostatic effect, and only some of them have a pronounced bactericidal effect against atypical mycobacteria and tuberculosis pathogens (Zavgorodnii et al., 2021; Uy et al., 2022).
These data indicate that the arsenal of disinfectants active against the causative agents of tuberculosis and atypical mycobacteria is limited, and the search for new disinfectants is an actual and promising area of veterinary medicine.
Traditional means of disinfection are gradually losing their relevance due to the formation of resistance in field isolates of pathogens, so scientists are working on determining the bactericidal properties of other means used in various industries.
The aim of this study was to examine the bactericidal properties of several inorganic acids against mycobacteria.
Materials and methods
Two inorganic acids were used in the experiment: orthophosphoric (phosphoric) acid, nitric acid. Orthophosphoric acid (H3PO4) is an 85% aqueous solution of a syrup-like consistency that is both colourless and odourless. Nitric acid (HNO3) is a colourless liquid with a pungent, specific smell. The mass fraction of the main substance is ≥ 56.0%.
Experiments were carried out following the current methodological recommendations for the approval of disinfectants in agriculture (Wales et al., 2021).
The bactericidal properties of the acids were determined against the tuberculosis pathogens M. bovis and M. avium, and the fast-growing atypical mycobacteria M. fortuitum, cultured on Pavlovsky’s medium for 30-45 and 14-21 days, respectively, at a temperature of 37.0±0.5°C.
The bacterial mass of mycobacteria test cultures, which had typical cultural and biological properties, was used in the experiments.
The bactericidal effect of acids against the causative agent of tuberculosis Mycobacterium bovis (strain Vallee), Mycobacterium avium (strain IECVM UAAS) and atypical mycobacteria Mycobacterium fortuitum (strain 122) was tested at concentrations of 0.1; 0.5; 1.0; 1.5; 2.0% at exposures of 1, 5 and 24 hours.
Before the experiment, a suspension at a concentration of 2 billion bacterial bodies per cm3 of sterile isotonic solution was prepared from mycobacteria test cultures grown on Pavlovsky’s medium. To do this, the bacterial mass of mycobacteria test cultures was transferred with a bacteriological loop into sterile vials filled with beads with a capacity of 100–200 cm3, pre-weighed on analytical scales. The mass of mycobacteria introduced into them was determined by weighing, and then the necessary volume of sterile isotonic solution was introduced. Vials were shaken on a shuttle apparatus for 30 minutes until a homogeneous suspension of mycobacteria was obtained.
After that, working solutions of acids were prepared in the above concentrations, which were introduced in vials with a capacity of 20 cm3 (10 cm3 in each). Then, 0.2 cm3 of the suspension of the corresponding mycobacteria species was introduced into each test vial. The contents of the vials were thoroughly mixed and subjected to the specified exposure to the disinfectant. As a control of the bactericidal effect of the tested drug, a vial with a suspension of mycobacteria test cultures was used, in which 10 cm3 of a sterile isotonic solution was added instead of disinfectant solutions.
Then samples of 10 cm3 were taken from the experimental and control vials, transferred to centrifuge tubes, and centrifuged at 3000 rpm for 30 minutes.
To stop the action of the disinfectants in the test tubes, the sediment formed after centrifugation, as well as the control sample, were washed twice with a sterile isotonic solution by centrifugation.
After that, the sediment suspension was inoculated on a nutrient medium for the cultivation of mycobacteria. Test tubes with cultures were kept in a thermostat at a temperature of 37.0±0.5°C for 90 days, and after inoculation the growth of cultures was recorded every 3–5 days.
Determination of the bactericidal properties of inorganic acids was also carried out using test objects: wood, ceramic tile, batiste, glass, metal using the Mycobacterium bovis test culture and biological load (liquid manure).
A mixture containing 1 cm3 of the tuberculosis pathogen test culture suspension and 0.5 cm3 of sterile manure was applied to each test object. After that, the experimental test objects were treated with the working disinfectant solutions.
A sterile isotonic solution was applied separately to the control test objects instead of the disinfectant. After the given exposure, from each control and experimental test object, scrapings and swabs with a sterile isotonic solution in Petri dishes were made. The contents were transferred to centrifuge tubes and centrifuged at 3000 rpm for 30 minutes. To neutralise the effects of the drugs the sediment in the test tubes was washed twice with sterile isotonic solution by centrifugation. The obtained sediment of experimental and control samples was resuspended in 5 cm3 sterile isotonic solution and inoculated with a sterile pipette on a nutrient medium for the cultivation of mycobacteria. Guinea pigs were inoculated with this solution.
Test tubes with cultures were kept in a thermostat at a temperature of 37.0±0.5°C for three months, and every 3–5 days the growth of cultures was recorded.
The biological experiment was performed on 24 healthy experimental and 10 control guinea pigs weighing 300–350 g. Guinea pigs were subcutaneously injected in the groin suspension of the sediment obtained after processing the experimental and control test objects with culture Mycobacterium bovis at a dose of 1 cm3.
Laboratory animals were observed for 3 months. During this period, the animals were examined once a month with a tuberculin test. Animals that died during the experiment and were sacrificed after its completion were examined by pathoanatomical and cultural methods for tuberculosis.
The experiments conducted on animals do not contradict the current legislation of Ukraine (Article 26 of the Law of Ukraine 5456-VI of 16.10.2012 “On the Protection of Animals from Cruelty”) and international bioethical norms (the materials of the IV European Convention on the Protection of Vertebrate Animals, which are used for experimental and other purposes, Strasbourg, 1985) (Simmonds, 2017).
Results and discussion
The preliminary determination of the bactericidal effect of inorganic acids was carried out against atypical mycobacteria of the species Mycobacterium fortuitum using the suspension method. The results of the research are presented in Table 1.
Note: “–“ – there is no colony growth; “+” – up to 10 colonies of mycobacteria on the surface of the nutrient medium; “++” – from 10 to 20 colonies of mycobacteria on the surface of the nutrient medium; “+++” – from 20 to 50 colonies of mycobacteria on the surface of the nutrient medium; “++++” – more than 50 colonies of mycobacteria on the surface of the nutrient medium.
The analysis of the obtained results shows that orthophosphoric acid at exposure from 1 to 5 hours at a concentration of 0.1-0.5% does not show an antimicrobial effect against M. fortuitum, and the growth of mycobacterium cultures on the nutrient medium is similar to the control cultures. When the exposure was increased to 24 hours, as well as when using acid at a concentration of 1.0% at exposure of 1-5 hours and a concentration of 1.5% at exposure of 1 hour, the growth of mycobacteria on the nutrient medium decreased slightly, and amounted from 20 to 50 colonies on the surface of the nutrient medium. When exposure increased to 24 hours at a concentration of acid 1.0% and 5 hours at a concentration of 1.5%, the growth of mycobacteria decreased twice, compared to the control.
The lack of growth of mycobacteria cultures was observed after exposure to orthophosphoric acid at a concentration of 1.5% for 24 hours and at a concentration of 2.0% and exposure from 1 to 24 hours, which indicates the manifestation of the bactericidal effect of this compound.
As for nitric acid, when tested at a concentration of 0.1-0.5% at exposure of 1-5 hours; at a concentration of 0.1% at exposure of 24 hours; at a concentration of 1.0-1.5% at exposure of 1-5 hours, the growth of mycobacteria was similar to the effect of orthophosphoric acid.
When using nitric acid at a concentration of 0.5% at exposure of 24 hours, the same result was obtained as when using orthophosphoric acid at a concentration of 1.0%. Under the action of nitric acid at a concentration of 1.0-1.5% at 24-hour exposure and at a concentration of 2.0% at 1-hour exposure, sporadic growth of mycobacteria cultures on the nutrient medium was observed, which indicated the manifestation of the sub-bactericidal effect of this agent. When using acid at a concentration of 2.0% at exposure from 5 to 24 hours, mycobacteria growth on the nutrient medium was absent, proving the presence of a bactericidal effect.
At the next stage, the bactericidal effect of inorganic acids against the causative agents of tuberculosis M. bovis and M. avium was studied using the suspension method, taking into account the results of previous experiments. The results of the research are presented in Table 2.
Note: “–“ – there is no colony growth; “+” – up to 10 colonies of mycobacteria on the surface of the nutrient medium; “++” – from 10 to 20 colonies of mycobacteria on the surface of the nutrient medium; “++++” – more than 50 colonies of mycobacteria on the surface of the nutrient medium.
Data in Table 2 show that orthophosphoric acid at a concentration of 1.5% after exposure for 1 hour has a sub-bactericidal effect against the causative agent of tuberculosis M. bovis, while the growth of mycobacteria did not exceed 10 colonies on the surface of the nutrient medium.
When the exposure time was increased to 5-24 hours, as well as at a concentration of 2.0% after exposure of 1-24 hours, the growth of mycobacteria on the nutrient medium was not observed. Somewhat different results were obtained in experiments with the culture of the causative agent of tuberculosis M. avium. For example, at an acid concentration of 1.5% at exposure of 1-5 hours, the growth of cultures was up to 20 colonies of mycobacteria on the surface of the nutrient medium. Along with this, under the action of orthophosphoric acid at a concentration of 1.5% at exposure of 24 hours and at a concentration of 2.0% at exposure from 1 to 24 hours, a bactericidal effect was observed.
In experiments with nitric acid, it was established that the culture M. bovis completely died under the action of acid at a concentration of 2.0% after exposure for 1-24 hours, while the causative agent M. avium was resistant to the action of acid after exposure for 1 hour. When the exposure was increased to 5 and 24 hours, the bactericidal effect of nitric acid was also established.
After obtaining positive results of preliminary experiments in solutions, the final determination of the mode of bactericidal action of inorganic acids was carried out against Mycobacterium bovis using test objects: wooden bars, ceramic tiles, batiste strips, metal and glass plates.
The results of the conducted research are shown in Table 3.
Note: “–“ – absence of mycobacteria growth; “+” – mycobacteria growth.
Table 3 shows that orthophosphoric acid at a concentration of 1.5% after exposure for 1 hour did not disinfect test objects contaminated with M. bovis, the causative agent of tuberculosis, regardless of their physical properties. When the exposure was increased to 5 hours, the disinfecting effect on ceramic tiles, glass, and metal was observed, while the live causative agent of tuberculosis was isolated from wood and batiste. Similar results were obtained when using nitric acid at a concentration of 2.0% for 1 hour of exposure.
Disinfection of test objects contaminated with mycobacteria was detected when using orthophosphoric acid at a concentration of 1.5% after exposure for 24 hours and at a concentration of 2.0% after exposure from 1 hour, as well as nitric acid at a concentration of 2.0% after exposure of 5-24 hours.
A biological test on laboratory animals was the final stage in determining effective modes of application of inorganic acids for the devitalisation of mycobacteria. For this purpose, three groups of animals were formed, which were separately injected with a suspension obtained after treatment of test objects contaminated with the causative agent of tuberculosis M. bovis and the use of orthophosphoric acid (1.5%-24 hours) (experimental group I, n=12) and nitric acid (2.0%-5 hours) (experimental group II, n=12). The control (group III, n=10) was infected with a live pathogen of tuberculosis without the action of antimicrobial agents. The results of the experiment are presented in Table 4.
Note: “–“ – absence of tuberculosis infection; “+“ – presence of tuberculosis infection.
During a biological study of swabs from the test objects on laboratory animals, after their treatment with acids, the bactericidal properties of orthophosphoric acid at a concentration of 1.5% after a 24-hour exposure and nitric acid at a concentration of 2.0% after the exposure for 5 hours against the causative agent of tuberculosis M. bovis were confirmed. Only the animals of the control group responded to the administration of tuberculin, and during the pathological examination, lesions characteristic of tuberculosis were found in those animals. In the pathological material taken from the experimental and control animals, M. bovis was isolated only from animals of the control group.
Disinfectants for use in veterinary medicine and animal husbandry are essential elements for controlling infectious agents, including zoonotic and antimicrobial-resistant microorganisms in managed animal species (Paliy et al., 2019; Rodionova et al., 2021; Wales et al., 2021).
The arsenal of modern disinfectants is quite limited, therefore the constant search and improvement of antimicrobial agents for the purpose of sanitation of livestock facilities is an urgent task for veterinary science and practice (Chen et al., 2021; Scollo et al., 2023). Traditional means used for the control and prevention of animal tuberculosis are aldehydes, chlorine derivatives, oxygen compounds, etc. (Yoo, 2018; Xiao et al., 2022).
However, mycobacteria have developed increased resistance to most disinfectants and their use is ineffective in modern conditions.
Currently, due to the widespread use of disinfectants, especially due to the pronounced COVID-19 pandemic, resistance to disinfectants needs to be more clarified than antibiotics in this field (Tarashi et al., 2022).
To destroy tuberculosis pathogens in the environment, we tested two inorganic acids whose effectiveness has been recognised in the practice of disinfection (Ganesh et al., 2012; Didouh et al., 2022). These inorganic acids are used in the dairy industry (Ózsvári and Ivanyos, 2022) and in water sanitation (Gul et al., 2021).
During the study, we found that different types of mycobacteria have varying sensitivity to chemical compounds.
For instance, M. fortuitum, a fast-growing atypical mycobacteria, was most resistant, while M. bovis, the tuberculosis pathogen, was the least. Other researchers have also noted the high resistance of atypical mycobacteria to antimicrobial agents (Le Dantec et al., 2002; Chang et al., 2015). Moghaddam et al. (2022) showed a significant difference in resistance between M. fortuitum and other atypical mycobacteria.
Dauendorffer et al. (2000) demonstrated that M. xenopi is more resistant to disinfectants than M. tuberculosis or M. smegmatis and suggested that strains from the environment may be more resistant to disinfectants than strains isolated from pathological samples.
A biological test is mandatory to study the bactericidal properties of antimicrobial agents. Guinea pigs are the most sensitive model for anti-tuberculosis bioassay (Clark et al., 2014), and we considered this in our studies.
It is recommended that protocols for the use of disinfectants be tested under the specific conditions required to ensure their suitability for use (Uy et al., 2022).
The development of new inorganic acid-based disinfectants and their direct testing in production conditions is a prospect for further research.
Conclusions
Bacteriological research has shown that orthophosphoric acid can destroy atypical mycobacteria M. fortuitum in solution, and the causative agents of tuberculosis M. bovis, when applied to test objects at a concentration of 1.5% for 24 hours. Nitric acid also exhibited tuberculocidal properties at a concentration of 2.0% at exposure of 5 hours. It has been shown that atypical mycobacteria M. fortuitum species are more resistant to the action of inorganic acids, compared to the causative agents of tuberculosis M. bovis and M. avium. Orthophosphoric and nitric acids can be used for preventive and forced disinfection in cases of tuberculosis in farm animals.
Currently, most disinfectants offered by manufacturers are ineffective against mycobacteria. As a result, we urgently need to search for new and improved disinfectants with tuberculocidal properties.
Our research results broaden the range of anti-tuberculosis disinfectants, enabling us to rationally rotate disinfectants in the general complex of veterinary and sanitary measures.
References [… show]
Baktericidna svojstva nekih organskih kiselina u odnosu na mikobakterije
Anatoliy PALIY, Doctor of Veterinary Sciences, Professor, Laboratory of Veterinary Sanitation and Parasitology, National Scientific Center «Institute of Experimental and Clinical Veterinary Medicine», Kharkiv, Ukraine; Olena PAVLICHENKO, Candidate of Veterinary Sciences, Associate Professor, Doctor of Juridical Science, Department of Sanitation, Hygiene and Forensic Veterinary Medicine, State Biotechnological University, Kharkiv, Ukraine; Andrii BEREZOVSKYI, Doctor of Veterinary Sciences, Professor, Department of Veterinary Examination, Microbiology, Zoohygiene and Safety and Quality of Livestock Products, Sumy National Agrarian University, Sumy, Ukraine; Anatoliy FOTIN, Candidate of Veterinary Sciences, Associate Professor, Department of Veterinary Examination, Microbiology, Zoohygiene and Safety and Quality of Livestock Products, Sumy National Agrarian University, Sumy, Ukraine; Dmytro KISIL, Candidate of Veterinary Sciences, Teacher, Department of Virology, Pathanatomy and Poultry Diseases, Sumy National Agrarian University, Sumy, Ukraine; Oleksandr PANASENKO, Candidate of Veterinary Sciences, Associate Professor, Department of Virology, Pathanatomy and Poultry Diseases, Sumy National Agrarian University, Sumy, Ukraine
Danas, unatoč uspjehu u borbi protiv životinjske tuberkuloze, još uvijek se u životinja javljaju sporadični slučajevi infekcije s uzročnicima bolesti i s atipičnim mikobakterijama. Zbog činjenice da mikobakteriozu životinja nije moguće liječiti lijekovima, prvo mjesto za njihovu prevenciju i eliminaciju pripada nespecifičnim mjerama koje uključuju dezinfekciju. Jedinstvena struktura stanica mikobakterija određuje njihovu visoku otpornost na antimikrobna sredstva, stoga je stalna potraga za učinkovitim dezinfekcijskim sredstvima hitan zadatak veterinarske znanosti. Naš rad ima za cilj odrediti baktericidna svojstva nekih anorganskih kiselina u odnosu na mikobakterije. Provedeni su eksperimenti slijedeći moderne metodološke aspekte uporabom atipičnih mikobakterija M. fortuitum, patogena tuberkuloze M. bovis i M. avium.
Ortofosforna (fosforna) kiselina (H3PO4) i dušična kiselina (HNO3) su rabljene u eksperimentima. Sa suspenzijskom metodom istraživanja, otkriven je baktericidni učinak ortofosforne kiseline u odnosu na atipične mikobakterije M. fortuitum i patogen tuberkuloze M. avium kada je ispitana u koncentraciji od 1,5 % (24 sata) i 2,0 % (1-24 sata), protiv uzročnika tuberkuloze M. bovis u koncentraciji od 1,5 % (5-24 sata) i 2,0 % (1-24 sata). Dušična kiselina pokazala je baktericidni učinak kada je ispitana u otopini u odnosu na mikobakterije M. fortuitum i M. avium u koncentraciji od 2,0 % (5-24 sata) i u odnosu na M. bovis u koncentraciji od 2,0 % nakon izlaganja od 1 sata. Primijećena je različita razina intenziteta rasta mikobakterija u epruvetama nakon djelovanja anorganskih kiselina u subbaktericidnim i bakteriostatskim koncentracijama, što je izravno ovisilo o koncentraciji i izloženosti rabljenim kiselinama, kao i o vrsti ispitne kulture mikroorganizama. Kroz eksperimente je dokazano da anorganske kiseline mogu dezinficirati površine kontaminirane uzročnicima tuberkuloze. To se odnosi na različite površine poput: drva, pločica, tkanine, stakla i metala.
Otkriveno je da je moguće rabiti ortofosfornu kiselinu (1,5 % tijekom 24 sata) i dušičnu kiselinu (2,0 % tijekom 5 sati) za dezinfekciju u slučajevima infekcije tuberkulozom. Ova otkrića potvrđena su biološkim testovima na laboratorijskim životinjama.
Buduća istraživanja usredotočit će se na otkrivanje i razvoj novih lijekova sa snažnim baktericidnim svojstvima u odnosu na mikobakterije.Ključne riječi: ortofosforna kiselina, dušična kiselina, mikobakterija
