For many years, tuberculosis has remained relevant in veterinary and human medicine worldwide and in Ukraine. According to WHO, tuberculosis is one of the top ten causes of death in the world. In 2017, 10,000,000 people had a tuberculosis infection, and 1,600,000 died from the disease. In 2018, according to WHO, the overall incidence of tuberculosis in Ukraine was 36,000 (per 37,000,000 population).
Complexities of overcoming tuberculosis and mycobacteriosis can be interrelated to the variability of the bacterial cells that enable pathogens to survive both in hostile macrophages (Kumar and Sanyal 2012) and outside the organism. Mycobacteria have an extraordinary capacity to adapt to the changing environment (Velayati and Farni 2012). It has been shown that nutrient deficiency, hypoxia, temperature, pH, NaCl, and various exogenous stress can significantly influence the metabolism of mycobacteria and, at the same time, the microorganism’s morphology (Vera and Rettger 1940; Smeulders et al. 1999; Bentrup and Russell 2001; Shleeva et al. 2002; Young et al. 2005; Anuchin et al. 2009; Velayati et al. 2009; 2011; Velayati and Farnia 2012).
It has been established that mycobacteria have a high degree of variability, and changes in the biological properties of
Understanding the processes in a bacterial cell in response to the external factors could push the knowledge on the complex mechanism of mycobacterial adaptation and survival in macroorganisms and the environment.
There is no sufficient research on the impact of long-term storage of vaccine strains on the effectiveness of preventive immunization for infectious diseases. The BCG strain is a good example. Long-term storage of a parent strain in various countries led to its variability and the formation of BCG sub-strains: Russia (Moscow), Japan (Tokyo), Moreau (Brazil, Rio), Sweden (Gothenburg), Birkhaug, Danish (Denmark, Copenhagen), Prague (Czech Republic), Glaxo (London), China, Phipps (New York, Park, Philadelphia), Tice (Chicago), Frappier (Montreal), Pasteur (Paris), Bulgaria (Sophia), Connaught (Toronto), etc. BCG sub-strains slightly differ in genetic traits and immunogenicity. Therefore, considering potential changes in bacteria’s biological features after the long-term storage is essential for a comprehensive assessment of vaccines’ safety and efficiency (Ritz et al. 2008; Zhang et al. 2016).
Therefore, there was necessary to investigate the morphology, growth rate, tinctorial properties, biochemical activity, pathogenic, and sensitizing properties of mycobacteria in long-term conditions of hypoxia, nutrient deficiency, and low temperature (3°C).
Experiments were performed in a laboratory of the Department of Epizootiology and Infectious Diseases of Animals of the Dnipro State Agrarian and Economic University using the museum’s fast-growing virulent strain
Our study was approved according to the guidelines and roles of the Animal Researches Committee of the Dnipro State Agrarian and Economic University.
First, long-term storage of cultures (10–13 years at 3°C) in the test tubes was performed. The passages (variants of subculture, first-generation bacteria) No. 54, 115, 135, 135, 171, and 180 at pH 6.5, and passage No. 143 at pH 7.1 were grown in tubes covered with rubber stoppers. Each culture was stored in a nutrient medium in two tubes. It ensured the persistence of mycobacteria in an anoxic environment. Microscopy of smears was carried out, studying the tinctorial properties and morphology of mycobacteria. The
The culture purity was verified as follows: The bacterial suspension was divided into two parts, and one of them was heated at 130°C for 5 minutes. Then, both parts were inoculated on Lövenstein-Jensen medium at 37°C and Sabouraud agar at 30°C. Cultures were considered pure in the absence of growth of any foreign microorganisms on Lövenstein-Jensen nutrient medium after inoculation of heat-treated suspensions and the absence of colonies on Sabouraud agar.
At the second step, the pathogenic properties of mycobacteria were investigated. Seven guinea pigs were infected for each of six variants of subculture (a first passage). A control group of pigs was infected with the pathogenic parental strain of
During the experiments (within 90 days), animals were weighed, an ulcer formation was studied in the injection site, and the manifestation of allergic reaction to PPD for mammals at a dose of 25 IU in 0.1 cm3 isotonic solution was monitored. The allergic tests were performed at 30, 60, and 90 days after infection of the animals, and the reaction was recorded 24–48 hours after tuberculin injection.
The guinea pigs were then infected for a second time (a second passage) with the mycobacteria recovered from the organs of guinea pigs infected with the bacteria of the first passage. The microorganisms recovered from the animal organs infected with the second passage were used to infect the next group of guinea pigs (a third passage). In the second and third rounds of infections, 84 (42 + 42) guinea pigs were infected (the control group’s guinea pigs were not infected).
At the third step of our studies,
To determine the biochemical activity of
The polymerase chain reaction was used to confirm or refute the mycobacterial changes in genetic material. DNA was isolated from cultures of the original highly virulent fast-growing strain (at the second passage), long-term variants of subculture (passages), as well as Vallee strain (KMIEV-9 modification, provided by the Suny National Agrarian University) and BCG strain (sub-strain of Russia BCG-1, used for vaccine production). These strains were inoculated at a volume of 0,2 cm3 and a concentration of 2 × 105 CFU/ml and grown in a nutrient medium for 30 days.
For RT-PCR, an amplifier iCycler iQ5 (Bio-Rad, USA) and reagent kits for DNA isolation and PCR («MIKO-GEN», producer NGO DNA-Technology, Russian Federation) were used.
Initial cultures on a Lövenstein-Jensen nutrient medium were characterized by a predominantly moderate growth rate in the inoculation line (Fig. 1), the color of ivory. Some strains also grew as individually formed colonies. The colony surface was rough (R) in three subculture variants (passages No. 135, 171, and 180) or smooth (S) – also in three subculture variants (passages No. 54, 115, and 143).
Cultures of
The mycobacteria from passage 54 appeared as the acid-fast forms (thick rod with rounded ends and single grains) (Fig. 2a). All other subculture variants (passages No. 115, 135, 145, 171, and 180) displayed mixed morphological forms (Fig. 2b), i.e., acid-fast rods, non-acid-fast filamentous and rod-shaped forms, and grains.
Morphology of initial cultures of
After inoculation of the Lövenstein-Jensen nutrient medium, the mycobacteria were capable of growing at two temperatures (3°C and 37°C) (passages No. 115, 171), at one selected temperature 37°C (passages No. 54, 143, 180), or 3°C – one strain (passage No. 135). The subcultures obtained (Fig. 3) differed from the initial cultures, as shown in Fig. 1.
Subcultures of
At 37°C, the bacterial growth became visible on the fourth day – passages No. 115, 143, on the fifth day – passages No. 171 and 180, and on the twentieth day – the passage No. 54. The growth rate in all cultures was considerable. In four subculture variants (passages No. 54, 115, 171, and 180), the growth was characterized by an oily coating (fur) of yellow or orange colors (Fig. 3), in one subculture variant (passage No. 143) – small translucent ivory-colored S-colonies were located in clusters.
At 3°С, the growth was noted on the sixth day – passages No. 115 and 171, and on the fiftieth day – passage No. 135. In two cultures (passages No. 115, 171), there was considerable growth in the form of blue-green mucous ropy fur, and in one culture (passage No. 135) – yellow fur.
Microscopy of smears prepared from the isolated cultures revealed the difference between the parental strain of
Morphology of
At the end of the animal experiments and euthanasia, an autopsy was performed to reveal any pathological-anatomical changes characteristic for tuberculosis in the experimental animals. The infection of guinea pigs by all studied mycobacterial subcultures (passages) was not accompanied by sensitization of the macroorganism to PPD for mammals, the formation of ulcers at the site of mycobacteria suspension, or bodyweight decrease.
Guinea pigs infected with the parental pathogenic strain of
The spleen of the guinea pig: 1) normal, 2) with tuberculous foci.
Through bacteriological investigations of specimens from each guinea pig, eight cultures of mycobacteria were isolated: after four days of culture, there were three cultures at 3°C (passages No. 115, 135, and 171) and five cultures at 37°C (passages No. 54, 115, 143, 171, and 180). Non-acid-fast rod-shaped mycobacteria and grains were therefore visible under the immersion in the microscope.
In animals infected with a second passage, no allergic reactions to PPD for mammals, formation of ulcers in the injection site, or tuberculosis characteristic features were observed. However, the bacteria growth from tissue specimens at 37°C was observed on the 6th day (passages No. 54 and 180) and on the 10th day (passages No. 171 and 143). Under microscopy, the smears from cultures revealed acid fast-negative rod-shaped bacteria and grains.
The animals infected with a third passage did not suffer allergic reactions to the PPD for mammals, the formation of ulcers in the site of inoculation of material, decrease in body weight, or and symptoms of tuberculosis. No bacteria were grown from the animal tissues.
These mycobacteria could grow on nutrient media. In the nutrient broth, colony growth was observed for all passages except for passage No. 54. In the Lövenstein-Jensen medium with sodium salicylate at 3°C, colony growth was observed for all passages except for 54, 135, and 171 (Fig. 6).
Growth of pathogenic
Concerning the biochemical activity of the cultures studied, variations in the activity of mycobacterial enzymes were ascertained depending on the number of passages and the cultivation temperature. The tendency to change the biochemical activity of the microorganisms studied was established: catalase activity increased along with the decrease of the cultivation temperature in passages 115 and 171 (Fig. 7).
Catalase activity of
Peroxidase activity was observed only in the control samples of the highly virulent strain. Dehydrogenase activity in the first 15–30 minutes was not detected in any culture, but after 24 hours was observed in almost all cultures, except those on the 54 passage (Fig. 8).
Dehydrogenase activity of
Nitrate reduction was recorded in all cultures except for passages No. 143 and 171 grown at 37°C (Fig. 9).
Reduction of nitrates by
Hydrolysis of Tween-80 in the first 4 hours was not found in all subcultures (passages) of
When the biochemical activity of mycobacteria at subsequent passages in guinea pig was compared with the initial strain, the following phenomena become apparent: an increase in the activity of the enzyme dehydrogenase – passages No. 54, 135 (grown at 37°C) and 171 (grown at 3°C); loss of ability to reduce nitrates, especially in cultures grown at low temperature (3°C) – passages No. 115, 171, and at 37°C – for the passage No 195; increase of catalase activity – passages No. 115 and 171 at both cultivation temperatures (3°C and 37°C); the ability to hydrolyze Tween-80 in the vast majority of cultures was decreased for all passages except passage No. 54 (Fig. 10).
Tween-80 hydrolysis by
It should also be noted that the biochemical activity of one subculture strain No. 143 after passage through the body of guinea pigs remains unchanged.
Using PCR, the amplicons were detected in the initial pathogenic
The results of DNA amplification of mycobacteria
Type of mycobacteria | Strain of mycobacteria | Result of detection of amplification products |
---|---|---|
fast-growing (original) | + | |
after the long-term storage and reinoculation of cultures on the nutrient medium), passages 54, 115, 135, 135, 171, and 180 | – | |
Vallee strain | + | |
BCG strain | + |
+ – positive result; – – negative result
According to the experimental findings of this study, mycobacteria stored in the nutrient medium for 9–12 years manifested viability on the Lövenstein-Jensen medium. After long-term storage, it was possible to trace the non-acid-fast elements and L-forms of vesicular type in the subsequent cultures.
Other authors have also reported that the causative agent of tuberculosis of the horned presented highly adaptive properties (Djachenko et al. 2008). The transformation of mycobacteria may occur when environmental hostility leads to the change in their lifestyle (Gomez and McKinney 2004; Lewis 2007). To minimize the need for nutrients, bacteria may undergo hibernation and increase their resistance.
The phenotypic change that occurred in mycobacteria in our laboratory may stem from the decrease in the oxygen level in tubes due to its gradual use for bacterial metabolism during storage under strictly closed stoppers.
According to Kumar and Sanyal (2012), such a process of increasing resistance by reducing metabolism may be caused by the decrease in oxygen concentration in the habitat, which leads to anaerobiosis. It has appeared that the slight decrease in oxygen content in the tubes allows mycobacteria to survive. In contrast, the rapid decrease of oxygen concentration leads to the sharp decrease in the number of colony-forming units (Wayne and Lin 1982; Dick et al. 1998; Usha et al. 2002; Lewis and Falkinhan 2015).
The oxygen limitation causes some specific changes in mycobacteria. The microorganisms become thermotolerant, reduce overall protein synthesis, lose acid resistance, and their cell wall display a thickening process (Gillespie et al. 1986, Cunningham and Spreadbury 1998, Hu et al. 1998, Primm et al. 2000).
Adaptation to nutrient deficiency is also crucial for the long-term survival of mycobacterial cells.
The results of guinea pig infection in this study demonstrated that mycobacteria underwent a strong transformation during the years of storage because they became avirulent and lost their sensitizing properties. Even multiple direct passages through the laboratory animals failed to restore their pathogenicity. On the contrary, after the second passage through guinea pigs, only half of the mycobacterial subcultures were isolated from animals, and no cultures were grown after the third passage. No allergic reactions to tuberculin in any of the infected guinea pigs were observed for three passages. In our opinion, the multiple passages of mycobacterial subcultures through the nutrient medium and subsequent long-term preservation at 3°C affected their vital properties. Due to the influence of the above-named factors, mycobacteria adapted so much to the conditions of existence in vitro that the immune system overcame them when they enter the macroorganism.
Already described by many authors (Tkachenko 2004; Djachenko et al. 2009; Yavorska and Sybirna 2009; Glebenjuk and Telizhenko 2015; Lysenko et al. 2019), the high variability of mycobacteria is confirmed by our investigations of the biochemical activity.
On this basis, we can assume that the changes that have occurred in
After the passage of
It has been reported (Li et al. 1998) that catalase promotes
According to some assertions, catalase and peroxidase activity acts as a virulence factor of mycobacteria (Manca et al. 1999; Kondratjuk and Sybirna 2008).
However, our results indicate that mycobacteria’s final typing and species identification based only on biochemical tests are impossible. These characteristics may differ between members of the same species depending on the environmental conditions of the specific microorganism. (Torkko et al. 1998; Yavorska and Sybirna 2009; Lysenko et al. 2011). Thus, mycobacteria can rapidly alter metabolic processes and adapt to environmental conditions. To identify mycobacteria, researchers (Wayne and Sramek 1992; Torkko et al. 1998; Tkachenko 2004) recommend other tests, for instance, fatty acid analysis, detection of bacterial growth rate, sequence analysis, or PCR.
In the microbiological diagnosis of tuberculosis, PCR is recognized as a method of high specificity and sensitivity (Rodriguez et al. 1995; Portillo-Gómez et al. 2000). However, sequencing of the complete genome of
Repeatedly passaged