|Year : 2023 | Volume
| Issue : 1 | Page : 17-22
Evaluation of possibility of cultivation of acid resistant bacteria on solid egg-based and agar cultural media
Artem Lyamin1, Alena Ereshchenko2, Vladimir Antipov3, Andrey Kozlov2, Danir Ismatullin1, Tatyana Nikitina1
1 Research and Educational Professional Center for Genetic and Laboratory Technologies, Samara, Russia
2 Research and Educational Professional Center for Genetic and Laboratory Technologies; Department of Fundamental and Clinical Biochemistry with Laboratory Diagnostics, Samara State Medical University, Samara, Russia
3 Department of Fundamental and Clinical Biochemistry with Laboratory Diagnostics, Samara State Medical University, Samara, Russia
|Date of Submission||01-Nov-2022|
|Date of Decision||17-Dec-2022|
|Date of Acceptance||20-Jan-2023|
|Date of Web Publication||14-Mar-2023|
No. 89, Chapayevskaya Street, Samara 443099
Source of Support: None, Conflict of Interest: None
Background: The increase in the number of patients at risk for opportunistic infections caused by rare bacteria, which include individual representatives of acid-resistant bacteria (ARB), is a serious problem in modern health care. Significant difficulties in the etiological diagnosis of mycobacteriosis, nocardiosis, and actinomycosis are associated not only with the problem of identifying the main pathogens but also with certain difficulties in isolating pathogens from biological material. Methods: The research provides data on 402 strains of ARB, which were isolated from various biological materials obtained from patients during examination for tuberculosis. All samples of biological material were negative on the Mycobacterium tuberculosis complex. The isolates were identified on the MALDI-ToF mass spectrometer. The cultural characteristics of ARB were evaluated on the solid Löwenstein–Jensen egg-based culture media, universal chromogenic media, and 5% blood agar with lamb blood. Results: The studies carried out indicate the possibility of culturing ARB representatives on agar media. At the same time, based on the comparison of the growth properties of ARB, it was found that the universal chromogenic media provides more acceptable conditions for the isolation of nontuberculous mycobacteria (NTM) compared to blood agar. The comparison of the growth rate of bacteria did not reveal significant differences for fastly growing NTM. For slowly growing species, the growth rate on blood agar was lower than on chromogenic media and on the Löwenstein–Jensen media. Conclusion: Thus, the use of a universal chromogenic media during incubation makes it possible to isolate and preidentify representatives of the ARB under the conditions of standard operating procedures of the microbiological laboratory.
Keywords: Acid-resistant bacteria, agar media, egg-based media, nontuberculous mycobacteria
|How to cite this article:|
Lyamin A, Ereshchenko A, Antipov V, Kozlov A, Ismatullin D, Nikitina T. Evaluation of possibility of cultivation of acid resistant bacteria on solid egg-based and agar cultural media. Int J Mycobacteriol 2023;12:17-22
|How to cite this URL:|
Lyamin A, Ereshchenko A, Antipov V, Kozlov A, Ismatullin D, Nikitina T. Evaluation of possibility of cultivation of acid resistant bacteria on solid egg-based and agar cultural media. Int J Mycobacteriol [serial online] 2023 [cited 2023 Mar 30];12:17-22. Available from: https://www.ijmyco.org/text.asp?2023/12/1/17/371664
| Introduction|| |
Acid-resistant bacteria (ARB), as a large and heterogeneous group of prokaryotes, today attracts more and more attention from specialists in clinical and laboratory medicine. This is due to several factors, among which the dominant ones can be distinguished: an increase in the number of patients with risk factors for the development of diseases caused by this group of prokaryotes (mycobacteriosis, nocardiosis, and actinomycosis). As well as the improvement of methods for identifying bacteria that have become available to a wide range of laboratories.,, At the same time, the problem of isolating ARB from clinical material remains. At the moment, there is no unified system of procedures for the isolation of ARB, with the exception of nontuberculous mycobacteria (NTM), which in some countries are isolated and identified in laboratories of the antituberculosis departments. In other countries, there have been developed operation procedures that allow working with these bacteria at microbiological laboratories which are nonprofiled for tuberculosis.,, In this regard, the research was conducted to assess the possibility of cultivating ARB on agar media with a description of their property depending on the cultivation conditions.
| Materials and Methods|| |
The study included 402 ARB isolated from biological material (sputum, pleural fluid, bronchoalveolar lavage fluid, cerebrospinal fluid, urine, gastric wash water, wound discharge, and autopsy material) obtained from patients during tuberculosis examination. The initial inoculation of the biological material was carried out on solid culture media of Löwenstein–Jensen and Finn II after it is processed with 10% sodium phosphate trisubstituted solution. All samples of biological material included in the research were negative for the Mycobacterium tuberculosis complex.
Methods of identification of microorganisms
Identification of bacteria was performed by MALDI-ToF (Matrix-assisted laser desorption/ionization-time of flight) mass spectrometry using a Microflex LT spectrometer (Bruker Daltonik GmbH, Germany) [Figure 1]. For all samples, identification was carried out directly from tubes with solid and liquid media.
|Figure 1: ARB strains isolated from biological material; count of strains (n = 402). ARB: Acid-resistant bacteria|
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Methods for evaluation of ARB cultivation on agar media
The cultural characteristics of ARB were evaluated on solid Löwenstein–Jensen egg-based media. UriSelect universal chromogenic media (BioRad, USA) and 5% blood agar with lamb blood (HiMedia, India) were used to assess the possibility of cultivating ARB on agar media. Inoculum with an approximate bacteria concentration of 10 colony-forming unit/mL was prepared for the cultures, followed by incubation at 37°C for 40 days. The total count of colonies obtained during the entire incubation period was estimated. Nutrient media productivity criteria were estimated by counting the number of strains that gave more than 50% and more than 70% of colonies on the tested media; the timing of growth (the criteria for the beginning of growth was the appearance of the first colony on nutrient media, the assessment was carried out daily). Strains from the American Type Culture Collection (ATCC): Escherichia More Details coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as control cultures in the assessment of the growth properties of ARB on tested agarized media. The effect of various incubation temperature modes (28°C and 37°C) on the time of visible colony growth on the universal chromogenic media was also evaluated.
Statistical processing of the obtained results was carried out using StatTech version. 2.4.3 (Developer-StatTech LCC, Russia). The assessment of the microflora structure included calculation of the frequency of taxon occurrence in individual types of biological material and the relative average for each identified bacteria genus (the proportion of bacteria in the studied population of genera, depending on the biological material in consideration of other factors). The description of quantitative features which was different from the normal distribution is presented in the form of median and interquartile interval. Qualitative characteristics are presented in the form of percentages and absolute numbers. The Mann–Whitney test was quantitatively used to assess the differences between the two independent samples by the level of a certain trait. The association between the signs was statistically regarded as significant at the level of significance P < 0.05.
| Results|| |
Evaluation of culture properties of ARM culturing on media for Mycobacterium tuberculosis complex
Nowadays, the classification of NTM which is used in practical microbiology, based on their growth rate and cultural characteristics (pigmentation conditions) when cultivating on solid egg-based media. According to this classification, all NTM are divided into fastly growing and slowly growing. In turn, slowly growing NTM is divided into photochromogenic NTM (pcNTM), scotochromogenic (scNTM), and non-pcNTM (npcNTM).
To assess the possibility of using the principle of this classification for NTM, as well as other ARB, appropriate research was carried out to determine the cultural characteristics of bacteria and their comparison with species belonging. The final classification of ARB was also based on the growth rate and the presence of colony pigment. In this way, all ARBs were classified as fastly growing and slow growing and each of these subgroups were subdivided into pigmented and unpigmented.
On the Löwenstein–Jensen media, during the incubation, it was possible to obtain the growth of all strains of bacteria. The results of evaluating the growth rate and culture properties of the test isolates are presented in [Figure 2].
|Figure 2: Results of evaluation of growth rate and culture properties of ARB isolates on Löwenstein–Jensen media (n = 402). ARB: Acid-resistant bacteria|
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Slowly growing cultures were isolated in 56.5% (227/402) of all ARB. The prevalent bacteria – 37.4% (85/227) – among them were pcNTM, which form a pigment in the light. These included as classical representatives of pcNTM – Mycobacterium kansasii (48.2% [41/85] of all pcNTM) and Mycobacterium pseudoshottsii (2.4% [2–85] of all pcNTM). In addition strains of representatives of nonpigment forming species, which in our research gave a pigment – Mycobacterium avium (49.4% [42/85] of all pcNTM) was also determined. Colonies of photochromogenic M. avium had yellow pigmentation of varying degrees of severity.
Scotochromogenic NTM (scNTM) was isolated in 32.6% (74/227) of all slowly growing ARB. Isolates of this subclass were characterized by the formation of yellow pigment in the dark. The species composition of bacteria in this group was as follows: Mycobacterium gordonae (81.1% [60/74] of all scNTM), Mycobacterium szulgai (8.1% [6/74] of all sxNTM), Mycobacterium lentiflavum (4.1% [3/74] of all sxNTM) and isolated single strains of Mycobacterium scrofulaceum, Mycobacterium xenopi, Mycobacterium bohemicum, Mycobacterium interjectum, and Mycobacterium seoulense.
npcNTM characterized by the absence or weak pigmentation of colonies were isolated in 32.6% (68/227) of all slowly growing ARB. This subclass includes M. avium (63.2% [43/68] of all npcNTM) Mycobacterium chimaera/intracellulare (16.2% [11/68] of all npcNTM), Mycobacterium malmoense (11.8% [8/68] of all npcNTM), and strains of Mycobacterium marseillense (2/68), Mycobacterium celatum (1/68), and Mycobacterium gastri (1/68). Colonies of all cultures were light beige. At the same time, colonies of all strains of Mycobacterium malmoense had an atypical mucosal growth pattern for mycobacteria.
The group of fastly growing ARB was the most diverse in cultural characteristics [Figure 3].
|Figure 3: Structure of fastly growing ARB depending on cultural characteristics cultivated on Löwenstein–Jensen media. ARB: Acid-resistant bacteria|
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When cultivated some strains of streptomycetes and representatives of the genus Cellulosimicrobium (42/175), both significant deformation of the culture media (destruction and liquefaction) and the presence of scarce growth of bacteria with the formation of small colonies covered with air mycelium filaments also were noted. This fact makes it much more difficult to analyze the colony morphology of this fastly growing ARB.
Therefore, the subsequent evaluation of the cultural characteristics was carried out only for 76.0% (133/175) strains of fastly growing ARB. This group of bacteria was divided into pigment forming – 16.5% (22/133) and nonpigment forming – 83.5% (111/133) of all fastly growing ARB.
The species composition of fastly growing pigment-forming ARB is shown in [Figure 4].
|Figure 4: Species composition of fastly growing pigment forming ARB cultivated on Löwenstein–Jensen media (n = 22). ARB: Acid-resistant bacteria|
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The prevalent bacteria in this group were representatives of the genera Gordonia and Nocardia. In this case, strains of Gordonia spp. gave colonies of orange color, strains of Nocardia spp. – yellow, and NTM – yellow-orange, which is a characteristic cultural feature of M. gordonae, which is a classic representative of slowly growing NTM.
The species composition of fastly growing nonpigment-forming ARB is shown in [Figure 5].
|Figure 5: Species composition of fastly growing nonpigment forming ARB cultivated on Löwenstein–Jensen media (n = 111). ARB: Acid-resistant bacteria|
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The prevalent bacteria in this group were Mycobacterium fortuitum (35/111). At the same time, in 20% (7/35) of cases of cultivating of this representative of NTM, as well as Mycobacterium farcinogenes (1/111), mucoid-type colonies were obtained, which is an atypical feature for mycobacteria.
Evaluation of the possibility of nontuberculous mycobacteria cultivation on universal chromogenic medium and blood agar
Next, the possibility of culturing ARB on agar media was assessed. To do this, the prevalent bacteria from the NTM group (slowly growing M. avium, M. kansasii, and M. gordonae and fastly growing M. fortuitum, Mycobacterium peregrinum, Mycobacterium abscessus, and Mycobacterium septicum; a total of 255 strains) were incubated on blood agar and UriSelect universal chromogenic media. The results of the comparative evaluation of the growth properties of the studied media are presented in [Table 1].
|Table 1: Growth properties of nontuberculous mycobacteria on agar media obtained during 40 days of cultivation|
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It should be noted that in agar media, growth was not obtained in 5.9% (15/255) of the studied NTM strains (M. avium [12/255] and M. gordonae [3/255] strains). On both blood agar and universal chromogenic media, less revealed growth properties from slowly growing NTM were proper for M. kansasi strains. For fastly growing NTM, the average number of colonies grown on these media over the 40-day incubation period was generally comparable. In addition, it was noted that on the universal chromogenic media, the total number of colonies compared to blood agar is significantly higher for all researched bacteria from the NTM group, with the exception of M. peregrinum.
In addition, a comparison of the productivity criteria of the media was performed [Table 2].
|Table 2: Average values of the number of cultures in consideration of the media productivity criteria while cultivating on blood agar and universal chromogenic media|
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Significant differences in values for the productivity criteria were noted only for strains M. avium, M. fortuitum, M. abscessus, and M. septicum at a value of ≥70%. At the same time, an increase in the number of colonies was again noted for the universal chromogenic media. For the remaining strains, as well as when comparing the number of colonies at media productivity of 50%–69%, the results had no statistical differences.
The results of the comparison of the time of appearance of visible growth on agar media and Löwenstein–Jensen media are presented in [Table 3].
There were no statistically significant differences in these criteria for representatives of fastly growing NTM. On average, the appearance of colonies on both agar and Löwenstein–Jensen media was recorded on the 2nd day of incubation. For all types of slowly growing NTM, statistical differences in the average timing of growth were established depending on the culture media. The maximum duration of the average period of the beginning of colony growth on all three media was proper for M. avium (17 days on Löwenstein–Jensen media and universal chromogenic media, and 20 days on blood agar). Strains of M. kansasii and M. gordonae also demonstrated significantly higher growth rates on chromogenic media (average 12 days).
Effect of incubating temperature on nontuberculous mycobacteria growth rate on agar media
The results of evaluating the effect of different incubation temperature conditions on the timing of the appearance of visible growth of colonies of bacteria using a universal chromogenic media are presented in [Table 4].
|Table 4: Comparison of the timing of colony growth during cultivation on a universal chromogenic media at different temperature conditions|
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Thus, for almost all analyzed species of fastly growing NTM, as well as for M. gordonae, incubation at a temperature of 28°C significantly reduced the time of the appearance of visible growth on a universal chromogenic media by an average of 1 day, which generally coincides with other researches data that incubation at a temperature below 37°C is optimal for many types of NTM.
| Discussion|| |
It has been found that the use of NTM classification on culture media for MBTC according to E. Ranyon has restrictions. In our and a number of other researches,, it was found that representatives of one type of slow-growing NTM can demonstrate different cultural characteristics while incubating on classical nutrient media (belonging to the M. avium strains to both npcNTM and pcNTM). For fastly growing NTM and some representatives from other ARB groups that do not form pigment when cultivated on Löwenstein–Jensen media, it was also not possible to establish clear criteria for preliminary identification due to the similarity of the morphological properties of the colonies. At the same time, 5/22 representatives of fastly growing pigment forming NTM gave a yellow-orange colony, which is a more typical culture feature for slowly growing M. gordona. Identification of the similarity of the morphological properties of microbial colonies of fastly growing NTM with other representatives of ARB when cultivated on egg-based media can make it difficult to choose methods for their further identification.
Based on the comparison of the growth properties of ARB, it was found that the universal chromogenic media provides more acceptable conditions for the isolation of NTM compared to blood agar. The comparison of the growth rate of bacteria did not reveal significant differences for fastly growing NTM. For slowly growing species, the growth rate on blood agar was lower than on chromogenic media and on Löwenstein–Jensen media. In addition, it was found that when cultivating NTM representatives on a universal chromogenic media at 28°C, the timing of growth is reduced (with the exception of M. avium and M. kansasii, the growth of which did not differ under different temperature conditions).
The obtained data indicate the possibility of using a universal chromogenic media for cultivating most strains of NTM. At the same time, with an increase in the incubation time of samples in the laboratory incubator, in some cases, there were obvious signs of deformation of the culture media, which makes it difficult to carry out further stages of microbiological research. These changes in the chromogenic media were primarily associated with its dehydration. Potential methods for solving these problems can be the use of specialized laboratory utensils,, or the use of parafilm and/or containers with tightly fitting caps for packaging Petri dish More Detailses during incubation.
| Conclusion|| |
It has been found that the use of culture media designed to work with MBTC for the identification of ARB has a number of limitations due to the heterogeneity and the presence of similar cultural signs of representatives of this group of bacteria.
Of the 402 studied strains of ARB isolated during the examination for tuberculosis, more than 20% could be identified only using modern microbiological diagnostic methods, such as MALDI-ToF mass spectrometry. Since these methods are rarely used in the operations of tuberculosis laboratories, representatives of this group of bacteria are often not identified, and the corresponding biological material is rejected from further research as contaminated.
The research shows the possibility of cultivating ARB representatives on agar media. The use of a universal chromogenic media allows the isolation and preliminary identification of ARB under the conditions of routine operations of the microbiological laboratory.
We have not explored the possibility of cultivation of ARB on a wide list agarized media. Cultivation was evaluated only strains isolated from patients in tuberculosis screening. Species identification was limited by the MALDI-ToF method of mass spectrometry. For some genera, the study included a small number of species: Cellulosimicrobium spp., Rhodococcus spp., Tsukamurella spp., and Brevibacterium spp. It should also be noted that as a universal chromogenic medium only one of the many on the market was used. In connection with this, it should be carefully extrapolate the results obtained without additional opportunity for studies use of other chromogenic media. The lack of reliability in the growth properties of some representatives NTM on agarized media should be regarded as an important fact, determining the need for more research on more number of NTM species and more strains.
This study was approved by the Bioethics Committee at Samara State Medical University with Approval Number 196; on October 31, 2018.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Conville PS, Witebsky FG. Nocardia, rhodococcus, gordonia, actinomadura, streptomyces, and other aerobic actinomycetes. Man Clin Microbiol 2007;1:515-54.
Kendall BA, Winthrop KL. Update on the epidemiology of pulmonary nontuberculous mycobacterial infections. Semin Respir Crit Care Med 2013;34:87-94.
Rivero-Lezcano OM, González-Cortés C, Mirsaeidi M. The unexplained increase of nontuberculous mycobacteriosis. Int J Mycobacteriol 2019;8:1-6.
] [Full text]
Emikoglu Cerit SK, Tarhan GL, Ceyhan I. Investigation of species distribution of nontuberculosis mycobacteria isolated from sputum samples in patients with suspected pulmonary tuberculosis. Int J Mycobacteriol 2022;11:145-9.
Martin A, Colmant A, Verroken A, Rodriguez-Villalobos H. Laboratory diagnosis of nontuberculous mycobacteria in a Belgium hospital. Int J Mycobacteriol 2019;8:157-61.
] [Full text]
Moore JE, Millar BC. Comparison of four agar media for the enumeration of the Mycobacterium abscessus
complex. Int J Mycobacteriol 2020;9:289-92.
] [Full text]
Fang BZ, Han MX, Zhang LY, Jiao JY, Zhang XT, Zhang ZT, et al.
Nocardia aurea sp. nov., a novel actinobacterium
isolated from a karstic subterranean environment. Int J Syst Evol Microbiol 2019;69:159-64.
Intra-Laboratory Quality Control of Culture Media for Clinical Laboratory Research. Association of Specialists and Organizations of Laboratory Service Federation of Laboratory Medicine. Moscow: Federation of Laboratory Medicine; 2014.
Runyon EH. Anonymous mycobacteria in pulmonary disease. The Medical clinics of North America 1959;43:273-90.
Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C, Gordin F, et al.
An official ATS/IDSA statement: Diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 2007;175:367-416.
van Ingen J, Turenne CY, Tortoli E, Wallace RJ Jr., Brown-Elliott BA. A definition of the Mycobacterium
avium complex for taxonomical and clinical purposes, a review. Int J Syst Evol Microbiol 2018;68:3666-77.
Adelman MH, Addrizzo-Harris DJ. Management of nontuberculous mycobacterial
pulmonary disease. Curr Opin Pulm Med 2018;24:212-9.
Silveira Paro Pedro HD, Tonelli Nardi SM, Ule Belotti NC, Tegon de Freitas AC, de Souza NG, Chimara E. A laboratory-based analysis of rapidly growing mycobacteria
in Northwest Paulista, Sao Paulo, Brazil. Int J Mycobacteriol 2021;10:170-6.
Lyamin AV, Toneev IR, Kozlov AV, Ismatullin DD, Zhestkov AV, Khaliulin AV, et al
. Cup for Cultivation of Micro-Organisms with Two-Point Thread. Patent for the Invention of the Russian Federation RU 173302U1. Available from: https://patents.google.com/patent/RU173302U1/en
. [Last accessed on 2017 Aug 21].
Lyamin AV, Toneev IR, Kozlov AV, Ismatullin DD, Zhestkov AV, Kondratenko OV, et al
. Bottle for Cultivation of Microorganisms. Patent for the Invention of the Russian Federation RU 175134U1. Available from: https://patents.google.com/patent/RU175134U1/en
. [Last accessed on 2017 Nov 22].
Lyamin AV, Toneev IR, Kozlov AV, Ismatullin DD, Kondratenko OV, Ereshchenko AA, et al
. Bottle for Cultivation of Microorganisms with Removable Cover. Patent for the Invention of the Russian Federation RU 175863U1. Available from: https://patents.google.com/patent/RU175863U1/en
. [Last accessed on 2017 Dec 21].
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4]