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ORIGINAL ARTICLE
Year : 2023 | Volume
: 12
| Issue : 1 | Page : 49-54
Application of chromogenic media for preliminary identification of acid-resistant bacteria
Artem Viktorovich Lyamin1, Alena Anatolyevna Ereshchenko2, Oksana Anatolyevna Gusyakova3, Vladimir Alexandrovich Antipov3, Andrey Vladimirovich Kozlov2, Danir Damirovich Ismatullin1
1 Research and Educational Professional Center for Genetic and Laboratory Technologies, Samara State Medical University, 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
Correspondence Address:
Alena Anatolyevna Ereshchenko
443099, Chapayevskaya str., 89, Samara
Russia
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ijmy.ijmy_6_23
Background: The variety of morphological and cultural characteristics of acid-resistant bacteria (ARB) makes it possible to use microscopy and estimate the growth rate and pigment formation when cultivating on solid egg media for preliminary identification only as additional indicative methods. It is necessary to develop new approaches for the cultivation and primary identification of ARB isolated from the biological material. It will allow to obtain data on the prevalence, structure, epidemiological, and clinical features of infectious processes caused by opportunistic ARB. Methods: Three hundred and sixty strains of ARB were isolated from the various biological materials obtained from the patients during the examination for tuberculosis. All biological material samples were negative on Mycobacteria tuberculosis complex. Species identification of all bacteria was performed by matrix-assisted lazer desorption/ion-ization time-of-flight mass spectrometry. The cultural characteristics of ARB were evaluated on a universal chromogenic media. As a selective additive, a mixture of bacitracin and polymyxin sulfate which had no effect on ARB was tested to suppress concomitant Gram-positive and Gram-negative microflora. Results: Cultural characteristics were identified and described for all tested representatives of fast-growing nontuberculous mycobacteria (NTM), as well as for all types of nocardia, gordonia, and streptomycetes. Representatives of other genera of ARB on a universal chromogenic media gave meager growth or did not show it at all. When inoculated on a universal chromogenic media with a selective addition, 100% of the strains from the ARB group showed abundant or moderate growth. Incubation time for fast-growing species was up to 7 days; for slow-growing species, it was up to 28 days. Concomitant control strains of Gram-positive and Gram-negative bacteria on universal chromogenic media with selective growth additive did not show the growth. Conclusions: The use of a universal chromogenic media allows to preliminarily identify NTM and other ARB by cultural characteristics. The addition of bacitracin and polymyxin sulfate does not reduce the growth properties of ARB, which can be used when working with both biological materials and for the isolation of pure ARB cultures from mixtures with other bacteria.
Keywords: Acid-resistant bacteria, chromogenic media, nontuberculous mycobacteria
In medical microbiology, the initial identification of acid-resistant bacteria (ARB) is carried out using two of the simplest and most accessible methods: microscopic and microbiological. The acid resistance characteristic of most ARB makes it possible to use in routine practice available methods for identifying this feature. The determination of this characteristic of bacteria in the biological material significantly narrows the diagnostic search.[1],[2] Some authors in the scientific and educational literature provide information on the possibility of using a microscopic examination method for the preliminary species identification of ARB, although in practice, this technique is ineffective. Different morphology types of ARB cells, such as rods, branching rods, rods with thickening at the ends, cocci, as well as certain signs based on the mutual arrangement of cells in swabs, are described for many types of nontuberculous mycobacteria (NTM) and some other types of ARB and are not strictly specific.[3] In practice, the microscopic test method, having a high specificity (89%–100%), turned out to be an important but only an approximate method for determining ARB in the biological material.[4] Especially considering the accumulated data on acid resistance as a nonconstant, variable feature.[5]
Many laboratories use the principle of group identification of NTM according to E. Ranyon and J. M. Range, based on their growth rate and cultural characteristics, followed by their classification into fast- and slow-growing and forming and nonforming pigment.[4] However, this principle has a number of serious limitations, especially considering the accumulated data on the ability of slow-growing NTM to demonstrate various pigment-developing properties.[6],[7] The result is an erroneous choice of methods for identifying the obtained cultures and determining the sensitivity of these bacteria to antibacterial medications. An increase in the growth rate of NTM and ARB during the examination for tuberculosis is often regarded as contamination with foreign microflora and their subsequent rejection,[8] which leads to a low level of diagnosis of mycobacteriosis. Thus, it is necessary to analyze existing approaches and develop new ones for the cultivation and primary identification of ARB isolated from the biological material, which will provide data on the prevalence, structure, epidemiological, and clinical features of infectious processes caused by opportunistic ARB.
MethodsSampling
The studied strains of bacteria were isolated from the biological material obtained from patients during the tuberculosis examination. The initial inoculation of the biological material was made on dense culture media Löwenstein-Jensen and Finn-II. All samples of biological material included in the research were negative for mycobacterium of the tuberculosis complex. Species identification of bacteria was carried out by matrix-assisted lazer desorption/ion-ization time-of-flight (MALDI-ToF) mass spectrometry using a Microflex LT instrument (Bruker Daltonik GmbH, Germany). Thus, 360 strains of ARB were included in the research. Based on cultural characteristics when cultivating on Löwenstein-Jensen media, all ARBs were divided on photochromogenic NTM-43 strains, scotochromogenic NTM-74 strains, nonphotochromogenic NTM-110 strains, fast-growing NTM, and other bacteria from the ARB group-133 strains.
The possibility of preliminary identification of ARB on chromogenic media was evaluated using UriSelect universal chromogenic media (BioRad, USA). Inoculum with an approximate bacterial concentration of 1000 colony-forming unit (CFU)/1 ml was prepared for the cultivation, followed by inoculation at 28°C for 40 days.
The possibility of using a universal chromogenic media with a selective additive for culturing ARB was evaluated using UriSelect universal chromogenic media (BioRad, USA). As a selective additive to the universal chromogenic media, a mixture of two antibacterial preparations: Bacitracin and polymyxin sulfate, which have no effect on ARB, was used to suppress the concomitant Gram-positive and Gram-negative microflora. Concentrations of antibacterial drugs were selected on the basis of a similar selective additive oxidative-fermentative polymyxin B-bacitracin-lactose in the isolation of Burkholderia cepacia complex (HiMedia, India). The research included strains that gave growth on a universal chromogenic media in the previous stage of the research (fast-growing and slow-growing types of NTM, gordonia, nocardia, and streptomycetes). Testing of possible growth suppression using a selective additive was carried out by the presence of culture growth during inoculation on the agar surface of a suspension containing 1000–10,000 CFU/ml at incubation at a temperature of 28°C for 40 days. Strains from the American Type Culture Collection (ATCC): Escherichia More Details coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 were used as control cultures in assessing the growth properties of ARB.
ResultsAssessment of the possibility of preliminary identification of ARB when using a universal chromogenic media. For all photochromogenic NTM cultures, the following results were obtained: On a universal chromogenic media, dysgonic growth was detected, characterized by the appearance of small transparent or slightly pigmented colonies, 1-2 mm in size, easily removable from the surface of the media. The timing for the appearance of signs of visible growth was within 1−2 weeks, when transferred to a chromogenic media, followed by incubation at a temperature of 28°C. This growth was obtained for the 41/41 strains of Mycobacterium kansasii and 2/2 strains of Mycobacterium pseudoshottsii.
Scotochromogenic NTM cultures were characterized by the growth of dot-shaped, yellow, or orange-colored colonies 1−2 mm in size, which appeared on a universal chromogenic media during 1−2 weeks of cultivation at a temperature of 28°C. The described results of evaluation of cultural characteristics were obtained for 57/60 strains of Mycobacterium gordonae [Figure 1], 3/3 strains of Mycobacterium lentiflavum and 1/1 of Mycobacterium scrofulaceum, Mycobacterium bohemicum, Mycobacterium interjectum, and Mycobacterium seoulense. For 6/6 strains of Mycobacterium szulgai, 1/1 strains of Mycobacterium xenopi, and 3/60 strains of M. gordonae, growth under the described conditions was not obtained.
Figure 1: Colonies of Mycobacterium gordonae on a universal chromogenic media (colony size 1–2 mm)It was decided to include all the strains of Mycobacterium avium in the group of non-photochromogenic NTM, regardless of pigment formation on Löwenstein-Jensen media. Most of the strains, when transferred to a universal chromogenic media, produced small yellowish or cream-colored colonies 1–2 mm in size, with the appearance of visible growth for 7–28 days when cultivated at a temperature of 28°C [Figure 2].
Figure 2: Colonies of Mycobacterium avium on a universal chromogenic media (colony size 0.5–1 mm)It should be noted that the yellowish color of the colony was given by strains of Mycobacterium avium, which formed a pigment when cultured on the egg media. The same type of colony was obtained for Mycobacterium malmoense and Mycobacterium marseillense strains. Strains of Mycobacterium avium gave classic colony variant, strains of Mycobacterium chimaera/intracellulare, Mycobacterium celatum, and Mycobacterium gastri gave colonies of light beige color. In this group of bacteria, stains were also identified that did not give growth under the conditions. Of the 110 strains, no visible growth was obtained for the 12 strains of Mycobacterium avium with the classical colony variant and the 3 strains of Mycobacterium chimaera/intracellulare.
Fast-growing NTM and other bacteria from the ARB group gave colonies of 2−5 mm in size stained in various shades of blue on a universal chromogenic media [Figure 3], [Figure 4], [Figure 5].
Figure 3: Isolated colonies of Mycobacterium fortuitum on a universal chromogenic media (colony size 4–5 mm)
Figure 4: Colonies of Mycobacterium septicum on a universal chromogenic media (colony size 3–4 mm)
Figure 5: Colonies of Mycobacterium peregrinum on a universal chromogenic media (colony size up to 5 mm)In some cases, representatives of this ARB group formed complex consortia of large sizes [Figure 6].
Figure 6: Colonies of Mycobacterium fortuitum in the form of consortia on a universal chromogenic media (consortia size 9–12 mm)The growth of colonies was noted in the first 2−3 days of cultivation; however, the characteristic staining of the colony was acquired by the 15th to 17th days.
Both fast-growing NTM and other species from the ARB group gave equally colored colonies on the media. When analyzing the cultural characteristics, a feature characteristic only of nocardias and streptomycetes was revealed, which, unlike NTM, gave colonies grown into the media. Streptomycetes additionally acquired whitish plaque on the surface of the colonies on the 15th to 17th days of cultivation. Gordonias were characterized by light-colored wet S-colonies with the painted media under them. The figures show different colony variants of individual NTM species, nocardia [Figure 7], gordonium [Figure 8], and streptomycete [Figure 9] and [Figure 10].
Figure 7: Colonies of Nocardia carnea 
a universal chromogenic media (colony size 1–2 mm)
Figure 8: Colonies of Gordonia sputi universal chromogenic media (colony size 2–3 mm)
Figure 9: Colonies of Streptomyces violaceoruber on a universal chromogenic media (colony size up to 5 mm)
Figure 10: Colonies of Streptomyces phaeochromogenes on a universal chromogenic media (colony size 2–3 mm)The described properties were identified in all representatives of fast-growing NTM, as well as for all types of nocardia, gordonia, and streptomycetes. Representatives of other genera of ARB on a universal chromogenic media gave meager growth or did not show it at all.
Evaluation of the use of a universal chromogenic media with a selective additive for acid-resistant bacteria culture
The results of strain growth evaluation are presented in [Table 1].
Table 1: Evaluation of the growth rate of certain acid-resistant bacteria on a selective universal chromogenic mediaIncubation time for fast-growing species was up to 7 days, for slow-growing species up to 28 days. As a result, 100% of the strains from the ARB group, included in the second part of the research, gave abundant or moderate growth when inoculated on a universal chromogenic media with a selective additive.
For the control strains, E. coli ATCC 25922, P. aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25,923, no growth was obtained on the universal chromogenic media with selective additive.
DiscussionThe use of classical methods of cultivation on solid egg media using the subsequent classification of NTM by cultural characteristics can lead to an erroneous determination of NTM to an incorrect group of mycobacteria, which, in turn, can negatively affect both the choice of methods not only for the species identification of the resulting cultures; but also for determining the sensitivity of bacteria to antibacterial drugs. Assessment of the growth rate and pigmentation of ARB representatives for their preliminary identification can be used not only when working with classical media that are used in the laboratories of the anti-tuberculosis service but also when using chromogenic media.[9],[10]
In our work, the use of a universal chromogenic media for the preliminary identification of ARB was acceptable for 100.0% of photochromogenic NTM cultures, 86.5% (64/74) of scotochromogenic NTM cultures, and 86.4% (95/110) of nephotochromogenic NTM species. Thus, the universal chromogenic media used in the research can be used to pre-identify NTM groups, as well as to differentiate fast-growing NTM from other members of the ARB group. However, universal chromogenic media are optimal for the growth of a significant number of bacteria, which can be important when used for primary inoculation of biological material, since even fast-growing microorganisms have a certain growth delay when cultured on solid nutrient media. The use of a selective additive to the chromogenic media inhibits the growth of the “classical” microflora characteristic of non-sterile human body loci.
The possibility of isolating fast-growing NTM on solid selective media for Burkholderia cepacia complex with antibacterial additives is known with regard to the isolation of Mycobacterium abscessus from material from cystic fibrosis patients, and therefore, this approach needs to be analyzed in more detail with respect to other culture media containing similar additives.[11]
ConclusionsThe use of a universal chromogenic media allows preliminary identification of NTM and other ARB by cultural properties and the influence of the selective addition of bacitracin and polymyxin sulfate does not reduce the growth properties of ARB, which can be used when working with both biological material and for the isolation of pure ARB cultures from mixtures with other microorganisms.
Limitation of study
We have not studied the possibility of isolating ARB on a universal chromogenic media without a selective additive or with a selective additive directly from the biological material from patients. Growth was assessed on the analyzed media of previously isolated ARB strains. Species identification was limited by the MALDI-ToF mass spectrometry method. For some genera, the research included a small number of species. It should also be noted that only one universal chromogenic media of the many available for sale has been used. Consequently, it should be carefully extrapolated the results obtained without additional studies of the use of other chromogenic media. Not all strains of bacteria included in the research gave visible growth on the universal media used, which must be considered in further research in this direction.
Ethics statement
This research was approved by the Bioethics Committee Samara State Medical University with the Approval Number 196; October 31, 2018.
Patient declaration of consent statement
All the patients signed informed consent to participate in the research.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References
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