|Year : 2020 | Volume
| Issue : 3 | Page : 289-292
Comparison of four agar media for the enumeration of the Mycobacterium abscessus complex
John Edmund Moore, Beverley Cherie Millar
Department of Bacteriology, Northern Ireland Public Health Laboratory, Nightingale (Belfast City) Hospital; School of Biomedical Sciences, Ulster University, Coleraine; School of Medicine, Dentistry and Biomedical Science, The Wellcome-Wolfson Institute for Experimental Medicine, Queen's University, Belfast, Northern Ireland, United Kingdom
|Date of Submission||18-Jun-2020|
|Date of Decision||01-Jul-2020|
|Date of Acceptance||02-Jul-2020|
|Date of Web Publication||28-Aug-2020|
John Edmund Moore
Department of Bacteriology, Northern Ireland Public Health Laboratory, Belfast City Hospital, Belfast BT9 7AD
Source of Support: None, Conflict of Interest: None
Background: Traditional culture of nontuberculous mycobacteria (NTMs) has involved egg-based formulations (Lowenstein–Jensen medium) or defined media (Middlebrook formulations), which have disadvantages of composition complexity, availability, and cost. This study quantitatively compared three non-selective, non-blood based basal agars with Columbia blood agar (CBA), to enumerate Mycobacterium abscessus complex organisms in pure culture. Methods: M. abscessus subsp. massiliense, M. abscessus subsp. bolletii, and M. abscessus subsp. abscessus were employed. Inocula of each of these were counted on three basal agar media, including (i) standard plate count agar (SPCA), (ii) tryptone soya agar (TSA), and (iii) Mueller–Hinton agar (MHA) and compared to counts on CBA. Results: All NTM isolates of all subspecies grew successfully on all four media examined. The growth was most profuse on SPCA, with a mean colony diameter of 3 mm, whereas the mean colony diameter on all other media was 1 mm. Statistically, there was no significant difference in counts when comparing CBA with SPCA or MHA (P > 0.05), whereas there was a statistically significant difference between CBA and TSA (P = 0.01). There was no statistically significant difference between SPCA and MHA (P = 0.53). Conclusion: This study indicates that SPCA and MHA are equally effective as CBA, when enumerating of M. abscessus complex organisms. Employment of TSA gave significantly lower counts than CBA (P = 0.01) and therefore should not be employed when enumerating these organisms. SPCA yielded the most profuse growth of all media examined. In addition to these advantages, given that SPCA does (i) not require blood as a medium constituent, (ii) is simple to reconstitute, (iii) is relatively cheap, and (iv) is widely available commercially, this study endorses employment of SPCA for the nonselective culture of M. abscessus complex organisms, including enumeration.
Keywords: Culture, cystic fibrosis, enumeration, microbiology, Mycobacterium abscessus complex, nontuberculous mycobacteria
|How to cite this article:|
Moore JE, Millar BC. Comparison of four agar media for the enumeration of the Mycobacterium abscessus complex. Int J Mycobacteriol 2020;9:289-92
|How to cite this URL:|
Moore JE, Millar BC. Comparison of four agar media for the enumeration of the Mycobacterium abscessus complex. Int J Mycobacteriol [serial online] 2020 [cited 2020 Oct 1];9:289-92. Available from: http://www.ijmyco.org/text.asp?2020/9/3/289/293536
| Introduction|| |
Cystic fibrosis (CF) is a genetically inherited disease, most commonly associated with European populations. It is a multiorgan disease associated with mainly the gastrointestinal tract and the lungs, where due to the molecular defect in the CF transmembrane conductance regulator protein in the respiratory tract, sticky mucus accumulates trapping environmental bacteria in the airways, mainly Pseudomonas aeruginosa and other Gram-negative bacteria, leading to a vicious cycle of infection and inflammation. Recently, the nontuberculous mycobacteria (NTM), particularly the Mycobacterium abscessus complex, consisting of M. abscessus subsp. massiliense, M. abscessus subsp. abscessus, and M. abscessus subsp. bolletii, have emerged as significant clinical pathogens within this disease. For a seminal review of the clinical significance of NTMs in CF, please refer Degiacomi et al. and Richards and Olivier.
Once isolated from CF sputum using conventional cultural techniques employing Middlebrook media and Lowenstein–Jensen agar, there are several occasions when NTM organisms require laboratory subculture in pure culture, including for enumeration purposes. Traditionally, the culture of the NTMs, as well as M. tuberculosis complex organisms, has occurred on highly specialized medium, including those containing eggs (Lowenstein–Jensen medium), as well as in a variety of formulations of Middlebrook agars and broths, containing a combination of highly defined ions, as well as Middlebrook Oleic Albumin Dextrose Catalase (OADC) enrichment components, including sodium chloride, dextrose, bovine albumin (fraction V), catalase, and oleic acid. While many of these components are beneficial in the primary isolation of mycobacteria from the clinical specimens and when mixed with other commensal flora from the specimen, they are not subsequently required for the routine culture of mycobacteria in the laboratory. Once isolated from the clinical specimen, it is necessary to manipulate the culture in the laboratory through subculturing or to further characterize the isolate with enumeration studies in investigating the effect of certain processes or procedures, such as the effect of drying. Given that the organism is now safely isolated from the clinical specimen and that such investigations are performed in pure culture, there is no need to employ agar, with a complex list of constituents. Previously, Drancourt and Raoult demonstrated that blood agar was as effective in the isolation of mycobacteria, than traditional mycobacterial agar and broth formulations.
Unlike M. tuberculosis, the M. abscessus complex are rapid growers and are adept at utilizing a diverse range of substrates and metabolites, thus are capable of utilizing many components in nonselective basal agar media, which could simplify their routine culture in the laboratory. It was, therefore, the aim of this study to compare the sensitivity and growth characteristics of members of the M. abscessus complex of three nonselective basal agars with Columbia blood agar (CBA), in order to determine the effectiveness of nonblood-based basal agar in the mundane culturing/subculturing of the M. abscessus complex.
| Methods|| |
Bacterial strains employed
Isolates (n = 5) from the three members of the M. abscessus complex were employed in this study, as detailed in [Table 1]. These included (i). M. abscessus subsp. massiliense (n = 1), (ii). M. abscessus subsp. bolletii (n = 2), and (iii). M. abscessus subsp. abscessus (n = 2). All isolates were recovered on CBA (Oxoid CM0031, Oxoid Ltd., Basingstoke, UK), supplemented with 5% (v/v) defibrinated horse blood for 5 days at 37°C, under aerobic conditions and passaged a further three times, prior to use. Their identification was confirmed by employing the Hain Genotype Mycobacterium assay (NTM-DR VER 1.0) (Hain Lifescience GmbH, Nehren, Germany). With regard to antibiotic resistance, no mutations to macrolides (MA)/aminoglycosides (AMG) were detected in isolate NTM-E. All other NTM isolates harbored erm41 resistance to MA, but no mutations to AMG.
|Table 1: Description of nontuberculous mycobacteria organisms employed in this study|
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Enumeration of Mycobacterium abscessus complex organisms
Inocula of each NTM isolate, as detailed in [Table 1], were prepared in sterile 0.1% (w/v) peptone saline diluent (Oxoid CM0733), equating to a McFarland 3.0 (9.0 × 108 colony-forming units [cfu]/ml). Enumeration of each of these prepared inocula was performed using the spread plate technique on four agars, including (i) CBA (Oxoid CM0031) (CBA), supplemented with 5% (v/v) defibrinated horse blood, (ii) tryptone soya agar (Oxoid CM0131) (TSA), (iii) standard plate count agar (Oxoid CM0463) (SPCA), and (iv) Mueller–Hinton agar (Oxoid CM0337) (MHA). The constituents of each agar are detailed in [Table 2]. All agars were reconstituted in accordance with the manufacturer's instructions. Serial dilutions (to 10−6) of each isolate were performed in sterile 0.1% (w/v) peptone saline diluent (Oxoid CM0733) (9 ml). Dilutions, 10−4, 10−5, and 10−6, were chosen to enumerate, and 100 μl of each dilution was inoculated onto each agar medium in triplicate. Inoculated plates were incubated aerobically at 37°C for 1 week, prior to counting. Following incubation, the serial dilution yielding approximately 100 cfu/plate was selected for enumerating. The mean of triplicate plate counts was determined, and the mean counts were recorded for each isolate on each agar medium.
|Table 2: Description of four agar media and their constituents employed in this study|
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Statistical analysis was performed employing the Student's t-test, and probability values <5% (<0.05) were considered statistically significant. As this was a service development study, not requiring the involvement of patients nor animals, it did not require ethical approval.
| Results|| |
All NTM isolates of all subspecies grew successfully on all four media examined [Figure 1]. Growth was most profuse on SPCA, with a mean colony diameter of 3 mm, whereas the mean colony diameter on all media was 1 mm. Comparison of enumerative counts is shown in [Figure 2]. Error bars represent the standard error of the mean. Statistically, there was no statistically significant difference in counts when comparing CBA with SPCA or MHA (P > 0.05), whereas there was a statistically significant difference in the counts between CBA and TSA (P = 0.01). There was no statistically significant difference in the counts between SPCA and MHA (P = 0.53).
|Figure 1: Comparison of colonial growth (count + colony size) of Mycobacterium abscessus subsp. bolletii on four agar media|
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|Figure 2: Mean enumerative counts of Mycobacterium abscessus complex organisms on four agar media|
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| Discussion|| |
Historically, several reports have endorsed employment of nonclassical medium (Lowenstein–Jensen and Middlebrook) for the culture of Mycobacterium spp.,,, More recently, Drancourt and Raoult have shown that M. abscessus grew equally well on blood agar, compared to growth on Middlebrook 7H10 agar, as did all other forty reference NTM isolates, with the exception of M. ulcerans. While this is a welcome departure from the requirement to grew M. abscessus only on traditional mycobacterial media, it is still problematic, particularly due to the requirement for blood. The preparation of agar plates with blood is complicated due to the availability of blood, its shelf life, and the associated cost of blood. Therefore, we wished to validate if blood agar could be substituted with other nonselective and nonblood-based basal agars, for the mundane culture of M. abscessus complex organisms in pure culture.
The M. abscessus complex of organisms remains a challenge in terms of microbiological cultural manipulation, environmental persistence in the clinical setting, as well as clinical treatment/outcomes., This study indicates that SPCA and MHA are equally effective as CBA, when enumerating M. abscessus complex organisms. Employment of TSA gave significantly lower counts than CBA (P = 0.01) and therefore should not be employed when enumerating these organisms. SPCA yielded the most profuse growth of all media examined (mean colony diameter = 3 mm). In addition to these advantages, given that SPCA does (i) not require blood as a medium constituent, (ii) is simple to reconstitute, (iii) is relatively cheap, and (iv) is widely available commercially, this study endorses employment of this agar medium for the nonselective culture of the M. abscessus complex organisms, including enumeration.
| Conclusions|| |
Standard Plate Count Agar (SPCA) was shown to be valuable non-selective agar medium for the culture of the M. abscessus complex in pure culture.
The authors wish to thank Mr. Alan Murphy, Northern Ireland Public Health Laboratory, Nightingale Hospital, Belfast, for his help in media preparation. In addition, the authors wish to thank Mr. Mark Smyth, Northern Ireland Mycobacterial Reference Laboratory, Royal Victoria Hospital, Belfast, Northern Ireland, for the preparation of isolates employed in this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]