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Year : 2015  |  Volume : 4  |  Issue : 1  |  Page : 25-30

Microaerobic growth and anaerobic survival of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium scrofulaceum


Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0406, USA

Date of Web Publication21-Feb-2017

Correspondence Address:
Joseph O Falkinham III
Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0406
USA
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Source of Support: None, Conflict of Interest: None


DOI: 10.1016/j.ijmyco.2014.11.066

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  Abstract 


Representative strains of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium scrofulaceum (MAIS) grew at equal rates in laboratory medium at 21% (air) and 12% oxygen. Growth in 6% oxygen proceeded at a 1.4–1.8-fold lower rate. Colony formation was the same at 21% (air) and 6% oxygen. The MAIS strains survived rapid shifts from aerobic to anaerobic conditions as measured by two experimental approaches (Falkinham (1996) [1]). MAIS cells grown aerobically to log phase in broth were diluted, spread on agar medium, and incubated anaerobically for up to 20 days at 37 °C. Although no colonies formed anaerobically, upon transfer to aerobic conditions, greater than 25% of the colony forming units (CFU) survived after 20 days of anaerobic incubation (Prince et al. (1989) [2]). MAIS cells grown in broth aerobically to log phase were sealed and vigorous agitation led to oxygen depletion (Wayne model). After 12 days anaerobic incubation, M. avium and M. scrofulaceum survival were high (>50%), while M. intracellulare survival was lower (22%). M. avium cells shifted to anaerobiosis in broth had increased levels of glycine dehydrogenase and isocitrate lyase. Growth of MAIS strains at low oxygen levels and their survival following a rapid shift to anaerobiosis is consistent with their presence in environments with fluctuating oxygen levels.

Keywords: Mycobacterium, Oxygen, Microaerobic, Anaerobiosis


How to cite this article:
Lewis AH, Falkinham III JO. Microaerobic growth and anaerobic survival of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium scrofulaceum. Int J Mycobacteriol 2015;4:25-30

How to cite this URL:
Lewis AH, Falkinham III JO. Microaerobic growth and anaerobic survival of Mycobacterium avium, Mycobacterium intracellulare and Mycobacterium scrofulaceum. Int J Mycobacteriol [serial online] 2015 [cited 2019 Dec 11];4:25-30. Available from: http://www.ijmyco.org/text.asp?2015/4/1/25/200585




  Introduction Top


Members of the species Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (MAIS) are opportunistic pathogens of humans, animals, and birds whose source of infection is the environment [1]. Particularly important are M. avium and M. intracellulare (the M. avium complex, MAC), which cause pulmonary disease in older, slender women [2], cervical lymphadenitis in children [3], and bacteremia in immunodeficient individuals and those with AIDS [4].

Oxygen levels appear to be determinants of MAIS numbers in different habitats. High numbers of MAIS were correlated with low oxygen levels in acidic, brown water coastal swamps [5] and estuaries [6], and waters and soils of the southeastern United States [7]. High numbers of M. avium were found in swimming pools and whirlpool baths of low oxidation–reduction potential [8]. Biofilms in drinking water distribution systems, which have anaerobic regions [9], yielded high numbers (600 colony forming units [CFU] per cm2) of M. intracellulare [10]. Finally, M. avium was shown to grow equally well in human macrophages at low and high oxygen levels [11].

As rapid shifts in the level of oxygen and prolonged periods of anaerobiosis can occur in the habitats occupied by MAIS (e.g., plumbing biofilms), they ought to be able to survive anaerobiosis and shifts from aerobic to anaerobic conditions. Rapid depletion of oxygen leads to dramatic loss of colony-forming ability in cultures of Mycobacterium tuberculosis and Mycobacterium smegmatis, whereas slow oxygen depletion permits survival [12],[13],[14]. One adaptation of M. tuberculosis to low oxygen is increased levels of both isocitrate lyase and glycine dehydrogenase activities, perhaps to provide NAD for metabolic needs [12].

Based on those observations, the present study sought to test the hypothesis that members of MAIS are able to tolerate changes in oxygen concentration; perhaps even to survive rapid shifts to anaerobiosis that might be encountered in the environment.


  Materials and methods Top


Mycobacterial strains

The following strains were used to study the effects of oxygen concentration on mycobacterial growth and survival: transparent colonial variants of M. avium strains A5, 5502, 5002, 1508, 1060 and 101; M. intracellulare strains TMC 1406T, TMC 1405, and TMC 1403; and M. scrofulaceum strains TMC 1323T, TMC 1312, and TMC 1306.

Growth of mycobacteria

Stock cultures were maintained on Middlebrook M7H10 agar slants containing 0.5% (vol/vol) glycerol and 10% (vol/vol) oleic acid-albumin (OAA) enrichment. Growth from the slants was streaked to M7H10 agar plates to confirm purity. Single isolated colonies were used to inoculate 2 ml of Middlebrook M7H9 broth containing 0.2% (vol/vol) glycerol and 10% (vol/vol) OAA (M7H9) or Nitrogen Test (NT) medium [15]. Cultures were incubated for 7 days at 37 °C. To prepare cultures of the stock cultures for experiments (starter cultures), 1 ml of the 2 ml M7H9 or NT cultures was added to 10 ml of M7H9 broth of NT medium and incubated for 7 days at 37 °C. The cultures were refrigerated and could be used for up to two months.

Adjustment of oxygen concentration

For experiments requiring adjustment of the oxygen concentration within flasks, the vessels were evacuated and pre-purified nitrogen gas–specified to contain less than three parts per million of oxygen was used to replace the air. The volume of air evacuated from the container, in inches of Hg, based on normal atmospheric oxygen content of 21%, was calculated by: [(21−desired oxygen level)/21]×(barometric pressure). To achieve anaerobic conditions in flasks, they were evacuated to a pressure of 25inches of Hg and then refilled with nitrogen to 0inches of Hg. This procedure was repeated five times. To achieve anaerobic conditions inside anaerobe jars containing palladium catalysts, the jars were evacuated to 25inches of Hg three times, followed by replacement with nitrogen gas twice and, finally, hydrogen gas. A methylene blue anaerobic indicator strip (Becton Dickinson Microbiology Systems, Sparks, Maryland) was included in each jar to verify that an anaerobic atmosphere was created, turning from blue to white.

Anaerobic survival on agar

Starter cultures were used to inoculate M7H9 broth in sidearm nephelometer flasks. Cells were grown to log phase with aeration (rotation at 60rpm) at 37 °C. Samples were collected, dilutions prepared in sterile buffered saline gelatin (BSG; per liter, 0.1g gelatin, 8.5g NaCl, 0.3g KH2PO4, and 0.6g Na2HPO4), and 0.1 ml was spread onto M7H10 agar (stored anaerobically). Plates were incubated at 37 °C under anaerobic conditions. Every two days, 3 plates were removed and incubated aerobically for 12 days at 37 °C and colonies counted.

Anaerobic survival in broth (Wayne model)

Starter cultures were used to inoculate M7H9 broth in sidearm nephelometer flasks and cells were grown to log phase with aeration (rotation at 60rpm) at 37 °C. Cells were diluted in M7H9 to an absorbance (580 nm) of 0.008 and 11 ml were transferred to 16×125 mm screw-capped tubes. The tubes had a capacity of 16.5 ml, resulting in a 0.5 headspace ratio, as described [11]. Tubes were then tightly or loosely capped. Methylene blue was added to one tightly capped tube at a final concentration of 1.5μ/ml to detect anaerobiosis. The tubes were incubated at 37 °C with vigorous agitation at 250rpm to produce rapid oxygen depletion in the tightly capped tubes [12]. Tubes were removed every 2 days, diluted in BSG, and 0.1 ml spread on M7H10 agar in triplicate. Colonies were counted after 12 days incubation at 37 °C.

Growth, harvest, and breakage of M. avium strain A5 for enzyme activity measurements

Cells for enzyme activities were grown in paired cultures of 500 ml in M7H9 broth at 37 °C at 120rpm in air. Inoculum volumes were one-tenth the culture medium volume. After 3 days incubation, one of the pair was transferred to anaerobic conditions and incubation continued without agitation for an additional 7 days at 37 °C. Incubation under aerobic conditions for 7 days at 37 °C (120rpm) was continued for the other member of the pair. By limiting growth to a total of 10 days, cells remained in the exponential phase of growth and did not enter the stationary phase. The cells were harvested, broken by sonication, and the crude and soluble fractions isolated as described [16]. The crude extract was the source of enzyme for catalase [17], and the soluble fraction was used as the source of enzyme for glycine dehydrogenase [18] and isocitrate lyase [19]. Protein in each fraction was measured as described [20].


  Results Top


Microaerobic growth and colony formation

All three representative MAIS strains grew at 21% (air), 12% and 6% oxygen ([Table 1] ). At all three oxygen levels, biphasic exponential growth was observed as illustrated for M. avium strain A5 ([Figure 1]) as has been described for other M. avium strains [21]. Generation times for the second exponential phase were equal for 21% and 12% oxygen, but were decreased 1.4- to 2.8-fold in 6% oxygen ([Table 1], [Figure 1]). MAIS strains failed to grow in M7H9 broth under anaerobic conditions ([Figure 1]). Colony counts of the aerobic cultures were the same whether incubated in 21% (air) or 12% and 6% oxygen ([Table 2]).
Table 1: MAIS growth at 21%, 12% and 6% oxygen.

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Table 2: Efficiency of colony formation under aerobic and microaerobic conditions.

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Figure 1: Biphasic growth of M. avium strain A5 in M7H9 broth at 21%, 12% and 6% O2.

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Anaerobic survival on agar

To assess MAIS anaerobic survival on agar medium surfaces, cells from an aerobic culture were diluted and spread on M7H10 agar, incubated anaerobically at 37 °C, and at intervals of 2–20 days of anaerobic incubation at 37 °C; 3 plates were removed and incubated aerobically for 12 days at 37 °C and colonies counted. Surviving colony counts were ≥100% for the initial 8 days and only reached 25% after 20 days of anaerobic incubation ([Table 3], [Figure 2]).
Table 3: Aerobically grown MAIS cells on solid media survive a rapid shift to anaerobiosis.

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Figure 2: Aerobically grown M. avium A5 cells, spread onto pre-reduced agar plates, survive a rapid shift to anaerobiosis.

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Anaerobic survival in broth

Following growth under aerobic conditions, cultures were sealed, incubated with vigorous agitation to deplete oxygen, and CFU/ml measured over time. M. avium and M. scrofulaceum survival was >50% at 12 days ([Figure 3]), while that for M. intracellulare was near 20% ([Table 4]). Survival was influenced by medium composition, as cells of M. avium strain A5 rapidly lost the ability to form colonies in the NT minimal medium ([Table 5]). Compared to M7H9 broth, the NT medium lacks l-glutamic acid.
Table 4: MAIS survive rapid self-depletion of oxygen in sealed broth cultures.

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Table 5: M. avium strain A5 anaerobic survival influenced by medium composition.

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Figure 3: M. avium A5 survival in sealed broth cultures following rapid self-depletion of oxygen.

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Catalase, glycine dehydrogenase, and isocitrate lyase activities

The enzyme activities of M. avium strain A5 cells were measured in cells collected after 1week aerobic growth and a further 2 weeks of anaerobic incubation. Whereas the catalase activity in the crude extract of the shifted, anaerobic culture was half that in the aerobic cells, the activities of both glycine dehydrogenase and isocitrate lyase were 1.6- and 2.5-fold higher (respectively) in cells from the shifted, anaerobic cultures ([Table 6]).
Table 6: Enzyme activities of aerobic and anaerobic shifted cells of M. avium strain A5.

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  Discussion Top


The results document the ability of M. avium, M. intracellulare, and M. scrofulaceum to grow under microaerobic conditions and to survive a rapid shift to anaerobiosis. Such evidence is consistent with the recovery of high numbers of MAIS from habitats of reduced oxygen [5],[6],[7] and those of reduced redox potential [8]. Further, the ability of MAIS to grow at reduced oxygen provides an explanation for the growth of M. avium in biofilms [22],[23]. Biofilms have anaerobic zones [9], yet M. avium is capable of growth in biofilms that model those found in drinking water distribution systems [22].

Unlike cells of M. tuberculosis [12] and M. smegmatis [13],[14], cells of M. avium, M. intracellulare and M. scrofulaceum were relatively resistant to rapid shifts to anaerobiosis ([Table 3] and [Table 4], [Figure 2] and [Figure 3]). Measurements of survival in broth and survival on agar medium yielded similar results; namely, greater than 25% of cells survived after 10–20 days ([Table 3] and [Table 4]). Those survival values are substantially higher than the 0.1% survival of M. tuberculosis after 10 days [11] and the 5%–10% survival of M. smegmatis 5 days following the shift to anaerobiosis [13],[14]. Relatively high survival of cells of these three species of opportunistic pathogens is consistent with their ecology. MAIS cells need to be able to survive rapid shifts in oxygen levels; otherwise they would be rapidly killed upon transfer to anaerobic environments.

To attempt to gain an understanding of the basis for the increased survival of M. avium upon transfer to anaerobic conditions compared with M. tuberculosis and M. smegmatis, the activities of a number of enzymes was measured. M. tuberculosis cells slowly adapted to microaerobic conditions that had increased glycine dehydrogenase and isocitrate lyase activities [12], while glycine dehydrogenase activity was elevated in cells of M. smegmatis [14]. Both enzyme activities were moderately increased in cells of M. avium shifted to anaerobiosis ([Table 6]). Although the increases for M. avium were not as high as measured in M. tuberculosis [12], the cells of M. avium had been rapidly shifted to anaerobic conditions, not slowly adapted. Thus, those increases occurred rapidly and under conditions of rapid onset of anaerobiosis. A comparison of the activities of glycine dehydrogenase and isocitrate lyase indicates that the level of their activities in aerobically grown M. tuberculosis and M. smegmatis cells [24] was approximately 10-fold lower than the activities of M. avium ([Table 6]). Perhaps during the rapid shift to anaerobiosis, the relatively higher activities of glycine dehydrogenase and isocitrate lyase in M. avium cells is sufficient to ensure survival by maintaining a suitable redox potential [25]. The loss of colony-forming ability of M. avium on the glutamate-free NT-minimal agar medium may be related to a need for glutamate to provide intermediates for functioning of the TCA cycle for glyoxylate metabolism [12].

In spite of demonstrating that microaerobic growth and survival following a rapid shift to anaerobiosis is a characteristic of the MAIS group, the results of the enzyme assays do not illuminate the mechanism of this unique behavior amongst members of the genus Mycobacterium. Like other Mycobacterium species tested, exposure to anaerobic conditions results in enzymatic adaptation in M. avium. Quite possibly, the presence of a functional oxyR gene in M. avium, unlike the nonfunctional oxyR gene in M. tuberculosis [26], is involved in the survival of M. avium after anaerobiosis. The objective of these continuing investigations will be to identify those genes and characteristics of M. avium resulting in higher levels of survival following a rapid shift to anaerobiosis.


  Conflict of interest Top


None declared.


  Acknowledgments Top


The authors acknowledge the technical assistance of Ms. Myra Williams on the enzyme assays. Financial support was provided by Applied Sciences and Microbiology.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]


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