Formation and resuscitation of ‘non-culturable’ cells of Rhodococcus rhodochrous and Mycobacterium tuberculosis in prolonged stationary phase

Abstract

After growth of Rhodococcus rhodochrous in Sauton's medium, and further incubation for about 60 h in stationary phase, there was a transient (up to 5 log) decrease in the c.f.u. count, whereas the total count remained similar to its initial value. At the point of minimal viability, the most probable number (MPN) count was 10 times greater than the c.f.u. count. This difference was further magnified by 3-4 logs (giving values close to the total count) by incorporating supernatant taken from growing cultures. A small protein similar to Rpf (resuscitation-promoting factor of Micrococcus luteus) appeared to be responsible for some of the activity in the culture supernatant. The formation of ' non-culturable ' cells of the ' Academia ' strain of Mycobacterium tuberculosis was similarly observed following growth in Sauton's medium containing Tween 80 in sealed culture vessels, and further incubation for an extended stationary phase. This resulted in the formation, 4-5 months postinoculation, of a homogeneous population of ostensibly ' non-culturable ' cells (zero c.f.u.). Remarkably, the MPN count for these cultures was 10 5 organisms ml N1 , and this value was further increased by one log using supernatant from an actively growing culture. Populations of ' non-culturable ' cells of Mycobacterium tuberculosis were also obtained by the filtration of ' clumpy ' cultures, which were grown in the absence of Tween 80. These small cells could only be grown in liquid medium (MPN) and their viability was enhanced by the addition of culture supernatant or Rpf. The ' non-culturable ' cells that accumulated during prolonged stationary phase in both the R. rhodochrous and the Mycobacterium tuberculosis cultures were small ovoid and coccoid forms with an intact permeability barrier, but with undetectable respiratory activity. The authors consider these non-culturable bacteria to be dormant. The observed activity of culture supernatants and Rpf with ' non-culturable ' bacterial suspensions invites the speculation that one, or more, of the cognate Mycobacterium tuberculosis Rpf-like molecule(s) could be involved in mechanisms of latency and reactivation of tuberculosis in vivo.

Formation of ‘non-culturable’ cells of Mycobacterium smegmatis in stationary phase in response to growth under suboptimal conditions and their Rpf-mediated resuscitation

“…We have shown previously that a variety of different cultivation regimes may be employed to induce 'nonculturability' of various actinobacteria during the stationary phase of growth in batch culture (Mukamolova et al, 1995;Shleeva et al, 2002). In the present study, using a new model organism, Myc.…”

“…smegmatis cells grown in mHdeB medium and taken from the period of 'non-culturability' was performed as described previously for Mcc. luteus and R. rhodochrous in Sauton's medium supplemented with 0?05 % yeast extract using a MPN protocol in 48-well plates (Mukamolova et al, 1998a;Shleeva et al, 2002). The best resuscitation was obtained when the NC cells were incubated in the presence of supernatant (50-100 %) taken from a culture of viable cells grown in Sauton's medium for 26 h (Fig.…”

“…luteus (Mukamolova et al, 1998a) and R. rhodochrous (Shleeva et al, 2002), rather strictly defined culture conditions must be maintained to establish a rapid transition of significant numbers of cells to an NC state. Apart from medium composition (see Fig.…”

“…luteus, Rhodococcus rhodochrous and Mycobacterium tuberculosis) can enter a NC state when grown to and maintained in stationary phase in batch culture Kaprelyants et al, 1994;Shleeva et al, 2002). These NC organisms may be described as dormant, since they have extremely low metabolic activity and they require specialized treatment to promote their resuscitation.…”

New insights into the function of granulomas in human tuberculosis

“…In vitro studies by Zhang et al describe several phospholipids and mycobacterial proteins whose presence ensured transition from dormancy to a metabolically active state [54]. Culture of M. tuberculosis in Sauton's medium and repeated subcultures in sealed tubes to stationary phase resulted in 'non-culturable' cells, considered as dormant [55]. These bacteria could be resuscitated by addition of Rpf (resuscitation promoting factor of Micrococcus luteus) to the media.…”