Acid Tolerance of Streptococcus macedonicus as Assessed by Flow Cytometry and Single-Cell Sorting

Abstract

An in situ flow cytometric viability assay employing carboxyfluorescein diacetate and propidium iodide was used to identify Streptococcus macedonicus acid tolerance phenotypes. The logarithmic-phase acid tolerance response (L-ATR) was evident when cells were (i) left to autoacidify unbuffered medium, (ii) transiently exposed to nonlethal acidic pH, or (iii) systematically grown under suboptimal acidic conditions (acid habituation). Stationary-phase ATR was also detected; this phenotype was gradually degenerated while cells resided at this phase. Single-cell analysis of S. macedonicus during induction of L-ATR revealed heterogeneity in both the ability and the rate of tolerance acquisition within clonal populations. L-ATR was found to be partially dependent on de novo protein synthesis and compositional changes of the cell envelope. Interestingly, acidhabituated cells were interlaced in lengthier chains and exhibited an irregular pattern of active peptidoglycan biosynthesis sites when probed with BODIPY FL vancomycin. L-ATR caused cells to retain their membrane potential after lethal challenge, as judged by ratiometric analysis with oxonol [DiBAC 4 (3)]. Furthermore, F-ATPase was important during the induction of L-ATR, but in the case of a fully launched response, inhibition of F-ATPase affected acid resistance only partially. Activities of both F-ATPase and the glucose-specific phosphoenolpyruvate-dependent phosphotransferase system were increased after L-ATR induction, distinguishing S. macedonicus from oral streptococci. Finally, the in situ viability assessment was compared to medium-based recovery after single-cell sorting, revealing that the culturability of subpopulations with identical fluorescence characteristics is dependent on the treatments imposed to the cells prior to acid challenge.The existence of common strategies employed by gram-positive bacteria, particularly lactic acid bacteria, in response to acid challenge has emerged only recently (14, 53). However, generalizations about specific mechanisms involved in such responses could prove premature, since it has been shown that these mechanisms can be species or even subspecies specific (14). A typical example relevant to this report is the current knowledge about streptococcal responses to acid stress. Linkages of acid production (acidogenicity) or tolerance (aciduricity) with pathogenicity have fueled recent interest in the basic physiology of oral streptococci (44). Today, Streptococcus mutans is probably the best-studied Streptococcus species in terms of acid stress physiology, but it is uncertain whether the behavior of this species is truly representative of the genus. For instance, a recent report on S. sobrinus revealed key differences from S. mutans concerning the involvement of F-ATPase and glucose-specific phosphoenolpyruvate-phosphotransferase system (PEP-PTS) activities in the manifestation of acid adaptation (35). Additionally, comparison of the recently completed genome sequence of S. thermophilus with those of other streptococci sh...

Viability states of bacteria—Specific mechanisms of selected probes

“…Nevertheless, E. coli was found to not present elevated PI values in the same experiments suggesting that this false positive staining may not only be substrate but also species dependent (65). Nevertheless, PI is a good probe for detecting dead cell numbers in bacterial populations, which is proven by numerous publications (86)(87)(88)(89). However, the user should be aware that the integrity of the bacterial surface can be also disturbed by cell growth and the connected rearrangement of membranes and cell walls.…”

Stress Physiology of Lactic Acid Bacteria

“…However, there is at least one additional physiological state after exposure of a population of cells to stress, namely, that of the "injured" cell (51). Injured cells are metabolically active but have suffered damage to a degree that causes a transient or even permanent loss of proliferation capacity (34). They sometimes require extended recovery times or can recover only under special conditions.…”

“…First, cells may be left under suboptimal conditions much longer than needed to induce adaptation. This adaptive procedure is probably best described as habituation, a term used only scarcely for LAB (34). The molecular mechanisms underlying transient adaptation and habituation to a specific stress may overlap to a degree, but they are not completely identical (34)(35)(36).…”

“…This adaptive procedure is probably best described as habituation, a term used only scarcely for LAB (34). The molecular mechanisms underlying transient adaptation and habituation to a specific stress may overlap to a degree, but they are not completely identical (34)(35)(36). This may explain a number of contradictory results reported for some LAB species, since the two responses have sometimes been considered identical (37, 38).…”

“…Cells enter stationary phase due to the exhaustion of nutrients and/or the accumulation of toxic metabolic products in their environment during growth. The environmental conditions of stationary phase become so stressful that the death rate of cells is accelerated (34,39). Transition from the exponential to the stationary phase is accompanied by the induction of multiple regulons, resulting in the ability to cope with a number of different stresses.…”