The viable but nonculturable (VBNC) state is a survival mechanism adopted by many bacteria (including those of medical interest) when exposed to adverse environmental conditions. In this state bacteria lose the ability to grow in bacteriological media but maintain viability and pathogenicity and sometimes are able to revert to regular division upon restoration of normal growth conditions. The aim of this work was to analyze the biochemical composition of the cell wall of Enterococcus faecalis in the VBNC state in comparison with exponentially growing and stationary cells. VBNC enterococcal cells appeared as slightly elongated and were endowed with a wall more resistant to mechanical disruption than dividing cells. Analysis of the peptidoglycan chemical composition showed an increase in total cross-linking, which rose from 39% in growing cells to 48% in VBNC cells. This increase was detected in oligomers of a higher order than dimers, such as trimers (24% increase), tetramers (37% increase), pentamers (65% increase), and higher oligomers (95% increase). Changes were also observed in penicillin binding proteins (PBPs), the enzymes involved in the terminal stages of peptidoglycan assembly, with PBPs 5 and 1 being prevalent, and in autolytic enzymes, with a threefold increase in the activity of latent muramidase-1 in E. faecalis in the VBNC state. Accessory wall polymers such as teichoic acid and lipoteichoic acid proved unchanged and doubled in quantity, respectively, in VBNC cells in comparison to dividing cells. It is suggested that all these changes in the cell wall of VBNC enterococci are specific to this particular physiological state. This may provide indirect confirmation of the viability of these cells.
Aims:The viable but non-culturable (VBNC) state is a survival strategy adopted by bacteria when exposed to environmental stress. When in this state bacteria are no longer culturable on conventional growth media, but cells display metabolic activity and maintain pathogenicity factors/genes and, in some cases, resuscitation from the VBNC state has been shown. This state has been described for both human pathogens and faecal pollution indicators. In this study, we present evidence for entry of different enterococcal species into the VBNC state in an oligotrophic microcosm. Methods and Results: The duration of the viability of the cells in the VBNC state was measured either by detecting the presence of pbp5 mRNA or by quantifying their resuscitation capability. Enterococci showed different behaviours. Enterococcus faecalis and Enterococcus hirae entered into the VBNC state within 2 weeks and remained in that state for 3 months. In the experiments described the resuscitation rate was 1 : 10 000 cells as soon as the cells entered the VBNC state and decreased gradually to undetectable levels over the following 3 months. Enterococcus faecium, however, remained culturable up to 4 weeks. After this time period, when the population was totally unculturable, the cells were far less resuscitable than other enterococci and only over a narrow time interval (2 weeks). Conclusions: These results suggest that Ent. faecalis and Ent. hirae enter the VBNC state but that Ent. faecium, in an oligotrophic laboratory environment, tends to die instead of entering the VBNC state. Signi®cance and Impact of the Study: These experiments may mimic what happens when enterococci are released by humans and animals in natural environments.
The protein expression patterns of exponentially growing, starved, and viable but nonculturable (VBNC) Enterococcus faecalis cells were analyzed to establish whether differences exist between the VBNC state and other stress responses. The results indicate that the protein profile of VBNC cells differs from that of either starved or exponentially growing bacteria. This demonstrates that the VBNC state is a distinct physiological phase within the life cycle of E. faecalis, which is activated in response to multiple environmental stresses.
Daptomycin at the MIC allowed the cell mass increase of enterococcal strains and Bacilus subtilis to continue for 2 to 3 h at rates comparable to those of the controls. During this time the cell shape of the former changed to a rod configuration and that of the latter changed to long rods. In these bacteria, in which cell mass continued to increase, the MIC of daptomycin inhibited peptidoglycan synthesis by no more than 20% after 20 min of incubation and by roughly 50% after 2 h of incubation. Other macromolecules, such as DNA, RNA, and proteins, were only slightly affected. In contrast, incorporation of [14C]acetate into lipids was reduced by about 50% in the various strains after 20 min of treatment with daptomycin at the MIC. When the effect of the major lipid-containing polymers on synthesis was evaluated in detail, it was found that under conditions in which peptidoglycan and the other macromolecules mentioned above were inhibited only slightly (20%) and total lipid synthesis was inhibited by 50%, synthesis of teichoic and lipoteichoic acids was inhibited by 50 and 93%, respectively. Daptomycin was not found to enter the cytoplasm of either bacterial or mammalian cells. It bound, in the presence of calcium ions only, to whole bacterial cells, cell walls (both those that contained and those that did not contain membranes), and isolated membranes of bacterial and mammalian cells. Washing with EDTA removed daptomycin from all cells mentioned above and cell fractions except the bacterial membrane. It is concluded that lipoteichoic acid is most likely the primary target of daptomycin.Daptomycin (LY146032) is an acidic lipopeptide antibiotic that is active against gram-positive bacteria (F. T. Counter,
The two-competing-sites model for peptidoglycan assembly for bacterial cell shape regulation suggests that in rods, bacterial cell shape depends on the balance between two reactions (sites), one responsible for lateral wall elongation and the other responsible for septum formation. The two reactions compete with each other so that no lateral wall can be formed during septum formation and vice versa. When the site for lateral wall elongation overcomes that for septum formation, long rods or filaments are formed and cell division may be blocked. When the reaction leading to septum formation is hyperactive compared with the other, coccobacilli or cocci are formed. Other bacteria carry only one site for peptidoglycan assembly and can grow only as cocci. The two-competing-sites model predicts that two different types of cocci exist (among both morphology mutants and wild-type strains); one carries only the site for septum formation, whereas the other also carries the site for lateral wall elongation, the former site predominating over the latter. As a consequence of the inhibition (by antibiotics or by mutations) of septum formation in wild-type cocci of various species and in coccoid morphology mutants, some cocci are expected to undergo transition to rod shape and others are not. We have evaluated these predictions and show that they are in agreement. In fact, we found that among wild-type cocci belonging to 13 species, those of 6 species formed rods, whereas the remaining organisms maintained their coccal shape when septa were inhibited by antibiotics. Some coccoid morphology mutants of rod-shaped bacteria underwent coccus-to-rod transition after septum inhibition by antibiotics, whereas others maintained their coccal shape. When a mutation that causes septum inhibition was expressed in a morphology mutant of Klebsielia pneumoniae grown as a coccus, transition to rod shape was observed. A total of 914 mutants unable to form colonies at 42TC were isolated from the coccoid species mentioned above. Between 75 and 95% of the mutants isolated from the species that formed rods when septum formation was inhibited by antibiotics but none of those isolated from the others underwent coccus-to-rod transition upon incubation at the nonpermissive temperature.In previous papers, we proposed a model for shape regulation in bacteria and presented a large body of experimental data in support of it (33,34,37). In this study, we have extensively tested predictions concerning the effects on cell shape of septum inhibition by antibiotics and mutations in various wild-type and mutant coccoid bacteria (see predictions 6 and 7 of Table 1, which have previously been evaluated only preliminarily). We show that all predictions are fulfilled and demonstrate that some coccoid species but not others undergo transition to rod shape when septa are specifically blocked by antibiotics and that mutants undergoing coccus-to-rod transition can be isolated in some coccoid species but not in others.The two-competing-sites (TCS) model for peptidoglycan ...
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