INTRODUCTION

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It has been clearly demonstrated that many bacteria can enter the viable but nonculturable (VBNC) state when faced with adverse environmental conditions (1, 24, 26, 32). When in this state, bacteria lose culturability on growth media but remain viable and demonstrate metabolic activity (3, 19, 33). These considerations stem from the results of many experiments, performed in vitro, in which an exponentially growing culture is transferred to a low-nutrient medium (usually freshwater, tap water, or seawater). With time (a few days or a couple of weeks, depending on the bacterial species, but probably also with the single strain) the count of culturable bacteria declines to undetectable levels, while the total count (evaluated as particles) remains constant. At the same time, the active count (i.e., the number of cells that display metabolic activity) declines very slowly (1). It has also been demonstrated that VBNC forms of medically important bacteria may conserve their pathogenicity genes (27, 30) and that some are capable of resuming active growth when optimal environmental conditions are restored (20, 25, 27, 37, 40). This survival strategy has been described for many gram-negative bacteria (whether pathogenic or saprophytic bacteria). Only very recently have we proven for the first time that a gram-positive species, namely, Enterococcus faecalis, can also activate the VBNC state. In this state, enterococcal cells are metabolically active and can resume active growth when normal growth conditions are restored (23). This could be of interest, in that this microorganism is used as an indicator of fecal contamination of water. Thus, it can be concluded that transmission of infection and public health monitoring procedures may be the areas in which the VBNC phenomenon appears to be most immediately relevant.

When rod-shaped gram-negative bacteria enter the VBNC state, they acquire a coccal (or very short rod) morphology and are reduced in size (6, 13, 20). Alterations of their envelopes have been shown by electron microscopy. Thin-section micrographs of Vibrio cholerae show that some parts of the outer membrane are separated and a gap is formed between the inner and outer membrane with the formation of blebs (20). In addition, polymer-like filaments have been seen in Vibrio spp., and an exopolysaccharide nature was suspected (20). The same types of alterations, together with the tendency of cells to aggregate in clusters, have also been described in Helicobacter pylori (13). Very recently, Hartke et al. (18) studied E. faecalis cell morphology upon incubation in a nutrient-poor microcosm (tap water). Within 3 to 7 weeks cells were seen to develop a rippled cell surface with irregular shapes. However, the same authors reported that, after 85 days of incubation in this microcosm, 10 to 30% of the cells were still culturable (18), thus indicating starvation, which is a situation very different from the VBNC state. These dramatic shape alterations, observed in both gram-negative and gram-positive bacteria, might be compatible with changes in the biochemical composition of the cell envelopes. The crucial role played by the bacterial envelope during cell growth and the division process has long been known (for a recent review, see reference 34). In particular, among the various wall polymers, peptidoglycan must be regarded as the main macromolecule involved in cell shape determination and maintenance (39). Moreover, alterations in its biochemical composition have been linked to shape alterations (36), growth rate (14, 28, 38), and cell division inhibition during the stationary growth phase (2). Very little information is available about the biochemical composition of the cell envelope during VBNC state acquisition. Only very recently, Costa et al. (9) have shown important differences in the peptidoglycan chemical composition of metabolically active but nonculturable coccus-shaped H. pylori in comparison to rod-shaped dividing cells. It seems plausible, then, that alterations in the chemical composition of the wall could also be observed in gram-positive bacteria in the VBNC state.

In this study we examine the biochemical composition of the cell wall of E. faecalis in the VBNC state. The state of the enzymes involved in peptidoglycan metabolism is also considered. The results are consistent with specific changes in the cell wall, thus suggesting that VBNC is a physiological state in response to particular environmental conditions and not only a stage preceding or associated with cell death.

	MATERIALS AND METHODS

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Bacterial strains and growth conditions. E. faecalis 56R was used (35). This strain was grown at 37°C in tryptic soy (TS) broth (Difco). Cell growth was monitored either by measuring the optical density at 640 nm (OD₆₄₀) of the culture with an LKB spectrophotometer or by determining the number of cells with a Coulter Counter as previously described (23). To obtain an exponentially growing culture, an overnight culture (OD₆₄₀ = 1.3) was 20-fold diluted in 2 liters of fresh prewarmed medium and incubated until an OD₆₄₀ of 0.7 was reached. Half of this culture was used as such, while the second half was reincubated for an additional 20 h to obtain a stationary 12-h-old culture. For preparing VBNC cells of E. faecalis 56R, an early exponentially growing culture (OD₆₄₀ = 0.3) was resuspended at a concentration of 1 × 10⁶ to 2 × 10⁶ CFU/ml in a microcosm consisting of water collected from Lake Garda (Verona, Italy) and prepared as previously described (23). Cultures were then incubated at 4 ± 0.5°C. After 16 days of such incubation cells were metabolically active but nonculturable (23).

Evaluation of cell wall resistance. A pellet (from exponentially growing, stationary, VBNC cultures and UV-killed cells [see below]) was resuspended in 0.01 M phosphate buffer (pH 7.2), and cells were broken by several vigorous shakings in the presence of glass beads in a Braun MSK homogenizer under nitrogen flow to keep the suspension chilled. Each shaking lasted 30 s, and a sample was then taken to evaluate the rate of cell lysis as a decrease in OD₆₄₀.

Cell wall preparation. One liter each of an exponentially growing, a stationary, and a UV-killed culture was collected by centrifugation at 8,000 × g at 4°C for 10 min. To collect the culture in the VBNC state, centrifugation was increased to 15,000 × g at 4°C for 10 min in view of the fact that the cell population was highly diluted in the microcosm (roughly 10⁶ cells/ml). The resulting pellets were washed in cold 0.01 M phosphate buffer (pH 7.2), and the cells were boiled in 4% sodium dodecyl sulfate (SDS) for 30 min. Cells were collected by centrifugation at room temperature (20,000 × g for 30 min), and the pellet was washed six times with distilled water to remove all SDS. Cells were then broken with glass beads with a Braun MSK homogenizer under nitrogen flow to keep the suspension chilled. Unbroken cells and glass beads were removed by low-speed centrifugation (2,500 × g for 10 min), and the resulting supernatant containing cell walls was centrifuged at 30,000 × g for 30 min.

Preparation of peptidoglycan and separation of muropeptides by high-performance liquid chromatography (HPLC). The protocol described by de Jonge et al. (11, 12), as modified by Signoretto et al. (35), was used. Bacterial walls, prepared as described above, were treated enzymatically (100 µg each) with -amylase, DNase, RNase, and trypsin in sequence and then with 8 M LiCl before extensive (four times) washes with distilled water, as described previously (11, 12). To remove the teichoic acids (TA), the walls were treated with hydrofluoric acid (49% final concentration) for 2 days at 4°C and then centrifuged at 30,000 × g for 30 min. Pellets were washed four times with distilled water, neutralized with 100 mM Tris-HCl (pH 7.5), and washed again twice with water. Finally, the walls were treated with alkaline phosphatase in 100 mM (NH₄)₂CO₃ overnight at 37°C. After boiling to inactivate the enzyme, pure peptidoglycan was washed twice with distilled water and stored at 20°C.

Identification and quantitation of peaks obtained by HPLC analysis. Peaks were identified on the basis of their retention times according to the method of de Jonge et al. (11), and quantitation (expressed as percentage of the total) was calculated as integration of the UV response corrected with the conversion factor obtained by applying a mathematical formula similar to that described by Glauner (16): C = D / 0.6 × D + 0.1 × A + 0.1 × L, where C is the conversion factor, D is the number of disaccharide units, A is the number of amide bonds, and L is the number of alanine residues. The conversion factor for the various muropeptides ranged from 0.7 to 1.2.

Preparation and quantitation of TA. TA were selectively extracted from bacterial walls, prepared as described above, by treatment with 5% trichloroacetic acid (TCA) (20 ml per g of purified wall) for 24 h at 4°C (17). Insoluble material was removed by centrifugation at 20,000 × g for 30 min. The polymer was then precipitated from the TCA extract by mixing with five volumes of absolute ethanol for 16 h at 4°C and was recovered by centrifugation at 30,000 × g for 15 min. The precipitate was redissolved in 5 ml of 5% TCA, and any insoluble material was removed by centrifugation. TA was again precipitated with cold ethanol and recovered by centrifugation. The pellet was washed with ethanol and then with diethyl ether and was dried under a vacuum. Quantitation of TA was performed by estimating the organic phosphorus using the method of Chen et al. (7).

Preparation and quantitation of LTA. Bacteria harvested as described above were extracted twice with chloroform-methanol (2:1 vol/vol) (20 mg of pelleted bacteria/ml) at room temperature for 2 h. Lipoteichoic acid (LTA) was extracted from these whole defatted cells with 45% aqueous phenol at 68°C (for 45 min with stirring) as described by Kessler and Shockman (21). Phenol was removed by dialysis against six changes of 40 volumes of 0.1 M sodium acetate, pH 5.0, at room temperature. Nucleic acids were degraded by extensive treatment with DNase and RNase (100 U/ml; each for 24 h at 37°C) in the presence of 0.05% sodium azide to prevent microbial contamination. Additional phenol extraction and extensive dialysis, as described above, were used to remove the nuclease proteins and nucleic acid fragments. The absence of nucleic acid was confirmed at an absorbance at 260 nm of <0.1 (values ranged from 0.01 to 0.03) and the absence of amplification products by PCR with an E. faecalis-specific set of primers (23). The resulting LTA was quantitated by measurement of phosphate (7).

Evaluation of autolysis rate of E. faecalis 56R whole cells and walls. The cell wall autolysis rate was evaluated according to the method of Pooley and Shockman (29). An exponentially growing culture, a stationary culture, a UV-killed culture, and 20 liters of VBNC cells were collected by centrifugation at 4°C, as stated above. Pellets were then washed in 10 mM phosphate buffer (pH 7.2) and resuspended in 2 ml of the same buffer. Each sample was subdivided into two parts. One was immediately incubated at 37°C and was used to evaluate the amount of active muramidase-1 (mur-1). To the second sample, trypsin (50 µg/ml, final concentration) was added before incubation at 37°C. This sample served to evaluate mur-1 in the latent form. Every 10 min each sample was read spectrophotometrically at 640 nm. The rate of lysis was calculated as a percentage of OD₆₄₀ decrease per hour.

Analysis of PBPs. Penicillin binding protein (PBP) analysis was conducted as previously described (4, 5). Briefly, an exponentially growing culture, a stationary culture, a UV-killed culture, and 20 liters of VBNC E. faecalis 56R cells were collected and the cells disrupted cold with glass beads as described above. The resulting walls and membranes were collected by centrifugation at 30,000 × g for 30 min at 4°C. Pelleted material was resuspended in phosphate buffer 0.01 M (pH 7.2) (10-mg/ml protein concentration), and to 100 µl of this was 20 nmol of [³H]benzylpenicillin added; the mixture was incubated for 60 min at 37°C. After binding of radioactive penicillin to the membranes, proteins were solubilized with hot SDS and separated by SDS-polyacrylamide gel electrophoresis (4, 5). PBPs were then visualized by fluorography as previously described (4, 5). The amount of radioactivity bound to PBPs was estimated by microdensitometry of the fluorograms using an Image Master-VDS apparatus (Pharmacia).

Chemicals. The [³H]benzylpenicillin (specific activity, 629 GBq/mmol) used was from Amersham. Protein concentrations were determined by a Bio-Rad protein assay. All the reagents used were commercially purchased reagent grade (Merck), and the chemicals used for HPLC were HPLC grade (Baker).

Statistical analysis. The data presented in this study are the means of three distinct experiments. The coefficient of variation is indicated in brackets in each table. Data were analyzed using the analysis of variance procedure, followed by Tukey's test, with the aid of the SPSS 8.0 statistical package (SPSS Inc., Chicago, Ill.).

	RESULTS

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Dimension of E. faecalis cells in the VBNC state. Light microscope observations of E. faecalis 56R cells in the VBNC state in comparison with exponentially growing or stationary cells were used to measure cell dimensions. Stationary cells were slightly larger than the exponentially growing cells, due to the thickening of their cell walls (10), with cell diameters which were 1.38 ± 0.31 µm and 1.29 ± 0.35 µm, respectively. VBNC cells appeared slightly elongated, with a cell length which was 1.48 ± 0.31 µm. However, these observed differences were not significant from a statistical point of view.

Mechanical resistance of cell wall of E. faecalis in the VBNC state. The mechanical resistance of E. faecalis 56R in the VBNC state in comparison with exponentially growing, stationary, or UV-killed cells was determined by analysis of the mechanical disruption of the cells with glass beads in a homogenizer. Figure 1 shows that E. faecalis 56R VBNC cells were twice as resistant to mechanical disruption as exponentially growing, stationary, and UV-killed cells. This was established by the fact that ten 30-s shakings were necessary to decrease the absorbance of the cell suspension by 90%, while only five to six shakings were necessary for all other types of cells to obtain the same lysis.

View larger version (13K): [in this window] [in a new window]   FIG. 1.   Hardness of cell wall of E. faecalis 56R VBNC cells in comparison with exponentially growing or stationary cells, evaluated as decrease in OD during 30-s shakings with glass beads. Points are mean values from three distinct experiments. The coefficient of variation for each point was less than 5%. Symbols: , VBNC cells; , exponentially growing cells; , stationary cells; , UV-killed cells.

Peptidoglycan chemical composition of E. faecalis VBNC cells. The yield of peptidoglycan purified from the batch containing VBNC cells was 13.2 mg/g of cells (wet weight) which was very similar to the yield obtained from an exponentially growing culture and less than that obtained from a stationary or overnight culture (12.1 and 17.5 mg/g (wet weight), respectively). The UV-treated cells yielded 13.8 mg of peptidoglycan/g of cells. Table 1 shows the relative amount and biochemical identification (12, 35) of each peak of the muropeptide profiles obtained by reversed-phase HPLC of exponentially growing, stationary, UV-killed, and VBNC cells of E. faecalis 56R. The table also presents the same results with the values grouped in families of muropeptides (monomers, dimers, trimers, tetramers, pentamers, and higher oligomers, as specified in Materials and Methods). It is evident that in exponentially growing cells about 60% of the peptidoglycan consisted of cross-linked muropeptides and about half were dimers (12, 35). No major differences were seen between peptidoglycans purified from E. faecalis 56R stationary cells and exponentially growing ones, the only difference being a slight increase in dimers and trimers which was offset by an equivalent reduction in the monomer family. However, the differences observed were not significant from a statistical point of view. In the peptidoglycan recovered from VBNC cells, an abnormal biochemical composition was observed. No new peak was seen, but there were differences which were confined to some changes in the relative amounts (Table 1). These changes, however, were more noticeable when families of muropeptides were considered. In fact, all families underwent changes. In particular, a substantial increase in the families containing cross-linked muropeptides of a higher order than dimers was observed when compared with both exponentially growing or stationary cells (Table 1): 24% increase in trimers, 37% increase in tetramers, 65% increase in pentamers, and a doubling (95% increase) in the family containing higher oligomers. This increase in cross-linked peptidoglycan, despite a slight decrease in the dimer family, was more than offset by a sharp decrease (24%) in the monomer family, in such a way that the total cross-linking, calculated according to the equation of Fordham and Gilvar (15), rose from 39% in growing cells to 48% in VBNC cells. All these values were statistically significant in that the differences among the single families of muropeptides were greater than 3 standard deviations. As far as the monomer family of the VBNC cells was concerned, it is significant (Table 1) that the decrease (significant differences) was mainly confined to compounds 7 (33% decrease) and 9 (47% decrease), which corresponded to the pentapeptide monomers, thus indicating a consumption of substrate required for the transpeptidation reaction. When, however, peptidoglycan biochemical composition was evaluated in a control model which consisted of UV-killed cells prior to incubation at 4°C for 20 days, a reduction in total oligomers higher than dimers was observed, and, thus, the total cross-linking was reduced from 39 (exponentially growing cells) to 31%. These differences were again statistically significant.

Evaluation of enzymes involved in peptidoglycan metabolism. It has long been known (34) that peptidoglycan synthesis during the cell cycle is the result of a balance between polymerizing and hydrolytic enzymes. Among the polymerizing enzymes involved in terminal stages of peptidoglycan assembly, some are capable of penicillin binding (PBPs), and thus their presence and function can be simply evaluated by determination of this activity (10). Table 2 shows the PBP pattern of E. faecalis 56R in the exponentially growing, stationary, and VBNC states. In this experiment, penicillin binding was prolonged to 60 min in order to also evaluate the state of the low-affinity PBP, which in this case corresponds to PBP 5 (4, 5, 35). Table 2 shows that no significant differences (P > 0.05) could be seen in the PBP pattern of exponentially growing and stationary cells and that, as expected, PBPs 5 and 6 account for over 50% of the bound radioactivity (4, 5, 35). Interesting results were observed when the PBP pattern of VBNC cells was compared with that of growing or stationary cells. PBPs 1 and 5 in VBNC cells increased significantly (P < 0.05), while PBPs 3 and 6 decreased significantly (P < 0.05). By contrast, PBPs 2 and 3 did not change (P > 0.05).

Quantitation of wall TA and LTA. Table 4 shows the results of the quantitation of the two wall polymers by phosphate measurement in exponentially growing, stationary, VBNC, and UV-killed E. faecalis 56R cells. As expected, most of the organic wall phosphate was due to TA (10). No significant differences (P > 0.05) were observed in TA in the four kinds of cells, while LTA was more than doubled in VBNC compared with exponentially growing, stationary, and UV-killed cells (P < 0.05).

	DISCUSSION

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The VBNC state is a particular condition that bacteria may undergo when environmental conditions are not suitable for normal cell growth and division. This state was first described in gram-negative bacteria (1, 3, 19, 20, 26, 31, 41) and was only very recently in a gram-positive organism such as E. faecalis (23). Under these conditions, bacteria are unable to form colonies in normal growth media but are still viable and endowed with metabolic activity. Moreover, under certain conditions they are capable of resuming active cell growth. That many bacteria of medical interest enter into the VBNC state may be a very relevant consideration for all those involved in determining the microbiological quality of specific environments, in that pathogenic bacteria in this state may no longer be evaluable in environmental samples when classic techniques for their detection (enumeration of CFU/milliliter) are used. At present, however, the real meaning of the VBNC state is still unclear: it can be regarded either as (i) a premortem status or (ii) a specific physiological state in response to environmental stresses which lead to the death of the bacterial cell unless it reverts to normal division in a reasonable period of time. On the basis of the data reported in the literature, hypothesis ii appears more tenable. As regards the changes which bacteria may undergo during entry into the VBNC state, very little is known. What we do know is only that gram-negative bacteria which are in the VBNC state are much smaller than their normally growing counterparts and have a rounded morphology if they are rod-shaped when normally dividing. Very recently, Costa et al. (9) have shown specific alterations of the peptidoglycan biochemistry attributable to an increase in disaccharide-dipeptides in coccal VBNC cells of H. pylori. Nothing is known, however, about which morphological and biochemical alterations of gram-positive bacteria occur in the VBNC state, essentially because the state has only recently been identified (23). In this paper, we show no reduction in cell size of E. faecalis, as seen with gram-negative bacteria, but on the contrary, cells appeared slightly elongated as previously shown when enterococci were treated with antibiotics that block septum formation or in mutants which are thermosensitive for cell growth or division (22). For the first time, we show biochemical alterations involving more than one component of the cell wall displayed by bacteria in the VBNC state. In particular, peptidoglycan of VBNC E. faecalis 56R cells appears to be more cross-linked (48 versus 39%), and this is mainly due to an increase in oligomers of a higher order than dimers. These alterations in the chemical composition of the wall polymer seem to be specific to the VBNC state. In fact, analysis of the biochemical composition of peptidoglycan of UV-killed cells aged for a period corresponding to entry into the VBNC state yields opposite results, namely, a less cross-linked peptidoglycan which may be the consequence of destructive (lytic) enzyme action. This indicates the need for production of additional amounts of the enzymes involved in peptidoglycan synthesis. What is more, PBP evaluation in E. faecalis VBNC cells has clearly indicated that these proteins have maintained a penicillin binding capability close to 60% compared to that of growing cells. This property is conserved essentially by PBPs 1 and 5, which become the prevalent ones in the PBP pattern. Particularly interesting seems to be the role of PBP 5. This enterococcal PBP, which, when overproduced, is also involved in penicillin resistance (4, 5), has previously been shown to be important for cell growth under suboptimal but not under optimal environmental conditions and has been defined as an SOS protein (4, 35). The fact that, in the VBNC state, PBP 5 continues to maintain its function, penicillin binding capability, and presumably its activity, as also previously shown (35), not only lends further support to its hypothetical role but also indicates that VBNC cells are still alive and, therefore, in a precise physiological state. In a previous paper we showed that PBP 5 alone is capable of synthesizing only a peptidoglycan in which dimers predominate strongly over higher cross-linked oligomers (35). The presence of higher oligomers in peptidoglycan of E. faecalis VBNC cells may be explained by the concomitant presence of an additional PBP, such as PBP1, a high-molecular-weight PBP for which both transpeptidase and transglycosylase activity may be suspected (34). The higher cross-linking creates a harder wall than that of dividing cells, as demonstrated by the increased resistance to cracking by shaking with glass beads. These changes in the cell wall take place in spite of the increase in the autolytic system: 25 to 50% for active mur-1 and up to a threefold increase for inactive mur-1. Inactive mur-1 was shown to be located essentially (about 85%) in the cytoplasm of exponentially growing cells and exported towards the cell wall, which is precisely where it is activated (29). VBNC cells of E. faecalis, by contrast, build up large amounts of this enzyme in the cell wall. This suggests that the bulk of inactive mur-1 is transferred from the cytoplasm to the cell wall, but only a small proportion of it is activated (Table 3). However, the autolysis rate is slightly higher in VBNC than in exponentially growing or stationary cells. This does not necessarily mean that VBNC walls possess greater amounts of active mur-1: the increase in autolysis observed in these cells may be due simply to the change in peptidoglycan chemical composition, which, in turn, acts as a more efficient substrate for mur-1. The fact that the cells in the VBNC state do not lyse but rather present highly cross-linked peptidoglycan may be due to the increase in LTA (Table 4), the role of which in controlling cellular autolysis has long been known (8). Finally, the increase in the cell wall autolytic complex during entry into the VBNC state may be explained speculatively in terms of the need to remove hyper-cross-linked peptidoglycan when the cell reverts to active growth. Confirmation of the activation of a specific cell wall enzyme(s), at least during the transition to the VBNC state, has recently been provided by Costa et al. (9), who show an accumulation of disaccharide-dipeptides and the activation of a ()-glutamyl-diaminopimelate endopeptidase in H. pylori when cells stop dividing and become committed to morphological transition (9).