FEMS Microbiology Letters 25 (1984) 21-25 21 Published by Elsevier

FEM 01899

Characterization of penicillin-resistant Streptococcus faecium mutants

(Penicillin resistance; penicillin-binding proteins; cell morphology; ultrastructure, autolysis)

R obe r t a Fon tana , Giul io Bertoloni , G u i d o Amal f i t ano and Pie t ro Canepar i *

lstituto di Microbiologia, Universith di Padova, Via Aristide Gabelli 63, 35100 Padova and * Istituto di Microbiologia, Universita di Genova, Viale Benedetto XV, 16132 Genova, Italy

Received 26 June 1984 Accepted 29 June 1984

1. SUMMARY

The characteristics of three spontaneous mutants of Streptococcus faecium PS which over- produce the penicillin-binding protein (PBP) 5 and are resistant to penicillin were investigated and compared with those of the parent.

The mutants grew a little more slowly than the parent, had a smaller cellular size, did not grow as filaments in the presence of penicillin minimal inhibitory concentration (MIC), and were signifi- cantly more prone to autolysis. The relationship between the characteristics of the mutants and the overproduction of PBP 5 is discussed.

concentrations saturating all PBPs but not PBP 5, and stopped growing in the presence of the minimal concentration of antibiotic saturating this protein [2,31.

In an attempt to learn more about this unusual mechanism of penicillin-intrinsic resistance, we have undertaken a further characterization of the above-mentioned mutants of S. faecium.

We found that the mutants grew slower than the parent only marginally, had a smaller cell diameter, did not grow as filaments in the presence of the penicillin MIC, and were significantly more prone to autolysis.

2. INTRODUCTION

It was recently shown that enterococci syn- thesize a PBP which shows an unusually low affin- ity for penicillin and was proposed to be responsi- ble for both natural insensitivity and acquired resistance of enterococci to penicillin [1-3]. This hypothesis was supported by the findings that penicillin-resistant mutants of S. faecium overpro- duced the low-affinity PBP (the No. 5, in this strain), grew normally in the presence of penicillin

3. MATERIALS AND METHODS

3.1. Bacterial strains and growth conditions Spontaneous mutants resistant to different

penicillin concentrations were isolated from a penicillin-sensitive (PS) S. faecium (MIC: 0.25 /xg/ml) by serial cultivation in a medium contain- ing increasing penicillin concentrations [2]. S. faeciurn PS was the same strain used in previous studies and indicated as S. faecium (faecalis) ATCC9790 [1-4]. The penicillin-resistant strains were indicated as R5 (MIC: 20/xg/ml), R20 (MIC:

0378-1097/84/$03.00 © 1984 Federation of European Microbiological Societies

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40 /~g/ml), and R40 (MIC: 80 t~g/ml). Bacteria were grown in SB medium [2] at 37°C and the growth was monitored turbidimetrically at 550 nm in a Perkin-Elmer spectrophotometer. Cultures in balanced exponential growth were obtained by inoculating a medium at low turbidity (0.05 units, equivalent to about 5 . 1 0 6 colony-forming units ( C F U ) / m l and allowing them to undergo 4 to 5 mass doublings in the phase of exponential growth prior to each experiment.

3.2. Morphology studies The turbidity of cultures in exponential growth

was adjusted to 0.2 units by dilution in a fresh prewarmed medium and rapidly added to small predisposed volumes of penicillin to obtain final concentrations corresponding to the MIC and its fractions. At intervals, the cell shape was examined by interference phase-contrast microscopy. For electron microscopy studies the cells were fixed by adding glutaraldehyde solution to a final con- centration of 3% in a 0.1 M cacodylate buffer (pH 7.2). After 2 h of incubation at 4°C, the cells were washed, postfixed in 1% osmium tetroxide in a 0.1 M phosphate buffer (pH 7.2) embedded in epoxy resin (DOW Chemical Co.), sectioned, stained with uranylacetate, and photographed with a Hitachi HS9 electron microscope.

3.3. Determination of cellular autolytic activity 10 ml of broth cultures in balanced exponential

growth (turbidity 0.8-1.0 units) were rapidly chilled and filtered (Millipore filter, 0.45 t~m pore size diameter), washed three times with distilled water at 4°C, resuspended in 10 ml of 0.3 M sodium phosphate (pH 7) and incubated in a 37°C water bath [5]. At intervals, turbidity was monitored at 550 nm.

4. RESULTS A N D DISCUSSION

The growth rates of S. faecium PS and penicil- lin-resistant mutants (R5, R20, and R40) were similar. The R5 grew as fast as the parent strain, whereas the more resistant strains (R20 and R40) grew slower. The doubling times were 28 min for the parent, 30 min for R5, and 36 min for R20 and

R40. All mutants were rather stable and the resis- tance was not lost after 50 serial transfers in a medium not containing penicillin, suggesting that the overproduction of PBP 5 had very little effect upon the growth of the cells and did not influence the ability of resistant cells to compete with the wild type.

The morphology of resistant cells did not differ from that of the parent, but these showed a de- crease in cell diameter (Figs. 1 and 2). By measur- ing the maximum cell diameter of 400 untreated cells for each strain, obtained from the same stage of exponential growth, it was found that the aver- age diameter was 1.06 (_+0.2) #m for the parent and 0.77 (-t-0.1)/~m for all resistant strains. Simi- lar results were obtained when the maximum cell diameter were measured in 50 longitudinal thin sections of cells showing tribanded walls profiles of each strain. The diameter of the parent and resistant cells resulted to be 0.93 (_+ 0.05) and 0.69 (+0.06)/~m, respectively (see also Fig. 2A and B). From these results it appears that the resistant cells were significantly smaller than sensitive cells, since they showed average cell diameters that were between 26 and 28% shorter. Such reduced cell dimensions are unlikely to depend on the slightly lower growth rate, since the R5 mutant, which had exactly the same dimensions as strain R20, and R40 showed a generation time that was 2 rain longer than that of the parent, but 6 min shorter than that of mutants R20 and R40. A more likely possibility is that such a reduced cell size was due to an altered cell wall metabolism associated with the overproduction of PBP 5.

The degree of chaining was also investigated by evaluating the percentage of single cells, doublets and chains (4 and more cells) in 200 microscope fields of parent and resistant strain cultures. No significant differences were found because the exponential growth cultures of all strains con- tained approx. 10% single cells, 60% doublets and 30% chains.

S. faecium PS formed filaments in the presence of MIC, as other enterococcal strains [4,6]. Figs. 1 and 2 show that resistant mutants, in the presence of the respective MIC, did not form filaments but only ovoidal or very short rod-shaped cells. More- over, electron microscope studies showed that

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Fig. 1. Morphological changes produced by penicillin in sensitive and resistant strains of S. faeeium. Exponential phase cells were grown in the presence of the respective MIC of penicillin. Microphotographs were taken after 2 h, the incubation time which allowed maximum cellular elongation. (A) Parent, untreated cells; (B) R5, untreated cells; (C) R20, untreated cells; (D) R40, untreated cells; (E) parent grown in the presence of 0.25 /~g/ml of penicillin (F) R5 grown in the presence of 20 /~g/ml; (G) R20 grown in the presence of 40 t~g/ml; (H) R40 grown in the presence of 80 t~g/ml. Bar represents 1 tl.

penicillin MIC-t rea ted sensitive cells contained a higher number of nascent septa than the R40 cells under similar conditions. This different morpho- logical response could be the consequence of the different mechanism by which penicillin inhibits growth in sensitive and resistant S. faecium cells [2,3]. It has been shown that penicillin MIC saturates all PBPs in R40 but only PBPs 1, 2 and 3 in the parent. Thus the activity of the unsaturated PBPs (4, 5 and 6) in sensitive cells might allow filament formation and be responsible for the nascent septa observed.

Since a decrease in autolytic activity has been described as interfering with cell response to penicillin inhibitory effect [7], we evaluated the autolysis rate in both the parent and resistant mutants. As shown in Fig. 3, the mutants auto-

lyzed more quickly than the parent. A more or less direct relationship between the autolysis rate and the resistance level was also observed; R5 and R20 were the least prone to autolysis and R40 the most prone. Such an increase in autolytic activity may depend on the fact that the pept idoglycan of re- sistant cells was more sensitive to autolysis or that PBP 5 had an autolytic activity. The former hy- pothesis is stimulating because it was proposed that PBP 5, under certain conditions, could take over the functions of the other PBPs (1, 2, 3). Thus, it may be possible that PBP 5 possesses both pept idoglycan polymerizing and depolymerizing activities to ensure the correct growth and division of the cells.

In conclusion, these results show that the peni- cillin resistance in S. faecium is accompanied by

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Fig. 2. Ultrastructure of penicillin-treated cells of sensitive and resistant strains of S. faecium. Exponential-phase cells were grown in the presence of the respective MIC of penicillin, and harvested after 2 h of incubation at 37 o C. (A) Parent, untreated cells; (B) R40, untreated cells; (C) parent grown in the presence of 0 . 2 5 / t g / m l of penicillin; (D) R40 grown in the presence of 80/ , tg/ml. Part (C) shows only a portion of a filament like that shown in Fig. 1E. Bar represents 0.5 #.

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0.6

0 115 130 145 160

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Fig. 3. Cellular autolysis of penicillin-sensitive and -resistant strains of S. faecium. Exponential phase cells (A 0.8 units) were resuspended in buffer and incubated at 37 o C. At intervals, the rate of reduction of turbidity was determined. A, Parent; ©, R5; O, R20; m, R40.

several changes in cell physiology. Most of them, if not all, could be due to the overproduction and the activity of PBP 5. Further studies are required to obtain definitive information on the biochemi- cal reactions catalyzed by this multifunctional pro- tein and its evolutionary relationship with the other PBPs.

A C K N O W L E D G E M E N T

This research was supported by the grant No. 83.00651.52 from the Consiglio Nazionale delle Ricerche.

REFERENCES

[1] Fontana, R. and Cerini, R. (1981) in Current Chem- otherapy and Immunotherapy (Periti, P. and Gialdroni Grassi, G., Eds.), 12th International Congress Chemother., pp. 225-226, American Society for Microbiology, Washing- ton, DC.

[2] Fontana, R., Cerini, R., Longoni, P., Grossato, A. and Canepari, P. (1983) J. Bacteriol. 155, 1343 1350.

[3] Fontana, R., Canepari, P. and Satta, G. (1980) in The Target of Penicillin (Hakenbeck, R , H61tje, J.V. and Labischinski, U., Eds.), pp. 531-536. Walter de Gruyter, Berlin.

[4] Fontana, R., Canepari, P., Satta, G. and Coyette, J. (1983) J. Bacteriol. 154, 916-923.

[5] Cornett, J.B., Redman, B.E. and Shockman, G.D. (1978) J. Bacteriol. 133, 631-650.

[6] Lorian, V. and Atkinson, B. (1978) J. Infect. Dis. 138, 865-871.

[7] Tomasz, A. (1979) Annu. Rev. Microbiol. 33, 114-137.