ANTIMICROBIAL AGENT8 AND CHEMOTHERAPY, Mar. 1977, p. 559-562 Copyright © 1977 American Society for Microbiology
Vol. 11, No. 3 Printed in U.S.A.
NOTES Ampicillin-Induced Morphological Alterations of Haemophilus influenzae Type b RICHARD D. KLEIN AND G. H. LUGINBUHL* Department of Microbiology, North Carolina State University, Raleigh, North Carolina 27607
Received for publication 29 October 1976
Inhibitory levels of ampicillin induced filamentation of growing Haemophilus influenzae ATCC 19418 within 30 min. Filaments became swollen and interrupted by regular periodic saccular outpouchings along the major axis. The degree of filamentation was dependent upon ampicillin concentration and time. The beta-lactam antibiotics, which inhibit peptidoglycan biosynthesis, have been demonstrated to induce a variety of morphological changes in gram-positive and gram-negative bacteria (3, 4, 7). These changes include spheroplasting, filament formation, and lysis. Haemophilus influenzae has been reported to form spheroplasts in the presence of bacteriostatic concentrations of ampicillin (1). In the present study the morphological effects of ampicillin on H. influenzae type b were examined by light and scanning electron microscopy. H. influenzae type b (ATCC 19418), an ampicillin-susceptible, beta-lactamase-negative strain (minimum inhibitory concentration [MIC], 0.1 ,g/ml), was grown overnight at 37°C in brain heart infusion broth (Difco) with 1% Difco supplement C (BHIC) on a rotary shaker. The culture was diluted 1:10 in BHIC, and 0.45 ml ofthis dilution was added to 8.55 ml of fresh BHIC and incubated for 2 h at 37°C on a rotary shaker. One milliliter of appropriate concentrations of ampicillin (Parke Davis oral suspension) was added to give final ampicillin concentrations of 0.1, 1, and 10 times the MIC. No ampicillin was added to one tube as a control. At 30 min and 4 h, samples were removed and processed for light and scanning electron microscopy. Bacterial suspensions on glass slides were air dried, heat fixed, and stained with crystal violet. Samples for scanning electron microscopy were filtered through 0.45-gm Nucleopore filters, dehydrated through 100% ethanol, critical point dried, and observed in an ETEC scanning electron microscope. Light microscopy of the untreated control culture revealed a uniform population of coccobacilli. When incubated for 30 min with ampicillin at a concentration 10 times the MIC, bacillary cells two to three times larger than the
control were observed. After 4 h at this concentration, large, swollen, elongated cells were prevalent. These cells were irregular and displayed circular distortions or saccular outpouchings of apparent cellular material along the major axis of the cell. As examined by scanning electron microscopy (Fig. 1), the untreated control cells were coccobacillary in nature and smooth surfaced and ranged from 0.7 to 1 ,um in length. After 30 min of incubation with 10 times the MIC of ampicillin (Fig. 2), the cells displayed a rough surface and were elongated, ranging from 1 to 3 ,um in length. Obvious septum formation had taken place between some cells (Fig. 3). After 4 h of incubation with 10 times the MIC of ampicillin (Fig. 4), elongated filaments were present. These filaments were smooth surfaced and frequently intertwined. No evidence of septum formation was observed. Periodic saccular outpouchings were present along the filaments (Fig. 5). The diameter of these spherical saccular outpouchings was not consistent. At concentrations of 0.1 and 1 times the MIC of ampicillin the morphological changes were less evident. At both concentrations, after 30 min, 4 h, and 24 h of exposure, the cells were not appreciably elongated, ranging from 1 to 3 ,tm in length. The only dramatic change noted was an increased roughness of the cell surface. At 24 h, ampicillin at 10 times the MIC is bactericidal, killing greater than 99.9% of the cells, whereas the lower concentrations of ampicillin, 0.1 and 1 times the MIC, are not bactericidal. A variety of gram-negative organisms have been demonstrated to form filaments in the presence of beta-lactam antibiotics. Proteus mirabilis (6) and Proteus vulgaris (3) produce filaments in response to carbenicillin and penicillin, respectively. The same phenomenon has 559
560
NOTES
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 1. Scanning electron micrograph of untreated control cells. Bar, 1 um.
FIG. 2. H. influenzae after 30 min of incubation with 10 times the MIC ofampicillin. Cells are elongated. Bar, 1 .tm.
been reported for Enterobacter cloacae (7) and Escherichia coli (5) with cefoxitin and cephalexin, respectively. In addition, filaments of H. influenzae have been reported by light micros-
copy after exposure to bacteriostatic concentrations of ampicillin (1). In each of these studies the presence of saccular outpouchings of the filament was noted.
VOL. 11, 1977
Saccular outpouching of H. influenzae filaments may result from the inhibition of septum formation at ampicillin concentrations greater than the MIC. This is suggested by the pres-
NOTES
561
ence of obvious septation between cells after a 30-min exposure to 10 times the MIC (Fig. 3) in contrast to the absence of septation after a 4-h exposure to this concentration (Fig. 4, 5). In
FIG. 3. H. influenzae after 30 min of incubation with 10 times the MIC of ampicillin. Septum formation has been completed between some cells. Bar, 1 ,im.
FIG. 4. H. influenzae after 4 h of incubation with 10 times the MIC of ampicillin. Filamentous forms predominate. Bar, 1 jum.
562
NOTES
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 5. H. influenzae after 4 h of incubation with 10 times the MIC of ampicillin. Periodic saccular outpouchings along the filaments. Bar, 1 ,um.
addition, transmission electron microscopy of E. cloacae treated with cefoxitin revealed that the resulting filaments and saccular outpouchings were multinucleate and nonseptate (7). Similar results were noted when E. cloacae was treated with benzylpenicillin or cephalothin. In a report on the effects of carbenicillin on Pseudomonas pseudomallei, Dilworth et al. (2) observed by transmission electron microscopy the presence of saccular outpouchings on the resulting filaments at sites where transverse septa would occur. A similar situation is reported here in H. influenzae with ampicillin. Dilworth et al. (2) further suggest that the presence of beta-lactam antibiotics preferentially inhibits the formation of transverse septa, as opposed to cell wall peptidoglycan in general. Although conclusive evidence has not been obtained, this process could readily result in the formation of multinucleate, nonseptate filaments. This investigation was supported in part by the Faculty Research and Professional Development Fund of North Carolina State University and by the North Carolina Agriculture Experiment Station.
LITERATURE CITED 1. Bottone, E. J., Z. Brandman, and S. S. Schneierson.
2.
3.
4. 5.
6.
7.
1976. Spheroplasts of Haemophilus influenzae induced by cell wall-active antibiotics and their effect upon the interpretation of susceptibility tests. Antimicrob. Agents Chemother. 9:327-333. Dilworth, C. R., M. Franklin, and G. N. Bance. 1974. Morphological and cytological effects of carbenicillin on Pseudomonas pseudomallei. Can. J. Microbiol. 20:1229-1233. Fleming, A., A. Voureka, I. R. H. Kramer, and W. H. Huges. 1950. The morphology and motility of Proteus vulgaris and other organisms cultured in the presence of penicillin. J. Gen. Microbiol. 4:257-269. Lorian, V., and B. Atkinson. 1976. Effects of subinhibitory concentrations of antibiotics on cross walls of cocci. Antimicrob. Agents Chemother. 9:1043-1055. Nishino, T., and S. Nakazawa. 1973. Cephalexin-induced morphological alterations in the surface structures of Staphylococcus aureus and Escherichia coli demonstrated by scanning electron microscopy. Jpn. J. Microbiol. 17:383-392. Prior, R. B., and R. L. Perkins. 1974. Artifacts induced by preparation for scanning electron microscopy in Proteus mirabilis exposed to carbenicillin. Can. J. Microbiol. 20:794-795. Zimmerman, S. B., and E. 0. Stapley. 1976. Relative morphological effects induced by cefoxitin and other beta-lactam antibiotics in vitro. Antimicrob. Agents Chemother. 9:318-326.
Vol. 11, No. 3 Printed in U.S.A.
NOTES Ampicillin-Induced Morphological Alterations of Haemophilus influenzae Type b RICHARD D. KLEIN AND G. H. LUGINBUHL* Department of Microbiology, North Carolina State University, Raleigh, North Carolina 27607
Received for publication 29 October 1976
Inhibitory levels of ampicillin induced filamentation of growing Haemophilus influenzae ATCC 19418 within 30 min. Filaments became swollen and interrupted by regular periodic saccular outpouchings along the major axis. The degree of filamentation was dependent upon ampicillin concentration and time. The beta-lactam antibiotics, which inhibit peptidoglycan biosynthesis, have been demonstrated to induce a variety of morphological changes in gram-positive and gram-negative bacteria (3, 4, 7). These changes include spheroplasting, filament formation, and lysis. Haemophilus influenzae has been reported to form spheroplasts in the presence of bacteriostatic concentrations of ampicillin (1). In the present study the morphological effects of ampicillin on H. influenzae type b were examined by light and scanning electron microscopy. H. influenzae type b (ATCC 19418), an ampicillin-susceptible, beta-lactamase-negative strain (minimum inhibitory concentration [MIC], 0.1 ,g/ml), was grown overnight at 37°C in brain heart infusion broth (Difco) with 1% Difco supplement C (BHIC) on a rotary shaker. The culture was diluted 1:10 in BHIC, and 0.45 ml ofthis dilution was added to 8.55 ml of fresh BHIC and incubated for 2 h at 37°C on a rotary shaker. One milliliter of appropriate concentrations of ampicillin (Parke Davis oral suspension) was added to give final ampicillin concentrations of 0.1, 1, and 10 times the MIC. No ampicillin was added to one tube as a control. At 30 min and 4 h, samples were removed and processed for light and scanning electron microscopy. Bacterial suspensions on glass slides were air dried, heat fixed, and stained with crystal violet. Samples for scanning electron microscopy were filtered through 0.45-gm Nucleopore filters, dehydrated through 100% ethanol, critical point dried, and observed in an ETEC scanning electron microscope. Light microscopy of the untreated control culture revealed a uniform population of coccobacilli. When incubated for 30 min with ampicillin at a concentration 10 times the MIC, bacillary cells two to three times larger than the
control were observed. After 4 h at this concentration, large, swollen, elongated cells were prevalent. These cells were irregular and displayed circular distortions or saccular outpouchings of apparent cellular material along the major axis of the cell. As examined by scanning electron microscopy (Fig. 1), the untreated control cells were coccobacillary in nature and smooth surfaced and ranged from 0.7 to 1 ,um in length. After 30 min of incubation with 10 times the MIC of ampicillin (Fig. 2), the cells displayed a rough surface and were elongated, ranging from 1 to 3 ,um in length. Obvious septum formation had taken place between some cells (Fig. 3). After 4 h of incubation with 10 times the MIC of ampicillin (Fig. 4), elongated filaments were present. These filaments were smooth surfaced and frequently intertwined. No evidence of septum formation was observed. Periodic saccular outpouchings were present along the filaments (Fig. 5). The diameter of these spherical saccular outpouchings was not consistent. At concentrations of 0.1 and 1 times the MIC of ampicillin the morphological changes were less evident. At both concentrations, after 30 min, 4 h, and 24 h of exposure, the cells were not appreciably elongated, ranging from 1 to 3 ,tm in length. The only dramatic change noted was an increased roughness of the cell surface. At 24 h, ampicillin at 10 times the MIC is bactericidal, killing greater than 99.9% of the cells, whereas the lower concentrations of ampicillin, 0.1 and 1 times the MIC, are not bactericidal. A variety of gram-negative organisms have been demonstrated to form filaments in the presence of beta-lactam antibiotics. Proteus mirabilis (6) and Proteus vulgaris (3) produce filaments in response to carbenicillin and penicillin, respectively. The same phenomenon has 559
560
NOTES
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 1. Scanning electron micrograph of untreated control cells. Bar, 1 um.
FIG. 2. H. influenzae after 30 min of incubation with 10 times the MIC ofampicillin. Cells are elongated. Bar, 1 .tm.
been reported for Enterobacter cloacae (7) and Escherichia coli (5) with cefoxitin and cephalexin, respectively. In addition, filaments of H. influenzae have been reported by light micros-
copy after exposure to bacteriostatic concentrations of ampicillin (1). In each of these studies the presence of saccular outpouchings of the filament was noted.
VOL. 11, 1977
Saccular outpouching of H. influenzae filaments may result from the inhibition of septum formation at ampicillin concentrations greater than the MIC. This is suggested by the pres-
NOTES
561
ence of obvious septation between cells after a 30-min exposure to 10 times the MIC (Fig. 3) in contrast to the absence of septation after a 4-h exposure to this concentration (Fig. 4, 5). In
FIG. 3. H. influenzae after 30 min of incubation with 10 times the MIC of ampicillin. Septum formation has been completed between some cells. Bar, 1 ,im.
FIG. 4. H. influenzae after 4 h of incubation with 10 times the MIC of ampicillin. Filamentous forms predominate. Bar, 1 jum.
562
NOTES
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 5. H. influenzae after 4 h of incubation with 10 times the MIC of ampicillin. Periodic saccular outpouchings along the filaments. Bar, 1 ,um.
addition, transmission electron microscopy of E. cloacae treated with cefoxitin revealed that the resulting filaments and saccular outpouchings were multinucleate and nonseptate (7). Similar results were noted when E. cloacae was treated with benzylpenicillin or cephalothin. In a report on the effects of carbenicillin on Pseudomonas pseudomallei, Dilworth et al. (2) observed by transmission electron microscopy the presence of saccular outpouchings on the resulting filaments at sites where transverse septa would occur. A similar situation is reported here in H. influenzae with ampicillin. Dilworth et al. (2) further suggest that the presence of beta-lactam antibiotics preferentially inhibits the formation of transverse septa, as opposed to cell wall peptidoglycan in general. Although conclusive evidence has not been obtained, this process could readily result in the formation of multinucleate, nonseptate filaments. This investigation was supported in part by the Faculty Research and Professional Development Fund of North Carolina State University and by the North Carolina Agriculture Experiment Station.
LITERATURE CITED 1. Bottone, E. J., Z. Brandman, and S. S. Schneierson.
2.
3.
4. 5.
6.
7.
1976. Spheroplasts of Haemophilus influenzae induced by cell wall-active antibiotics and their effect upon the interpretation of susceptibility tests. Antimicrob. Agents Chemother. 9:327-333. Dilworth, C. R., M. Franklin, and G. N. Bance. 1974. Morphological and cytological effects of carbenicillin on Pseudomonas pseudomallei. Can. J. Microbiol. 20:1229-1233. Fleming, A., A. Voureka, I. R. H. Kramer, and W. H. Huges. 1950. The morphology and motility of Proteus vulgaris and other organisms cultured in the presence of penicillin. J. Gen. Microbiol. 4:257-269. Lorian, V., and B. Atkinson. 1976. Effects of subinhibitory concentrations of antibiotics on cross walls of cocci. Antimicrob. Agents Chemother. 9:1043-1055. Nishino, T., and S. Nakazawa. 1973. Cephalexin-induced morphological alterations in the surface structures of Staphylococcus aureus and Escherichia coli demonstrated by scanning electron microscopy. Jpn. J. Microbiol. 17:383-392. Prior, R. B., and R. L. Perkins. 1974. Artifacts induced by preparation for scanning electron microscopy in Proteus mirabilis exposed to carbenicillin. Can. J. Microbiol. 20:794-795. Zimmerman, S. B., and E. 0. Stapley. 1976. Relative morphological effects induced by cefoxitin and other beta-lactam antibiotics in vitro. Antimicrob. Agents Chemother. 9:318-326.
