REVIEWS OF INFECTIOUS DISEASES. VOL. I, NO.5 .• SEPTEMBER-OCTOBER 1979 © 1979 by The University of Chicago. 0162-0886/79/0105-0014$00.75

Drug-Inactivating Enzymes of Bacteria Grown in Subminimal Inhibitory Concentrations of Antibiotics G. Gialdroni Grassi

From the Faculty of Medicine and Surgery, University of Pavia, Pavia, Italy

The effects of subminimal inhibitory concentrations (sub-MICs) of antibiotics on bacteria have been examined from both morphological [1-8] and functional [9, 10] points of view. We have demonstrated that with a single exposure to sub-MICs of antibiotics, both grampositive and gram-negative bacteria are delayed in reaching the stationary phase, and that· the final concentration of cells at the end of growth is either the same as for untreated cells or one log unit below [11]. We also have studied the one-step mutations that appear after bacteria are exposed to subMICs of antibiotics. We observed that neither previous exposure of bacteria to sub-MICs of the same or other antibiotics nor the simultaneous treatment of bacteria to high selecting concentrations of another antibiotic modified this mutation rate. The exceptions we observed to this behavior were with trimethoprim, rifampicin, and fosfomycin: in many instances, the mutation rate of bacteria exposed to sub-MICs of one of these drugs was. reduced by previous exposure of these bacteria to sub-MICs of one of the drugs [12].

We have conducted further experiments in which bacteria were repeatedly exposed to subMICs of antibiotics. When some strains of Escherichia coli were submitted to serial transfers in sub-MICs of chloramphenicol, a certain degree of resistance to the drug was attained. From initial MICs of 0.8-12.5 Ilg of chloramphenicol/ml, MICs of 25-50 Ilg of chloramphenicol/ml were reached after 50 transfers. In parallel cultures that were transferred daily into increasing concentrations of chloramphenicol, MICs of the drug reached 400 Ilg/ ml and 800 Ilg/ ml after 10 transfers. It was found that the resistant strains were able to reduce the N0 2 group of the antibiotic molecule; this capacity was proportional to the degree of resistance of the bacterial strain. On the other hand, it is not clear what part this reducing capacity plays in the production of resistance, since acetylating enzymes appear to playa major role in the development of resistance [13-15]. In order to get more information about the mechanisms by which resistance is obtained following the repeated exposure of bacteria to subMICs of antibiotics, we exposed strains of E. coli, either producing or not producing aminoglycoside-inactivating enzymes, and Staphylococcus aureus strain 209 to either sub-MICs or increasing concentrations of gentamicin. At the end of the experiment, both the degree of resistance to anti-

Please address requests for reprints to Prof. G. Gialdroni Grassi, Faculty of Medicine and Surgery, University of Pavia, Pavia, Italy.

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Repeated transfers of strains of Escherichia coli and Staphylococcus aureus in medium containing subminimal inhibitory concentrations (sub-MICs) of gentamicin caused a moderate increase in the minimal inhibitory concentration of gentamicin. At the end of such transfers, E. coli K12 produced aminoglycoside phosphotransferase(3 ')-1 [APH(3 '»), and E. coli R112, which carries the plasmid-coded enzyme APH(3 ')-1, also produced the acetylating enzyme aminoglycoside acetyltransferase(2 ') [AAC(2 ')]. E. coli R148, which produces aminoglycoside phosphotransferase(3 ')-11 [APH(3 ')-11), did not change its output of enzymes. Repeated transfers to media containing increasing concentrations of gentamicin resulted in the development of complete resistance to all aminoglycosides without the concurrent development of any demonstrable new enzyme activity. With repeated transfers in drug-free medium, a complete reversal to susceptibility to gentamicin, but not to other aminoglycosides, was obtained for strains that had previously been transferred in sub-MICs of gentamicin, whereas strains that had been transferred in increasing concentrations of gentamicin did not revert to their original sensitivity to aminoglycosides despite repeated transfers in drug-free medium.

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biotics and the output of aminoglycoside-inactivating enzymes of the strains were determined. The possibility of reverting resistant strains to their original antibiotic susceptibility by repeatedly transferring cultures in drug-free medium was also investigated. Materials and Methods

Results

After repeated subculture in sub-MICs of gen-. tamicin, the three strains of E. coli tested showed only a four- to eightfold increase in the MIC of gentamicin; the final value still indicated a low level of resistance (table 1). Enzyme assays done at the end of the experiment showed that there was APH(3 ')-1 activity in E. coli KI2. In E. coli RII2, we could demonstrate the presence of aminoglycoside acetyltransferase(2') [AAC(2 ')] as well as the original plasmid-coded enzyme, APH(3 ')-I. On the other hand, in E. coli RI48, which carries the plasmidcoded enzyme APH(3 ')-11, only the original enzyme was found at the end of the experiment (ta-

Table 1. Effect of repeated subculture (n = 20) in subminimal inhibitory concentrations of gentamicin on the susceptibility of some strains of Escherichia coli and Staphylococcus aureus to gentamicin.

Microorganism

Aminoglycoside-inactivating enzyme produced by original strain

Before subculture

E. coli K12 E. coli R112 E. coli R148 S. aureus 209P

None APH(3 ')-1 APH(3 ')-11 None

0.5 2.0 0.5 0.2

Experiments were done in trypticase soy broth. APH

=

NOTE.

MIC of gentamicin (J.lg/ml) After 20 subcultures 4

8 4

0.4 aminoglycoside phosphotransferase.

No. of subcultures in drug-free medium for reversal to original susceptibility

12 18 12 2

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Microorganisms. The following strains of bacteria were employed in the experiments: E. coli KI2; E. coli RII2, which produces aminoglycoside phosphotransferase(3 ')-1 [APH(3 ')-1]; E. coli RI48, which produces aminoglycoside phosphotransferase(3 ')-11 [APH(3 ')-11]; and S. aureus 209P. Production ofresistance. Trypticase soy broth was used in all studies. In one set of experiments, bacteria were transferred daily to medium that contained gentamicin at a concentration equal to one-quarter of the MIC for the strain. In the other set, microorganisms were transferred daily to medium that contained a concentration of gentamicin that was increased daily. Simultaneously, a control series was tested in which broth with no added antibiotic was used. The inoculum consisted of 106 cfu of an overnight culture grown at 37 C in trypticase soy broth. The experiment was concluded after 20 transfers. Assessment of resistance. At the beginning and at the end of the experiment, the susceptibility of bacteria to gentamicin and other aminoglycoside antibiotics (neomycin, kanamycin, paromomycin, tobramycin, lividomycin, dideoxykanamycin B, amikacin, butirosin, and ribostamycin) was determined both by broth dilution using trypticase soy broth and by agar dilution in Mueller-Hinton agar as described by Price et al. [16].

The susceptibility of bacteria to ampicillin, chloramphenicol, and tetracycline also was determined by broth dilution in trypticase soy broth. Preparation of enzyme. The shock method of Nossal and Heppel [17] was used to release enzymes. The starter broths were prepared by inoculating 10 ml of trypticase soy broth containing gentamicin at the highest concentration that permitted bacterial growth. Enzyme assays. The methods described by Benveniste et al. [18], Davies et al. [19], and Haas and Davies [20] were used. Residual antibiotic activity. The residual antibiotic activity in the medium was determined by the cup-plate method [21] with the use of Bacillus subtilis ATCC 6633 (American Type Culture Collection, Rockville, Md.). Elimination ofplasmids. To eliminate R plasmids from gram-negative bacteria, the sodium dodecyl sulfate method was used [22].

Gialdroni Grassi

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Table 2. Effect of repeated subculture (n = 20) in subminimal inhibitory concentrations of gentamicin on resistance and production of aminoglycoside-inactivating enzymes of some strains of Escherichia coli and Staphylococcus aureus. Before subculture Microorganism

MIC of gentamicin (I-lg/ml)

After 20 subcultures

Enzymes produced

MIC of gentamicin (I-lg/ml)

E. coli KI2 E. coli RII2

2 4

None APH(3 ')-1

8

E. coli RI48

I 0.5

APH(3 ')-11 None

4 0.5

S. aureus 209P

8

Enzymes produced

Resistance profile·

APH(3 ')-1 APH(3 ')-1, AAC(2 ') APH(3 ')-11 APH(5")

Lv, Kn, Nm, Pm AIIAmG except Ak Kn, Nm, Pm, Bs Rm

ble 2). Treatment with sodium dodecyl sulfate abolished all of these enzyme activities. The resistance profiles determined by the agar dilution method follow quite well the enzymatic activity displayed by the tested strains. (The common definitions of resistance were used; Le., organisms were considered resistant to kanamycin, amikacin, butirosin, lividomycin, ribostamycin, and paromomycin when the MICs of these antibiotics were> 20 tJg/ml and resistant to gentamicin, tobramycin, dideoxykanamycin B, and neomycin when the MICs were> 8 tJg/ml.) According to these definitions of resistance, neither E. coli K12 nor E. coli R148 could be considered resistant to gentamicin after serial transfers in subMICs of that antibiotic. In the case of E. coli Rl12, gentamicin was utilized as a substrate for the final enzymes, and the resistance profile indi-

cated that the strain was resistant to all aminoglycosides except amikacin (table 2). In all cases, aminoglycosides were inactivated very little by the different strains of E. coli: the range of inactivation was ru 12070-25070. The repeated transfer of S. aureus 209P to subMICs of gentamicin did not produce any resistance to gentamicin; however, at the end of the experiment, phosphotransferase activity was found that accounted for the acquired resistance to ribostamycin (table 2). With regard to resistance acquired through serial transfers in increasing concentrations of gentamicin, the results were uniform for all strains tested. A high degree of resistance to gentamicin was obtained, with a 128- to 640-fold increase in the original MIC (table 3). In addition, complete resistance to all tested

Table 3. Effect of repeated subculture (n = 20) in increasing subminimal inhibitory concentrations of gentamicin on the susceptibility of some strains of Escherichia coli and Staphylococcus aureus to gentamicin.

Microorganism E. coli K12 E. coli R112 E. coli R148

S. aureus 209P

Aminoglycoside-inactivating enzyme produced by original strain None APH(3 ')-1 APH(3 ')-11 None

Before subculture

After 20 subcultures

No. of subcultures in drug-free medium for reversal to original susceptibility

0.5 2.0 0.5 0.2

128 256 128 128

NR NR NR NR

MIC of gentamicin (I-lg/ml)

NOTE. Experiments were done in trypticase soy broth. Abbreviations: APH = aminoglycoside phosphotransferase; NR = no reversal after 20 subcultures.

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NOTE. Resistance was determined by the agar dilution method [16J. Abbreviations: APH = aminoglycoside phosphotransferase; AAC = aminoglycoside acetyltransferase. AmG = aminoglycosides tested, a group that included gentamicin, tobramycin, and the following: Ak = amikacin; Bs = butirosin; Lv = lividomycin; Kn = kanamycin; Nm = neomycin; Pm = paromomycin; and Rm = ribostamycin. • Organisms considered resistant to Kn, Ak, Bs, Lv, Rm, and Pm when MICs of these antibiotics were >20 I-lg/ml and resistant to gentamicin, tobramycin, dideoxykanamycin B, and Nm when MICs were >8 I-lg/ml.

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Table 4. Effect of repeated subculture (n = 20) in increasing subminimal inhibitory concentrations of gentamicin on resistance and production of aminoglycoside-inactivating enzymes of some strains of Escherichia coli and Staphy-

lococcus aureus. After 20 subcultures

Before subculture Microorganism

MIC of gentamicin (~g/ml)

Enzymes produced

E. coli K12 E. coli R112 E. coli R148 S. aureus 209P

2 4 1 0.5

None APH(3 ')-1 APH(3 ')-II None

MIC of gentamicin (~g/ml) Enzymes produced 128 256 128 128

None APH(3 ')-1 APH(3 ')-II None

Resistance to AmG* Complete Complete Complete Complete

aminoglycosides was obtained. Neither significant new enzyme activity nor a significant increase in preexisting enzyme activity was demonstrated in the resistant strains obtained by this procedure (table 4). Susceptibility to ampicillin, chloramphenicol, and tetracycline remained unchanged throughout the experiment. After passages in drug-free medium, strains subjected previously to repeated transfers in sub-MICs of gentamicin reverted to their original susceptibility to gentamicin (table 1). However, only partial reversal occurred to the original susceptibility to the other aminoglycosides to which resistance, with concomitant production of inactivating enzymes, had been obtained. Strains subcultured in increasing concentrations of gentamicin did not revert to the original susceptibility to aminoglycosides after daily transfers in drug-free medium for three weeks (table 3). Conclusion and Discussion

The present experiments show that repeated exposure to sub-MICs of gentamicin can produce a low degree of resistance to the inducing agent. This resistance seems to be accompanied by the production of inactivating enzymes that, however, are often unrelated to the inducing agent. In fact, enzymes that do not have gentamicin as a substrate have been found to be produced by bacterial strains exposed to gentamicin. A reversal to initial sensitivity to gentamicin is obtained through passage of resistant bacteria in antibiotic- free medium, whereas there is no reversal to sensitivity

to the other aminoglycosides to which there exists an apparently enzyme-mediated resistance. The enzymic activity seems to be plasmid related, since treatment with sodium dodecyl sulfate abolishes the inactivating property. At present, we have no data that explain the mechanism by which exposure to an aminoglycoside can evoke the activity of an enzyme for which the aminoglycoside does not act as a substrate. In fact, it has been postulated that in some cases an episome may function by depressing a chromosomal determinant rather than by dictating the synthesis of a new protein [14, 23]. We observed that usually only a small percentage of the antibiotic towards which resistance was demonstrated was inactivated and that sometimes the degree of resistance was not correlated with the degree of drug inactivation. Our findings reflect well-known observations, and many models have been proposed to explain this behavior. It has been supposed that a modification in only a small part of the drug molecule is sufficient either to block the transport system for the drug itself and for some related antibiotics or to stimulate a mechanism by which the drug is pumped out of the cell or hindered in its access into the cell [24]. Our findings seem to add another question to those posed previously. By what mechanism does repeated exposure to sub-MICs of an antibiotic determine a low degree of resistance in vitroto the inducing agent and an even higher degree of resistance, which seems plasmid mediated, to aminoglycosides other than the inducing agent? The resistance obtained by subculturing of bac-

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NOTE. Resistance was determined by the agar dilution method [16]. Abbreviations: APH = aminoglycoside phosphotransferase; AmG = aminoglycosides tested. * See footnote to table 2 for the aminoglycosides tested and the definitions of resistance used.

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References 1. Gardner, A. D. Morphological effects of penicillin on bacteria. Nature 146:837-838, 1940. 2. Hughes, W. H. The structure and development of the induced long forms of bacteria in bacterial anatomy. In Sixth Symposium of the Society for General Microbiology, London, April 1956. Cambridge University Press, Cambridge, 1956, p. 341-360. 3. Lederberg, J., St. Clair, J. Protoplasts and a-type growth of Escherichia coli. J. BacterioI. 75:143-160, 1958. 4. Lorian, V., Sabath, L. D. Penicillins and cephalosporins: differences in morphologic effects on Proteus mirabilis. J. Infect. Dis. 125:560-607, 1972. 5. Lorian, V., Atkinson, B. Abnormal forms of bacteria produced by antibiotics. Am. J. Clin. Pathol. 64:678-688, 1975. 6. Lorian, V., Atkinson, B. Effects of subinhibitory concentrations of antibiotics on cross walls of cocci. Antimicrob. Agents Chemother. 9:1043-1055, 1976. 7. Lorian, V., Atkinson, B. Effects of subinhibitory concentrations of fosfomycin on bacteria. G. Hal. Chemioter. 23:65-74, 1976.

8. Lorian, V., Atkinson, B. Comparison of the effects of mecillinam, a 6-aminopenicillanic acid on Proteus mirabilis, Escherichia coli, and Staphylococcus aureus. Antimicrob. Agents Chemother. 11:541-552, 1977. 9. Greenwood, D. Differentiation of mechanisms responsible for inoculum effect in the response of Escherichia coli to a variety of antibiotics. J. Antimicrob. Chemother. 2:87-95, 1976. 10. Shah, P. M., Heetderks, G., Stille, W. Activity of amikacin at subinhibitory levels. J. Antimicrob. Chemother. 5:97-100, 1976. 11. Gialdroni Grassi, G. Effect of subinhibitory concentrations of antibiotics on the emergence of drug resistant bacteria in vitro. In W. Siegenthaler and R. Luthy [ed.]. Current chemotherapy. Proceedings of the 10th International Congress of Chemotherapy, Zurich, 1977. American Society for Microbiology, Washington, D.C. 1978, p. 77. 12. Gialdroni Grassi, G. Evaluation of in vitro effects of subinhibitory concentrations of antibiotics as function of their possible role in antibiotic combinations. In W. Brumfitt, L. Curcio, and L. Silvestri [ed.]. Workshop on combined antimicrobial therapy. ISEDI, Milan, 1978 (in press). 13. Miyamura, S. Inactivation of chloramphenicol by chloramphenicol-resistant bacteria. J. Pharm. Sci. 53:604607, 1964. 14. Shaw, W. V. Enzymatic chloramphenicol acetylation and R factor induced antibiotic resistance in Enterobacteriaceae. Antimicrob. Agents Chemother. 1966:221226, 1967. 15. Shaw, W. V. The enzymatic acetylation of chloramphenicol by extracts of R-factor-resistant E. coli. J. BioI. Chern. 25:687-690, 1967. 16. Price, K. E., Pursiano, T. A., DeFuria, M.D., Wright, G. E. Activity of BB-K8 (amikacin) against clinical isolates resistant to one or more aminoglycoside antibiotics. Antimicrob. Agents Chemother. 5: 143-152, 1974. 17. Nossal, N. G., Heppel, L. A. The release of enzymes by osmotic shock from Escherichia coli in exponential phase. J. BioI. Chern. 241:3055-3062, 1966. 18. Benveniste, R., Yamada, T., Davies, J. Enzymatic adenylation of streptomycin and spectinomycin by R-factor resistant Escherichia coli. Infec. Immun. 1:109-119, 1970. 19. Davies, J., Brzezinska, M., Benveniste, R. R factors: biochemical mechanisms of resistance to aminoglycoside antibiotics. Ann. N.Y. Acad. Sci. 182:226-233, 1971. 20. Haas, M. J., Davies, J. Enzymatic acetylation as a means of determining serum aminoglycoside concentrations. Antimicrob. Agents Chernother. 4:497-499, 1973. 21. Grove,D. C., Randall, W. A. Methods of antibiotic assay: a laboratory manual. Medical Encyclopedia, New York, 1955. 238 p. 22. Adachi, H., Nakano, M., Inuzuka, M., Tomoeda, M. Specific role of sex pili in the effective eliminatory action of sodium dodecyl sulfate on sex and drug resistance factors in Escherichia coli. J. Bacteriol. 109: 1114-1124, 1972. 23. Datta, N., Kontomichalou, P. Penicillinase synthesis con-

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teria in increasing concentrations of drugs has some different characteristics from that obtained by subculturing of bacteria in sub-MICs of these drugs; the former type of resistance does not seem to be related to production of inactivating enzymes or to the enhancement of activity of preexisting ones. It seems, instead, to be related to the emergence of resistant mutants obtained through the selective pressure exerted by increased concentrations of drug. The resistance in these mutants could be attributed to modified permeability to antibiotics (the so-called permeability mutants) [24, 25]. In the present experiments, the bacterial strains that were transferred repeatedly in media containing increasing concentrations of gentamicin showed resistance to all aminoglycosides but not to ampicillin, chloramphenicol, and tetracycline. Therefore, the modification in permeability seems to be specific for aminoglycosides and does not extend to all antibiotics. Many aspects of the present data need to be clarified in order to assess the relevance of subMICs of antibiotics to the determination of resistance in vitro. It will be even more difficult to determine the role of sub-MICs in vivo, where many different factors can interfere with the production and expression of resistance and where the condition of "subinhibition" cannot be rigorously maintained.

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trolled by infectious R factors in Enterobacteriaceae. Nature 208:239-241, 1965. 24. Davies, J., Kagan, S. A. What is the mechanism of plasmid-determined resistance to aminoglycoside antibiotics? In J. Drews and G. H6genauer [ed.]. R factors: their properties and possible control. Symposium, Baden near Vienna, April 27-29, 1977. Springer-Verlag, New York, 1977, p. 207-219.

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25. Bryan, L. E., van den Elzen, Shahrabadi, M. S. The rela-

tionship of aminoglycoside permeability to streptomycin and gentamicin susceptibility of Pseudomonas aeruginosa. In S. Mitsuhashi and H. Hashimoto [ed.]. Microbial drug resistance. University Park Press, Baltimore, 1975, p. 475-490.

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Drug-inactivating enzymes of bacteria grown in subminimal inhibitory concentrations of antibiotics.

REVIEWS OF INFECTIOUS DISEASES. VOL. I, NO.5 .• SEPTEMBER-OCTOBER 1979 © 1979 by The University of Chicago. 0162-0886/79/0105-0014$00.75 Drug-Inactiv...
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