resistance mechanism of chloramphenicol in streptococcus

Microbiol. Immunol. Vol. 21 (2), 69-76, 1977

Resistance Mechanism of Chloramphenicol in Streptococcus haemolyticus, Streptococcus

pneumoniae and Streptococcus faecalis

Sadao MIYAMURA, Hiroshi OCHIAI, Yoshiyuki NITAHARA, Yoji NAKAGAWA, and Michinori TERAO

Department of Bacteriology, Niigata University School of Medicine, Niigata

(Received for publication, June 30, 1976)

Abstract The chloramphenicol resistance of Streptococcus haemolyticus, Streptococcus pneumoniae and Streptococcus faecalis isolated from clinical materials was proved to be due to an inactivating enzyme produced by these bacteria. The inactivated products of chloramphenicol were identified as 1-acetoxy, 3-acetoxy and 1,3-diacet-oxy derivatives by thin-layer chromatography and infrared spectroscopy. The responsible enzyme was thus confirmed to be chloramphenicol acetyltransferase. The enzyme was inducible. It was partially purified by ammonium sulfate pre-cipitation, DEAE-cellulose chromatography and gel filtration on Sephadex G-150. The enzymes obtained from S. haemolyticus, S. pneumoniae and S. faecalis have been compared with the conclusion that they are identical with respect to molecular weight (approximately 75,000-80,000), optimum pH and heat stability.

It has generally been considered that streptoccocci seldom acquire resistance to chemotherapeutic agents. However, recently, strains resistant to chloramphenicol, tetracycline or erythromycin have been isolated and consequently chemotherapy against infectious diseases caused by streptococci has been greatly affected (4, 6, 7).

Chloramphenicol resistance by staphylococci and gram-negative bacteria carrying R plasmid has been shown to be due to the production of the chloramphenicol in-activating enzyme, chloramphenicol acetyltransferase, by these bacteria (2, 3, 8).

Recently we derived chloramphenicol-resistant strains of Streptococcus haemo-lyticus, Streptococcus pneumoniae and Streptococcus faecalis successively from various clinical materials in this district. This paper is concerned with the confirmation and com-

parison of the mechanism of chloramphenicol resistance in the strains of Streptococcus.

MATERIALS AND METHODS

Bacterial strains. Streptococcus haemolyticus 0-78, Streptococcus pneumoniae N-77 and Streptococcus faecalis N-117 were used in this experiment. S. haemolyticus 0-78 is one of 52 strains isolated from pharyngeal swabs of patients with scarlet fever which broke out in Niigata city in the autumn of 1974, and belongs to type 12 in group A. S.

pneumoniae N-77 was isolated from sputa of a pneumonia patient and S. faecalis N-117

69

70 S. MIYAMURA ET AL

was derived from the urine of a cystitis patient in the Niigata University Hospital. Assay of antibiotic susceptibility. For determining the minimal inhibitory con-

centration of antibiotics, a serial dilution method with the nutrient agar medium containing 10% sheep blood was used. Results were obtained after incubation at

37 C for 24 hr.Determination of chloramphenicol inactivation. Organisms to be tested for ability to

inactivate the antibiotic were inoculated into 5 ml of heart infusion broth and in-cubated at 37 C for 24 hr. To each culture was added an equal volume of 0.01 M

phosphate buffer solution (pH 7.0) containing various concentrations of chloram-phenicol, and the tubes were placed in a water bath at 37 C. After 3 hr, the tubes were heated to 60 C for 30 min to stop the enzymatic reaction, the majority of cells were spun down in a centrifuge (8,000 rpm for 20 min), and the potency of the re-sidual chloramphenicol in the supernatant was determined by the thin-layer cup method, using Bacillus subtilis ATCC 6633 as the test organism.

For the cell extracts expected to have chloramphenicol acetyltransferase, acetyl CoA was added in addition to the sample and chloramphenicol in 0.01 M Tris-HC1 buffer (pH 7.8), and the degree of inactivation ofchloramphenicol was assayed by the same method.

Assay of chloramphenicol acetyltransferase. The enzyme was assayed spectropho-tometrically using 5, 5'-dithiobis-2-nitrobenzoic acid (DTNB) (1). Reactions were

performed at 37 C in a final volume of 1 ml which contained 0.01 M Tris-HC1 buffer (pH 7.8), 0.5 mm DTNB, 0.3 mm acetyl CoA, 0.15 mm chloramphenicol and 0.1 ml of enzyme solution. The enzyme activity was measured by following the change in adsorbance at 412 nm resulting from the release of thionitrobenzoic acid.

Extraction and purification of the inactivating enzyme. Test strains were grown in heart infusion broth with 1% glucose at 37 C with shaking. After 24 hr, the cells were harvested by centrifugation at 8,000 rpm for 20 min, washed twice with 0.01 m Tris-HC1 buffer (pH 7.8), resuspended in the same buffer and disrupted by a cell homogenizer (Braun Co.) for 10 min. After centrifugation at 18,000 rpm for 30

min, the supernatant was brought to 90% saturation of ammonium sulfate and the precipitate was collected and dialyzed overnight against the same buffer. The resulting extract was applied to a diethylaminoethyl (DEAE)-cellulose column and eluted with 0.01 M Tris-HC1 buffer containing a linear gradient of zero to 0.5 M sodium chloride. The fractions with the highest activity were collected and applied to gel filtration on Sephadex G-150. The resultant pooled eluate with the activity was concentrated under reduced pressure and stored at 10 C.

Identification of inactivated products of chloramphenicol. Products of chloramphenicol inactivated by cell extracts were analyzed by thin-layer chromatography using silica

gel as the absorbent. Cell extract was added to Tris-HC1 buffer containing 0.2 mm chloramphenicol and 0.2 mm acetyl CoA, and allowed to react at 37 C for 1 hr. The reaction mixture was extracted with ethyl acetate and the extract was chroma- tographed with a solvent system of chloroform: methano1=95: 5. Chemically syn-thesized monoacetyl and diacetyl derivatives of chloramphenicol were ,used as reference substances. 1, 3-diacetoxy chloramphenicol was prepared by reacting

CHLORAMPHENICOL RESISTANCE IN STREPTOCOCCI 71

chloramphenicol with acetic anhydride in dry pyridine and 3-acetoxy and 1-acetoxy chloramphenicol were prepared by reacting chloramphenicol with acetyl chloride

in dry pyridine and separating by thin-layer chromatography after ethyl acetate extraction (5). The chromatograms were surveyed for ultraviolet absorption. Intrared absorption spectra of inactivated products of chloramphenicol were obtained after thin-layer chromatographic separation.

Induction effects. Test strains were cultivated in media with and without the addition of 0.5 iug/m1 of chloramphenicol. Cells were harvested at intervals and chloramphenicol acetyltransferase activities per mg of protein of the cell extracts were determined.

RESULTS

Susceptibilities qf the Test Organisms to Antibiotics In vitro susceptibilities of the test strains to seven antibiotics are shown in Table

1. Three strains tested were resistant to chloramphenicol and the minimal inhibitory concentrations were 31.2 1ug/m1 against S. haemolyticus and S. pneumoniae and 125

,ug/m1 against S. faecalis.

Chloramphenicol-Inactivating Activity of the Test Organisms The inactivating activities of the three strains were measured by the bioassay

mentioned above. As a result, 1 ml of the respective broth of S. haemolyticus and S.

pneumoniae inactivated 12 ,ug of chloramphenicol and that of S. faecalis inactivated 31 eug of chloramphenicol.

Comparative Studies of Purified Enzymes from S. haemolyticus, S. pneumoniae and S. faecalis

Figure 1 depicts the effect of pH on enzyme activity as measured in Tris-maleate buffer over the range of 6.0 to 8.5. All three preparations showed an optimal pH of 7.8.

The heat stability of enzyme solutions allowed to stand at various temperatures

Table 1. Susceptibility of test strains to antibiotics

CP, chloramphenicol; TC, tetracycline; EM, erythromycin; SM, streptomycin;

KM, kanamycin; PC, penicillin-G; CER, cephaloridine.


for 20 min was determined. As shown in Fig. 2, all preparations lost their activity at 75 C.

Calibration of the preparative Sephadex G-150 column with appropriate purified

protein markers (aldolase, bovine serum albumin, egg albumin and cytochrome c) gave values of 75,000-80,000 for the molecular weights of active enzymes from the three test strains.

Identification of Inactivated Products The inactivated products of chloramphenicol were identified by the procedures

Fig. 1. Effects of pH on the activities of enzymes from S. haemolyticus 0-78 (., S. pneumoniae N-77

(0), and S. faecalis N-117 (A). The initial reac-tion rates were determined spectrophotomerically in the presence of 0.05 M Tris-maleate buffer at the specified pH.

Fig. 2. Heat stability of inactivating enzymes from S. haemolyticus 0-78 (., S. pneumoniae N-77 (0), and S. faecalis N-117 (A).


described in MATERIALS AND METHODS. In each case, three spots were detected on a thin-layer chromatogram as shown in Fig. 3. The products showed the same chromatographic mobilities as 1-acetoxy, 3-acetoxy and 1, 3-diacetoxy chloram-

phenicol. In addition, the infrared absorption spectra of the products (Fig. 4) showed in every case carbonyl absorption of the acetyl group at 1,745 cm-1, absorption of acetate at 1,225 cm-1 and absorptions at 3,320 cm-1and 1,680 cm-1 corresponding to NH and CONH. The inactivated products from all the three test strains were thus proved to be acetylated chloramphenicol.

From the results obtained in the present study, the inactivation of chloram-

phenicol was concluded to be due to the enzymatic actions of chloramphenicol acetyltransferase produced by these organisms.

Fig. 3. Thin-layer chromatogram of the inacti-

vated products of chloramphenicol and the au-

thentic samples observed by ultraviolet absorp-

tion. The solvent system was chloroform : methanol＝ 95):5.(A)Thc inactivatcd produc.ts ofchloram一

phenicol by S. faecalis N-117, (B) by S. haemoly-ticus 0-78, and (C) by S. pueumaniae N-77. (D) Au-thentic samples of 1,3 diacetoxy, 3-acetoxy, and 1-acetoxy chloramphenicol, and chloramphenicol.


Induction of the Enzyme The effect on the production of the enzyme of the addition of 0.5 ,ugiml of

chloramphenicol to heart infusion broth at the time of cultivation was investigated. As indicated in Fig. 5, all the test strains showed approximately 10 times as much activity with the addition of chloramphenicol.

DISCUSSION

Recently, antibiotic-resistant streptococci have been isolated from patients with various infectious diseases and increasing attention has been paid to the mechanism of the resistance. In this paper, we revealed that chloramphenicol-resistant strepto-cocci, S. haemolyticus, S. pneumoniae and S. faecalis, produced chloramphenicol acetyl-transferase and inactivated chloramphenicol. The chloramphenicol acetyltrans-ferase has already been shown to play an important role in the chloramphenicol resistance of various gram-negative bacteria and staphylococci, especially in plasmid-derived resistance. Although there was no direct testing on the plasmids in this experiment, the role of chloramphenicol acetyltransferase in the resistance suggests their participation.

Fig. 4. Infrared absorption spectra of chloramphenicol, inactivated product of chlorampheni-col by the enzyme from S. pneumoniae N-77 and the authentic sample of 3-acetoxy chloram-phenicol (KBr disks).


Hemolytic streptococci and pneumococci cause many serious infectious diseases,

and enterococci occupy an important position as enteric bacteria. The finding

that chloramphenicol resistance of these three species was due essentially to chloram-

phenicol acetytransferase indicates that drug resistance by the same mechanism is spreading among a wide range of bacteria.

REFERENCES

1) Alpers, D.H., Appel, S.H., and Tomkins, G.M. 1965. A spectro-photometric assay for thiogalac-toside transacetylase. J. Biol. Chem. 240: 10-13.

2) Miyamura, S. 1964. Inactivation of chloramphenicol by chloramphenicol-resistant bacteria. J. Pharm. Sci. 53: 604-607.

Fig. 5. Induction of chloramphenicol acetyltransferase by chloramphenicol. S. haemolyticus 0-78 (., S. pneumoniae N-77 (0) and S. faecalis N-117 (A) were cul-tured overnight under two different conditions : broken lines represent the absence of chloramphenicol andsolid lines represent the presence of 0.5,ug/m1 of chloramphenicol. The crude enzymes were extracted as described in MATERIALS AND METHODS and prepared as 1 mg of protein per ml.


3) Okamoto, S., and Suzuki, Y. 1965. Chloramphenicol, dihydrostreptomycin, and kanamycin-inactivating enzymes from multiple drug-resistant Escherichia coli carrying episome "R". Nature

208: 1301-1303. 4) Okubo, N., Kashiwagi, Y., Shibata, M., and Tokue, S. 1973. A six year study of the antibiotic

sensitivities of group. A streptococci. J. Japan. Assoc. Infect. Dis. 47: 506-509 (in Japanese). 5) Robestock, M.C., Crooks, H.M., Jr., Controulis, J., and Bartz, O.R. 1949. Chloramphenicol

(Chloromycetin). IV. Chemical studies. J. Amer. Chem. Soc. 71: 2458-2462. 6) Ubukata, K., Konno, M., and Fujii, R. 1973. Macrolides, lincomycin, chloramphenicol and

tetracycline resistance in group A streptococci. J. Pediat. Prac. 26: 1451-1458 (in Japanese). 7) Ubukata, K., Takahashi, Y., Konno, M. Fujii, R., and Sasaki, U. 1975. Isolation and the drug-

resistance of pneumococci in acute respiratory infections in children. J. Pediat. Prac. 28: 1292 - 1297 (in Japanese) .

8) Winshell, E., and Shaw, W.V. 1969. Kinetics of induction and purification of chloramphenicol acetyltransferase from chloramphenicol-resistant Staphylococcus aureus. J. Bacteriol. 98: 1248-1257.

Requests for reprints should be addressed to Dr. S. Miyamura, Department of Bacteriology, Niigata University School of Medicine, Niigata, Japan.

resistance mechanism of chloramphenicol in streptococcus

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