Vol. 28, No. 8


0095-1137/90/081867-03$02.00/0 Copyright (O 1990, American Society for Microbiology

Use of Commercially Available Rapid Chloramphenicol Acetyltransferase Test To Detect Resistance in Salmonella Species LORENA DE LA MAZA,' SAMUEL I. MILLER,2'3'4 AND MARY JANE FERRAROl.3* Francis Blake Bacteriology Laboratories' and Infectious Diseases Unit,2 Massachusetts General Hospital, Boston, Massachusetts 02114, and Departments of Medicine3 and Microbiology and Molecular Genetics,4 Harvard Medical School, Boston, Massachusetts 02115 Received 22 February 1990/Accepted 30 April 1990

Chloramphenicol resistance among Salmonella spp. has important public health and clinical implications, especially in areas of the world where these strains are endemic. The availability of rapid and sensitive screening methods for detection of antibiotic resistance is important. Therefore, we tested 33 strains of Salmonella for chloramphenicol acetyltransferase (CAT) activity using two rapid techniques. Evaluation of a 1-h tube method and a 30-min commercial disk procedure demonstrated that they are as accurate as standardized susceptibility techniques. Both the 1-h tube and 30-min disk methods detected CAT enzymatic activity produced by one CAT gene copy per cell.

(ATCC 14028) of strains CSO19, CS015, and CS003 was used as a control in all experiments. Stock cultures of Enterococcusfaecalis ATCC 29212, Escherichia coli ATCC 25922, and Staphylococcus aureus ATCC 29213 were obtained from the American Type Culture Collection, Rockville, Md., and were used for quality control of the agar dilution reference method. A chloramphenicol-susceptible strain (MIC, 0.5 ,ug/ml) of H. influenza lacking CAT activity and CATproducing strain TD-1 (MIC, 16 ,ug/ml), which were obtained from G. V. Doern, University of Massachusetts Medical Center, Worcester, were used as controls for the CAT assays. Strains were inoculated onto MacConkey and blood agar media (BBL Microbiology Systems, Cockeysville, Md.) and were incubated at 35°C in an ambient atmosphere for 24 h. Thereafter, a single colony was transferred to a second blood agar plate and incubated under identical conditions; growth from this plate was used to prepare inocula for all subsequent tests. In order to prevent the possible loss of plasmids, the genetically defined strain with multiple plasmid copies was grown in Luria broth with ampicillin at 50 ,ug/ml. MICs were determined by use of an agar dilution technique, as recommended by the National Committee for Clinical Laboratory Standards (14), which uses MuellerHinton II agar (BBL). Chloramphenicol solutions were prepared from standard reference powders (Parke-Davis, Morris Plains, N.J.), as recommended previously (1, 14), by using twofold concentration increments from 0.125 to 512.0 pg/ml. The MIC was defined as the lowest concentration of antibiotic that did not allow macroscopic evidence of growth after 18 h of incubation. The final inoculum on the agar was ca. 104 CFU. All determinations were performed in dupli-

Typhoid fever is still an endemic disease in developing countries (7), and other Salmonella spp. have had a resurgence in immunosuppressed individuals (4). The emergence of chloramphenicol resistance among Salmonella spp. throughout the world (3, 5, 15, 16, 18) has been an important public health problem. Chloramphenicol resistance conferred by the constitutive production of chloramphenicol acetyltransferase (CAT) is mediated by R plasmids among Salmonella strains (6, 10, 17). Since its discovery in 1947, chloramphenicol has been widely used as therapy for Salmonella infections and is the treatment of choice for typhoid fever, because of its easy administration and its clinical efficacy. Because of rare potential adverse reactions (6, 9, 17, 21), its use in the United States has been limited to those life-threatening infections, such as typhoid fever, other serious Salmonella infections, and meningitis caused by Haemophilus influenza (11, 20). However, in many areas of the world, chloramphenicol remains the antimicrobial agent of choice for serious Salmonella infections as well as a variety of other syndromes. Therefore, we evaluated the adequacy of two rapid screening methods for the detection of chloramphenicol resistance in Salmonella spp. Thirty-three strains of Salmonella were studied: S. typhi (n = 15), S. typhimurium (n = 10), and other Salmonella spp. (n = 8). Twenty-nine strains were recovered from clinical specimens submitted to the Hospital Infantil de Mexico, Mexico City, Mexico, during an outbreak of infections caused by chloramphenicol-resistant strains in the 1970s (15). Three strains, all derivatives of the chloramphenicolsusceptible strain S. typhimurium ATCC 14028, have been genetically defined (13). Two strains (CS019, CS015) each possess a transposon TnlOd-Cam (8) that encodes chloramphenicol resistance in different locations on the chromosome; one strain contains approximately 200 copies of a plasmid, pSMO03 (13), a derivative of pBR328 (19), that contains genes for CAT and P-lactamase. The parent strain *

cate. CAT activity was investigated by a 1-h visual tube CAT (t-CAT) assay performed as described by Azemun et al. (2), using the reagents sodium dodecyl sulfate, EDTA, trizma hydrochloride, trizma base, acetyl coenzyme A, and nitrobenzoic acid (Sigma Chemical Co., St. Louis, Mo.) and sodium chloride (Mallinckrodt, Inc., Paris, Ky.). In addi-

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TABLE 1. In vitro assay results for chloramphenicol resistance detection No. of isolates

Identification (no. of isolates)

MIC (ktg/ml) of chloramphenicola

Clinical strains S. typhi (2) S. typhi (6) S. typhimurium (1) Salmonella spp. (8) S. typhimurium (4) S. typhi (1) S. typhi (5) S. typhimurium (1) S. typhi (1)

Genetically defined strains S. typhimurium (1) (one transposon) S. typhimuirum (1) (one transposon) S. typhimurium (1) (200 copies of plasmids) S. typhimurium (1) (parent strain) a Susceptible, -8

ptg/ml; resistant,

demonstrating CAT activity by: t-CAT assay

d-CAT assay

512.0 256.0 128.0 8.0 8.0 8.0 4.0 4.0 2.0

2 6 1 0 0 0 0 0 0

2 6 1 0 0 O 0 0 0















tion, a 30-min disk CAT (d-CAT) assay was evaluated by using a commercial CAT reagent kit (Remel, Lenexa, Kans.). The results from this study are given in Table 1. Of 29 clinical strains, 9 strains were resistant to chloramphenicol (MICs, -128.0 ,ug/ml). The MICs of chloramphenicol for the genetically defined strains with determinants for chloramphenicol resistance were .128.0 ,utg/ml, while the MIC for the parent strain was 4.0 ,ug/ml. All CAT assay results correlated with those observed with standardized susceptibility methods, as did those for the control H. influenza strains. The d-CAT and the t-CAT detection methods yielded identical results, with no difficulty in their interpretation. Similar results were observed by Matthews et al. (12) for 91 H. influenza and 113 S. pneumoniae isolates, although the only two chloramphenicol-resistant (MIC, 32 ,ug/ml) Aerococcus spp. in their study were negative for CAT, as determined by the d-CAT assay. These strains may have been resistant to chloramphenicol by another mechanism or, perhaps, may have possessed a class of CAT enzyme inhibited by dithionitrobenzoic acid, the colorimetric indicator in the test (2). Enzyme induction was not required to demonstrate CAT activity in Salmonella spp. The commercial d-CAT method is preferable because it eliminates the necessity of preparing and storing several complex reagents. Reagents for either the d-CAT or t-CAT method must be stored frozen but are stable for approximately 1 year. The genetically defined strains were included as controls to determine the sensitivity of the methods used for detection of CAT activity. Our results indicated that both the d-CAT and the t-CAT methods are highly sensitive, since enzyme activity produced by only one gene copy per cell was detected. Based on these data, it appears that the d-CAT method is useful for sensitive and rapid detection of chloramphenicol

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Use of commercially available rapid chloramphenicol acetyltransferase test to detect resistance in Salmonella species.

Chloramphenicol resistance among Salmonella spp. has important public health and clinical implications, especially in areas of the world where these s...
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