Plasmid-Mediated Chloramphenicol Resistance in Haemophilus influenzae

Abstract

A clinical isolate ofHaemophilus influenzae HC234 was found to be resistant to chloramphenicol and tetracycline. It was shown that both resistance markers are tranferable as one unit to other Haemophilus influenzae strains and also to Escherichia coli. Data are presented which indicate that conjugation is the most likely mechanism of resistance transfer. HC234 was shown to carry a single plasmid species with a molecular weight of 38 x 106.Recent investigations have shown the existence of plasmid-mediated drug resistance in Haemophilus influenzae (2,3,5,17). Transfer of resistance, probably by conjugation, was demonstrated for resistance to ampicillin (13,15) and for resistance to kanamycin (3). Recently, we reported on a chloramphenicol (Cm)-resistant strain of H. influenzae isolated from the pharynx of a patient with lymphatic leukemia (10). The resistance to Cm appeared to be due to the production of an enzyme, presumably an acetyltransferase, inactivating this antibiotic. The strain was also resistant to tetracycline (Tc).This report describes experiments on the transfer of the resistance determinants to susceptible strains ofH. influenzae and to a strain of Escherichia coli. Also, the isolation and characterization of plasmid deoxyribonucleic acid (DNA) from the resistant strain are reported. MATERIALS AND METHODSBacterial strains. The strains used in this study are listed in Table 1. The rifampin-and streptomycin-resistant mutant strains HC215-1, HC217-1, and HC218-1 were obtained by selection on plates containing these antibiotics. All strains of H. influenzae investigated in this study were untypable according to Pittman's agglutination method (12). The strain ofE. coli K-12 (SC181) was kindly provided by E. Lederberg (Stanford University).Media. Nutrient broth was prepared in this institute from fresh meat and contained in addition 0.5% NaCl, 1% peptone (Difco) and 0.069% Na2CO3; the pH was 7.5. Nutrient agar contained, in addition, 2% agar (BBL).NXV broth was nutrient broth, supplemented with 4% X factor and 4% V factor. X and V factors were freshly prepared from lysed horse blood and lysed yeast cells, respectively. NXV agar was prepared by adding 2% agar to NSV broth.Stock cultures of H. influenzae were maintained and transferred every 2 weeks in semisolid NXV medium containing 1% agar (BBL).Susceptibility tests were usually carried out on NXV agar. Wellcotest agar, supplemented with X and V factors was used for establishing the susceptibility to cotrimoxazole.Determination of MICs. The minimum inhibitory concentrations (MICs) were determined by agar dilution, using a Steers replicator. As inocula, undiluted overnight cultures (10k to 109 colony-forming units/ml) and also the dilutions 10-', 10-2, and 1-3 were used. The antibiotics used were: Cm (Globenicol, Mycofarm), Tc (Tifaco), ampicillin sodium (Amfipen, Mycofarm), erythromycin (Erythrocin, Ab-

Molecular Characterization of a Small Haemophilus influenzae Plasmid Specifying -Lactamase and its Relationship to R Factors from Neisseria gonorrhoeae

“…There are obviously at least two groups of R factors in H. influenzae: the 30 to 38 Mdal R plasmids (Elwell et al, 1977b;Van Klingeren et al, 1977;Laufs & Kaulfers, 1977) and the 2.5 to 4.4 Mdal R plasmids (De Graaff et al, 1976;Saunders & Sykes, 1977). The large 30 to 38 Mdal R factors which specify ampicillin, tetracycline or chloramphenicol resistance are all closely related to each other.…”

“…These H. influenzae R plasmids probably carry the ampicillin resistance transposon TnA or the tetracycline resistance transposon TnD (Kaulfers et al, 1978). The plasmids were of 30 to 33 megadalton (Mdal) and were closely related to each other and to the 30 to 38 Mdal R plasmids found in H. inJuenzae isolates from other countries (Elwell et al, 1977 b;Van Klingeren et al, 1977). Besides these 30 to 38 Mdal plasmids, small H. in,uenzae R plasmids which specify ampicillin resistance have been described (De Graaff et al, 1976;Saunders & Sykes, 1977).…”

Tetracycline Antibiotics: Mode of Action, Applications, Molecular Biology, and Epidemiology of Bacterial Resistance

“…Tc r genes in both gram-positive and gramnegative species are often found on the same units (plasmids, transposons, or integrons) as other antibiotic resistance genes. For example, all chloramphenicol-resistant (Cm r ) Haemophilus influenzae strains isolated in the 1970s and 1980s were also Tc r (111,168,282,304 (111). In another, the ampicillin transposon was integrated into the inverted repeats of the tetracycline transposon (111).…”

Haemophilus influenzae: antibiotic susceptibility

“…Thornsberry (53,117). There rapidly followed reports of strains resistant to both chloramphenicol and ampicillin (70,97,116 It is important for the laboratorian to be aware of the specific antibiotics which might be used as alternatives to ampicillin and chloramphenicol, since this knowledge may influence susceptibility test strategies.…”