β-Lactamase Genes of the Penicillin-Susceptible Bacillus anthracis Sterne Strain

Penicillin G, doxycycline, and ciprofloxacin have long been the drugs of choice for treatment of all forms of anthrax disease in humans (27). Combinations of these drugs were used to treat the recent U.S. cases (28, 13); yet, historically, penicillin has been the antimicrobial agent most commonly used for treating anthrax worldwide (29, 62). Susceptibility to penicillin and other β-lactam agents is often listed as a defining characteristic of B. anthracis (48, 62). However, antimicrobial susceptibility profiles of clinical and nonclinical isolates have revealed various prevalences of penicillin G-resistant strains. Tests of 50 historical Bacillus anthracis isolates and 15 isolates from the recent human anthrax cases in the United States indicate widespread susceptibility to β-lactam-containing compounds with one exception. Of the 65 strains tested, one strain, isolated from a human case of anthrax in 1974, was β-lactamase positive and penicillin resistant (47). In another survey, 7 of 44 isolates from carcasses and soil in an area of South Africa to which anthrax is endemic were resistant to penicillin G (51). A third survey of isolates recovered in France, including 1 isolate from a human, 28 from animal sources, and 67 from other environmental sources, revealed resistance to penicillin G and amoxicillin in 11.5% of the isolates (12). The susceptibility of most B. anthracis strains to β-lactam agents is intriguing considering that closely related species are characteristically β-lactamase positive (10). B. anthracis is a member of the Bacillus cereus group species, which includes B. anthracis, B. cereus, Bacillus thuringiensis, and Bacillus mycoides. Numerous phylogenetic studies, including 16S rRNA gene comparisons, multienzyme electrophoresis, and amplified fragment length polymorphism analysis, indicate that B. anthracis is most closely related to B. cereus (6, 24, 61). The most obvious phenotypic differences between B. anthracis and B. cereus are related to pathogenicity. B. anthracis, the causative agent of anthrax, is a highly virulent mammalian pathogen (46), while B. cereus is better known as a causal agent of mild food poisoning (21). The virulence of B. anthracis has been attributed primarily to the presence of two plasmids that are not found in B. cereus. B. anthracis plasmid pXO1 carries the structural genes for the anthrax toxin proteins, while plasmid pXO2 harbors the biosynthetic genes for the antiphagocytic poly-d-glutamic acid capsule (42, 52). Unlike those of B. anthracis, B. cereus strains commonly exhibit resistance to penicillin and other β-lactam agents. Three different β-lactamases, named β-lactamase I, II, and III, have been reported for various B. cereus strains. B. cereus strain 569/H is resistant to penicillins and cephalosporins and contains genes encoding all three enzymes. According to the classification scheme proposed by Bush (11), which groups β-lactamases on the basis of specific substrate and inhibitor profiles, B. cereus β-lactamase I and β-lactamase III are group 2A enzymes that prefer penicillin substrates and are inhibited by clavulanic acid (15, 16, 33, 50). B. cereus β-lactamase II is a group 3 enzyme, a heat-stable metallo-β-lactamase, that is not inhibited by clavulanic acid (8, 18, 25, 33, 34, 39). A β-lactamase I enzyme is also produced by the B. cereus group species B. thuringiensis (40, 65). Most B. mycoides strains are resistant to penicillin (J. Mahillon, personal communication), and a β-lactamase I gene has been cloned and sequenced (GenBank, accession no. {"type":"entrez-nucleotide","attrs":{"text":"X62244","term_id":"39612","term_text":"X62244"}}X62244). In work presented here, we report the presence of two β-lactamase genes in the Sterne strain of B. anthracis. The Sterne strain is a well-studied attenuated strain that serves as a live vaccine for animals (58). The Sterne strain does not produce capsule due to the absence of pXO2. With the exception of being cured of pXO2, this strain is considered to be a prototypical B. anthracis strain (59). It is sensitive to penicillin and does not exhibit β-lactamase activity. Our data indicate that the β-lactamase genes carried by this strain are not expressed in the Sterne strain but encode functional enzymes when cloned in other species.