Ten major antigens from Mycobacterium bovis culture filtrate of 39, 32, 30, 25, 24, 22 (a and b forms), 19, 15, and 12 kDa have been purified and characterized by classical physicochemical methods. With monoclonal antibodies and/or N-terminal amino acid sequencing data, it was found that the antigens of 32, 30, 24, 22 (a), 19, and 12 kDa are related to M. bovis or M. tuberculosis antigens P32, MPB59, MPB64, MPB70, 19 kDa, and 12 kDa, respectively. The 39-, 25-, 22 (b)-, and 19-kDa antigens showed concanavalin A-binding properties and were positive in a glycan detection test, suggesting that they are glycoproteins. The 25and 22 (b)-kDa proteins were found to be glycosylated forms of MPB70. * Corresponding author. ammonium sulfate and subjected to either chromatofocusing or anion-exchange chromatography. Chromatofocusing was performed on a PBE-94 column (1.5 by 40 cm; Pharmacia) essentially by a scaled-up version of the procedure described for the purification of the M. bovis protein antigen MPB70 (16). Anion-exchange chromatography was performed on a DEAE-Sepharose CL-6B column (2.5 by 30 cm; Pharmacia) equilibrated with 30 mM Tris-HCl (pH 8.8) containing 2% butanol and eluted with a 0 to 200 mM NaCl gradient as described by Harboe et al. (17). In both procedures, the column was linked to a fast protein liquid chromatography system (Pharmacia) to maintain the flow rate at 0.5 ml/min and to generate the gradients. For each run, fractions enriched for particular antigens were pooled, concentrated, and processed as described below. Columns and buffers. Two gel filtration columns were used, a Biogel P-60 (1.6 by 100 cm) and a Biogel P-100 (0.9 by 50 cm); these were equilibrated with 1% NH4HCO3, pH 8.2. A Pharmacia Mono Q HR 5/5 anion-exchange column was equilibrated with 30 mM Tris-HCl (pH 7.8). The proteins, unless otherwise indicated, were eluted with a 0 to 300 mM NaCl gradient in the equilibration buffer. The Pharmacia Mono P 5/20 chromatofocusing column was equilibrated with 0.025 M piperazine (pH 5.5) adjusted with iminodiacetic acid (IMDDA). The proteins were applied to the column in a 1/10 dilution of Polybuffer PB-74 (Pharmacia) adjusted to pH 4.0 with IMDDA and eluted from the column with a pH gradient from 5.5 to 4.0 generated by washing the column with a 1/10 dilution of PB-74-IMDDA at pH 4.0. A Pharmacia concanavalin A (ConA)-Sepharose column was equilibrated with 20 mM Tris-HCl-0.5 M NaCl-1 mM Mn2+-1 mM Ca2+ at pH 7.4. The proteins were eluted with 1% aD -methylglucoside in the equilibration buffer. Purification of the 39-kDa antigen. Fractions off the DEAE-Sepharose column enriched for the 39-kDa protein were pooled, concentrated, and further fractionated on the P-60 gel filtration column. Fractions containing the antigen were freeze dried and applied to the ConA-Sepharose column. The bound material was eluted with 1% CX-D-methylglucoside, dialyzed, freeze dried, and then put on the Mono Q column. The fractions containing the 39-kDa protein were

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IS6100 is an insertion sequence of the IS3 family and it is present in multiple copies in the chromosome of Mycobacterium tuberculosis. Four to 15 copies are present in various strains of M. tuberculosis. In this study, the value of IS6110 as an epidemiological marker of tuberculosis was examined. Unrelated clinical strains from Greek patients presented, in restriction fragment length polymorphism analysis, a high degree of polymorphism, whereas patterns of related clinical strains from familial outbreaks were identical. Since RFLP analysis with acetylaminofluorene labeled IS6110 as the probe gave satisfactory results, it is suggested that this non-radioactive probe can be used in hospitals and health centres for the epidemiological survey of M. tuberculosis infections.

The in vitro antibacterial activity of eight newer β-lactam antibiotics (mecillinam, piperacillin, mezlocillin, cefoxitin, cefotaxime, moxalactam, ceftriaxone and ceftazidime) was determined against 87 cephalothin-resistant strains of Enterobacteriaceae isolated during 6 months in a general hospital. Ceftriaxone, cefotaxime, moxalactam and ceftazidime proved to be highly active; only a minority of strains required higher concentrations than 0.125 μg/ml for inhibition of growth. Cefoxitin, mecillinam, mezlocillin and piperacillin were less active. Mecillinam displayed greater efficacy against Escherichia coli, Klebsiella and Enterobacter spp., while the same was the case for piperacillin against Proteus mirabilis and Serratia marcescens, and for cefoxitin against indole-positive Proteus spp. The production of β-lactamase was correlated with a reduced activity of mecillinam, mezlocillin and piperacillin but not of cefoxitin or the other β-lactamase-stable cephalosporins. However, some strains, mainly those of Proteus, Enterobacter and Serratia, though resistant to mecillinam, mezlocillin and piperacillin did not produce β-lactamases. This observation might indicate that ceftriaxone, moxalactam, cefotaxime and ceftazidime, besides their indifference to β-lactamases, are characterized also by a high degree of intrinsic activity.