An extended multiplex PCR method was established to rapidly identify and classify Bacillus thuringiensis strains containing cry (crystal protein) genes toxic to species of Lepidoptera, Coleoptera, and Diptera. The technique enriches current strategies and simplifies the initial stages of large-scale screening of cry genes by pinpointing isolates that contain specific genes or unique combinations of interest with potential insecticidal activities, thus facilitating subsequent toxicity assays. Five pairs of universal primers were designed to probe the highly conserved sequences and classify most (34 of about 60) genes known in the following groups: 20 cry1, 3 cry2, 4 cry3, 2 cry4, 2 cry7, and 3 cry8 genes. The DNA of each positive strain was probed with a set of specific primers designed for 20 of these genes and for cry11A. Twenty-two distinct cry-type profiles were identified from 126 field-collected B. thuringiensis strains. Several of them were found to be different from all published profiles. Some of the field-collected strains, but none of the 16 standard strains, were positive for cry2Ac. Three standard and 38 field-collected strains were positive by universal primers but negative by specific primers for all five known genes of cry7 and cry8. These field-collected strains seem to contain a new gene or genes that seem promising for biological control of insects and management of resistance.

No abstract

Sulfamethoxazole-trimethoprim and three oral cephalosporins, cefaclor, cephalexin, and cephradine, were evaluated in vitro as possible alternatives to chloramphenicol in the treatment of non-central nervous system infections due to ampicillin-resistant Haemophilus influenzae. Sixty-four isolates of H. influenzae, including 31 ,f-lactamase-positive strains, were tested by the agar dilution method.All strains were inhibited by 0.78/0.039 jig sulfamethoxazole-trimethoprim per ml and by 0.78 jig of chloramphenicol per ml. At 6.25 jig/ml, 100, 11, and 3% of all strains were inhibited by cefaclor, cephalexin, and cephradine, respectively. Thus, on the basis of drug concentrations presumably achievable in serum, 100% of strains were susceptible to sulfamethoxazole-trimethoprim, chloramphenicol, and cefaclor. However, a considerable inoculum effect was noted with both ,B-lactamase-positive and -negative strains, when tested with sulfamethoxazole-trimethoprim; the miniInal inhibitory concentrations of cefaclor were only slightly affected. Also, synergistic effects of sulfamethoxazole-trimethoprim, sulfamethoxazole-erythromycin, and sulfamethoxazole-cefaclor were seen when combinations were tested against both ,8-lactamase-positive and -negative strains, as determined by minimal inhibitory concentrations measured by the broth dilution method and by killing curve analyses. These results support further evaluation of these combinations and of cefaclor alone for the treatment of non-central nervous system infections due to H. influenzae. were adjusted to contain 2 x 105 CFU/ml, and 0.05 ml was added to an equal volume of drug suspension. The plates were incubated at 35°C for 18 h. The lowest concentration of drug with no visible turbidity was used to define the minimal inhibitory concentration (MIC) of ampicillin, chloramphenicol, erythromycin, TMP, and cefaclor, whereas a sudden reduction in growth was used for SMZ MIC determinations. Effect of inoculum concentration. The effect of inoculum size on MIC was tested (i) by the agar dilution technique, with all strains using inocula of 10' and 107 CFU/ml, or (ii) by the microtiter broth dilution technique with one strain each of ,B-lactamasepositive and -negative strains, using final inocula of 103, 104, 105, and 106 CFU/ml. The effect of inoculum size on the synergism of antimicrobials used in combination was also tested by the microtiter broth dilution technique using the four different inocula described above.Tests for synergism. Synergism was measured by two techniques against one strain each of,B-lactamasepositive and -negative strains: (i) by checkerboard microtiter broth dilution and (ii) hibited by 0.78/0.039 ig of SMZ-TMP per ml and by 0.78 ,ug of chloramphenicol per ml. At 6.25 ug/ml, 100, 11, and 3% of all strains were inhibited by cefaclor, cephalexin, and cephradine, respectively. With ampicillin, 100% of the /3-lactamase-negative strains were inhibited by a concentration of 0.78 ug/ml; in contrast, f8-lactamase-positive strains exhibited a wide range of susceptib...