The infectivity of Agrobacterium tumefaciens strain B6 was inhibited about 50% when these bacteria were inoculated on potato discs with equal viable cell counts of a weakly virulent strain of A. tumefaciens (B-48) The fact that crown gall host systems differ with respect to the degree of bacterial virulence shown by a given strain, the time it takes for tumor formation, and the size and number of tumors formed has been well established (1, 6, 7).The necessity for bacterial attachment to a specific site as a prerequisite for the formation of crown gall tumors on the primary leaves of Pinto beans and the time it takes for such attachment have been shown (5). Studies as to whether a similar type of attachment is necessary in other crown gall host systems have been limited due to the lack of another suitable quantitative bioassay for tumor formation, although the necessity for site attachment has been established for some nonquantitative host systems (8,10). With the recent development of the potato disc bioassay by Anand and Heberlein (1). we have undertaken studies which show that attachment to a specific site, which occurs in a short period of time, is a necessary prerequisite for tumor formation on potato discs.With the recent discovery that infectivity is due to the presence of a plasmid (11) found within tumor-inducing strains of Agrobacterium tumefaciens, experiments to determine the biochemical nature of the transformation process should increase in number. The need to understand more completely the nature of specific host systems for studying tumor formation takes on great importance. 2To whom reprint requests should be sent. MATERIALS AND METHODSof Northwestern University. The bacterium and yeast (which was obtained from the culture collection at the USDA Lab in Peoria) were grown for 48 hr in a medium containing 0.8% (w/v) nutrient broth, 0.1% (w/v) yeast extract, and 0.5% (w/v) sucrose on a New Brunswick Scientific Shaker at 27 C.Tumors were induced on potato discs by the method first described by Anand and Heberlein (1), and modified slightly in our laboratory (3). Red potatoes (Solanum tuberosum var. Red Russett) were purchased from the local market and soaked in 20%o Clorox (v/v) for 20 min. Potato discs were then obtained by placing a sterilized cork borer (20-mm diameter) through the potato and cutting 5-mm discs from the resulting plug. Five potato discs were then placed on Petri dishes containing 1.5% (w/v) agar and inoculated with 0.5 ml of bacterial suspension containing a known quantity of viable cells. The Petri dishes were placed at 27 C and exposed to 15 hr of room light and 9 hr of darkness daily. Twelve days following inoculation the tumors were counted with the aid of a dissecting scope. The tumors at this time, however, were clearly visible with the human eye. Each experimental sample consisted of three Petri dishes which contained five potato discs. Sterile techniques were employed throughout the above procedure.In order to insure that a maximum competition for attachment site...

Although all of 14 clinical isolates of Haemophilus influenzae type b strains demonstrated rifampin susceptibility in vitro (minimal inhibitory concentration c0.4 ,ug/ml) when an inoculum of 104 colony-forming units (CFU) was used, 10 of the 14 strains manifested resistance to this agent when an inoculum of 108 CFU was tested. The mutation rate for rifampin resistance ranged from 1 resistant colony per 3.5 x 106 CFU to 1 per 4 x 107 CFU. The emergence of rifampinresistant mutants was prevented when trimethoprim was combined with rifampin. This finding suggests that when used alone for prophylaxis of H. influenzae type b nasopharyngeal carriers, rifampin is likely to lead to the emergence of resistant strains.The association between nasopharyngeal carriage of H. influenzae type b (HIB) and cases of secondary systemic infections due to this bacterium in closed pediatric populations prompted antibiotic trials in attempts to eradicate this agent from the nasopharynx. Recent data suggested rifampin to be the most promising chemoprophylactic agent for treatment of close contacts of individuals with HIB infections (3, 7, 9). However, other bacteria, such as Staphylococcus aureus, have been reported to develop both in vitro (1, 6) and in vivo (5) resistance to rifampin when this drug is used alone. The present in vitro study was designed to examine whether clinical isolates of HIB will develop resistance to rifampin. MATERIALS AND METHODSFourteen strains of HIB isolated from blood (5), cerebrospinal fluid (6), and nasopharynx (3) of pediatric patients were studied. Ten strains were ampicillin susceptible, and four were ampicillin resistant. Minimal inhibitory concentrations (MIC) of rifampin (kindly supplied by Ciba Pharmaceutical Co.) for these strains were determined by using supplemented Mueller-Hinton broth (sMH) containing 10 ,ug of hemin per ml and 1% IsoVitaleX. Ten milligrams of rifampin was dissolved in methanol and diluted with sMH broth to obtain final concentrations ranging from 0.1 to 12.5 p.g/ml. From an overnight growth of HIB on a chocolate agar plate, three colonies were inoculated into 5 ml of sMH and shaken for 4 h at 35°C. Dilutions were made for a final inoculum of lo' colony-forming units (CFU) per ml. One milliliter of inoculum was added to each antibiotic tube, with a final volume of 2 ml. The tubes were incubated overnight at 350C with 5% CO2. The MIC was considered to be the lowest concentration which prevented visible growth. The minimal bactericidal concentration (MBC) was read as the lowest concentration of drug in which there was no growth of the organism after plating 10 ,ul from each tube into chocolate agar and incubating for 18 h at 35°C with 5% CO2.To study the development of resistance to rifampin, two experiments were performed. In one experiment, the effect of subinhibitory concentrations of rifampin on growth of HIB was evaluated by overnight incubation in the presence of one-quarter to one-half MIC of rifampin for a given strain. After overnight incubation, the MIC was determined ...

The MICs for 90% of the organisms tested (MIC%0s) of 11 antibiotics against 24 clinical isolates of Actinobacillus actinomycetemcomitans were determined by the MIC 2000 system. The lowest MICgos (16 ,ug/ml) were observed with ceftriaxone and rifampin. The next lowest MIC90s were found with cephapirin, tetracycline, and chloramphenicol (3.12 ,ug/ml). The MIC90s of penicillin, ampicillin, ticarcillin, piperacillin, and amikacin were each .12.5 ,ug/ml. Antibiotic synergy was studied by the killing curve method and was defined as a .12 logl0 reduction in CFU when two antibiotics were used in combination at one-fourth the MBC for each compared with the effect of each antibiotic alone at one-half the MBC. Synergism between rifampin and penicillin, cephapirin, or ceftriaxone was tested for with 12 A. actinomycetemcomitans strains. In 7 of 37 instances, synergism was demonstrated for the combinations rifampin plus ceftriaxone (n = 3) or rifampin plus penicillin (n = 4); in 9 instances, an additive effect was noted, and impaired killing with drug combinations compared witb the effect of a single antibiotic was suggested in 4 strains. The majority of strains were indifferent to the combinations. Similarly, variable results were observed when the combination of trimethoprim and cephapirin was tested against eight A. actinomycetemcomitans strains. Our data suggest that rifampin and cephapirin are the most active of the 11 antibiotics studied against A. actinomycetemcomitans. In addition, in vitro synergism between rifampin and other antibiotics or between trimethoprim and cephapirin was not consistently demonstrable.

A total of 42 strains of Haemophilus influenzae type b isolated from pediatric patients were sensitive in vitro to temocillin (90% minimal inhibitory concentration = 0.25 ,ug/ml). No difference in mean minimal inhibitory concentration between ,B-lactamase producer (0.25 ,ug/ml) and nonproducer (0.23 jig/ml) strains was found. Various dosages of ampicillin or temocillin for the treatment of infant rats with ampicillin-resistant H. influenzae bacteremia and meningitis yielded no difference in cure rates. These results suggest that temocillin may not be as effective as other new cephalosporins for the treatment of H. influenzae type b infections.Temocillin (BRL 17421), a new semisynthetic P-lactam antibiotic, has recently been shown to be active in vitro against most aerobic gramnegative organisms, including a wide range of 1B-lactamase producers (4). In addition, the bactericidal activity of this compound against Haemophilus influenzae was found to be good (3). These properties suggest that temocillin might be suitable for the treatment of serious H. influenzae type b infections in childhood. The purpose of this study was to evaluate in vitro the activity of temocillin against clinical isolates of H. influenzae type b and to compare in vivo temocillin and ampicillin against ampicillin-resistant H. influenzae type b bacteremia and meningitis in the infant rat model. A total of 42 H. influenzae type b strains isolated from the blood or cerebrospinal fluid (CSF) of pediatric patients were studied. Identification, serotyping, and biotyping were performed by methods previously described (8). 1-lactamase activity was determined by pH changes due to penicilloic acid production from penicillin (2). The minimal inhibitory concentration (MIC) and the minimal bactericidal concentration of temocillin against each strain were determined, as previously described (6). Dilu inhibited at a concentration of 0.25 ,.g/ml. No difference in MIC between 3-lactamase-positive (14 strains) and -negative (28 strains) strains was found (mean ± standard deviation was 0.25 ± 0.12 and 0.23 + 0.07 ,ug/ml, respectively). The minimal bactericidal concentrations were identical to or one tube higher than the MICs for all strains. Susceptibility to temocillin was not influenced by increasing the inoculum size from 104 to 106 CFU per ml. These results confirm previous reports that H. influenzae is inhibited by temocillin at a concentration of <0.5 ,ug/ml (4, 5) and that there is no difference in MIC against either 3-lactamase-positive or -negative strains (5).In vivo experiments used the infant rat model of bacteremia and meningitis as previously described (7). Animals were injected intraperitoneally with 104 CFU of ampicillin-resistant H. influenzae type b (strain Bloch) in 0.1 ml (MIC = 12.5 ,ug/ml). Blood and CSF cultures were taken 36 h after inoculation by procedures described previously (7). By the method used, the lower limit of detectable bacteria was 200 CFU per ml. Twelve infected animals were injected intraperitoneally with a single dose of...

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