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The sensitivity and reproducibility of the radioenzymatic adenylylating assay for spectinomycin were improved by modifications of the assay procedure and by partial purification of the enzyme. The presence of Bacillus subtilis ribosomes or S-150 fraction in the enzymatic reaction mixture interfered with the ability to measure spectinomycin by this method.

Mecillinam (formerly called FL 1060), a novel 6β-amidinopenicillanic acid derivative, showed markedly higher activity than ampicillin against clinical isolates of Enterobacteriaceae . The mode of action of mecillinam is different from that of ampicillin and consequently no intrinsic cross-resistance between the two antibiotics was observed. Mecillinam is inactivated by β-lactamases but is generally more stable than ampicillin; thus an apparent cross-resistance exists for strong β-lactamase producers. The protein binding was low and the activity was relatively independent of pH between 5.5 and 8.0. Slow-growing, insensitive variants of Escherichia coli could be selected but these were not stable. The frequency of insensitive variants was affected by the sodium chloride concentration in the medium.

Electron microscopic studies with Escherichia coli revealed that bicyclomycin inhibits septum formation and converts the cells to filamentous forms. The antibiotic induced high undulation and numerous blebs of the outer membrane. Sometimes cytoplasmic contents leaked into the lumen of the bleb through a disrupted region of the membrane. Breakage of the outer membrane or blebs led to cell lysis. Electron-dense masses of amorphous material and vesicles were found in the cytoplasm.

Human cell culture-derived interferon was shown to inhibit human cytomegalovirus in vitro. A prototype strain, Davis, and six clinical isolates of cytomegalovirus were tested. All six isolates showed uniform susceptibility to interferon, exceeding that of the Davis strain by two- to fourfold. The latter virus was found to be 32 to 4 times less susceptible than the sensitive indicator, vesicular stomatitis virus. However, the laboratory finding of susceptibility to an antiviral material may not relate to its clinical effectiveness.

The activity of azlocillin and mezlocillin, new semisynthetic ureido penicillins, was investigated and compared with that of other known β-lactam antibiotics. At a concentration of 25 μg/ml, azlocillin inhibited 74% of Enterobacter , 97% of Proteus mirabilis , 64% of Citrobacter , 91% of Pseudomonas aeruginosa , and 82% of Bacteroides strains tested. Mezlocillin inhibited 86% of Shigella , 96% of Enterobacter , 80% of indole-positive Proteus , 88% of Bacteroides , and 63% of Pseudomonas strains tested. Azlocillin was more active against Pseudomonas than was ticarcillin, carbenicillin, or mezlocillin. Mezlocillin was more active than carbenicillin and ampicillin against Escherichia coli, Klebsiella, Enterobacter, Citrobacter, Acinetobacter, Serratia , and Bacteroides . Azlocillin and mezlocillin were less active than cefazolin against β-lactamase-producing E. coli and Klebsiella strains but more active than cefazolin against Enterobacter , indole-positive Proteus, Acinetobacter, Citrobacter , and Serratia strains. Both compounds showed activity equivalent to that of cefoxitin against Bacteroides isolates. Both agents were destroyed by many of the β-lactamases from gram-negative organisms.

A new product, the 3M Brand Microbial Control System for monitoring the reproducibility of Bauer-Kirby susceptibility tests, was compared with standard ATCC strains of Escherichia coli, Staphylococcus aureus , and Pseudomonas aeruginosa . Results are presented from four independent laboratories in which all obvious protocol variables, excluding media and personnel, were identical. Evaluation of the system revealed that the commercial procedure was simple to implement and efficacious and produced results equivalent to those derived from ATCC control strains of E. coli, S. aureus , and P. aeruginosa .

The susceptibilities of T-mycoplasmas ( Ureaplasma urealyticum ) to minocycline, demeclocycline, doxycycline, tetracycline, and erythromycin were determined by a direct tube dilution test. T-mycoplasma-positive urine sediments of 105 patients with a history of reproductive failure were used as inocula. Minocycline was found to be the most active of the group of antibiotics commonly used to eradicate T-mycoplasma infection. Based on the median initial minimum inhibitory concentration, minocycline was the lowest with 0.03 μg/ml, followed by demeclocycline and doxycycline with 0.125 μg/ml, tetracycline with 0.25 μg/ml, and erythromycin with 2.0 μg/ml. Six T-mycoplasma isolates which had been cloned three times were also tested for susceptibility to the same five antibiotics. The same susceptibility pattern was found. Strains resistant to high concentrations of all antibiotics occurred. Strong positive correlation was seen in 21 patients between in vitro highly resistant strains and positive posttreatment cultures. These results indicate that empirical treatment of genital mycoplasma infections is not justified. Cultures should be taken pretreatment, susceptibility testing performed prior to treatment, and follow-up cultures done posttreatment.

Thirteen independent isolates of Serratia marcescens associated with nosocomial urinary tract infections were obtained from the clinical microbiology laboratory at Hines Veterans Administration Hospital. The isolates were resistant to at least ampicillin, carbenicillin, gentamicin, and tobramycin. They could be divided into two groups on the basis of their antibiotypes. Group I (9 strains) showed resistance to 13 antibiotics, including 3 beta-lactams, 6 aminoglycosides, tetracycline, sulfonamide, trimethoprim, and polymyxin B. Group II (4 strains) was resistant to 11 antibiotics, including 3 beta-lactams, 5 aminoglycosides, sulfonamide, trimethoprim, and polymyxin B. Donors from both groups transferred resistance traits to Escherichia coli . Transconjugants from matings with group II donors all acquired resistance to nine antibiotics, including the three beta-lactams, five aminoglycosides, and sulfonamide. Transconjugants from matings with group I donors were of varied antibiotypes, inheriting resistance to up to 11 of the 13 antibiotics. Resistances to trimethoprim and polymyxin B were never observed to transfer. E. coli transconjugants of each group were capable of transferring multiple-antibiotic resistance to several other members of the family Enterobacteriaceae . All group II S. marcescens and E. coli donors and all group I S. marcescens donors transferred carbenicillin, streptomycin, kanamycin, gentamicin, tobramycin, and sisomicin resistance to Pseudomonas aeruginosa . The results suggest that these S. marcescens strains harbor R factors of a broader host range than previously reported.

Previous methods using low- or high-pressure liquid chromatography and UV absorbance for quantitation of arabinosides in plasma can practically detect only >/=200 ng of adenine arabinoside and >/=100 ng of hypoxanthine arabinoside per ml, and they require expensive equipment and expert technical assistance. We describe in this report a simple quantitative microbioassay for arabinosides in human plasma based on their ability to inhibit the cytopathic effect of vaccinia virus in an adenosine deaminase-free cell culture system. Using prior separation of nucleosides in plasma by thin-layer chromatography, followed by the microbioassay, we quantitated adenine arabinoside with a sensitivity of approximately 4 ng/ml and hypoxanthine arabinoside at approximately 625 ng/ml. This assay method is simple, sensitive, and reproducible, requires small plasma samples, measures biological activity, and is adaptable to routine use. It is an important tool for evaluating the pharmacology of adenine arabinoside and Ara-AMP in patients in current clinical trials.

The comparative bactericidal activity of penicillin G, carbenicillin, clindamycin, and metronidazole against eight susceptible strains of Bacteroides fragilis and four strains of Clostridium perfringens was determined by performing colony counts anaerobically of cultures incubated in brucella broth. With the B. fragilis strains, there was a lag phase of growth of approximately 8 h, during which time metronidazole did not reduce the colony counts. However, within 4 h of the onset of exponential growth, metronidazole caused an abrupt decrease in counts to less than 100 colonies per ml in all strains tested. Moreover, in two strains in which the bactericidal rate was followed hourly, a 3- to 6-log decrease occurred over 1 h or less. In contrast, penicillin G and carbenicillin caused a gradual decline in colony counts from the start of approximately 1 log for each 8-h interval and were bactericidal for all strains tested. Clindamycin demonstrated the slowest bactericidal activity and for 25% of the strains was only bacteriostatic. With the C. perfringens strains, after a lag phase of 4 h, an abrupt decrease in colony counts also occurred with metronidazole, whereas penicillin and clindamycin again demonstrated more gradual killing effects. These studies showed a unique, time-related bactericidal action of metronidazole as compared with the other three antimicrobial agents.

The minimal inhibitory concentration of 10 antibiotics for 244 isolates of group B streptococci was determined. Susceptibility to penicillin G, ampicillin, cephalothin, chloramphenicol, and carbenicillin was uniform. Tetracycline and bacitracin resistance among these isolates was frequent (87.5 and 97.9%, respectively). Three strains (1.2%) failed to be inhibited by 100 μg of lincomycin or clindamycin per ml. Susceptibility of these 244 strains to the agents tested was unrelated to source of the isolate, year of isolation, or strain serotype. No apparent change in the suceptibility of group B streptococci to penicillin G has occurred during the past 2 decades.

The two components of virginiamycin, virginiamycin M (VM) and virginiamycin S (VS), were used to explore the life cycle of symbiosis-dependent and -independent strains of Bdellovibrio bacteriovorus during multiplication in a two-membered system with either living or heat-inactivated Escherichia coli or in axenic cultures. Relatively high concentrations of these inhibitors separately were required to stop growth under all the conditions, but the minimum inhibitory concentration of the single components was reduced 1,000-fold by the association of VM and VS. No dissociation between mass growth and cell division was observed with VM; VS specifically halted cell division without affecting the kinetics of macromolecules formation and overall growth. This effect on cell division was only obtained when the antibiotic was added during the first half of the multiplication cycle and was reversible at any time.

The agar dilution method of in vitro susceptibility testing of fungi was analyzed with a Steers-type inoculum replicator, ten strains, and three drugs. The replicator reproducibly delivered the same inoculum to each series of plates. The minimal inhibitory concentrations of ketoconazole (an imidazole) and 5-fluorocytosine, but not that of the polyene nystatin, were dependent on the initial inoculum size. With the former two drugs, but not with the latter, minimal inhibitory concentrations were highly variable depending on the time of reading. Results with agar and broth dilution methods were divergent, and the differences in minimal inhibitory concentrations were variable in serial comparisons by the two methods. If the agar dilution minimal inhibitory concentrations were determined at first appearance of control growth, a commonly used end point, small variations in the time of reading (as could occur by variation in observer perception of when initial growth appears) induced large variations in the minimal inhibitory concentrations of 5-fluorocytosine and ketoconazole, particularly with rapidly growing strains. Results at 35 and 30°C were similar. The differences in results with the three drugs suggest different mechanisms of action. The variability quantitated with the agar dilution method could result in variability in results between laboratories and even observers in the same laboratory.

An accurate and sensitive assay for vancomycin in serum and body fluids has not been available. This paper reports an assay for vancomycin that can detect serum and fluid levels as low as 0.8 μg/ml. A disk diffusion technique was designed employing buffered glucose minimal salts agar and Bacillus subtilis as an indicator strain. A linear relationship was obtained between zone diameter and concentration for vancomycin standards from 0.8 to 50 μg/ml prepared in pooled human serum. Results were accurate (

The in vitro antibacterial effects of cefoxitin, a semisynthetic cephamycin, cefuroxime, a new cephalosporin antibiotic, and cephaloridine were compared. With gram-positive bacteria, marked differences were found only in the effects against Streptococcus faecalis , where cephaloridine and cefuroxime were superior to cefoxitin. With gram-negative aerobic bacteria, cefoxitin, which is known to be more resistant to beta-lactamases from gram-negative bacteria than any cephalosporin, was found to be more effective than cefuroxime and cephaloridine against ampicillin-resistant strains of Escherichia coli and indole-positive strains of Proteus. Haemophilus influenzae was found to be more susceptible to cefuroxime than to cefoxitin and cephaloridine. When ampicillin-resistant strains of H. influenzae were tested, markedly higher minimal inhibitory concentration values were obtained with cephaloridine in comparison to those obtained with ampicillin-susceptible strains. These increases in the minimal inhibitory concentration values were not observed with cefoxitin and cefuroxime, probably due to the resistance of these two compounds to beta-lactamases. Strains of Bacteroides fragilis were found to be much more susceptible to cefoxitin than to cefuroxime, which in turn was superior to cephaloridine. The results obtained indicate that cefoxitin and cefuroxime both are superior in their antibacterial spectra to the cephalosporins that are now in clinical use.

Hospital isolates of Escherichia coli resistant to tetracycline (TC) were studied to identify mechanisms which regulate TC resistance levels and ability to transfer TC resistance. Antibiotic resistance patterns, resistance levels to TC, and ability to transfer TC resistance were determined for the isolates. Similar data were obtained for the transferable plasmids after transfer to several new host strains of E. coli . Of the 110 isolates, 50% were able to transfer TC resistance by conjugation. There was a nearly linear relationship between the minimum inhibitory concentration (MIC) of TC for the hospital strains and the percentage of strains at a given MIC that could transfer TC resistance. The strains that were simultaneously resistant to tetracycline, streptomycin, and ampicillin had relatively high MICs of TC and high ability to transfer TC resistance. These results and surveys of TC-resistant E. coli by others suggest that TC resistance levels and transmissibility may be influenced by other resistance markers. The isolates which did not transfer TC resistance by conjugation were tested for the presence of TC resistance plasmids by mobilization or by transformation with deoxyribonucleic acid from the isolates. Evidence for plasmid-mediated TC resistance was found in 92 (84%) of the 110 hospital strains.

Bay k 4999 and piperacillin, two new substituted ampicillins, were compared with other β-lactam antibiotics, including carbenicillin, azlocillin, mezlocillin, benzylpenicillin, ampicillin, and cefoxitin, against a wide range of gram-positive and -negative organisms. Bay k 4999 and piperacillin were extremely active against Pseudomonas aeruginosa (50% inhibited by 2 μg/ml), being about 16-fold more active than carbenicillin. Bay k 4999 was the most active drug against Escherichia coli (50% inhibited by 0.5 μg/ml) and Klebsiella spp. (50% inhibited by 2 μg/ml). Piperacillin and Bay k 4999 were equally active against Proteus spp., and piperacillin had high activity against Bacteroides fragilis (50% inhibited by between 1 and 2 μg/ml).

Although it is generally believed that bactericidal and bacteriostatic drugs should not be combined in vivo, in vitro experiments using the checkerboard dilution technique revealed no antagonism between penicillin/cephalothin and rolitetracycline but rather additive or synergistic activity of either drug combination in 40 to 50% of 20 Escherichia coli , and 14 Staphylococcus aureus strains. Slight antagonism occurred only between 3 and 8 h after combining penicillin/cephalothin and rolitetracycline in either bacteriostatic or bactericidal concentrations, but not after 24 h of incubation, nor was antagonism found with combinations of these drugs in bacteriostatic concentrations. Neither bacteriostatic nor bactericidal activity of penicillin/cephalothin and rolitetracycline was inhibited by pretreatment of one E. coli strain with bacteriostatic rolitetracycline or bacteriostatic penicillin/cephalothin concentrations. Penicillin and cephalothin could exert a bactericidal effect after 2-h exposure of the E. coli strain to bacteriostatic rolitetracycline concentrations. Combined action of subinhibitory penicillin and rolitetracycline concentrations resulted in more pronounced inhibition of growth than either drug alone. The higher activity of penicillin/cephalothin in combination with rolitetracycline on some E. coli and S. aureus strains might be due to a better access of rolitetracycline into bacterial cells whose cell walls have been weakened by cell wall-active, bactericidal drugs. Thus, growth of penicillin-induced spheroplasts of E. coli and stable staphylococcal L-forms was inhibited by much lower concentrations of rolitetracycline than were the corresponding parent cells with intact cell walls.

The in vitro activity of mecillinam, a 6 β-amidinopenicillanic acid derivative, was investigated. Mecillinam is not active against most gram-positive coccal or bacilliary forms. Many members of the Enterobacteriaceae are inhibited, with 86% of Escherichia coli , 71% of Klebsiella, 62% of Enterobacter , 75% of Salmonella , 69% of Shigella , and 70% of Citrobacter inhibited by 6.3 μg/ml. Indole-positive Proteus and Serratia were generally resistant as are Pseudomonas strains. Although mecillinam is hydrolyzed by gram-negative β-lactamases, the compound inhibits β-lactamase-producing organisms, particularly E. coli . The conductivity of medium used to determine minimal inhibitory concentration and inoculum size produce markedly different values. In medium of high conductivity, 10 mS, mecillinam is inactive against many strains of bacteria. In all media there is a great difference between the minimal inhibitory and minimal bactericidal levels.

An antibiotic susceptibility test (AST) device is described. Minimum inhibitory concentration results on 1,294 clinical isolates with seven antibiotics were rapid, reliable, and reproducible. Agreement with the disk diffusion test was 90%. Interlaboratory agreement for the AST was 94%. The AST provides minimum inhibitory concentration capability without the necessity of prior weighing and dilution of antibiotics.

Minimal inhibitory concentration and disk diffusion susceptibility studies were carried out with oxolinic acid to determine a recommended disk content for disk susceptibility testing. Regression curve analyses were performed with disks containing 1, 2, and 5 μg of oxolinic acid. Data suggest that the 5-μg disk more satisfactorily fulfills requirements for susceptibility testing than the 1- or 2-μg disks evaluated. Ultraviolet spectral analysis studies for absorption maxima and extinction coefficients were done to verify the authenticity and concentration of oxolinic acid in the aqueous solutions studied.

The activity of amphotericin B methyl ester was compared with that of amphotericin B, using Candida albicans (34 isolates), Torulopsis glabrata (12 isolates), Filobasidiella neoformans (stat. conid. Cryptococcus neoformans ) (14 isolates), and Coccidioides immitis (37 isolates) and tube dilution in a totally synthetic, completely defined medium (SAAMF) with inocula of 10 4 colony-forming units per ml. Minimal inhibitory concentrations were read after 24 h at 34°C for C. albicans and T. glabrata , and after 48 h at 34°C for C. immitis and F. neoformans . Minimal lethal concentrations were determined by subculture of 10% of the volume of the cultures without evident growth onto Sabouraud glucose agar medium. Overall, amphotericin B methyl ester was slightly less active than amphotericin B, with the differences attaining statistical significance for: (i) inhibition of C. albicans and T. glabrata and (ii) killing of T. glabrata .

The O-antigen types of 600 independently isolated Escherichia coli strains from human feces have been determined, and the types have been related to the antibiotic resistance patterns of the strains. The relative abundance of each O-antigen type differed in the susceptible and resistant series of strains. The majority (86%) of the resistant strains carried R plasmids. Resistant E. coli (20.3%) were found associated with O-antigen types 8, 9 and 101, whereas the susceptible strains covered a wide range of O-antigen types. Examination of 174 resistant strains isolated from calf feces also showed a prevalence of O-antigen types 8, 9, 101 (24.1%), and it seems probable that strains expressing these three O-antigen types commonly carry R plasmids in the alimentary tracts of man and calves. The number of strains not typeable with the O sera available were similar in the human (12.5%) and the calf (11.5%) series. There are no grounds for distinguishing “human” from “calf” E. coli on the basis of their O-antigen reactions.

Forty recent clinical isolates of three different Candida sp. were tested in the microtiter system for susceptibility to two new 2,4-diaminoquinazoline (DAQ) compounds, amphotericin B and flucytosine. The two DAQ preparations showed activity similar to amphotericin B and flucytosine. The geometric mean minimal inhibitory concentrations for these four drugs were as follows: DAQ 1A, 0.64 μg/ml; DAQ 2A, 1.39 μg/ml; amphotericin B, 1.03 μg/ml; and flucytosine, 0.72 μg/ml. An additional seven DAQ compounds were tested but showed less or no activity against 17 Candida isolates. Forty-eight-hour viability studies with DAQ 2A alone or in combination with amphotericin B, flucytosine, or sulfamethoxazole were carried out with one isolate of intermediate susceptibility to each of these agents except sulfamethoxazole. For this isolate the combination of DAQ 2A and sulfamethoxazole was synergistic, and the combination of DAQ 2A and AMB was either synergistic or additive, whereas the combination of DAQ 2A and flucytosine was antagonistic. Although regrowth of cultures exposed to DAQ 2A was noted over a 48-h period, neither degradation of the drug nor development of resistance to the drug could be detected. Swiss white mice receiving DAQ 1A at a dose of 6 mg/kg for 5 days showed no obvious signs of toxicity, including weight loss.

A total of 91 multiply resistant bacterial strains, including Klebsiella pneumoniae (32 strains), Pseudomonas aeruginosa (16 strains), and Serratia marcescens (43 strains), were collected during hospital epidemics of nosocomial infection from 1975 to 1979. These strains were resistant to gentamicin, tobramycin, cephalothin, chloramphenicol, and ampicillin. Their susceptibility to three new broad-spectrum β-lactams, LY127935 (a 1-oxa-β-lactam), cefotaxime (HR 756), and cefoperazone (T 1551), was compared with the susceptibility of random strains of nine species of aerobic gram-negative bacilli collected in the same hospital in 1979. Susceptibility to cefamandole and ticarcillin was also determined. Strains of staphylococci and streptococci from that hospital and two nearby city-county hospitals were also compared for the three new cephalosporins and other effective antibiotics. The agar dilution method was used to measure the minimum inhibitory concentration for each antibiotic. The multiply resistant strains (minimum inhibitory concentration for gentamicin ≥ 8 μg/ml) usually were as susceptible to the three new broad-spectrum β-lactams as were non-multiply resistant strains. Both Klebsiella pneumoniae and Serratia marcescens , including multiply resistant and non-multiply resistant strains, were most susceptible to the 1-oxa-β-lactam LY127935 and cefotaxime. P. aeruginosa (both multiply resistant and non-multiply resistant strains) were most susceptible to cefoperazone. All three new β-lactams were active against non-multiply resistant strains of Escherichia coli, Enterobacter spp., Proteus spp., and Citrobacter spp. Providencia stuartii were most susceptible to cefotaxime and the 1-oxa-β-lactam LY127935. The three new β-lactams were all less active against staphylococci (especially methicillin-resistant Staphylococcus aureus ) than cephalothin. Streptococcus pyogenes and S. pneumoniae were very susceptible to cefotaxime and cefoperazone, though less susceptible to LY127935. None of the three new β-lactams was active against S. faecalis . All were very active against both penicillinase-positive and -negative strains of Neisseria gonorrhoeae .

A newly described 6β-amidinopenicillanic acid derivative, mecillinam (formerly called FL 1060), showed a high in vitro activity against Enterobacteriaceae . The effect on Escherichia coli was bactericidal and was due to lysis of the cells. The longer the culture grew under the influence of mecillinam or the lower the inoculum, the greater the bactericidal effect. The morphology of the cells changed towards large spheric forms (2 to 5 μm) under the influence of mecillinam. Consequently a great discrepancy between the optical density and the viable count was seen. The morphologically abnormal cells could be protected against lysis in vitro by addition of ionized compounds such as sodium chloride. Abnormal cells were more sensitive to ampicillin than normal cells. As expected synergy could be demonstrated between mecillinam and ampicillin. This was marked under experimental conditions where the abnormal cells were protected against lysis.

Inter- and intralaboratory reproducibility of susceptibility testing requires stable control strains. The Food and Drug Administration diffusion procedure recommends the Seattle strains of Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) for this purpose. It was of interest to determine the present reproducibility of control cultures maintained in various laboratories over several years. Fifteen cultures each of S. aureus and E. coli were obtained from laboratories in different parts of the country. Their performance was compared with strains directly derived from ATCC. Diffusion susceptibility tests using a modified overlay technique were made with four replicates. Seven of the eight statistically significant differences in responses of the staphylococci were to penicillin, methicillin, or cephalothin. One culture was a penicillinase producer with a zone 15 mm less than the standard strain. Eleven of the 15 cultures showed no significant deviations or differences greater than 2 mm from the results with the strain derived directly from ATCC. All except the penicillinase producer were of identical phage type. Among 150 organism-antibiotic combinations tested with E. coli , all but one reading were within 2 mm of the standard. Four of the six statistically significant differences were in a culture from one laboratory. The stability of the cultures appears to have been influenced by the method of storage. Cultures that were kept frozen during extended storage were remarkably stable. Significant differences were found in cultures from four of five laboratories that maintained cultures in refrigerators or at ambient temperature.

The results of drug susceptibility tests were found to be affected by changes that occur spontaneously in populations of Mycobacterium avium maintained in the laboratory. Because the transparent colony type variant was resistant to antituberculosis chemotherapeutic agents and the opaque colony type variant was usually susceptible to these agents, the transition of transparent to opaque colony type was investigated. The rate of the transition was found to be temperature dependent and, in agreement with a previous report, was found to be about 10 −4 to 10 −5 per generation at 37 C. Reversion was found to occur at a rate of 10 −6 to 10 −7 at 37 C. The mutation rate from susceptibility to resistance to rifampin, kanamycin, and erythromycin was about 10 −8 to 10 −9 mutations per bacterium per generation. Judged from our data, the high rate of the transparent to opaque variation was not caused either by mutator effects or by the occurrence of extrachromosomal genes in these bacteria, but could have been due to selective mechanisms still incompletely understood.

Susceptibility disks impregnated with 35 different antibiotics were assayed by the current Code of Federal Regulations method and by a new method employing an agar overlay. The new method, a simplified analytical procedure, exhibited improvement in zone definition and an increase in dose response. Assays performed by the new method were accurate and precise. The agar overlay technique appears to be superior to the current Code of Federal Regulations method.

A number of characteristics were determined with a new automated method for a partially purified β-lactamase from Vibrio parahaemolyticus . The enzyme had a molecular weight of 28,000 by gel filtration, a pH optimum between 6.5 and 7.0, and a temperature optimum at 36°C. With penicillin G as the substrate, the K m value for the β-lactamase was 54.4 μM. The β-lactamase was inhibited by cloxacillin but not by p -chloromercuribenzoate. The enzyme was similar but not identical to β-lactamases from gram-negative, “nonhalophilic” organisms described by other workers. The microiodometric assay to measure β-lactamase activity was automated with the use of a centrifugal analyzer that permitted 14 simultaneous determinations. Within-run precision was tested by putting the same reaction mixture in each well, and the coefficient of variation was only about 3%. Four extracts from different strains of halophilic vibrios were used to demonstrate that reaction rates were linear with enzyme concentration. The correlation coefficient of activity by the automated method with activity by the spectrophotometric method was 0.9721, demonstrating that the methods compared favorably with each other. The automated method greatly facilitated the characterization of the β-lactamase.

Cefamandole minimum inhibitory concentrations (MICs) of 10 strains of Enterobacter were determined by the ICS agar dilution and broth dilution procedures. Agar dilution MICs ranged from 1 to 8 μg/ml, with an inoculum of 10 4 organisms/spot. Broth dilution MICs were consistently higher, with an inoculum of approximately 7 × 10 5 organisms/ml. Seven strains showed MICs of ≥64 μg/ml. There was a marked inoculum effect in broth, and skipped tubes were often observed. Variants resistant to 32 μg/ml or more were isolated by direct selection and were shown to occur at a frequency of approximately 10 −6 to 10 −7 . A mutant showing a 16-fold increase in agar dilution MIC was also isolated by indirect selection. These variants and others isolated from broth in the presence of cefamandole were tested for ability to inactivate the antibiotic, using both a biological and a chemical procedure. Two distinct classes of variants were seen. Twelve of 28 were shown by both methods to inactivate the antibiotic, whereas the others, including the indirectly selected mutant, did not. The wild types were also negative by both tests. The higher cefamandole MICs of Enterobacter in broth, thus, appeared to reflect a high frequency of resistant variants that were not detected with the inoculum and end point criteria usually used in agar dilution methods. The ability of some variants to inactivate cefamandole may have resulted from a mutation that extended the activity of Enterobacter cephalosporinase to include this antibiotic.

The in vitro activity of cefmenoxime (SCE-1365) was evaluated in a multiphased collaborative investigation. Over 7,500 consecutive clinical isolates were tested in five laboratories, and greater than 90% of the following organisms were inhibited by cefmenoxime at the following concentrations: Enterobacteriaceae and non-enterococcal streptococci, ≤0.125 μg/ml; Staphylococcus aureus , ≤2.0 μg/ml; and nonfermenting gram-negative bacilli and Bacteroides fragilis group, ≤32 μg/ml. Both beta-lactamase-producing and -nonproducing Haemophilus influenzae and Neisseria gonorrhoeae were inhibited by cefmenoxime at ≤0.03 μg/ml. The spectrum of cefmenoxime was similar to that of other, newer cephalosporins, particularly cefotaxime. A pronounced inoculum effect was found with some species upon increasing inocula from 10 5 to 10 7 colony-forming units per ml, resulting in an approximate eightfold increase in minimum inhibitory concentrations. Cefmenoxime was bactericidal when tested with inocula of 10 5 colonyforming units per ml, and mean differences between the minimum inhibitory concentration and the minimum lethal concentration were less than one log 2 dilution. No significant hydrolysis of cefmenoxime by five different beta-lactamases was detectable, and cefmenoxime exhibited marked inhibition of type I beta-lactamases. Regression and error rate-bounded analyses of results of disk diffusion and reference broth microdilution susceptibility tests were performed on 421 bacterial isolates, and the following tentative zone size breakpoints are proposed: ≥22 mm, susceptible; ≤14 mm, resistant; and 15 to 21 mm, moderately susceptible (indeterminate). These data and cross-resistance studies with other newer cephalosporins indicate marked similarity of in vitro activity within this group of drugs, particularly between cefmenoxime, moxalactam, and cefotaxime. Any one of these could serve as the representative for the disk diffusion testing of this group of drugs if comparable minimum inhibitory concentration breakpoints were used for each drug.

We have shown that it is possible to screen for penicillinase-producing Neisseria gonorrhoeae directly from primary culture plates. Experiments involving the cocultivation of four genera of beta-lactamase-positive (β-lac + ) bacteria and a beta-lactamase-negative (β-lac − ) N. gonorrhoeae on modified Thayer-Martin medium indicated that suppressed or inhibited β-lac + bacteria did not give false-positive results when isolated β-lac − colonies of gonococci were tested. Colonies were assayed for β-lac production by an adaptation of a microacidometric method in which bacteria could be tested before or after the addition of oxidase reagent.

Polyene antifungal antibiotics produce various degrees of membrane damage in sheep erythrocytes in vitro. Mediocidin, filipin, amphotericin B, and candicidin were found to result in greater damage than nystatin, pimaricin, and amphotericin B methyl ester. The degree of sensitivity of the cells varied by 100-fold for mediocidin verus amphotericin B methyl ester as measured by curves of hemoglobin release versus drug concentration. In erythrocytes prelabeled with radioactive chromium, release of the isotope through polyene-damaged cell membranes was found to occur at lower drug concentrations than measurable hemoglobin release, and the percentage of isotope released at the highest drug dose was consistently greater than the percentage of hemoglobin released. Thus, the isotope assay is a more sensitive indicator of polyene-induced membrane damage in the test system. These significant differences in release of molecules through polyene-induced membrane lesions indicate the complex nature of the binding and further interactions of this class of drugs with the plasma membrane.

The in vitro activity of chephaloridine, cephalexin, cefatrizine (BL-S640), and cephapirin (BL-P-1322) was evaluated by the serial dilution method against pathogenic gram-positive and gram-negative bacteria isolated from bovine udders and neonatal calf diseases. Cephapirin showed the comparatively greatest activity against the most common streptococcal species associated with bovine mastitis, whereas cephaloridine exhibited the best activity against Staphylococcus aureus . Cefatrizine was more active than the other cephalosporins against the gram-negative bacteria studied. In general, the minimal bactericidal concentration of each cephalosporin was two- to fourfold lower than the comparative value reported in the literature against the same type of pathogen of human origin.

Due to the emergence of cephalothin- and gentamicin-resistant Klebsiella pneumoniae at this institution, we investigated the in vitro activity of two new cephalosporin compounds—cefoxitin and cefamandole. Whereas both drugs were active against cephalothin- and gentamicin-susceptible isolates of Klebsiella , only cefoxitin exhibited significant activity against cephalothin- and gentamicin-resistant isolates. Seventeen of 20 (85%) of the cephalothin- and gentamicin-resistant isolates were inhibited by ≤12.5 μg of cefoxitin per ml. The minimum bactericidal concentration in broth of representative isolates equaled the agar dilution minimum inhibitory concentration. Kirby-Bauer disk susceptibility testing correlated well with the agar dilution minimum inhibitory concentration. Cephalothin and cefamandole resistance of isolates could be correlated with antibiotic degradation by β-lactamase. Cephalothin and cefamandole evoked only a transient decrease in viable bacterial cell count with rapid inactivation of antibiotics, and full regrowth of the organisms. Cefoxitin, on the other hand, was quite effective in vitro against multiply resistant Klebsiella . No β-lactamase degradation of cefoxitin was detected. Growth curves with antibiotics indicated rapid killing of cephalothin- and gentamicin-resistant isolates by cefoxitin.

SCE-129 [3-4-carbamoyl-1-pyridiniomethyl-7β- ( d -α-sulfophenylacetamido)-ceph-3-em-4-carboxylate] is a cephalosporin that inhibits Pseudomonas aeruginosa and Staphylococcus aureus . SCE-129 is tenfold more active than carbenicillin in inhibiting P. aeruginosa . SCE-129 has poor activity against Enterobacteriaceae compared with other cephalosporins and is 16-fold less active than the cephalosporins against streptococci. The activity of SCE-129 does not correlate with β-lactamase stability, although SCE-129 is resistant to hydrolysis by gram-positive and gram-negative bacteria. The compound does not inhibit the hydrolysis of other cephalosporins. Although SCE-129 acts synergistically with gentamicin to inhibit some Pseudomonas , this cannot be predicted based on knowledge of resistance to one or more compounds.

Bacampicillin (proposed international nonproprietary name), 1′-ethoxycarbonyloxyethyl 6-( d -α-aminophenylacetamido)penicillanate, is a new orally well-absorbed penicillin, highly active in vivo due to rapid transformation into ampicillin. The compound is stable in vitro at gastric pH and hydrolyzed slowly to ampicillin at neutral pH but very rapidly in the presence of biological fluids, e.g., tissue homogenates or serum. In vivo the transformation into ampicillin is so rapid that no unchanged compound could be detected in the blood after oral administration of bacampicillin to rats, dogs, and humans. On oral administration to mice, rats, and dogs, bacampicillin was found to be better absorbed than ampicillin, giving higher and earlier peak blood levels of ampicillin. The bioavailability of bacampicillin in rats and dogs was three to four times higher than that of an equimolar amount of ampicillin. On oral administration to rats, bacampicillin was found to give higher levels of ampicillin in organs such as the kidney, liver, and spleen than ampicillin itself. In “tissue cages” in rats, higher transudate levels of antibiotic were found after oral administration of bacampicillin than after ampicillin. On oral treatment of experimentally infected mice, bacampicillin was found to be more active than ampicillin.

The antifungal activity of a lyophilized powder containing aloe leaf homogenate (whole-leaf powder) against Trichophyton mentagrophytes was investigated. The minimal inhibitory concentration was 25 mg/ml by the agar dilution method, using Sabouraud glucose agar medium. At subinhibitory concentrations, the powder exerted its main effect on colony growth by prolongation of the lag phase and inhibition of growth rate. Homogenates of fresh whole leaf were filtered through Whatman GF/A paper, and the filtrate was dialyzed and concentrated by molecular filtration using an Amicon hollow-fiber dialyzer concentrator DC-2, and a powder containing components with molecular weights higher than 10,000 (high-molecular-weight component powder) was prepared by lyophilization. The minimal inhibitory concentrations against three strains of T. mentagrophytes were all 10 mg/ml. The inhibitory activity was fungicidal and was lost by heating at 100°C for 30 min. Both the whole-leaf powder and the high-molecular-weight component powder induced various morphological abnormalities in spores and hyphae by the inhibition of spore germination and development of hyphae.

Six anionic and five nonionic surfactants were tested for their effect on the fungistatic action of clotrimazole against Candida albicans . All of the anionic agents that did not contain an ethylene oxide group were capable of potentiating the anti- Candida activity of clotrimazole, whereas all five members of the polyoxyethylene surfactant group, including four nonionic agents and one anionic agent, acted in an antagonistic fashion. The combination of clotrimazole and the anionic surfactant dioctyl sodium sulfosuccinate was the most potent in synergy and, thus, more precise studies were made with this combination. Although appropriate combinations of the two drugs showed a potent fungicidal activity against proliferating cultures, none of these combinations tested was lethal when cell growth was restricted by nutritional deficiency. The lethal effect of the combined drugs was partly reversed when growing cultures were treated in the presence of an osmotic stabilizer. Whether cells were treated with moderate and higher concentrations of clotrimazole and dioctyl sodium sulfosuccinate, alone or in combination, there was little change in cell wall content of total protein, carbohydrate, or lipid from that in untreated control cells. However, there was a significant decrease in the cell wall content of phospholipid when moderate concentrations of the two drugs were combined.

The in vitro activity of a new semisynthetic penicillin, piperacillin, was determined against 577 clinical isolates of gram-positive cocci and gram-negative bacilli. A concentration of 12.5 μg/ml inhibited 92% of isolates of Pseudomonas aeruginosa , 82% of Serratia marcescens , 73% of Escherichia coli , 61% of Klebsiella spp., and 42% of Enterobacter spp. Most Proteus spp. were extremely susceptible; over 85% were inhibited by 0.10 μg/ml. Piperacillin failed to inhibit the growth of gram-negative bacilli when large inocula were used. The type of media and pH had variable effects on the activity of piperacillin, depending upon the organism. Piperacillin was generally less active than PC-904 against gram-negative bacilli, but was consistently more active than carbenicillin and ticarcillin.

Quantitative bactericidal activities of β-lactam antibiotics were determined by the agar plate method. Broth cultures, of which the colony-forming units were counted before the study, were inoculated on antibiotic-containing agar plates, utilizing a 10 −3 , 10 −2 , or 10 −1 dilution or undiluted culture plated with each 0.001-ml calibrated loop. These plates were incubated at 37°C overnight, and the minimal drug concentration at which no bacterial growth was observed on the plates was defined as minimal inhibitory concentration. After this procedure, the agar surface was treated with β-lactamase spray to inactivate the antibiotic. These plates were incubated again at 37°C overnight. The minimal drug concentration at which no evidence of bacterial growth was visible on the plates (resulting in a 100% kill) was defined as minimal bactericidal concentration. The lowest concentration which reduced the number of colony-forming units to 1/1,000 that in the original inoculum (resulting in a 99.9% kill) was defined as minimal lethal concentration. When compared for Escherichia coli, Klebsiella pneumoniae, Enterobacter sp., Serratia marcescens, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis , alpha-hemolytic streptococcus (non-enterococcal), beta-hemolytic streptococcus, and enterococcus, the minimal bactericidal concentrations were generally several fold higher than the minimal inhibitory concentrations. Minimal lethal concentrations were virtually the same as minimal inhibitory concentrations for gram-negative strains; however, for some gram-positive strains, minimal lethal concentrations were higher than minimal inhibitory concentrations.

A rapid method for determining the minimal inhibition concentrations (MICs) of ampicillin using Haemophilus influenzae as the test organism is described. This semiautomated technique using the Autobac system was compared with the agar dilution MIC method. Fifty-seven isolates of both susceptible and resistant H. influenzae were tested. There was exact quantitative agreement with 65% of the isolates, and 91% of the isolates showed no more than a one-dilution-interval difference when MICs were determined by both methods. Total testing time was 3 to 4 h.

Triton X-100 treatment or freeze-thawing damages the membranes of sporulating or vegetative cells as seen by protein leakage from cells. A 40% increase in the specific [ 14 C]benzylpenicillin-binding capacity of detergent-treated or frozen sporulating cells was observed. Neither freezing nor Triton X-100 treatment of vegetative cells produced a detectable effect on their [ 14 C]benzylpenicillin-binding capacity. These data indicate the presence of penicillin-binding sites in intact sporulating bacilli not accessible to penicillin in routine binding assays. The chemical specificity of [ 14 C]benzylpenicillin binding to detergent-treated sporulating cells is similar to that observed with untreated vegetative or sporulating cells.

5-episisomicin (Sch 22591) is a novel semisynthetic aminoglycoside with a spectrum and potency similar to gentamicin in its activity against susceptible bacterial strains, but with increased potency against Pseudomonas, Providencia , and Proteus rettgeri . It is also more active than tobramycin and amikacin against these last-mentioned species. Against resistant strains, Sch 22591 is significantly more active than gentamicin or tobramycin. Against resistant gram-negative bacteria other than Pseudomonas , Sch 22591 has activity similar to that of amikacin, but Sch 22591 is more potent. Against Pseudomonas strains, it is active against most gentamicin- and tobramycin-resistant strains and is more active than the other three antibiotics. Some Pseudomonas strains are resistant to Sch 22591, but susceptible to amikacin. Against a selection of aminoglycoside-resistant staphylococci, Sch 22591 has very good activity against strains resistant to tobramycin, amikacin, and gentamicin. The superior in vitro potency of Sch 22591 against Pseudomonas has been confirmed in vivo in experimental infections in mice. Absorption in dogs is similar to that of other aminoglycoside antibiotics. The acute toxicity of Sch 22591 in mice is greater than that of gentamicin; its vestibular toxicity potential and nephrotoxicity potential in cats and rats appear to be similar to those of gentamicin.

Thirty-five strains each of Klebsiella and Proteus were tested for susceptibility to mecillinam alone and in combination with ampicillin, carbenicillin, cephalothin, and cefazolin. Antibiotics were considered to be synergistic when there was a ≥fourfold reduction in minimum inhibiting concentration of both antibiotics in the combination as compared with that of each antibiotic alone. Synergy of mecillinam with ampicillin, carbenicillin, cephalothin, and cefazolin was demonstrated against 2, 3, 7, and 8 of the 35 strains of Klebsiella and against 14, 14, 19, and 24 of the 35 strains of Proteus , respectively. Synergy against the isolates of Proteus was related to species, whereas against Klebsiella it was related to susceptibility of the isolates to mecillinam. Tests of combinations of mecillinam with other antibiotics on the same and different species indicated that synergy was related to the antibiotic, the species, and the strains of organisms tested.

Cefaclor (CCL), a new cephalosporin, was tested in vitro against 602 (271 anaerobic and 331 aerobic) clinical isolates in comparison with cephalothin, cefazolin, cephradine, and cefamandole. Sixteen micrograms of CCL per ml inhibited 68% of all aerobes tested and 80% of the 211 enteropathogenic organisms ( Escherichia coli, Salmonella , and Shigella ) isolated from cases of infantile diarrhoea. CCL inhibited 88% of gram-positive anaerobic cocci and 72% of Bacteroides other than B. fragilis at a concentration of 16 μg/ml. B. fragilis and Clostridia were resistant to CCL. Increased inoculum of E. coli from 10 5 to 10 9 increased the minimal inhibitory concentration of CCL and cefamandole by fourfold against 7 of the 64 strains tested. All seven were beta-lactamase negative. No antimicrobial synergism was noted between CCL and penicillin. The in vitro efficacy of CCL, an oral cephalosporin, against enteropathogenic E. coli , if proven safe, may be tested in vivo against such infections.

β-Lactamase from strains of Bacteroides melaninogenicus and Bacteroides oralis hydrolyzed penicillin more rapidly than ampicillin or carbenicillin. Cephalothin and a chromogenic cephalosporin (87/312) were also hydrolyzed by the enzyme. Activity was found only in β-lactam-resistant strains, but there was considerable variation in activity among strains having the same minimal inhibitory concentrations of antibiotic. β-Lactamase activity was cell bound and appeared to be tightly associated with the cell envelope since detergents were required to elute this activity.

CP-45,899 {3,3-dimethyl-7-oxo-4-thia-1-azabicyclo(3.2.0)heptane-2-carboxylic acid, 4,4-dioxide, [2S-(2α,5α)]} is an irreversible inhibitor of several bacterial penicillinases and cephalosporinases. In the presence of low concentrations of CP-45,899, ampicillin and other β-lactams readily inhibit the growth of a variety of resistant bacteria that contain β-lactamases. CP-45,899 used alone displays only weak antibacterial activity, with the notable exception of its potent effects on susceptible and resistant strains of Neisseria gonorrhoeae . CP-45,899 appears to be somewhat less potent but markedly more stable (in aqueous solution) than the recently described β-lactamase inhibitor clavulanic acid. The spectrum extensions provided by the two compounds are similar. A 1:1 mixture of CP-45,899 and ampicillin displays marked antimicrobial activity in mice experimentally infected with ampicillin-resistant Staphylococcus aureus, Haemophilus influenzae, Klebsiella pneumoniae , and Proteus vulgaris .

The anaerobic broth disk (AnBD) method of Wilkins and Thiel and a new modification, designated the thioglycolate broth disk method, were compared with an agar dilution technique. The thioglycolate broth disk method was incubated aerobically (AeTBD) or anaerobically (AnTBD). One hundred anaerobic bacteria representing 15 species were tested with clindamycin, chloramphenicol, erythromycin, penicillin, and tetracycline. Agreement of results by the two methods with minimal inhibitory concentration determinations were: AnBD, 95.2%; AnTBD, 91.5%; AeTBD, 94.5%. With clindamycin, chloramphenicol, and penicillin, the agreement of the AeTBD and agar dilution results was 100%, 100%, and 95%, respectively. Using the AeTBD method, only 1.1% of all tests gave false susceptible readings, whereas 4.4% gave false resistant readings. All susceptibility testing errors occurred with tetracycline, erythromycin, and, to a lesser extent, penicillin. For each method, the changes in designation of bacteria as being susceptible or resistant to an antibiotic between trials primarily involved strains with minimal inhibitory concentrations which were ± one dilution of the respective breakpoint value. The same situation was true for most bacteria that yielded false resistant readings within each trial. False resistant readings with tetracycline were determined to be unrelated to excess cation content of test media. These results reaffirm the reliability of the AnBD method and indicate that the AeTBD modification is equally reliable. The greater convenience and lower cost of the AeTBD method should make possible more widespread performance of susceptibility testing for anaerobic bacteria in hospital laboratories.