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The feces of five patients admitted to a hospital during an outbreak of cholera in Melbourne, Australia, in November 1972, were examined for the presence of tetracycline-resistant coliforms and tetracycline-resistant strains of Vibrio cholerae . Despite the abundance of tetracycline-resistant coliforms able to transfer this resistance to other strains of Escherichia coli , no tetracycline-resistant strains of V. cholerae were detected. In vitro transfer experiments using the V. cholerae strain responsible for the outbreak as recipient revealed that it was a particularly poor host for most R plasmids.

Sodium polyanethol sulfonate inhibits aminoglycoside and polymyxin classes of antibiotics in direct proportion to its concentration. Aminoglycoside and polymyxin class antibiotics are selectively inactivated; penicillin, including the semisynthetic penicillins, cephalothin, chloramphenicol, clindamycin, tetracycline, erythromycin, and vancomycin are not inhibited. By incorporating sodium polyanethol sulfonate directly into the test medium it is possible, in a 4-h antibiotic blood level assay, to selectively obviate the activity of the aminoglycosides and polymyxins to determine the concentration of other antibiotics present in the same serum sample.

The ability of some exogenously supplied purines, pyrimidines, and their nucleosides to antagonize the in vitro fungistatic activity of 5-fluorocytosine (5-FC) in Aspergillus species was examined. All compounds tested except thymidine were capable of altering the minimal inhibitory concentration of 5-FC for seven strains of aspergilli tested. In most instances, there was a reciprocal correlation between the ability of a compound to antagonize the fungistatic activity of 5-FC and the quantity of 5-FC taken up by cells in the presence of the compound over a 72-h period.

The antiviral effect of the nucleoside analog 5-iodo-5′-amino-2′,5′-dideoxyuridine (AIU) was tested with three isolates of varicella-zoster virus (VZV). AIU concentrations of 10 to 800 μM (3.5 to 288 μg/ml) reduced the number of plaques produced by VZV-infected cells and cell-free VZV from approximately 30 to 95%. Smaller plaque size was also observed in the presence of AIU. AIU was less effective than arabinofuranosylthymine in reducing VZV-induced plaques since as little as 5 μg of arabinofuranosylthymine per ml completely blocked plaque formation by cell-free VZV. Toxicity assays with human diploid embryo fibroblast cells were also carried out. Drug concentrations as high as 800 μM were not toxic to human diploid embryo fibroblast cells as determined by radiolabeling of cell deoxyribonucleic acid, ribonucleic acid, and protein.

To perform microdilution antimicrobial susceptibility tests efficiently, broth dilutions of the antimicrobial drugs should be prepared in large batches and stored frozen until needed. Studies were carried out to document the stability of ampicillin, benzylpenicillin, carbenicillin, methicillin, oxacillin, nafcillin, and cephalothin in microdilution trays during storage at −20 and −60 C. No significant deterioration was noted after 21 days of storage, as detected by bioassay and by replicate minimal inhibitory concentration determinations with control organisms. No significant differences were noted moreover between trays stored for three weeks at −20 C and those stored at −60 C. The microdilution technique was found to be a highly reproducible method for quantitative determination of antimicrobial susceptibility.

T-1220, sodium 6-[ d -(-)-α-(4-ethyl-2,3-dioxo-1-piperazinylcarbonyl-amino)- α-phenylacetamido] penicillanate, is a new semisynthetic penicillin derivative that possesses a broad spectrum of in vitro antibacterial activity against gram-positive and gram-negative bacteria. T-1220 is more effective than carbenicillin (CB-PC) against Pseudomonas aeruginosa, Klebsiella pneumoniae, Proteus species, and Serratia marcescens . Addition of human serum to culture media did not significantly alter the antibacterial activity of T-1220. Greater bactericidal activity toward various strains of gram-negative bacteria was demonstrated with T-1220 than with CB-PC. T-1220, like penicillin G, was hydrolyzed by penicillinase, but was sable to a type IV penicillinase produced by P. aeruginosa strains. In vivo antibacterial activities of T-1220, ampicillin (AB-PC), and CB-PC were compared, using systemic infections of mice with P. aeruginosa, K. pneumoniae , and Escherichia coli . The 50% effective doses (milligrams per kilogram) of T-1220 were consistently lower than those of AB-PC and CB-PC.

Micro-Media Systems (MMS) has developed a procedure by which microdilution trays can be filled with dilutions of antimicrobial agents, frozen, and distributed to clinical laboratories. The trays are prepared in various distribution centers throughout the United States to supply clinical laboratories in the vicinity of each center. For use, trays are removed from the freezer, allowed to thaw, and then inoculated with inocula prepared as for any other susceptibility test, using a convenient disposable inoculator (ca. 5 μl per well). A collaborative study was planned to evaluate microdilution trays prepared in three Micro-Media Systems distribution centers. Microdilution minimal inhibitory concentrations (MICs) were compared to standard tube dilution tests (the international collaborative study group method). With gram-positive cocci, the two techniques gave essentially equivalent results. With gram-negative bacilli, the microdilution MICs were generally one doubling dilution lower than the standard tube dilution MICs. Similar results were seen with microdilution trays prepared with a Cooke Dynatech MIC 2000. Inter- and intralaboratory reproducibility with the macro- and microdilution techniques were quite satisfactory, i.e., at least 96% of the end points were within a range of ±1 log 2 dilution intervals.

The in vitro activity of netilmicin (Sch 20569), a new semisynthetic derivative of gentamicin, was compared with that of gentamicin and amikacin. One hundred and ninety-two clinical isolates of Enterobacteriaceae, Pseudomonas aeruginosa , and Staphylococcus aureus were tested using both agar and broth dilution techniques. Netilmicin was comparable to gentamicin, with the following exceptions: (i) for Serratia marcescens and P. aeruginosa , gentamicin was more active than netilmicin; (ii) all strains of Escherichia coli, Klebsiella, Enterobacter, Proteus mirabilis , and Citrobacter freundii , which were resistant to gentamicin, were susceptible to netilmicin; (iii) some strains of S. marcescens , indole-positive Proteus , and Providencia , which were resistant to gentamicin, were susceptible to netilmicin. Netilmicin was more active than amikacin for all Enterobacteriaceae and S. aureus and equal to amikacin in activity against gentamicin-susceptible strains of P. aeruginosa . All strains of P. aeruginosa , resistant to gentamicin, were also resistant to netilmicin but were susceptible to amikacin. Minimal inhibitory concentrations (MICs) obtained with broth and agar showed no significant differences except for P. mirabilis , where broth MICs were twofold greater than agar MICs, and for P. aeruginosa , where agar MICs were twofold higher than broth MICs. The minimal bactericidal concentration (MBC) was either identical to or within one twofold dilution of the MIC for the strains tested. A 100-fold increase in inoculum size produced less increase in MIC and MBC with netilmicin than with gentamicin or amikacin.

In vitro activity of BL-S786, a new parenterally semisynthetic cephalosporin, was investigated against 570 bacterial isolates. BL-S786 inhibited most Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis , and Salmonella . It inhibited some Enterobacter and indole-positive Proteus , but it was less active against these later species than was cefamandole, cefuroxime, or cefoxitin. It was not active against Serratia marcescens, Pseudomonas aeruginosa , or Bacteroides fragilis . BL-S786 was the least active new cephalosporin tested against staphylococci and was less active than cephalothin against streptococcal species. The activity of BL-S786 was not altered by the type of assay medium nor by 50% serum. The size of the test inoculum altered the minimal inhibitory and bactericidal concentrations for inhibition of some organisms, particularly those with Richmond type I β-lactamases. BL-S786 was not hydrolyzed by the R-factor-mediated, Richmond type III β-lactamase, but it was hydrolyzed by type I β-lactamases.

The overall prognosis of primary amoebic meningoencephalitis remains poor. The results of this study support previous finding that amphotericin B is the most efficacious drug against the Naegleria species in in vitro testing. In addition, the methyl ester of amphotericin B, a new derivative, also appears to be an effective agent. Of the drug combinations studied, amphotericin B plus minocycline and amphotericin B plus tetracycline showed synergy. The clinical significance of these findings remains to be determined.

The effects of supplementing Mueller-Hinton broth with calcium and magnesium on the minimal inhibitory concentrations (MICs) of eight aminoglycosides, colistin, tetracycline, and carbenicillin for 11 nonfermenters other than Pseudomonas aeruginosa were studied and compared with the effects for Escherichia coli and P. aeruginosa . MICs were simultaneously performed in unsupplemented Mueller-Hinton broth and Mueller-Hinton broth supplemented to contain 5 mg of calcium and 2.5 mg of magnesium per dl. Changes in MICs were expressed as the increases in the number of log 2 concentrations caused by supplementation. The usual increases in MICs of aminoglycosides caused by supplementation were: zero concentrations for E. coli , one to six concentrations for P. aeruginosa , and one to two concentrations for most other nonfermenters. The largest increases (five to six concentrations) were observed with gentamicin and P. aeruginosa . The usual increases in MICs of colistin were: zero concentrations for E. coli , two concentrations for P. aeruginosa , and one to two concentrations for other nonfermenters. Increases in MICs of tetracycline were: one to five concentrations for all organisms tested. The usual increases in MICs of carbenicillin were: zero concentrations for E. coli and P. aeruginosa and zero to two concentrations for other nonfermenters. These observations indicated that supplementation of Mueller-Hinton broth to contain recommended concentrations of calcium and magnesium had little effect on MICs of aminoglycosides and colistin for E. coli but increased MICs for most nonfermenters, increased MICs of tetracycline for E. coli and all nonfermenters, and had little effect on MICs of carbenicillin for E. coli and P. aeruginosa but increased the MICs for several nonfermenters other than P. aeruginosa .

Thirty clinical isolates of Staphylococcus aureus were tested for tolerance to oxacillin, cephalothin, and gentamicin by using killing curves and broth dilution susceptibility tests. Tolerance was defined as the presence of≥100 colony-forming units per ml remaining after 24 h of incubation of the killing curve suspensions. Nineteen of 30 isolates were tolerant to oxacillin and cephalothin, but none was tolerant to gentamicin. Among the tolerant isolates, there was a wide spectrum of tolerance, with from 0.2 to 43% of the colony-forming units in the starting inoculum remaining after 18 to 24 h of incubation. Tolerance was unrelated to phage type or to any of several other characteristics studied.

Early response of Escherichia coli to minimal inhibitory concentrations of 112883, BL-P1654, and carbenicillin was determined by [ 14 C]leucine uptake and scanning electron microscopy morphology studies. [ 14 C]leucine uptake was inhibited later by carbenicillin than by BL-P1654 or 112883. Cellular swelling at 30 min, septal region swelling, and lysis were the progressive morphological changes with BL-P1654 and 112883. Carbenicillin-treated cells showed septal region swelling, beaded chains, and lysis.

Kinetic data on potassium release from and killing of Candida albicans by the four polyene antibiotics amphotericin B, amphotericin B methyl ester hydrochloride, nystatin, and nystatin methyl ester hydrochloride are presented. The nystatins were relatively more effective than the amphotericins in causing potassium release rather than killing. These data suggest that the aqueous channels or pores formed by the polyene antibiotics are not central to the lethal action of the drugs.

Cefuroxime, a new parenteral cephalosporin was compared with cephalothin by broth microdilution susceptibility testing against 5,887 routine clinical bacterial isolates in four large clinical laboratories. The minimal inhibitory concentrations (MICs) of cefuroxime against the Enterobacteriaceae were consistently lower than those of cephalothin. This was most striking among the Enterobacter species, which were generally susceptible to cefuroxime (MIC ≤ 8 μg/ml), but resistant to cephalothin. Similar results occurred with Haemophilus species, Acinetobacter anitratus , meningococci, and Aeromonas hydrophilia , but Pseudomonas species and enterococci were resistant to high concentrations of both drugs. Streptococci showed slightly greater susceptibility to cefuroxime than to cephalothin. By contrast, staphylococci were more susceptible to cephalothin. Bacteroides fragilis was resistant to cefuroxime, but other anaerobes were generally susceptible.

Fifty strains of herpes simplex virus, isolated from patients with herpetic keratitis, were examined in vitro for susceptibility to polyinosinic acid-polycytidylic acid [poly(I:C)] in the presence of a constant concentration of diethylaminoethyl-dextran. The minimal inhibitory concentration of poly(I:C) for 44 of these strains ranged from 0.0001 to 0.1 μg/ml; for the remaining six strains, the minimal inhibitory concentration stood at 1 to 2 μg/ml. Fifteen isolates from primary infections were more susceptible to poly(I:C) than 35 isolates from recurrent infections. Isolates acquired at different points of a given clinical episode showed similar susceptibilities to poly(I:C). In two patients, isolates from consecutive recurrences of infection exhibited reduced susceptibilities. The implications of the above observations for the therapeutic use of poly(I:C) are discussed.

The effect of amphotericin B on CO 2 production was studied using a Bactec 225. A radiometric procedure for yeast susceptibility testing that requires 3 h of incubation was developed. A total of 48 yeast isolates was tested in this system and the results were correlated to tube dilution studies. The drug concentration causing at least a 44% decrease in CO 2 production correlated to the tube dilution minimal inhibitory concentration in 85% of isolates tested and was within one tube dilution of the minimal inhibitory concentration in over 95% of yeasts tested.

In vitro evaluation of 66 epidemiologically distinct, gentamicin-resistant, gram-negative isolates from four hospitals revealed that 92% were kanamycin resistant, 44% were netilmicin resistant, 41% were tobramycin resistant, and 6% were amikacin resistant. Combined resistance to gentamicin, tobramycin, and netilmicin occurred in 30% of the strains. Although the resistance percentage to amikacin was the lowest of the three newer agents, two strains were resistant to all of the aminoglycosides tested.

The minimal inhibitory concentrations of 30 antibiotics were determined by the agar dilution method for 17 penicillinase-positive and 50 penicillinase-negative strains of Neisseria gonorrhoeae . The latter included 42 strains that were penicillin susceptible (pen S) (minimal inhibitory concentration

Resistance plasmids isolated from Streptococcus agalactiae (group B) and S. faecalis (group D) have been compared in regard to resistance markers, molecular weight, and DNA-DNA homology. Three of them (pIP501, pIP612, and pIP613) have been found to confer identical (or very similar) resistance patterns (erythromycin, lincomycin, and streptogramin B, respectively) and to have similar molecular weights (19.8 × 10 6 , 22.7 × 10 6 , and 17.6 × 10 6 , respectively) and a high level of DNA-DNA homology in hybridization experiments (90 to 100%). These results are compatible with the view that these plasmids may derive from one common ancestor, and/or that they can be transferred between unrelated Streptococcus strains belonging to the same or different groups.

Methicillin-resistant Staphylococcus aureus (MRSA) strains show an unusual type of resistance at 37°C; only a small subpopulation of cells is resistant to the β-lactam antibiotics. Incubation in the presence of methicillin (MET), however, results in the emergence of a homogenous population highly resistant to MET. The purpose of the present study was to determine whether MET, despite its lack of killing effect on MRSA, would promote the bactericidal effect of gentamicin (GM), as demonstrated for MET-susceptible strains of S. aureus . Eleven epidemiologically distinct strains of MRSA were incubated with MET at various concentrations, in the presence or absence of sub-bactericidal concentrations (1/5 or 1/10 of the minimal bactericidal concentration) of GM, and tested for the synergistic action of both antibiotics by three different methods. Population analysis of the 11 strains in the presence of high concentrations of MET showed that the addition of GM at 1/5 or 1/10 of its minimal bactericidal concentration resulted in marked killing of the 11 strains. Time-kill curves obtained with 32 μg of MET per ml and GM at 1/5 or 1/10 of its minimal bactericidal concentration confirmed this synergistic killing at 24 h. These results were further documented by the checkerboard method on two strains. We conclude that the synergism between MET and GM, previously demonstrated for MET-susceptible S. aureus , holds true for MRSA as well and that it can be demonstrated at sub-bactericidal concentrations of GM.

Because of the need for non-β-lactam antimicrobics with antistaphylococcal activity, 14 antimicrobics (3 penicillinase-resistant penicillins, 7 aminocyclitols [5 new], 2 macrolides [1 new], clindamycin, and a new polysaccharide, everninomicin) were tested in vitro for activity against 22 penicillin-susceptible, 51 penicillin-resistant, and 47 methicillin-resistant strains of Staphylococcus aureus . Gentamicin, tobramycin, sisomicin, and verdamicin inhibited almost all strains at concentrations less than 1 μg/ml. The methicillin-resistant strains were as susceptible as other strains to the aminocyclitols and everninomicin. In contrast, many of the methicillin-resistant strains were also resistant to erythromycin, rosamicin, and clindamycin at attainable serum concentrations. The use of the bacterial synthetic amino acid medium, in comparison with Mueller-Hinton broth, resulted in an increase in the observed resistance of methicillin-resistant strains to the penicillinase-resistant semisynthetic penicillins.

The bactericidal efficacy of seven cationic detergents was studied in vitro against intact performed plaques of representative strains of four oral dental plaque-forming microorganisms: Streptococcus mutans, Streptococcus sanguis, Actinomyces viscosus , and Actinomyces naeslundii . Both absolute (plaque bactericidal index) and relative (chlorhexidine coefficient) indexes of antiplaque activity were established. Only cetyltrimethylammonium bromide was more efficacious than the reference agent, the bisbiguanide chlorhexidine, for the apparently more potent tooth-adherent cariogenic and periodontopathic elements, respectively, of the in vivo plaque microflora, S. mutans and A. viscosus .

The susceptibilities of 284 anaerobic bacteria, including 55 strains of the Bacteroides fragilis group, were determined by an agar dilution technique to metronidazole and two newer nitroimidazoles, ornidazole and SC-28538. All three agents showed marked in vitro activity against virtually all anaerobic bacteria tested. At concentrations ≤1 μg/ml, SC-28538 was significantly more active than either metronidazole or ornidazole. At concentrations of >1 μg/ml, the activities of all three agents were comparable. Propionibacterium and Actinomyces showed significant resistance to all three agents. Anaerobic and microaerophilic members of the genus Streptococcus were also often resistant, in contrast to Peptococcus and Peptostreptococcus strains. In addition, the bactericidal activities of ornidazole and SC-28538 were determined against 27 strains of the B. fragilis group by a broth dilution technique. The minimal inhibitory and minimal bactericidal concentrations of each agent were very close. At concentrations of ≤0.5 μg/ml, SC-28538 showed greater bactericidal activity; at concentrations of ≥2 μg/ml, the activies of both agents were similar.

Cephapirin was utilized to examine the interaction of β-lactam antibiotics with growing Bacillus subtilis cells and the biological effects simultaneously produced. Saturation binding and quantitative cell death were observed at the cephapirin concentration of 0.1 μg/ml. Cephapirin bound to all penicillin-binding proteins except the d -alanine carboxypeptidase. A specific [ 14 C]benzylpenicillin-binding assay was developed for the d -alanine carboxypeptidase. At the lowest saturating concentration of antibiotic (0.1 μg/ml), cephapirin inhibited formation of the d -alanine carboxypeptidase. Upon incubation with cephapirin, 18% of the membranous d -alanine carboxypeptidase was released into the media. The data suggest that β-lactam antibiotics may affect the formation of bacterial cytoplasmic membranes in addition to their effect on cell wall synthesis.

A rapid method of antimicrobial susceptibility testing has been developed, which uses a modified microdilution procedure and an inoculum of 10 7 bacteria per ml. Results are determined within 4 h with an indicator consisting of 2( p -iodophenyl)-3( p -nitrophenyl)-5-phenyltetrazolium chloride. The precipitation of a red formazan by bacteria uninhibited by antimicrobials is accelerated by the addition of phenazine methosulfate. Isolates are classified as resistant, indeterminate, or susceptible, based on growth in up to two antimicrobial concentrations which conform closely to concentrations which correlate with the millimeter breakpoints used in the Bauer-Kirby method. Results of testing 10 antimicrobial agents against 1,126 isolates were compared with results obtained when the Bauer-Kirby method and the agar dilution procedure were used as reference methods. Enterococci were excluded because of false resistance. Discrepancies were classified as very major (false susceptibility), major (false resistance), and minor (combinations of susceptibility or resistance with indeterminate results). The rapid method versus the agar dilution method yielded 2.3% very major, 0.7% major, and 2.9% minor discrepancies, for a total of 6.0%. Of 58 organism-antimicrobial agent combinations tested, 23 displayed 1% very major discrepancies between the rapid method and the agar dilution method. Six were not therapeutically important. The remainder involved Staphylococcus aureus, Staphylococcus epidermidis, Acinetobacter sp., and most organisms tested with chloramphenicol. It is suggested that adjustments in antibiotic concentrations and/or inoculum size may eliminate these discrepancies. The rapid method appeared economical when compared with Autobac 1 and the Bauer-Kirby procedure.

Beagle dogs were fed a diet containing 0, 2, or 10 μg of dihydrostreptomycin (DSM) per g of feed. The 2-μg/g level was selected to represent a residue level of the antibiotic. In both treatment groups, medicated feed resulted in a shift from a predominantly streptomycin (SM)-susceptible coliform fecal population to an SM-resistant population. The proportion of resistant organisms was significant ( P < 0.01) for both treatment groups. A definitive response did not occur with animals maintained on DSM-free diets. An increase in the prevalence of DSM-resistant organisms was observed after 15 days of DSM-supplemented feeding and persisted during the posttreatment phase of the study. The predominant pattern of resistance was SM-sulfamethoxypyridazine. Fifty-nine percent of SM-resistant strains transferred resistant determinants by conjugation to Escherichia coli K-12 recipients.

Piperacillin, a new semisynthetic penicillin, was compared with other semisynthetic penicillins, cephalosporins, and aminoglycosides by the agar dilution method against 3,600 isolates of facultative gram-negative bacilli, Bacteroides fragilis , and enterococci. At 64 μg/ml, piperacillin inhibited 90% of the isolates in each group of organisms tested except for Escherichia coli (83% inhibited by 64 μg/ml). Compared with carbenicillin, piperacillin had a 16-fold increase in activity by weight against Pseudomonas aeruginosa and the enterococcus, an 8-fold increase against Serratia marcescens , and a 4-fold increase against B. fragilis and Enterobacter species. Piperacillin was highly active against carbenicillin-resistant Klebsiella pneumoniae and inhibited many aminoglycoside-resistant organisms. Except for P. aeruginosa , the minimum bactericidal concentration of piperacillin was usually within one tube dilution of the minimum inhibitory concentration. Approximately one-third of the gram-negative bacilli were inhibited synergistically by piperacillin plus amikacin, but no synergy could be demonstrated against enterococci. Piperacillin's in vitro activity against gram-negative bacilli was similar to gentamicin's except that it also included B. fragilis , and piperacillin was decidedly superior to presently available penicillins against K. pneumoniae .

Checkerboard bactericidal studies were performed with two strains each of Streptococcus mitis, S. salivarius , and S. sanguis , and the results were analyzed by the use of isobolograms. Synergy between penicillin and streptomycin or gentamicin was demonstrated with four strains, representing two of S. salivarius , one of S. sanguis , and one of S. mitis ; the remainder were either too susceptible or too resistant to be analyzed. Clindamycin with streptomycin or gentamicin acted synergistically with three strains, representing two of S. sanguis and one of S. salivarius ; the remainder were either too susceptible or too resistant to be analyzed.

The in vitro activities of the new ureidopenicillins piperacillin, mezlocillin, azlocillin, and Bay k 4999 were compared with those of ampicillin and ticarcillin against 336 Enterobacteriaceae , 109 nonfermenters, 55 Neisseria , and 28 Haemophilus influenzae isolates. Bay k 4999 displayed the largest spectrum of activity and had lower minimal inhibitory concentrations than any of the other penicillins against all of the species tested. Piperacillin showed the same spectrum but was slightly less active than Bay k 4999; it was slightly more effective than mezlocillin against Enterobacteriaceae and fully as active as azlocillin against Pseudomonas . All ureidopenicillins were substantially more active than ampicillin and ticarcillin. Isolates highly resistant to ampicillin or ticarcillin were also less susceptible to the ureidopenicillins.

Fifty-one clinical isolates of ticarcillin-resistant, gram-negative bacilli were tested for susceptibility to combinations of ticarcillin and clavulanic acid (BRL 14151), a potent beta-lactamase inhibitor. Minimal inhibitory concentrations (MICs) were measured by a microdilution method, and minimal bactericidal concentrations for selected strains were measured by the broth dilution method. Ticarcillin MICs were ≥128 μg/ml for all and ≥512 μg/ml for 38 (75%) of the strains. Thirty-nine strains of Enterobacteriaceae tested included Escherichia coli (14), Klebsiella pneumoniae (16), Citrobacter sp. (3), Enterobacter sp. (3), Salmonella enteritidis (1), Serratia marcescens (1), and Proteus mirabilis (1). Ticarcillin MICs for 34 strains (88%) were lowered at least threefold by the addition of 1.0 μg of clavulanic acid per ml. Against 33 strains (85%), the MICs were 64 μg or less per ml in the presence of 5 μg of clavulanic acid per ml. In contrast, the MICs for seven of eight strains of Pseudomonas aeruginosa were unaffected by the addition of up to 10 μg of clavulanic acid per ml. Ticarcillin with 5 μg of clavulanic acid per ml was bactericidal against ticarcillin-resistant (MIC ≥ 2,048 μg/ml) E. coli, K. pneumoniae, Enterobacter , and P. mirabilis .

Antimicrobial susceptibility to cefamandole versus cephalothin and cefazolin versus cephalothin was compared by the broth microdilution method against 3,000 and 5,895 clinical bacterial isolates, respectively. Cefamandole and, to a lesser degree, cefazolin showed greater activity than cephalothin against Enterobacteriaceae , but the three drugs were comparable against gram-positive cocci.

The in vitro susceptibility of 145 anaerobic clinical isolates and 96 gram-positive aerobic clinical isolates to josamycin, a new macrolide antibiotic, was studied using the agar dilution technique. Ninety-five of the aerobes were susceptible to 1.56 μg or less of josamycin per ml. The median minimal inhibitory concentration of these organisms was ≤0.39 μg/ml. The in vitro activity of josamycin against the anaerobes was compared with that of erythromycin, clindamycin, penicillin, and chloramphenicol. At concentrations ≤3.12 μg/ml, 100% of strains of Bacteroides species and Bacteroides fragilis were susceptible to josamycin. At low concentrations (≤0.39 μg/ml), clindamycin was more active than josamycin against the anaerobes. However, at concentrations ≤3.12 μg/ml, the activities of josamycin and clindamycin were similar except against the Fusobacterium species, which was quite resistant to josamycin.

Bacillus subtilis, Neisseria subflava , and LYT coccus were found to undergo massive lysis after growth in media containing 0.01 to 10 mM NaF. When cells of these organisms were transferred from late-exponential-phase cultures to 0.02 M sodium phosphate buffer plus 0.1 M KCl, they underwent spontaneous autolysis. Cells grown in media with fluoride were more liable to autolysis, and walls isolated from them also showed enhanced autolytic sensitivity, even though added fluoride did not directly stimulate autolysins. Sporadic or partial lysis occurred in populations of Streptococcus sanguis and Streptococcus mutans BHT or LM-7 after growth in fluoridated media. Most bacteria that were tested did not undergo fluoride-induced lysis. However, cells of all test bacteria were found to have reduced amounts of peptidoglycan per unit of cell weight when grown in the presence of fluoride. Incorporation of labeled lysine or glucosamine into peptidoglycan (Park-Hancock residue) was stimulated, instead of inhibited, by fluoride. However, fluoride also stimulated the loss of radioactivity from Park-Hancock residues of cells that had previously incorporated labeled lysine or glucosamine. Thus, fluoride appeared to enhance peptidoglycan turnover, and this turnover reduced the peptidoglycan contents of all bacteria tested, but induced lysis in only those bacteria that normally have highly active autolytic systems.

Rifampin was incorporated into Middlebrook 7H10 medium either by adding an aliquot of the antibiotic into melted agar (final concentration 1.0 and 3.0 μg/ml) or by submerging a 5 or 15 μg of rifampin paper disk into 5 ml of melted agar contained in one quadrant of a Felson “X” plate. At intervals, plugs of agar were removed from the stored plates and assayed. Plates stored at 5 C for 28 days showed no loss of potency; at 37 C, the half-life of rifampin was 9 days. Stability of rifampin at these concentrations in 7H10 medium was independent of the method used for incorporation. Using the disk method, uniform rifampin concentrations of 0.75 μg/ml on day 5 for the 5-μg disk and 2.7 μg/ml on day 6 for the 15-μg disk were observed. Results indicated that the rifampin concentrations within the agar dilution and disk diffusion plates were equivalent at these times.

Six clinical isolates of Staphylococcus aureus were compared for their relative susceptibilities to the killing effects of oxacillin. Three of the strains had minimum bactericidal concentrations which were >10 times the minimum bacteriostatic concentration for this antibiotic and were designated tolerant (Tol + ). The other strains had minimum bactericidal concentrations which were comparable to the minimum bacteriostatic concentration (Tol − ). Lysis curves of these strains revealed that the Tol + strains exhibited a diminished rate of lysis when inhibited by oxacillin. This reduced rate of lysis was reflected also in a reduced rate of viability loss when the cells were exposed to oxacillin. During log growth the uptake of [ 14 C]glycerol by Tol + cells was 1.5-fold greater than that by Tol − cells. Glycerol-labeled cells of each phenotype secreted radioactivity when inhibited by oxacillin. However, the Tol + strains released over twice as much label as the Tol − strains. No difference in the proportion of lipid secreted by the two phenotypes was found. The behavior of 60 to 65% of the labeled material released by inhibited cells during both sodium dodecyl sulfate gel electrophoresis and Sepharose 6B chromatography corresponded to that of lipoteichoic acid. When the major component of secreted material was added to oxacillin-inhibited Tol − strains, an inhibition of the lytic response was observed. These results suggest that oxacillin tolerance in S. aureus could be related to the enhanced secretion of an autolysin inhibitor, such as lipoteichoic acid.

Sporulation of Saccharomyces cerevisiae G2-2 was inhibited by the antibiotic cerulenin which is known to be a specific inhibitor of fatty acid and sterol synthesis. This inhibition was reversed by various fatty acids, especially by oleic acid (C 18:1 ) and pentadecanoic acid (C 15:0 ). Ergosterol showed only slight reversibility of this inhibition. When cerulenin was added to the sporulation medium later than 12 h after the start of incubation, the marked inhibition disappeared. When the fatty acid fraction extracted from the sporulated yeasts was added to the cells pretreated with cerulenin for more than 6 h, sporulation became evident 6 h after the fatty acid fraction addition. Therefore, sufficient synthesis of fatty acids required for sporulation was assumed to occur during the first 6 h in phase I of yeast sporulation.

The activity of furazlocillin (Bay k 4999) was compared with those of mezlocillin, piperacillin, and standard beta-lactam antibiotics against a number of gram-positive and gram-negative organisms. These new expanded-spectrum penicillins were less active than penicillin G against most gram-positive organisms. Furazlocillin, mezlocillin, and piperacillin showed activity comparable to ampicillin and penicillin G against Haemophilus influenzae and penicillin-susceptible neisseriae, respectively. None of the drugs tested was effective against penicillin-resistant gonococci. The activity of furazlocillin was greater than that of mezlocillin, piperacillin, ampicillin, or carbenicillin against many Enterobacteriaceae . However, certain beta-lactam-resistant strains among these organisms were not highly susceptible to any of the three new penicillins. Furazlocillin was less active than piperacillin against Pseudomonas aeruginosa but was more active than carbenicillin or mezlocillin. Inoculum effects and discrepancies between minimal inhibitory concentrations and minimal bactericidal concentrations were observed with furazlocillin, mezlocillin, and piperacillin against several genera. The kinetics of bacterial killing by the new penicillins were often slow and incomplete over 24 h, especially in tests with Enterobacter and P. aeruginosa . Synergy was demonstrated between furazlocillin and aminoglycosides against a variety of gram-negative bacilli and Streptococcus faecalis .

Several different methods of performing susceptibility tests on six clinical isolates of Aspergillus are described. Some of the conditions that affected the level of susceptibility to drugs were: the type of media used, temperature and time of incubation, and the initial inoculum size. For amphotericin B susceptibility testing, the effectiveness of the polyene antibiotic as measured by visual growth was equivalent to the effectiveness as measured by inhibition of ribonucleic acid synthesis and dry-weight increase. For 5-fluorocytosine and rifampin, the visual-turbidity method gave minimum inhibitory concentrations that were much higher than those determined by effects on ribonucleic acid synthesis and dry weight. The reason for these discrepancies in susceptibility testing with 5-fluorocytosine and rifampin are unknown. We conclude that the most relevant test of this fungus to antifungal agents will have to be determined by the correlation of in vitro data with animal experiments and clinical results.

The antibacterial activity of UK-18,892, a new semisynthetic aminoglycoside, was examined against aminoglycoside-susceptible and aminoglycoside-resistant clinical isolates of gram-negative bacilli and Staphylococcus aureus . UK-18,892 had a similar degree of activity to those of amikacin and kanamycin A against aminoglycoside-susceptible bacteria but was less potent than gentamicin against all isolates except Providencia spp. UK-18,892 was highly active against aminoglycoside-resistant bacteria, inhibiting 93% of the 268 isolates examined at 12.5 μg/ml. Amikacin was similarly active, whereas gentamicin inhibited only 14% of these isolates at 12.5 μg/ml.

Disk diffusion antibiotic susceptibility tests as performed by the hospital diagnostic microbiology laboratory were read at 4, 8, and 12 h of incubation and compared to the traditional 18-h readings. Both the zone size in millimeters and a judgement of susceptible or resistant were recorded by the investigator at each time interval for each drug tested against the various clinical isolates. Considerable variation of zone sizes existed for certain organisms (e.g., staphylococci and Proteus ) and certain antibiotics (e.g., trimethoprim-sulfamethoxazole) when zone sizes in millimeters were examined. However, 8 to 60% of isolates demonstrated visible growth at 4 h of incubation and could be placed in susceptible or resistant categories with a fair degree of accuracy (74 to 95% agreement with 18-h readings). By 8 h of incubation, 83 to 100% of the cultures showed visible growth and almost all the organisms had accurate readings (89 to 98% agreement). The ability to determine antimicrobial susceptibility patterns early could influence favorably the prognosis in serious infections.

A qualitative and quantitative investigation was undertaken to study the susceptibility of unicellular eucaryotic organisms (yeasts) to metallic cations generated by low levels of direct current. Results were characteristic of effects obtained previously using clinical and standard bacteria test organisms. The present study demonstrated that anodic silver (Ag + ) at low direct currents had inhibitory and fungicidal properties. Broth dilution susceptibility tests were made on several species of Candida and one species of Torulopsis . Growth in all isolates was inhibited by concentrations of electrically generated silver ions between 0.5 and 4.7 μg/ml, and silver exhibited fungicidal properties at concentrations as low as 1.9 μg/ml. The inhibitory and fungicidal concentrations of electrically generated silver ions are lower than those reported for other silver compounds.

The in vitro susceptibilities of 408 recent clinical isolates of anaerobic bacteria against cefaclor, cephalexin, cephalothin, cefazolin, cefamandole, and cefoxitin were compared by an agar dilution technique. Against gram-positive bacteria, especially peptococci, peptostreptococci, and propionibacteria, cephalexin and cefaclor were significantly less active than cephalothin ( P < 0.05). Cephalexin was also less active than cephalothin against clostridia and lactobacillus ( P < 0.05). Against gram-negative bacteria, major differences were observed primarily with Bacteroides fragilis , against which cephalexin, cefazolin and cefoxitin were all significantly more active than cephalothin ( P < 0.001). At concentrations of 16 μg per ml, however, all cephalosporins showed high in vitro activity, except against Lactobacillus species and B. fragilis . Cephalothin, cefazolin and cefamandole were considerably more active against the former, whereas cefoxitin was distinctly more active against the latter.

Though the primary action of the cationic antibiotic polymyxin B is against the membrane of susceptible bacteria, severe morphological changes are detected in the cytoplasm. Using fluorescence microscopy and a mono- N -dansyl-polymyxin B derivative, we could demonstrate aggregations of the antibiotic with cellular material, possibly nucleic acids and/or ribosomes. These aggregations were only produced by minimum inhibitory or higher concentrations of the antibiotic as shown with Salmonella and Escherichia strains differing in their polymyxin susceptibility. The outer membrane of Salmonella typhimurium revealed characteristic blebs when treated with polymyxin B. This was investigated by the gentle methods of spray-freezing and freeze-etching. The obtained electron micrographs suggest that the polymyxin-induced blebs are projections of the outer monolayer of the outer membrane. A possible mechanism of penetration of polymyxin B through the cell envelope of gram-negative bacteria is presented.

UK-18892 is a new aminoglycoside antibiotic, a derivative of kanamycin A structurally related to amikacin. It was found to be active against a wide range of pathogenic bacteria, including many gentamicin-resistant strains. The spectrum and degree of activity of UK-18892 were similar to those of amikacin, and differences were relatively minor. UK-18892 was about twice as active as amikacin against gentamicin-susceptible strains of Pseudomonas aeruginosa . Both amikacin and UK-18892 were equally active against gentamicin-resistant strains of P. aeruginosa . There were no appreciable differences in the activity of UK-18892 and amikacin against Enterobacteriaceae and Staphylococcus aureus . Cross-resistance between these two antimicrobials was also apparent.

Studies on the susceptibility of Candida albicans to 5-fluorocytosine (5FC) demonstrated a high rate of resistance (85%) for serogroup B strains, more frequently isolated in Africa, and a low incidence (Candida to 5FC. Electron microscopy studies revealed that susceptible strains had an additional granular layer dividing the inner stratum into two parts. After trypsin digestion of these strains, the disappearance of this layer was concomitantly associated with the emergence of resistance. Thus, it appears that the cell wall contains a proteinic material, which is located in the interior of the electron-transparent region and acts in the metabolism of 5FC.

A method for quantitative determination of tetracycline in serum, using high-pressure liquid chromatography, is reported. After extraction of the drug from serum, using methanol-trichloroacetic acid solution, tetracycline was separated by reverse-phase chromatography. Quantitation of tetracycline was based on a linear relationship between peak heights in the chromatograms and known concentrations of the drug in the original serum samples. Serum tetracycline concentrations as low as 0.3 μg/ml could be accurately measured. Serum samples obtained after a single intravenous injection of tetracycline to three human volunteers and six dogs were assayed by microbiological and chemical assays. Correlation coefficients of 0.95 and 0.97, respectively, were found. This chemical method is rapid (less than 30 min), accurate, sensitive, and reproducible, and it seems feasible for routine clinical use.

The effects of inocula and media on the activities of ampicillin, penicillin, chloramphenicol and co-trimoxazole against Haemophilus influenzae were examined in vitro. Two inocula and four media were tested by the disk diffusion, broth dilution, and agar dilution methods. Chloramphenicol activity versus H. influenzae was least affected by changes in inocula and media, whereas co-trimoxazole was most susceptible to these effects. Filde's and Levinthal's agar dilution tests were most satisfactory for ampicillin. Penicillin was less active on Levinthal's than on Filde's agar. Both ampicillin and penicillin were less active when tested against the higher inoculum. Co-trimoxazole was most active (H. influenzae was resistant) when tested at an inoculum of 10 6 colony-forming units/ml on diagnostic susceptibility test agar with 5% lysed horse blood added. The majority of H. influenzae appeared resistant to co-trimoxazole with increases in the test inocula and/or when tested on brain heart infusion with Filde's, Levinthal's or “low-thymidine” Mueller-Hinton medium.

Cefaclor solutions in pH 2.5 and 4.5 buffers contained at least 90% of their initial activity after 72 h at 4 degrees C. Samples in pH 6.0, 7.0, and 8.0 buffers contained 70, 46, and 34%, respectively, of their initial activity after 72 h at 4 degrees C. After 72 h at 25 degrees C, samples prepared with pH 2.5, 4.5, 6.0, 7.0, and 8.0 buffers contained 95, 69, 16, 5, and 3%, respectively, of their initial activity. After 72 h at 37 degrees C, cefaclor solutions in pH 2.5 buffer contained 80% of the initial activity, whereas samples prepared in pH 4.5, 6.0, 7.0, and 8.0 buffers contained less than 20%. Laboratory-prepared plasma and serum samples showed an 8% loss in activity when incubated for 6 h at 4 degrees C, a 51% loss when incubated for 6 h at 25 degrees C, and a 48% loss when incubated for 2 h at 37 degrees C. Clinical samples demonstrated a similar stability pattern. Degradation rates for cefaclor in commercially prepared serum increased from 4- to 10-fold in comparison to rates obtained when samples were made in human serum freshly prepared in our laboratory. Consequently, serum standards should be made in freshly prepared human serum.

Pirbenicillin, a semisynthetic penicillin, showed greater in vitro activity against 68 recent isolates of Pseudomonas aeruginosa than did ticarcillin or carbenicillin. The median minimum inhibitory concentration of each of these three compounds, respectively, was 3.1, 12.5, and 25 μg/ml when a 10 −4 dilution of an overnight culture (about 10 5 colony-forming units [CFU]/ml) was used as inoculum, but these differences were less striking when larger inocula were used: at 10 7 CFU/ml these values were 6.25, 12.5, and 50 μg/ml, and at 10 8 to 10 9 CFU/ml these values were 50, 50, and 100 μg/ml. All three compounds showed greater inhibitory activity at pH 6 than at pH 8. This pH effect was greatest with pirbenicillin, for 6.25 μg of pirbenicillin per ml inhibited 7, 11, and 57% of the strains at pH 6, 7, and 8, respectively; these values were 4, 4, and 11% with ticarcillin and 0, 0, and 7% with carbenicillin. At sufficient inhibitory concentrations, the rates of bacterial killing of the three compounds were similar. The observed differences in anti-pseudomonad activity were not due to differences in stability to pseudomonad β-lactamases, but all three compounds were more stable than were cefazolin, cephaloridine, and benzylpenicillin.