Ultrastructural study of polymyxin-resistant isolates of Pseudomonas aeruginosa.

Upon exposure to 6,000 U of polymyxin B sulfate per ml, cells of the polymyxin-sensitive PAO 1 strain of Pseudomonas aeruginosa displayed in thin sections long projections arising from the outer membrane of the cell wall and extensive cytoplasmic degradation with accumulation of cytoplasmic membrane infoldings. Polymyxin-resistant isolates derived from the PAO 1 strain, however, grew well in the presence of 6,000 U of polymyxin per ml and exhibited none of these effects, having instead the appearance of a typically healthy cell. Freeze-etching of cells of the sensitive strain grown in basal medium without polymyxin revealed a concave cell wall layer studded with numerous particles. Freeze-etching of cells of the resistant isolates grown in basal medium containing 6,000 U of polymyxin per ml revealed a concave cell wall layer (i.e., the outer half of the outer membrane) in which most of these particles were absent. Thus, acquisition of resistance to polymyxin was correlated with an alteration in the architecture of the outer membrane. When the resistant isolates were grown in the basal medium lacking polymyxin and then freeze-etched, the particle distribution in the concave cell wall layer resembled that of the sensitive parent strain. The cells had regained sensitivity to polymyxin upon suspension in medium containing 6,000 U/ml as determined by their failure to grow and by internal damages seen in thin sections. These cells also had acquired increased sensitivity to ethylenediaminetetraacetate, whereas the polymyxin-resistant cells grown in the presence of polymyxin were resistant to lysis by ethylenediaminetetraacetate. The polymyxin-resistant isolates were not stable mutants but instead represented an adaptive response to the presence of polymyxin in the medium.     

Effects of Aflatoxin on Germination and Growth of Lettuce

The relative susceptibility of 30 cultivars of lettuce to inhibition by aflatoxin was studied. Seed germination was not inhibited by concentrations as high as 1,000 mug/ml in cultivar Imperial 44 or by 100 mug/ml in the remaining cultivars. Hypocotyl elongation was inhibited by 46 to 68% at a concentration of 100 mug of aflatoxin per ml. Seedlings exposed to aflatoxin did not become chlorotic. The similarity between the morphological reaction of plants to coumarin and aflatoxin suggests a common mode of action, but further studies of the physiological basis for the inhibitory reactions induced by these compounds will be necessary before such conclusions will be valid.     

Susceptibility of Pseudomonas aeruginosa to Gentamicin Sulfate In Vitro: Lack of Correlation Between Disc Diffusion and Broth Dilution Sensitivity Data

Seventy-eight of 420 clinical isolates of Pseudomonas aeruginosa yielded zones of inhibition of less than 12 mm in diameter around 10-mug discs of gentamicin sulfate when tested by the standardized Bauer-Kirby disc diffusion method. Of 153 strains chosen from these isolates, one strain (0.65%) required 25 mug of gentamicin per ml for inhibition; the remainder (99.35%) were inhibited by 6 mug/ml or less of the antibiotic. It is recommended that those isolates of P. aeruginosa that yield zones of inhibition less than 12 mm in diameter be disc susceptibility-tested once more; those isolates that give zones of inhibition of less than 12 mm upon repeated examination should then be subjected to the broth dilution test before they are designated as sensitive or resistant to gentamicin.     

Synergism Between Hyperoxia and Antibiotics for Pseudomonas aeruginosa

Three strains of Pseudomonas aeruginosa were directly exposed on the surface of membrane filters to hyperoxic atmospheres. One of the three strains was found to be sensitive to oxygen. Colonies failed to appear during 18 hr of incubation in pure oxygen at 1 atm, but about 40% of the bacteria recovered to produce colonies upon reincubation in air. Similar killing was produced by 10 hr of oxygen exposure. No inhibition or killing was observed with two other strains. Streptomycin (1mug/ml) and kanamycin (5mug/ml) were more effective in killing the oxygen-sensitive strain in the presence of 60 to 70% oxygen than in air, but polymyxin B (5mug/ml) did not show a synergistic effect under such conditions.     

Optimal Combination and Concentration of Antibiotics in Media for Isolation of Pathogenic Fungi and Nocardia asteroides

Strains of Blastomyces dermatitidis, Sporothrix schenckii, Histoplasma capsulatum, Cryptococcus neoformans, Nocardia asteroides, and Coccidioides immitis were tested for in vitro susceptibility to polymyxin, gentamicin, kanamycin, chloramphenicol, and neomycin at concentrations of 1, 2, 4, 8, and 16 mug/ml. Polymyxin was the most inhibitory and gentamicin was the least inhibitory of the five antibiotics. Two Histoplasma mycelial strains were partially inhibited by 2 and 8 mug of gentamicin per ml and showed at least a 2+ growth at the higher antibiotic concentration. Kanamycin and neomycin produced significant inhibition of N. asteroides but otherwise were noninhibitory. A combination of chloramphenicol and kanamycin, each at 16 mug/ml, and gentamicin, at 4 mug/ml, was noninhibitory to the strains tested except for N. asteroides. Chloramphenicol at 16 mug/ml was not inhibitory for N. asteroides. The results suggest that the optimal antibiotic combination to use in the isolation of fungi and higher bacteria is chloramphenicol, 16 mug/ml, and gentamicin, 4 mug/ml. Addition of sheep blood (5%) had no effect on antibiotic susceptibility of the organisms studied.     

Factors contributing to the survival of poultry associated Pseudomonas spp. exposed to a quaternary ammonium compound

Resistance to benzalkonium chloride (BC) among Pseudomonas spp. isolated from poultry carcasses was determined and strategies for elimination of resistant strains evaluated. This investigation showed that resistance was quite common, about 30% of the isolates being able to grow in 200 μg ml⁻¹ BC. Pseudomonas fluorescens strains were generally less susceptible than strains of Ps. lundensis and Ps. fragi. An overnight incubation in medium containing 200 μg ml⁻¹ BC was sufficient to reduce the susceptibility of two Pseudomonas strains to the lethal effect of BC significantly. Adding EDTA enhanced the lethal effect of BC, but the effect was reduced after growing cells in medium containing BC and EDTA. Growth in medium with a quaternary ammonium compound (QAC) rendered the cells more susceptible to chlorine, phenolics and alkylaminoacetate. These results indicate that alternating use of QACs with these compounds can be used to avoid build-up of resistant strains. In addition, increased temperatures improved the lethal effect of BC and should be considered when planning disinfection routines.     

Susceptibility of Recent Isolates of Pseudomonas aeruginosa to Gentamicin, Polymyxin, and Five Penicillins, with Observations on the Pyocin and Immunotypes of the Strains

The susceptibilities of recently isolated strains of Pseudomonas aeruginosa to gentamicin, polymyxin B, carbenicillin, ampicillin, penicillin G, and two newer penicillins were tested with the inocula-replicating technique by using undiluted and 10(-3) dilutions of the cultures. With either inoculum, polymyxin B was the most active agent, and a comparison with previous data from this laboratory showed that the susceptibility of P. aeruginosa to this antibiotic had not changed over the past 20 years. Gentamicin was nearly as active as polymyxin, all but 2 of the 141 strains tested with the diluted inoculum being inhibited by 6.25 mug/ml or less. AB-2288, an agent resembling carbenicillin, was four times more active than carbenicillin or BLP-1654; the last two were equally active against the 10(-3) inoculum. A more marked inoculum effect was noted with the penicillin analogues tested, the increase in minimum inhibiting concentration with the undiluted culture being eight-fold for carbenicillin and at least 16-fold for AB-2288 and BLP-1654. Pyocin typing and serotyping failed to demonstrate any clearly predominating types.     

Resistance of Pseudomonas aeruginosa to amphoteric and quaternary ammonium biocides

Pseudomonas aeruginosa was able to grow in high levels of an amphoteric and a quaternary ammonium compound following repeated subculturing in increasing concentrations of the biocides. Resistance was acquired and lost gradually. Adaptation to both biocides resulted in cross resistance to biguanides but whereas quaternary adapted cells were resistant to a range of quaternary ammonium compounds, the amphoteric adapted organisms were not. Amphoteric adapted cells had increased hydrophobicity and exhibited ultrastructural modifications which suggested that the outer membrane might be involved in resistance. Both amphoteric and quaternary ammonium adapted organisms showed changes in their fatty acid profiles consistent with outer membrane modification but the changes were different in each case. The mechanisms involved in biocide resistance are discussed.     

Control of Salmonella enteritidis infections in poultry by polymyxin B and trimethoprim

Antimicrobial compounds were screened in vitro in Trypticase soy broth for antimicrobial activity against a virulent strain of Salmonella enteritidis. Of the several compounds tested, polymyxin B showed the strongest inhibition in vitro, preventing growth at a concentration of less than or equal to 10 micrograms/ml. Polymyxin B administered in the drinking water was effective in vivo for preventing infections in 1-day-old chickens but did not remove established infections in 1-week-old chickens. It was found that trimethoprim, which was not active in vitro, prevented colonization and removed existing infections in 1-day-old chickens when it was administered together with polymyxin B sulfate. Enrichment cultures in which selenite-cystine and tetrathionate broth media were used showed that chickens given a combination of 100 micrograms of polymyxin B sulfate per ml and 250 micrograms of trimethoprim per ml 24 h prior to oral inoculation with 10(8) to 10(9) CFU were negative for S. enteritidis after 7 days. Established infections (10(5) to 10(6) CFU/g of feces) in 1-week-old chickens were eliminated by treatment with the polymyxin-trimethoprim system. This antimicrobial agent treatment may be useful for preventing colonization in poultry and for eliminating S. enteritidis from infected flocks.     

Control of Salmonella enteritidis infections in poultry by polymyxin B and trimethoprim.

Antimicrobial compounds were screened in vitro in Trypticase soy broth for antimicrobial activity against a virulent strain of Salmonella enteritidis. Of the several compounds tested, polymyxin B showed the strongest inhibition in vitro, preventing growth at a concentration of less than or equal to 10 micrograms/ml. Polymyxin B administered in the drinking water was effective in vivo for preventing infections in 1-day-old chickens but did not remove established infections in 1-week-old chickens. It was found that trimethoprim, which was not active in vitro, prevented colonization and removed existing infections in 1-day-old chickens when it was administered together with polymyxin B sulfate. Enrichment cultures in which selenite-cystine and tetrathionate broth media were used showed that chickens given a combination of 100 micrograms of polymyxin B sulfate per ml and 250 micrograms of trimethoprim per ml 24 h prior to oral inoculation with 10(8) to 10(9) CFU were negative for S. enteritidis after 7 days. Established infections (10(5) to 10(6) CFU/g of feces) in 1-week-old chickens were eliminated by treatment with the polymyxin-trimethoprim system. This antimicrobial agent treatment may be useful for preventing colonization in poultry and for eliminating S. enteritidis from infected flocks.     

Outer membrane protein H1 of Pseudomonas aeruginosa: involvement in adaptive and mutational resistance to ethylenediaminetetraacetate, polymyxin B, and gentamicin.

It is well established that Pseudomonas aeruginosa cells grown in Mg2+-deficient medium acquire nonmutational resistance to the chelator ethylenediaminetetraacetate and to the cationic antibiotic polymyxin B; this type of resistance can be reversed by transferring the cells to Mg2+-sufficient medium for a few generations. Stable mutants resistant to polymyxin B were isolated and shown to have also gained ethylenediaminetetraacetate resistance. Both the mutants and strains grown on Mg2+-deficient medium had greatly enhanced levels of outer membrane protein H1 when compared with the wild-type strain or with revertants grown in Mg2+-sufficient medium. It was determined that in all strains and at all medium Mg2+ concentrations, the cell envelope Mg2+ concentration varied inversely with the amount of protein H1. In addition, the increase in protein H1 in the mutants was associated with an increase in resistance to another group of cationic antibiotics, the aminoglycosides, e.g., gentamicin. We propose that protein H1 acts by replacing Mg2+ at a site on the lipopolysaccharide which can otherwise be attacked by the cationic antibiotics or ethylenediaminetetraacetate.     

Quantitative Analysis of Polymyxin B Released from Polymyxin B-Treated Dormant Spores of Bacillus subtilis and Relationship between Its Permeability and Inhibitory Effect on Outgrowth

Polymyxin B, one of the cyclic polypeptide antibiotics, binds to the coat of Bacillus subtilis dormant spores and inhibits them from growing after germination. When about 2.8 × 10⁸ cells/ml of polymyxin B-treated dormant spores were incubated in heart infusion broth, 3.6 μg/ml of polymyxin B were released into the liquid medium during germination. Incubation of the same concentration of polymyxin B-treated ones in 100 mM CaCl₂ solution released 4.0 μg/ml of the antibiotic. The effect of various concentrations of polymyxin B on germination, outgrowth and vegetative growth of the dormant spores was investigated; the results showed that concentrations of 4.0 μg/ml and higher of the antibiotic inhibited their outgrowth and vegetative growth after germination. Young vegetative cells were less sensitive to the antibiotic than germinated spores. In addition to these results, immunoelectron microscopy with colloidal gold particles indicated that polymyxin B permeated into the core of the germinated spores and inhibited them from outgrowing.     

Effect of prostaglandins on cell function and multiplication of parainfluenza 3 viruses in WISH cells.

Cytotoxic effect of prostaglandins E2 and F2alpha on cells grown in vitro and the influence of these compounds on multiplication of myxovirus parainfluenza 3 were investigated. The prostaglandins were added to culture medium (0-01-10 mug/ml) 24 hr before virus infection, or for 2 and 48 hr after inoculation with viruses. WISH cells and monkey kidney cell cultures were used. No direct cytotoxic effect of prostaglandins at concentrations 0-01-1 mug/ml was found (viability, supravital staining, phase-contrast system, Nitro-BT reduction and succinic dehydrogenase tests), whereas the concentration of 10 mug/ml within 48 hr led to reduction and succinic dehydrogenase tests), whereas the concentration of 10 mug/ml within 48 hr led to partial injury of the cell population with symptoms of damage to mitochondria. Prostaglandins E2 and F2alpha inhibited multiplication of parainfluenza 3 virus at concentrations 0-1-10 mug/ml. The inhibitory effect was most pronounced if prostaglandins were added to medium for the whole period of virus multiplication i.e. for 48 hr but little or no effect was found if they were added prior to inoculation or for 2 hr after it. Inhibitory effect of prostaglandins on replication phase of viruses is suggested.     

Prophage Induction in Lysogenic Escherichia coli with Simple Hydroxylamine and Hydrazine Compounds

The prophage-inducing capability of hydroxylamine sulfate and 36 of its derivatives, and of hydrazine dihydrochloride and dihydrazine sulfate and 43 of their derivatives, was determined in Escherichia coli W1709 (lambda). Maximal nontoxic concentrations up to 1 mg/ml were tested. Hydroxylamine sulfate was active at 2.5 mug/ml and the following 17 derivatives were active at concentrations ranging up to 500 mug/ml: alpha-naphthylhydroxylamine, N-hydroxy-2-aminofluorene, oxamyl hydroxamic acid, O-carbamoyl hydroxylamine (isohydroxyurea), N-hydroxyurethane, N-methylhydroxylamine HCl, salicylhydroxamic acid, oxalohydroxamic acid, methoxyamine HCl, ethoxyamine HCl, N, N-diethylhydroxylamine oxalate, formaldoxime, formamidoxime, acetohydroxamic acid, acetaldoxime, acetone oxime, and hydroxyguanidine sulfate. Hydrazine dihydrochloride and dihydrazine sulfate were effective inducers at 5.0 and 2.5 mug/ml, respectively, and the following nine derivatives of them were active at concentrations ranging up to 500 mug/ml: phthalic acid hydrazide, phenylhydrazine HCl, p-nitrophenylhydrazine, p-chlorophenylhydrazine HCl, formylhydrazine, carbohydrazide, semicarbazide HCl, 1-methyl-1-phenyl-hydrazine sulfate, and acetic acid hydrazide. Nineteen hydroxylamine and 34 hydrazine derivatives were ineffective as inducers. Application of the prophage-induction system as a tool for detection of responsive hydroxylamino and hydrazino compounds which may be potential toxicological hazards in the environment is discussed.     

In Vitro Studies with 5-Fluorocytosine

5-Fluorocytosine, an antifungal agent with potential value as a chemotherapeutic agent, is being evaluated in the treatment of human cryptococcosis. In vitro studies with this agent have been hindered by the fact that it is inhibited significantly in the presence of partially degraded biological substances. This loss of activity is presumed to result from a competitive inhibition between the agent and its natural analogues. Procedures are described for in vitro studies with 5-fluorocytosine. These include methods for susceptibility testing and a bioassay for 5-fluorocytosine in biological fluids. Minimal inhibitory and minimal fungicidal concentrations of 5-fluorocytosine for Cryptococcus neoformans were usually in the range of 0.46 to 3.9 mug/ml and 3.9 to 15.6 mug/ml, respectively. Corresponding values for Candida albicans were 0.46 to 3.9 mug/ml and 15.6 mug/ml or greater, respectively. Strains of C. neoformans and C. albicans resistant to greater than 1,000 mug/ml were encountered both after exposure to the drug and in the absence of any known exposure. Bioassays of specimens from patients treated with 5-fluorocytosine indicated that serum and cerebrospinal fluid concentrations of 10 to 30 mug/ml and 8 to 20 mug/ml, respectively were readily achieved with a dosage of 100 mg per kg per day.     

Antibiotic Susceptibilities of Serratia marcescens and Enterobacter liquefaciens

Production of 5'-nucleotides by Serratia marcescens and Enterobacter liquefaciens correlates with deoxyribonuclease production, indicating the close relationship between these two organisms. To determine further relationships, susceptibilities of 279 strains of the tribe Klebsielleae were determined by the high-potency disc method, agar-dilution method, or both, by using 14 antibiotics. Ninety-seven per cent of S. marcescens (201 of 207 strains) and 100% of E. liquefaciens (17 strains) had minimum inhibitory concentration (MIC) of 100 mug/ml or greater with colistin and polymyxin B. With these two antibiotics, 93% of other Enterobacter species (28 strains) had MIC values of less than 1.6 mug/ml, and 100% of Klebsiella (27 strains) had MIC values less than 1.6 mug/ml. Consistent patterns were not noted with the other antibiotics tested, but the results with colistin and polymyxin B provide additional evidence of the close relationship of S. marcescens and E. liquefaciens.     

THE SANITATION OF FISH BOXES. II. THE EFFICIENCY OF VARIOUS SANITIZERS IN THE CLEANING OF COMMERCIAL WOODEN FISH BOXES

SUMMARY: An investigation of the effect of various sanitizers, including kinds containing hypo-chlorite, formaldehyde and quaternary ammonium compounds, alone and with detergents, and of a nonionic detergent alone, on the cleanability by various methods of commercial wooden fish boxes, showed that no major reductions in viable counts greater than 99·9% could be expected; this was due presumably to the high degree of contamination of the box and the porous nature of the wood. Of the materials investigated, hypochlorite solutions containing not less than 1,000 p/m of free chlorine and the quaternary ammonium compounds in 0·1–1% concentrations were most satisfactory. The implications of these investigations, to the fish industry in particular and to the cleanability of wooden surfaces in general, are discussed.     

Distribution and effects of 2,4,5-trichlorophenoxyacetic acid in cells of Bacillus megaterium.

Cell death in a resting population of an asporogenous Bacillus megaterium was accelerated by ambient concentrations of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) equal to or greater than 10 mug/ml or 5 mug/mg of cells (dry weight), but only after prolonged exposure. Conversely, populations of growing cells were not markedly influenced even at 100 mug/ml. Effects on cell respiration were not manifest until the ambient concentration reached 1,000 mug of 2,4,5-T/ml, or 500 mug/mg. Cells of B. megaterium did, however, accumulate 2,4,5-T passively to a level approximately twofold above the ambient concentration. Most of the accumulated compound was easily washed from the cells, but, of the firmly bound herbicide, about 0.5 mug/mg of cells (dry weight), nearly 60% by weight, was localized in the protoplast membrane. The foregoing results, obtained with a purified preparation of 2,4,5-T were also elicited by 2,4,5-T analytical standards. The extracted contaminants did not produce the results alone nor did they influence the results when present in combination with 2,4,5-T.