Antibiotic Susceptibility Patterns of Recent Isolates of Corynebacterium diphtheriae

A variety of factors which might affect zone sizes were studied with strains of Corynebacterium diphtheriae; a standard disc method for antimicrobial sensitivity testing was used. Moderate variations in inoculum size, inoculum preparation, and pH of Mueller Hinton agar (MHA) did not appreciably affect zone sizes. The addition of blood to MHA was necessary to insure the growth of all C. diphtheriae strains on all lots of MHA. Zone diameters on MHA with blood were consistently 4 to 9 mm smaller than on plain MHA; however, zone diameters were within the sensitive range for seven antibiotic discs used on both media. Minimal inhibitory concentration (MIC) values for penicillin, erythromycin, and rifampin were determined by a plate dilution method. The geographical source, toxigenicity, and type of the strains showed no significant correlation with MIC values or zone diameters for eight antibiotic discs. When MIC values were compared to obtainable blood levels, all of the strains appeared to be sensitive with MIC values of 相似文献   

Development and Evaluation of a Potency Index Screen for Detecting Mutants of Penicillium chrysogenum Having Increased Penicillin Yield

U.v.-treated conidia of an industrial strain of Penicillium chrysogenum were spread on a growth-limiting agar medium. Colonies arising from the survivors were surrounded with a spore suspension of Bacillus subtilis to which penicillinase had been added. After appropriate incubation, discrete zones of bacterial inhibition, with sizes limited by the penicillinase, appeared around each colony. Using the criterion of potency index (diameter of inhibition zone divided by diameter of colony) strains were selected that subsequently gave improved penicillin production in shake-flasks. The technique also ranked correctly four industrial strains in order of their known penicillin-producing capacity. Employing three operators, 5000 isolates could be screened in each experiment and approx. 15000 strains could be screened in a month.     

Large-Plate Method for the Assay of Neomycin in Serum

A large-plate method employing radial diffusion from small paper discs for assaying serum levels of neomycin is described. More than 120 discs placed on a loading plate and loaded with 20 muliters of sample could all be brought into contact with the agar plate at one time. The requirement for elaborate statistical design to compensate for time-dependent bias was thus eliminated. The dose-response curve was linear for a range of at least 0.5 to 2.5 mug/ml. The experimental limits for the actual zone width (distance from the edge of the paper disc to the outer edge of the inhibition zone) for 120 repetitions of the assay of neomycin in one and the same serum, carried out simultaneously on one large plate, were about +/- 6% (95% confidence interval). The 95% confidence interval for the distribution of difference between duplicate zones obtained for the assay of neomycin in 34 different sera, also carried out on one plate, was about +/- 10%. The dose-response lines for a standard and for three unknown sera, when carried out together on one plate, were parallel within the variability (+/- SD one) of the zone width. The large-plate method is considered to be more efficient than the use of the smaller petri dishes. The method is suitable for the assay of penicillin in serum and can most probably be used for the assay of a wide variety of substances for which radial diffusion from paper discs into agar is feasible.     

Permanent alterations of the L-forms of proteus and salmonella under various conditions

L-forms obtained from three strains of Proteus and from one strain of Salmonella have been kept for 15 to 20 years by weekly or monthly transfers on agar plates containing penicillin. The morphology and growth requirements of these strains have changed. They now grow abundantly on the surface of agar and in broth. The cultures consist of large bodies, small granules, and transitional forms. These organisms are more resistant to distortion and stain more deeply than organisms of the usual L-forms. In broth and to a lesser extent on agar, branching filaments develop, on the ends of which both the large, round organisms and small organisms are produced. The filaments are a transitional stage in the development of the cultures. Usual bacillary forms were not present in the culture and did not appear in successive transfers in the absence of penicillin. Bacilli reappeared on exposure of the L cultures to the influence of a spore-bearing bacillus. A similar transformation of L-forms has also been observed developing within a short time in recently isolated A and B type L cultures of one Proteus strain during the process of reversion to the bacterial form. The altered cultures are fixed in a stage of transition between the B type L-form and the regular bacteria.     

Role of haemagglutinins in adhesion of Erwinia carotovora to potato tissue

The adhesion of two strains each of Erwinia carotovora subsp. carotovora and Erwinia carotovora subsp. atroseptica to potato tuber discs, leaflets and tuber cell cultures was examined and found to occur independently of the presence of either mannose-sensitive (MSHA) or mannose-resistant (MRHA) haemagglutinins. Adhesion was generally greater when bacteria were grown in nutrient broth than on phosphate-buffered agar. The specific MSHA inhibitor α-methyl mannoside reduced the adhesion of two strains to tuber discs and leaflets and the specific MRHA inhibitor, asialofetuin inhibited strains only on leaflets. A reduction in adhesion of a MSHA-producing strain by α-methyl mannoside was observed by scanning electron microscopy which found that adhesion was localized at intercellular junctions.     

Penicillin resistance in Neisseria gonorrhoeae due to beta-lactamase production.

Two isolates of Neisseria gonorrhoeae from two patients who did not respond to repeated treatment with penicillin were found to contain an active beta-lactamase. This enzyme has not been previously found in other gonococci isolated in the United States. Compared to other gonococci, these isolates had higher penicillin minimal inhibitory concentrations, and gave very small or no zones of inhibition in the disc agar diffusion test. The enzyme was demonstrated with three different rapid tests for beta-lactamase, by disc diffusion assay methods, and by gas-liquid chromatography-mass spectrometry determination of penicilloic acid--the enzymatic end product from penicillin.     

Transformability of Group H Streptococcus Challis

From a wild type strain Challis of the group H streptococcus, greening (Challis α) and β-hemolytic (Challis β) colonies were isolated on horse blood agar. Both colonies formed greening on sheep blood agar, and no significant differences were found in their biological, serological and chemical analyses. They, however, showed clear differences on the transformability. Transformability, the producibility of competence-provoking factor (CPF) and competency which have been reported on the Challis strain were all found in Challis β strain. On the other hand, Challis α strain did not produce antibiotic-resistant transformants with the addition of CPF, and could not produce CPF even when the cells were cultured under various conditions of incubation or treated with lysozyme or detergents. The transformabilities of antibiotic-resistant mutants obtained from the Challis β strain were lower than those of the original Challis β strain, as pointed out by other investigators, while the Challis α strain became transformable on antibiotic resistance only when it acquired streptomycin resistance. In the Challis β strain and the antibiotic-resistant mutants of Challis α strain, the separate markers of streptomycin, penicillin, tetracycline, mitomycin C, as well as the combinations of these markers were found to be transformed at the highest rate in the strains having transformation of streptomycin resistance. The findings are discussed with respect to incorporation of deoxyribonucleic acid into recipient cells and to the reports of other workers.     

Electroantennogram responses of male Sphinx perelegans hawkmoths to floral and ‘green-leaf volatiles’

The effects of sublethal dosages of the chloronicotinyl insecticide imidacloprid on different strains of the tobacco whitefly, Bemisia tabaci Gennadius (Homoptera: Aleyrodidae), have been studied after leaf dip and systemic application. All bioassays were performed with the insecticide susceptible strain, SUD-S, and two Spanish biotypes, ALM-2 and LMPA-2, both resistant to conventional insecticides and with a lower susceptibility towards imidacloprid. Honeydew, excreted by all strains feeding on treated and untreated cotton leaf discs was quantified by photometric analysis of its carbohydrate content. EC50-values for the depression of honeydew excretion in female adults after systemic application of imidacloprid were calculated at 0.037 ppm, 0.027 ppm and 0.048 ppm for strains SUD-S, ALM-2 and LMPA-2, respectively, indicating no significant differences between strains in feeding behaviour throughout an 48 h testing period. Depending on the strain these EC50-values were 150- to 850-times lower than LC50-values calculated for mortality in the same bioassay. Starvation tests revealed mean survival times of >48 h for female adults placed on agar without leaf discs, indicating that sublethal dosages of imidacloprid which caused antifeedant responses, were probably not covered in common 48 h systemic bioassays, used to monitor resistance to imidacloprid. Effects of sublethal dosages on honeydew excretion after leaf dip application seem to be minor. In choice situations with systemically treated and untreated leaf discs in a single container, female adults of B. tabaci showed a clear preference for the untreated leaf discs. However, when using leaf discs treated by painting the surface with imidacloprid in the same bioassay, feeding activities on treated and untreated leaf discs were not significantly different. The results of the present study demonstrate the antifeedant properties of imidacloprid on B. tabaci, which might play an essential role after soil application or seed treatment under field conditions.     

Recombinant whole cell penicillin acylase biocatalyst: Production,characterization and use in the synthesis and hydrolysis of antibiotics

The production of a whole cell biocatalyst with high specific penicillin acylase activity using a recombinant strain is described. High activities were obtained using exponentially fed-batch fermentations to overproduce the enzyme. The cells were then entrapped in agar and the activity characterized in terms of particle size and stability. The technical feasibility of the catalyst in synthesis and hydrolysis reactions was demonstrated.     

A Novel Strategy for Control of Microbial Biofilms through Generation of Biocide at the Biofilm-Surface Interface

Biofilms of a mucoid clinical isolate of Pseudomonas aeruginosa (24 h; ca. 10(sup6) CFU/cm(sup2)) were established by immersion of polymer discs in nutrient broth cultures at 37(deg)C. Biofilms exposed for 30 min to various concentrations (0 to 3 mg/ml) of hydrogen peroxide or potassium monopersulfate were rinsed and shaken vigorously in sterile saline to detach loosely associated cells, and the residual viable attached population was quantified by a blot succession method on agar plates. Incorporation of copper and cobalt phthalocyanine catalysts within the polymers significantly enhanced the activity of these oxidizing biocides towards biofilm bacteria by several orders of magnitude. Biofilms established on the control discs resisted treatment with concentrations of either agent of up to 3 mg/ml. Enhancement through incorporation of a catalyst was such that concentrations of potassium monopersulfate of as low as 20 (mu)g/ml gave no recoverable survivors either on the discs or within the washings. Catalysts such as these will promote the formation of active oxygen species from a number of oxidizing agents such as peroxides and persulfates, and it is thought that generation of these at the surface-biofilm interface concentrates the antimicrobial effect to the interfacial cells and generates a diffusion pump which further provides active species to the biofilm matrix. The survivors of low-concentration treatments with these agents were more readily removed from the catalyst-containing discs than from the control discs. This indicated advantages gained in hygienic cleansing of such modified surfaces.     

Use of reporter genes to identify recessive trans-acting mutations specifically involved in the regulation of Aspergillus nidulans penicillin biosynthesis genes.

Starting from three amino acid precursors, penicillin biosynthesis is catalyzed by three enzymes which are encoded by the following three genes: acvA (pcbAB), ipnA (pcbC), and aat (penDE). To identify trans-acting mutations which are specifically involved in the regulation of these secondary metabolism genes, a molecular approach was employed by using an Aspergillus nidulans strain (AXTII9) carrying acvA-uidA and ipnA-lacZ gene fusions integrated in double copies at the chromosomal argB gene. On minimal agar plates supplemented with X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside), colonies of such a strain stained blue, which is indicative of ipnA-lacZ expression. After mutagenesis with UV light, colonies were isolated on agar plates with lactose as the carbon source, which produced only a faint blue color or no color at all. Such mutants (named Prg for penicillin regulation) most likely were defective in trans-acting genes. Control experiments revealed that the mutants studied still carried the correct number of gene fusions. In a fermentation run, mutants Prg-1 and Prg-6 exhibited only 20 to 50% of the ipnA-lacZ expression of the wild-type strain and produced only 20 to 30% of the penicillin produced by the wild-type strain. Western blot (immunoblot) analysis showed that these mutants contained reduced amounts of ipnA gene product, i.e., isopenicillin N synthase. Both mutant Prg-1 and mutant Prg-6 also differed in acvA-uidA expression levels from the wild type. Segregation analysis indicated that for both mutants the Prg phenotype resulted from mutation of a single gene. Two different complementation groups, which were designated prgA1 and prgB1, were identified. However, the specific activity of the aat (penDE) gene product, i.e., acyl coenzyme A:6-aminopenicillanic acid acyltransferase, was essentially the same for the mutants as for the wild-type strain, implying that the last step of the penicillin biosynthetic pathway is not affected by the trans-acting mutations identified.     

Evaluation of Rambach agar for the differentiation of Salmonella species from other Enterobacteriaceae

Rambach agar (Merck) was evaluated for its reliability as a selective diagnostic medium for the differentiation of Salmonella species from other Enterobacteriaceae. Twenty-five Salmonella strains were cultured on each of three agar media, Rambach (RAM), xylose lysine desoxycholate (XLD) and bismuth sulphite (BSA). Typical, easily interpreted reactions and colony morphologies were achieved for 23 strains on RAM and BSA and 17 on XLD. Of 135 other Enterobacteriaceae cultured on RAM, 134 gave characteristics which differentiated them from Salmonella . One strain which looked like Salmonella was identified as Citrobacter freundii . Rambach agar has potential as a supplementary agar in testing foods for Salmonella , but as with other selective diagnostic agars, it has limitations.     

Mathematical model for the control and standardization of discs]

Analysis of tetracycline and erythromycin diagnostic discs manufactured in the USSR showed their conformity with the requirements of the USA Federal Register. Comparison of the antibiotic amounts extracting and diffusing from the discs showed that as an average 92 and 90 per cent of the extracted amounts of erythromycin and tetracycline respectively diffused into the agar. Subsequently, it is desirable that on control testing of the disc quality both the similarity of the antibiotic content in the discs and the real amount of the antibiotic diffusing into the agar should be considered. A statistically reliable correlation between the values of the growth inhibition zones around the discs with definite and constant amounts of erythromycin, tetracycline or oxacillin and different resistance levels for every staphylococcal strain was found. On the basis of such a control system it is possible to divide the staphyloccal strains into the groups with high, low and intermediate resistance levels to the above antibiotics. However, it is not possible to use such data for accurate calculation of the value of the minimum inhibitory concentration of unknown strains because of a high value of the main error.     

Selection of Media for the Isolation of Common Bacterial L-Phase Organisms from a Clinical Specimen

The L-phase of 13 bacteria commonly associated with disease were induced by penicillin and inoculated into various solid and broth media; their growth was recorded for a period of 14 days. Plates containing highly purified agar and sucrose as the stabilizing agent and those incubated under aerobic conditions gave the best results. Magnesium seems to be necessary for growth in broth media on primary isolation, although it may not be necessary on multiple transfers after a more stable state has been reached. Growth in broth media is much more difficult to achieve. Reversion is aided by using a higher concentration of agar in plates, by decreasing the sucrose concentration, and by omitting the antibiotics and horse serum. A procedure has been outlined for the routine culture and identification of L-phase organisms from a clinical specimen.     

A new screening method for investigating microbial interactions

Interactions among Candida albicans, Staphylococcus aureus and Escherichia coli were investigated using a screening system in which test micro-organisms were incorporated in agar discs and effector micro-organisms in fluid growth media. Total as well as partial inhibition of test micro-organisms was observed in agar discs when these were incubated in broths containing effector micro-organisms. The ratio of numbers of test to effector micro-organisms was found to be of importance in the inhibition effect. The technique was found to be cheap, simple and versatile.     

Legionella anisa: a new species of Legionella isolated from potable waters and a cooling tower

Between March 1980 and June 1981, five strains of Legionella-like organisms were isolated from water. Four were recovered from potable water collected from hospitals in Chicago, Ill., and Los Angeles, Calif., during outbreaks of nosocomial legionellosis. The fifth strain was isolated from water collected from an industrial cooling tower in Jamestown, N.Y. The strains exhibited biochemical reactions typical of Legionella species and were gram-negative motile rods which grew on buffered charcoal-yeast extract agar but not on blood agar, required cysteine, and were catalase positive, urease negative, nitrate negative, hippurate negative, and nonfermentative. All strains were positive for oxidase and beta-lactamase and produced a brown, diffusible pigment. Of the five strains, four exhibited blue-white autofluorescence under long-wavelength UV light. The fatty-acid composition and ubiquinone content of these strains were consistent with those of other Legionella species. Direct fluorescent-antibody examination of the five strains with conjugates to previously described Legionella species demonstrated no cross-reactions except with the conjugates to L. longbeachae serogroup 2 and L. bozemanii serogroup 2. Four strains gave a 4+ reaction to the L. longbeachae serogroup 2 conjugate and the fifth strain gave a 1+ reaction. Each of the five strains gave a 4+ reaction with the conjugate to L. bozemanii serogroup 2. DNAs from the five strains were highly related (84 to 99%) and showed 5 to 57% relatedness to other Legionella species. These strains constitute a new species in the genus Legionella, and the name Legionella anisa sp. nov. is proposed. The type strain of L. anisa is WA-316-C3 (ATCC 35292).     

Legionella anisa: a new species of Legionella isolated from potable waters and a cooling tower.

Between March 1980 and June 1981, five strains of Legionella-like organisms were isolated from water. Four were recovered from potable water collected from hospitals in Chicago, Ill., and Los Angeles, Calif., during outbreaks of nosocomial legionellosis. The fifth strain was isolated from water collected from an industrial cooling tower in Jamestown, N.Y. The strains exhibited biochemical reactions typical of Legionella species and were gram-negative motile rods which grew on buffered charcoal-yeast extract agar but not on blood agar, required cysteine, and were catalase positive, urease negative, nitrate negative, hippurate negative, and nonfermentative. All strains were positive for oxidase and beta-lactamase and produced a brown, diffusible pigment. Of the five strains, four exhibited blue-white autofluorescence under long-wavelength UV light. The fatty-acid composition and ubiquinone content of these strains were consistent with those of other Legionella species. Direct fluorescent-antibody examination of the five strains with conjugates to previously described Legionella species demonstrated no cross-reactions except with the conjugates to L. longbeachae serogroup 2 and L. bozemanii serogroup 2. Four strains gave a 4+ reaction to the L. longbeachae serogroup 2 conjugate and the fifth strain gave a 1+ reaction. Each of the five strains gave a 4+ reaction with the conjugate to L. bozemanii serogroup 2. DNAs from the five strains were highly related (84 to 99%) and showed 5 to 57% relatedness to other Legionella species. These strains constitute a new species in the genus Legionella, and the name Legionella anisa sp. nov. is proposed. The type strain of L. anisa is WA-316-C3 (ATCC 35292).     

L form of Neisseria gonorrhoeae

Roberts, Richard B. (Walter Reed Army Institute of Research, Washington, D.C.). L form of Neisseria gonorrhoeae. J. Bacteriol. 92:1609-1614. 1966.-L forms were produced by the penicillin gradient plate technique from a recently isolated strain of Neisseria gonorrhoeae. To date, these L forms have had 30 serial passages on medium containing penicillin. Stabilized L forms developed on penicillin-free medium after 10 or more passages in the presence of penicillin. Morphological characteristics of these organisms were identical to L forms of meningococci. Medium and environmental conditions necessary for optimal growth included: Brain Heart Infusion of pH 7.2 to 7.4, 1.1 to 1.3% agar, 10 to 20% sucrose, 10 to 20% horse serum, temperature at 35 to 36 C, and increased CO(2) tension (candle jar). L forms were more resistant than the parent gonococcus to penicillin, ampicillin, methicillin, cycloserine, and cephalothin, whereas both organisms had similar sensitivities to bacitracin, vancomycin, ristocetin, novobiocin, tetracycline, and erythromycin. Revertant gonococci were produced on penicillin-free medium from L forms which had had 1, 5, and 10 serial passages. Morphology and fermentative reactions of revertant strains were identical to those of the parent gonococcus. Revertant strains produced L forms more readily than the parent organism; in fact, L forms from certain revertants did not require penicillin, but only serum and sucrose for their production and propagation on artificial medium.     

Cell fusion between L-forms and protoplasts of Staphylococcus aureus

Mixtures of various combinations of Lysostaphin protoplasts and stable L-forms of Staphylococcus aureus, which have different markers for drug resistance, were treated with polyethylene glycol (PEG) to examine the development of doubly resistant fusion products (fusants). To recover doubly resistant colonies as L-forms, they were incubated in 4.5% NaCl-brain heart infusion (BHI) broth containing penicillin G (PCG) for enrichment culture and cultured in PCG-4.5% NaCl-BHI agar medium (method 1), while to recover doubly resistant fusants as L-forms and coccal forms, they were grown on reversion medium (R medium) which causes reversion of protoplasts or fusants to parent type cells, and then cultured on assay media, i.e., R medium, BHI agar medium or PCG-4.5% NaCl-BHI agar medium (method 2). Under both experimental conditions, doubly resistant fusants developed as L-form cells by PEG treatment of pairs of protoplasts carrying the chloramphenicol (CP)-resistance plasmid and L-forms having chromosomal resistance to streptomycin (SM). In the reverse combinations, i.e., protoplasts showing chromosomal SM-resistance and L-form cells carrying the CP-resistance plasmid, the first method gave no doubly resistant colonies. By the second method, without enrichment culture on R medium, the latter combination gave doubly resistant fusants as L-form, coccal-type and mixed-type colonial forms, while when the PEG-treated mixture was enriched on R medium, fusants were obtained exclusively as the coccal type on either R medium or BHI agar assay medium. Neither of the methods yielded colonies of doubly resistant fusants on PEG-treatment of pairs of protoplasts and L-forms both of which were chromosomal, but with different drug resistances. These results show that PEG-induced cell fusion between protoplasts and L-forms of S. aureus, unlike the fusion between protoplasts or between L-forms, resulted in transfer of the drug resistance controlled by the plasmid to the fusion products. The fusants obtained were L-forms in method 1, and coccal type in the method 2.     

Enumeration of sublethally heated staphylococci in some dried foods.

The effect of 45 substances to restore the salt tolerance of sublethally heat-injured Staphylococcus aureus was tested. Sodium pyruvate, yeast extract, L-histine, casitone (Difco), adenosine triphosphate, and acetylphosphate were effective. For enumeration a repair medium was first used, containing sodium pyruvate and penicillin in 1% skim milk. This step was followed by counting on Baird-Parker agar with penicillinase. This method was selective; fewer than 100 staphylococci/g food could be enumerated and it gave counts about 8 times higher than the method of Giolitti and Cantoni used as a five-tube most probable number technique. Heat injury sensitized S. aureus to polymyxin.