Field Trial of a Microcolony Method for Testing the Antibiotic Sensitivity of Bacteria

The sensitivity of bacteria to antibiotics may be measured by a rapid method in which the criterion of sensitivity is inhibition of microcolony formation on agar which contains antibiotic. As this method allows a report on the antibiotic sensitivity of a pathogenic bacterium four hours after the test is set up, a trial of the method was carried out in a diagnostic laboratory. Two thousand five hundred and four strains of fast-growing bacteria were tested against 10 antibiotics. The overall correlation rate between the four-hour microscopic readings and the subsequent overnight readings on the same cultures was 98.1%. Seven per cent of the microscopical readings attempted gave indeterminate results, and reports on these tests were withheld until the following day.The time spent in making microscopical readings was considered fully justified by the fact that results of a high proportion of antibiotic sensitivity tests were available one day earlier than is usual with established methods.     

Tetanus: prophylaxis and treatment of the disease

Cleansing and debridement is paramount in dealing with tetanus-prone wounds (severe crushing injuries, piercing wounds, blisters and burns are outstanding examples, particularly if contaminated with dirt, grass or other debris). Prophylaxis then is relatively easy in persons who have been actively immunized by toxoid injections. For them, a "booster" injection is indicated. Use of antitoxin, however, is hazardous, whether for prophylaxis or for treatment of the disease. Since it may in itself cause severe disease, including anaphylactic reaction and serum sickness, decision to use it must be weighed against the possibility of the development of tetanus in each case. To prepare for use of it, careful history should be taken, with particular reference to sensitivity to horse dander. Dermal tests, and perhaps ophthalmic tests, for sensitivity to the serum should be carried out. Even the tests may be hazardous and precautions should be taken accordingly. If it is decided that the use of antitoxin is necessary even though the patient is sensitive to the material, desensitization must be carried out promptly, with adequate preparation for severe reaction. There is experimental evidence that antibiotics of the tetracycline group, given soon after injury, may have prophylactic effect against tetanus.     

Antibiograms of pathogenic bacteria isolated from laboratory rabbits in Ibadan, Nigeria

Antibiotic sensitivity patterns of 21 bacterial isolates from some clinically ill New Zealand rabbits were evaluated against 12 commonly used antibiotics by the Kirby-Bauer disk-diffusion sensitivity testing method. The 21 isolates consisted of six Bordetella bronchiseptica, eight Pasteurella multocida, four Staphylococcus aureus and three Pseudomonas alcaligenes that were associated with snuffles, pneumonia, otitis media, genital infections and conjunctivitis in these groups of caged rabbits. The four bacteria species were all sensitive to kanamycin, gentamycin and enrofloxacin, with variable sensitivity to the other antibiotics tested. The results of this antibiogram could serve as a field guide in the treatment of very acute bacterial diseases of rabbit prior to the availability of the results of local sensitivity tests. Such sensitivity tests should be reviewed yearly to update this antibiogram.     

A STATISTICAL METHOD TO COMPARE THE DEGREE OF MUSCLE CELL MULTIPLICATION IN DIFFERENT CULTURE DISHES

A method was developed for comparing two groups of numbers of cultured muscle cells which were counted under a microscope. Practically important problems for this purpose were: how many fields per dish should be observed, and how many dishes should be prepared under the same conditions, when given test criteria were set.
In the present experiment, 4 dishes were prepared under the same conditions. From each of the dishes, 20 fields were selected, and the numbers of muscle cells *** were counted and separately recorded. Since the purpose was to compare two groups of dishes, the design was a simple case of a nested one. From the experiment, the type of distribution seemed approximately a long-normal distribution with constancy of variance (homoscedasticity). Since the distribution of the cells in dishes belonging to the same group could be considered to be the same, the numbers from each dish could be pooled within a group. Therefore, if the test criteria for Student's t test and the sensitivity to descriminate the ratio of the number of groups are given, the number of fields to be observed per dish times that of dishes can be uniquely determined. This method can be applied for the same purpose to other kinds of cells with log-normal distribution.     

Synergism between antibiotics and plant extracts or essential oils with efflux pump inhibitory activity in coping with multidrug-resistant staphylococci

The alarming growth of the number of antibiotic resistant bacteria and in the same time limited possibilities to develop new antimicrobial compounds, lead to an urgent need to keep the sensitivity of bacteria against currently used antibiotics. Bacterial efflux pumps are an important mechanism of antibiotic resistance as the bacteria use efflux pumps for the extrusion of different types of antibiotics and chemicals. The knowledge about inhibitors of efflux pumps from natural sources suggests that this mechanism may be a good target for new drugs based on synergistic interactions of antibiotics with plant extracts, essential oils, or their constituents with efflux pump inhibitory activity. This review summarizes the current knowledge of staphylococcal efflux pumps and potential strategies to overcome them. Natural inhibitors of efflux pumps and their synergistic interactions with antibiotics are summarized.     

TETANUS—Prophylaxis and Treatment of the Disease

Cleansing and debridement is paramount in dealing with tetanus-prone wounds (severe crushing injuries, piercing wounds, blisters and burns are outstanding examples, particularly if contaminated with dirt, grass or other debris).Prophylaxis then is relatively easy in persons who have been actively immunized by toxoid injections. For them, a “booster” injection is indicated.Use of antitoxin, however, is hazardous, whether for prophylaxis or for treatment of the disease. Since it may in itself cause severe disease, including anaphylactic reaction and serum sickness, decision to use it must be weighed against the possibility of the development of tetanus in each case.To prepare for use of it, careful history should be taken, with particular reference to sensitivity to horse dander. Dermal tests, and perhaps ophthalmic tests, for sensitivity to the serum should be carried out. Even the tests may be hazardous and precautions should be taken accordingly.If it is decided that the use of antitoxin is necessary even though the patient is sensitive to the material, desensitization must be carried out promptly, with adequate preparation for severe reaction.There is experimental evidence that antibiotics of the tetracycline group, given soon after injury, may have prophylactic effect against tetanus.     

Antibiotic susceptibility patterns as aids in classification and characterization of gram-negative anaerobic bacilli

Patterns of susceptibility of gram-negative anaerobic bacilli to antibiotics have been found to be distinctive and of significant help in classification and identification. Five major groups of gram-negative anaerobic bacilli have been defined on the basis of morphological and biochemical criteria. Antibiotic sensitivity patterns conform to these groupings and provide additional taxonomic criteria. The Bacteroides fragilis group is resistant to penicillin G, whereas the other groups are generally sensitive. B. fragilis strains are relatively sensitive to erythromycin, whereas the Sphaerophorus necrophorus group is resistant. B. melaninogenicus strains, B. oralis, and Fusobacterium are all more sensitive to kanamycin and neomycin than the other two groups. Kanamycin is more active against Fusobacterium strains than neomycin, but less active against all other groups. Colistin or polymyxin B is useful for distinguishing between the resistant B. fragilis and the sensitive S. necrophorus. Antibiotic susceptibility determinations may be more readily performed in clinical laboratories than certain biochemical tests recommended for differentiation of the gram-negative anaerobic bacilli and may serve as helpful adjuncts to morphological and biochemical observations in classifying and characterizing these organisms. The use of standardized procedures for antibiotic susceptibility tests is essential if comparable results are to be obtained in different laboratories.     

In vitro activities of antibiotics and antimicrobial peptides against the plant pathogenic bacterium Xylella fastidiosa

AIMS: The objective of this study was to evaluate the effectiveness of antibiotics and antimicrobial peptides against 10 strains of Xylella fastidiosa. METHODS AND RESULTS: The minimal inhibitory concentration (MIC) of 12 antibiotics and 18 antimicrobial peptides were determined by agar dilution tests and growth inhibition assays. Antibiotics with the lowest MIC for X. fastidiosa strains were gentamicin, tetracycline, ampicillin, kanamycin, and novobiocin, chloramphenicol, and rifampin. Plate growth inhibition assays showed that four of the antimicrobial peptides (Magainin 2, Indolicidin, PGQ, and Dermaseptin) were toxic to all X. fastidiosa strains. CONCLUSION: All X. fastidiosa strains were sensitive to several groups of antibiotics, and minor differences in sensitivity to several antimicrobial peptides were observed among strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that antibiotics and antimicrobial peptides have some activity against the pathogen, X. fastidiosa and may have application in protecting plants from developing Pierce's disease.     

Detection,characterization, and phytotoxic activity of the nucleoside antibiotics,Blasticidin S and 5-Hydroxylmethyl-Blasticidin S

Screening for herbicidal compounds carried out on culture broths of Streptomyces strains isolated from soil resulted in the detection of potent phytotoxic activity. The active principles were isolated, and identified as the nucleoside antibiotics Blasticidin S (Bl-S) and 5-Hydroxymethyl-Blasticidin S (H-M-Bl-S). Bl-S was more active than H-M-Bl-S on seedling germination in petri dishes and in postemergence greenhouse tests. Moreover both antibiotics were more phytotoxic to dicotyledonous than to monocotyledonous plants. The increased sensitivity of dicots was confirmed in carrot and rice cell cultures. Both compounds also inhibited [¹⁴C]amino acid incorporation into proteins of rice and carrot cell cultures. Protein synthesis was more affected in carrot than rice.     

A Neural Mechanism for Time-Window Separation Resolves Ambiguity of Adaptive Coding

The senses of animals are confronted with changing environments and different contexts. Neural adaptation is one important tool to adjust sensitivity to varying intensity ranges. For instance, in a quiet night outdoors, our hearing is more sensitive than when we are confronted with the plurality of sounds in a large city during the day. However, adaptation also removes available information on absolute sound levels and may thus cause ambiguity. Experimental data on the trade-off between benefits and loss through adaptation is scarce and very few mechanisms have been proposed to resolve it. We present an example where adaptation is beneficial for one task—namely, the reliable encoding of the pattern of an acoustic signal—but detrimental for another—the localization of the same acoustic stimulus. With a combination of neurophysiological data, modeling, and behavioral tests, we show that adaptation in the periphery of the auditory pathway of grasshoppers enables intensity-invariant coding of amplitude modulations, but at the same time, degrades information available for sound localization. We demonstrate how focusing the response of localization neurons to the onset of relevant signals separates processing of localization and pattern information temporally. In this way, the ambiguity of adaptive coding can be circumvented and both absolute and relative levels can be processed using the same set of peripheral neurons.     

Observations on the in vitro sensitivity and resistance of Gram positive intestinal bacteria of farm animals to growth promoting antimicrobial agents

D utta , G.N. D evriese , L.A. 1984. Observations on the in vitro sensitivity and resistance of Gram positive intestinal bacteria of farm animals to growth promoting antimicrobial agents. Journal of Applied Bacteriology 56 , 117–123.
Results of qualitative in vitro sensitivity tests made with growth promoting antimicrobial agents on clostridia, lactobacilli and enteric streptococci, isolated from caeca of pigs, cattle and poultry, are summarized. The compounds were active in vitro against most of the 19 bacterial species studied. However, their activity spectra differed markedly. Only four bacterial species were naturally sensitive to all agents. The natural resistances, typical for many species and compounds, could be differentiated from the acquired resistances found with all growth promotors except avoparcin. High resistance and complex patterns of cross-resistance involving one or more of the macrolide, lincosamide and streptogramin antibiotics were detected.     

Antibiotic Sensitivities of Organisms Isolated from Mastitic and Nonmastitic Mammary Secretions

Antibiotic sensitivity tests to determine the minimal inhibitory concentrations (MIC) and the minimal lethal concentrations (MLC) for six antibiotics were conducted against mastitic isolates of staphylococci in skim milk and broth. The MIC and the MLC for all the antibiotics except benzylpenicillin were considerably higher in skim milk than in broth. Benzylpenicillin was the most effective antibiotic tested in either medium, and dihydrostreptomycin was the least effective against the organisms tested.     

Fabrication of silver nanoparticles by Phoma glomerata and its combined effect against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus

Aims:  We report extracellular synthesis of silver nanoparticles (Ag-NPs) from Phoma glomerata and its efficacy against Escherichia coli , Staphylococcus aureus and Pseudomonas aeruginosa . The bacteria exhibiting resistance to various antibiotics showed remarkable sensitivity, when used in combination of antibiotics and Ag-NPs.
Methods and Results:  Biosynthesis of Ag-NPs was carried out by challenging the fungal cell filtrate with 1 mmol l⁻¹ silver nitrate. The Ag-NPs were characterized with the help of UV–Visible spectrophotometer and Fourier transform infrared spectroscopy. Scanning electron microscopy was carried out to detect the size of Ag-NPs. Evaluation of the combined effect(s) was studied by disc diffusion method against E. coli , Staph. aureus and Ps. aeruginosa .
Conclusions:  The biosynthesis route seems to be eco-friendly and easy to scale up the process. Thus, these Ag-NPs may prove as a better candidate for drugs and can potentially eliminate the problem of chemical agents because of their biogenic nature.
Significance and Impact of the Study:  The bacterial resistance against antibiotics has been increasing with alarming rate. To overcome this problem, there is a pressing need to develop bactericidal agents. Ag-NPs may prove to be an answer to drug-resistant bacteria.     

Machine for Automatic Bacteriological Pour Plate Preparation

A fully automatic system for preparing poured plates for bacteriological analyses has been constructed and tested. The machine can make decimal dilutions of bacterial suspensions, dispense measured amounts into petri dishes, add molten agar, mix the dish contents, and label the dishes with sample and dilution numbers at the rate of 2,000 dishes per 8-hr day. In addition, the machine can be programmed to select different media so that plates for different types of bacteriological analysis may be made automatically from the same sample. The machine uses only the components of the media and sterile polystyrene petri dishes; requirements for all other materials, such as sterile pipettes and capped bottles of diluents and agar, are eliminated.     

Influence of antibiotic stability on the results of in vitro testing procedures

Wick, Warren E. (The Lilly Research Laboratories, Indianapolis, Ind.). Influence of antibiotic stability on the results of in vitro testing procedures. J. Bacteriol. 87:1162-1170. 1964.-Certain antibiotics undergo at least partial degradation under the conditions of in vitro testing procedures. With cephalothin used as an example, experimental evidence is presented to indicate the necessity for re-evaluation of results obtained from in vitro sensitivity testing methods for some antibiotics. The in vitro activity of cephalothin, tetracycline, and chloramphenicol against a variety of gram-negative bacteria is described. Plate counts demonstrate changes in the viable cell population over a 48-hr period in tubes of minimal inhibitory concentration (MIC) tests, with an abrupt rise in MIC value for cephalothin between the 12th and 24th hr. Data obtained by chromatographic methods, showing the degradation of cephalothin for the same time interval, indicated that instability of the antibiotic between the 12th and 24th hr might adversely affect the results obtained from standard 20- to 24-hr in vitro antibiotic sensitivity testing methods. Because repeated administration of an antibiotic to a patient at 4- to 8-hr intervals reinforces the original concentration, a more accurate estimate of antibacterial activity of that antibiotic might preferably be related to this time interval.     

Resistance of the honey bee, Apis mellifera to the acarian parasite Varroa destructor: behavioural and electroantennographic data

Abstract. One way in which Apis mellifera honey bees resist Varroa destructor is by detection and elimination of nestmates. This study uses behavioural tests and electroanntennography to assess the role of chemostimuli in recognition by honey bees of this acarian ectoparasite. Behavioural tests using living or dead parasites involved observation of honey bee grooming activity (antennation) under controlled conditions in Petri dishes, and removal behaviour (uncapping and elimination of parasitized and unparasitized control brood cells) under natural conditions. Some bees from colonies with both small and large parasite populations showed aggressive behaviour (biting). No difference was observed according to whether the mite was dead or alive. Under natural conditions, bees uncapped more parasitized cells than control cells. Electroantennographic tests were performed to measure sensitivity to various Varroa extracts at three concentrations (10, 20 and 30 Varroa Equivalents). Only 30 Varroa Equivalent methanol extracts made from Varroa collected from brood cells elicited significantly greater antennal response than controls (pure solvent). All three methanol extracts elicited significantly greater antennal response than controls. No response was observed using Varroa extracts made with acetone or hexane. These findings suggest that polar products may act as chemostimuli for recognition of V. destructor by honey bees. Further study will be necessary to determine which polar products are involved in this recognition and assess grooming and removal behaviour using these products.     

A microtechnique for the rapid analysis of macromolecule synthesis in minicultures of mammalian cells.

A new micromethod, called the Stanzen technique, is described for the rapid determination of DNA and protein content as well as the incorporation rates of radioactively labeled precursors into macromolecules in cells growing in replica minicultures on plastic petri dishes. The procedure yielded reproducible results assaying only minimal cell numbers per sample and was applied for studying both primary or early passaged cell cultures (mouse epidermal cells and fibroblasts) and a malignantly transformed epidermal cell line. In four well defined circular areas (called Stanzen) marked on the bottom of tissue-culture plastic petri dishes (by heated stamps), 0.2 to 4 x 10(5) cells per area were plated and grown as four individual cultures in one dish. Both treatment and labeling with radioactive precursors of these Stanzen cultures were performed as with normal petri dishes. After fixation and extraction of the cultures, the singular Stanzen areas (with the cells fixed onto them) were sawed out and transferred into vials for liquid-scintillation counting or determination of DNA and protein. The obtained values of specific activity corresponded well whether the samples compared were derived from the minicultures of the same dish or from several dishes. By modifications of the known colorimetric methods for DNA and protein determination, the sensitivity of these procedures was improved down to values of 1 microgram DNA or 5 micrograms protein per individual culture. These micromodifications yielded the same values as the standard methods whether applied to cell suspensions or to cell cultures. Finally, cell proliferation was not influenced by the growth conditions in the small Stanzen areas and proceeded as in normal dishes or larger areas similarly stamped on the bottom of petri dishes. Since this method proved valuable for biochemical studies using primary cultures of mouse epidermal cells (saving cell material by a factor of 10, test substances and time), it might also be advantageous for other purposes as well where the availability of cells or test substances are limiting factors for large test series.     

Isolation of 1,4-naphthalenedione, an antibacterial principle from the leaves of Holoptelea integrifolia and its activity against beta-lactam resistant Staphylococcus aureus

Antimicrobials derived from plants have been receiving increasing attention in recent years. Antimicrobial activities of a number of phytochemicals have been reported. Many present day antibiotics are ineffective against several pathogenic organisms. About 90% of Staphylococcus aureus isolates from clinical specimens is reported to have resistance against beta-lactam antibiotics. In the present study, the effect of hexane, diethyl ether, acetone and water extracts of leaves of a medicinal plant Holoptelea integrifolia has been tested against beta-lactam resistant strain of S. aureus in presence of antibiotics such as ampicillin, amoxicillin, cefotaxime and ceftriaxone. The diethyl ether extract has shown the maximum antibacterial activity and the active principle is found to be 1,4-naphthalenedione which is characterized by GC-MS and FTIR spectroscopy. The minimum inhibitory concentration (MIC) of the compound is found to be 4 mg/ml. Structural similarity of this compound with a functional group of a beta-lactamase-resistant antibiotic indicates that 1,4-naphthlenedione may be acting as an inhibitor to beta-lactamase.     

Oviposition of resistant and susceptible strains of Drosophila melanogaster in the presence of deltamethrin

Oviposition of three strains of Drosophila melanogaster in the presence of deltamethrin was observed. These strains had different levels of physiological susceptibility to deltamethrin. Two-choice tests were conducted with couples of flies in petri-dish arenas containing two oviposition dishes. On the first day of the experiment, females were given a choice between a treated oviposition dish and an untreated control dish. On the second day of the experiment, two control oviposition dishes were given to females. Although individual females showed a tendency to aggregate their eggs in one of the dishes, control experiments demonstrated an overall equal distribution of eggs between the dishes. When one of the two oviposition dishes in the arena was treated with deltamethrin, the percentage of females ovipositing and the mean number of eggs laid by females were reduced, compared with control arenas. Females avoided the treated oviposition dish and laid significantly more eggs on the control dish. Furthermore, when the deltamethrin concentration was increased on the first day, female flies postponed their oviposition and laid significantly more eggs on the second day. The resistant strain, SR, demonstrated the same capacity to select the untreated site for oviposition as the susceptible strain, but it showed a smaller oviposition reduction and egg retention. The relationship between physiological and behavioural susceptibility to deltamethrin is discussed.     

Cell detachment mediated by hyaluronic acid

Exogenous hyaluronic acid (HA) added to a confluent monolayer of 3T3 BALB cells facilitates cell detachment which can be enhanced by gently pipetting. When HA is added to a cell culture with the cell inoculum, the cells are able to grow and form a confluent monolayer, but the cellular density is lower than in the control cultures, in a concentration-dependent way. This difference seems due to the ease of detachment promoted by HA on the cells near confluency. In fact only near confluency is the amount of the detached cells greater in the culture plates containing HA than in controls. Culture dishes containing substrate-attached material (SAM) left behind by the confluent 3T3 BALB cells have been prepared by removing the cells with different detaching agents. The SAM-containing dishes have been incubated in the presence of HA for 24 h and, after washing, were used for cell cultures. The cells grown on such HA-treated dishes show a very low density at confluency and in some cases are prevented from forming a confluent monolayer. When the SAM-containing dishes are treated with Streptomyces hyaluronidase, the effect of HA is abolished and the cells are able to grow normally. Among the chondroitin sulphates, only chondroitin sulphate C shows the same effects as HA, whereas A and B are ineffective.