Abstract: The objective of this study was to analyze the antimicrobial effect of 2% sodium hypochlorite (NaOCl) and 2% chlorhexidine (CHX) by agar diffusion test and by direct exposure test. Five microorganisms: Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aernginosa, Bacillus subtilis, Candida albicans, and one mixture of these were used. These strains were inoculated in brain heart infusion (BHI) and incubated at 37 degrees C for 24 h. For the agar diffusion test (ADT), 18 Petri plates with 20 ml of BHI agar were inoculated with 0.1 ml of the microbial suspensions, using sterile swabs that were spread on the medium, obtaining growth injunction. Fifty-four paper disks (9 mm in diameter) were immersed in the experimental solutions for 1 min. Subsequently, three papers disks containing one of the substances were placed on the BHI agar surface in each agar plate. The plates were maintained for 1 h at room temperature, and then incubated at 37 degrees C for 48 h. The diameter of microbial inhibition was measured around the papers disks containing the substances. For the direct exposure test, 162#50 sterile absorbent paper points were immersed in the experimental suspensions for 5 min, and were then placed on Petri plates and covered with one of the irrigant solutions, or with sterile distilled water (control group). After intervals of 5, 1 0 and 30 min, the paper points were removed from contact with the solutions and individually immersed in 7 ml of Letheen Broth, followed by incubation at 37 degrees C for 48 h. Microbial growth was evaluated by turbidity of the culture medium. A 0.1 ml inoculum obtained from the Letheen Broth was transferred to 7 ml of BHI, and incubated at 37 degrees C for 48 h. Bacterial growth was again evaluated by turbidity of the culture medium. Gram stain of BHI cultures was used for verification of contamination and growth was determined by macroscopic and microscopic examination. The best performance of antimicrobial effectiveness of NaOCI was observed in the direct exposure test, and of CHX was observed in the agar diffusion test. The magnitude of antimicrobial effect was influenced by the experimental methods, biological indicators and exposure time.

Abstract: The purpose of the study was to investigate antibacterial activity of ripe and unripe Carica papaya on selected micro-organisms. Cultures of micro-organisms were routinely maintained in nutrient agar slants at 4 degrees C. Extracts of immature, mature and ripe Carica papaya fruit were obtained by separately grinding factions of the epicarp, endocarp and seeds and filtering them through gauze. Sensitivity tests were conducted by adding 0.06 ml of extract to agar wells (6 mm diameter) prepared from 20 ml agar seeded with 10(6) cells/ml suspension of one of the eight organisms per plate. The inoculated plates were allowed to equilibrate at 4 degrees C for 1 hour, incubated at 37 degrees C for 24 hours, and zones of inhibition measured in millimetres. Anti-bacterial activity was expressed in terms of the radius of zone of inhibition. Seed extracts from the fruit showed inhibition in the following order: B cereus > E coli > S faecalis > S aureus > P vulgaris > S flexneri. No significant difference was found in bacterial sensitivity between immature, mature and ripe fruits. No inhibition zone was produced by epicarp and endocarp extracts. Carica papaya seeds contain anti-bacterial activity that inhibits growth of gram-positive and gram-negative organisms. Observed activity was independent of stage of fruit maturity. Carica papaya has antibacterial effects that could be useful in treating chronic skin ulcers to promote healing.

Abstract: Antibiotic susceptibility of clinical mastitis pathogens has traditionally been determined using the agar diffusion method that was designed to reflect the antibiotic concentration in serum and interstitial fluid of human patients after receiving oral or intravenous administration. The validity of applying agar diffusion susceptibility breakpoints derived from humans to the treatment of bovine mastitis has not been established and is extremely questionable because (1) bovine milk pH and electrolyte, fat, protein, and leukocyte concentrations, growth factor composition, and pharmacokinetic profiles are different than those for human plasma and (2) human bacterial pathogens are often different from bovine mastitis pathogens. Also, antibiotics are distributed unevenly in an inflamed gland, and high antibiotic concentrations can alter neutrophil morphology or function in vitro and thereby inhibit bacterial clearance in vivo. The current cost of antibiotic susceptibility testing is $12 to $20 per test. Because the dairy industry is economically driven, any diagnostic test should be validated, have appropriate sensitivity and specificity, and have an acceptable economic return on the cost of testing before it can be routinely recommended. In the authors' opinion, antimicrobial susceptibility testing of mastitis pathogens has not been adequately validated for most mastitis pathogens and antibiotics; therefore, the authors do not currently recommend the use of susceptibility testing to guide treatment decisions for individual cows. Additional research is needed to further define the role, if any, that antimicrobial susceptibility testing should play in the treatment of clinical mastitis.

Abstract: Antibacterial activity of Acorus calamus rhizomes was evaluated in vitro. Different concentrations of petroleum ether extract (50-2000 μg) were tested and the antimicrobial activity was observed from 500 μg and the zone of inhibition increased with concentration. The maximum activity was observed at 2000 μg the highest concentration tested, beyond which the inhibition zone did not increase. Among the four types of bacteria tested, high inhibition zone was observed on Pseudomonas aeruginosa (1.62 cm) followed by Staphylococcus aureus (1.62 cm). Escherichia coli and Bacillus subtilis showed smaller zone of inhibition (1.34 and 1.04 cm, respectively). MIC test showed that the minimum inhibition concentration was 0.25 mg/mL for P. aeruginosa, S. aureus, B. subtilis and 0.5 mg/mL for E. coli.

Abstract: The essential oil of the aerial parts of Ammoides pusilla was analyzed using both GC and GC/MS. The results revealing no less than 46 constituents among which thymol (44.5%), γ-terpinene (32.9%) and p-eymene (13.5%) were the most abundant. The antimicrobial activity of A. pusilla oil was studied using the agar diffusion test on eight strains of bacteria, and against fungus and yeast. The two-fold oil solution showed an important antimicrobial activity against Serratia marcescens, Salmonella enteritidis, Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Staphyhloccus aureus (ATCC 25923), Klebsiella pneumoniae, Pseudomonas syringae pv. syringae, Pseudomonas syringae pv. mosprunorum, Aspergillus niger and Candida albicans.

Abstract: S . S A L V E T T I , F . C E L A N D R O N I , E . G H E L A R D I , A . B A G G I A N I A N D S . S E N E S I . 2003. Aims: Development of an agar-diffusion assay to measure vitamin B2 in biological samples and application of the method to determine the amount of vitamin B2 secreted by bacteria. Methods and Results: A riboflavin-auxotrophic mutant of Bacillus cereus was generated by mini-Tn10 insertion in the ribD gene. ribD mutant sensitivity to exogenous vitamin B2 was investigated by turbidimetric and agardiffusion assays. In turbidimetric assays, the B. cereus mutant displayed a similar level of sensitivity to vitamin B2 to that of Lactobacillus casei ATCC 7469, the reference organism used for microbiological vitamin B2 quantification. However, only the ribD mutant could be used as an indicator organism in agar-diffusion assays. A total of eight probiotic strains, from five different probiotic formulations, were analysed by the ribD mutant-based assay on agar plates in order to determine their ability to secrete vitamin B2 during growth. Conclusion: The agar diffusion method with the ribD mutant of B. cereus is highly reproducible, sensitive, rapid, inexpensive, and can be applied to measure the amount of vitamin B2 in different samples. Significance and Impact of the Study: The method developed in this study appears to be a good candidate for the screening of vitamin B2 secretion by bacteria growing on solid media.

Abstract: The purpose of this study was to investigate the antibacterial activity of newly developed amphiphilic lipids and DNA/lipid complexes against two types of oral bacteria and two types of hospital infection bacteria. Nine amphiphilic lipids were quantitatively prepared from the reaction of n-alkyl alcohol, alpha-amino acids, and p-toluenesulfonic acid. Nine DNA-lipid complexes were prepared by the simple mixing of DNA and amphiphilic lipids. The DNA-lipid complexes were insoluble in water. The antibacterial activity of lipids and DNA-lipid complexes against Porphyromonas gingivalis, Streptococcus mutans, Staphylococcus aureus, and Pseudomonas aeruginosa were evaluated by the disk-diffusion method. Seven artificial lipids showed antibacterial behavior; in particular, the lipids prepared from n-decyl alcohol and glycine and from n-decyl alcohol and L-alanine showed antibacterial activity against the four bacterial strains used in this study. On the other hand, the lipids of glutamic acid derivatives did not show any antibacterial activity against the four bacteria strains except for the lipid with an n-octyl group. Five DNA-lipid complexes also had an antibacterial effect. The complex prepared from DNA and glycine decyl ester p-toluenesulfonic acid salt exhibited antibacterial activity against the four types of bacteria strains. In this study it was found that lipids and DNA-lipid complexes with a mono-decyl group or a mono-dodecyl group have more favorable antibacterial activity.

Abstract: In order to find a disk diffusion method with both high sensitivity and specificity for determination of methicillin resistance primarily for S. aureus but also for coagulase-negative staphylococci we screened several methodological variants using a material of 66 S. aureus comprising of 11 methicillin-susceptible, 18 borderline-resistant, and 37 methicillin-resistant strains. Only four of the combinations studied performed with both high sensitivity and specificity. Two of these, the Columbia agar +4.5% NaCl and Mueller Hinton agar +2% NaCl combined with a 5 microg oxacillin disk, confluent inoculum and 24 h incubation at 35 degrees C were further evaluated using 105 MRSA and 91 mecA-negative S. aureus and 193 clinical isolates of coagulase-negative staphylococci. The Columbia agar +4.5% NaCl performed excellently for both S. aureus and coagulase-negative staphylococci. For Columbia agar +4.5% NaCl using a 5 microg oxacillin disk we suggest an interpretive zone diameter of R or =16 mm for S. aureus and R or =26 mm for coagulase-negative staphylococci. The Mueller Hinton agar +2% NaCl performed well for coagulase-negative staphylococci but for S. aureus at least three (3%) very major errors were found, making this method less attractive.

Abstract: Data obtained from studies on the antimicrobial properties of bonding agents are the subject of controversy, probably because of methodological differences. This study compared two commonly used in vitro methods, the disc agar diffusion test and the well agar diffusion test. Agar plates were seeded with Streptococcus sobrinus, Lactobacillus gasseri, or Actinomyces naeslundii. For the well diffusion test, wells cut out of the agar were filled with the test material, and for the disc method, discs impregnated with the test material were applied to the agar; the discs and wells were both 9 mm in diameter. After incubation, measurements of the zones of inhibition showed little agreement between the two methods when bonding agents were tested; the mean differences (+/- sdiff) in the zones of inhibition between the methods were 0.7 +/- 3.4 mm (P = 0.40, one sample t-test against zero), 4.9 +/- 4.4 mm (P = 0.97), and 0.8 +/- 4.3 mm (P = 0.47) for S. sobrinus, L. gasseri, and A. naeslundii, respectively. Mean differences were less contrasting when chlorhexidine and pure components were tested (P < 0.05 for S. sobrinus and L. gasseri). These results indicate the need for a gold standard method to evaluate the antimicrobial properties of bonding agents.

Abstract: This research expresses the potential of the bacterial activity present in the organic extracts obtained from Penicillium sp., isolated from the esponge Ircinia felix. This activity was evaluated through agar diffusion test and Minimal Inhibitory Concentration (MIC). The susceptibility trials of organic fractions were carried out against Staphylococcus aureus, S. epidermidis, Bacillus cereus and B. subtilis. The use of the chromatographic techniques (CLV and TLC), permitted to obtain bioactive organic extracts of different polarities, of which only the EtOAc and MeOH fractions inhibited the growth of the bacteria used. Of the EtOAc fractionation, only fraction number 3 EtOAc/Hex presented greatest activity against the Gram-positive bacteria. Number 1 EtOAc/Hex fraction increased its activity against S. aureus (24 mm) and S. epidermidis (25 mm), which can be explained by the loss of possible antagonistic effect during the fractionation process. The CMI trials were carried out for the EtOAc number 1 subfraction against S. aureus, S. epidermidis, B. cereus and B. subtilis, wich was clinical interest, and shows the potential of this organic extract as antimicrobial agent.

Abstract: Sir, Ketolides are a new class of semisynthetic macrolide derivatives that show excellent activity against Streptococcus pneumoniae, even against erythromycin-resistant isolates. Previous investigators have not found any telithromycin-resistant isolates among S. pneumoniae with the constitutive macrolide–lincosamide–streptogramin B (cMLSB) phenotype, even though staphylococci or Streptococcus pyogenes with the cMLSB phenotype can develop resistance to this drug.1–3 The lack of induction of methylase production by this drug is one reason for such a difference.4 Recently, Hamilton-Miller & Shah5 reported that resistance to the ketolide cethromycin (formerly ABT-773) could be induced in S. pneumoniae with the cMLSB phenotype by erythromycin or other related antibiotics. In this study, we examined whether 55 isolates of erythromycin-resistant S. pneumoniae (MIC ≥ 1 mg/L) carrying the mef(A) and/or erm(B) genes could develop resistance to telithromycin, a ketolide with different substitutions from cethromycin (ABT-773), when exposed to erythromycin. Fifty-five clinical isolates of erythromycin-resistant S. pneumoniae (obtained from the sputum of patients with lower respiratory tract infections between 1998 and 2000) were used. These isolates were identified by their sensitivity to optochin and the bile solubility test, and by PCR amplification of the lytA gene. S. pneumoniae ATCC 6305 was used as the quality control strain. Reference samples of the following antimicrobial agents of known potency were kindly supplied in powder form by the indicated manufacturers: erythromycin (Shionogi Pharmaceutical Co., Osaka, Japan), clarithromycin (Taisho Pharmaceutical Co., Tokyo, Japan), azithromycin (Pfizer Laboratories, Groton, CT, USA), rokitamycin (Asahi Kasei, Tokyo, Japan), telithromycin (Nippon HoechstMarion-Roussel, Tokyo, Japan) and clindamycin (Upjohn, Tokyo, Japan). All isolates were tested for susceptibility to the six antibiotics listed above at concentrations between 0.015 and 128 mg/L. MICs were determined by the two-fold agar dilution method using susceptibility test agar (Mueller–Hinton agar medium; Eiken Chemicals, Tokyo, Japan) with 8% Strepto Haemo supplement (SHS; Eiken Chemicals). DNA was obtained as previously reported, and the presence of macrolide resistance genes was investigated by PCR using primers and amplification conditions that have been described previously.6 Induction of telithromycin resistance was examined by the following two methods. (i) Agar dilution method: induction of telithromycin resistance was evaluated by comparing the MICs for telithromycin in the presence and absence of a subinhibitory concentration of erythromycin (0.1 mg/L). Two susceptibility agar plates were prepared for each isolate: one contained only telithromycin and the other contained telithromycin plus erythromycin. The MIC was determined in accordance with the susceptibility test. (ii) Disc diffusion method: A bacterial suspension (2 mL of ∼108 cfu/mL) was inoculated onto 10 mL of susceptibility test agar containing 8% SHS and spread over the surface. After excess suspension was removed, paper discs (8 mm diameter high discs; Tokyo Roshi Kaisha, Tokyo, Japan) containing erythromycin or rokitamycin at 20 μg/disc or telithromycin at 5 μg/disc were placed on the surface of the agar plate. Then the plates were incubated overnight at 35°C and induction of telithromycin resistance was assessed from the shape of the zone of inhibition around the telithromycin disc nearest either the erythromycin or the rokitamycin disc. Fifteen isolates carrying only the mef(A) gene had the M phenotype. Among the other 40 isolates, 25 carried the erm(B) gene, and 15 had both the mef(A) and erm(B) genes. All 40 isolates showed a high

Abstract: Achyranthes bidentata Blume belonging to the family Amaranthaceae was investigated for antibacterial activity against Bacillus subtilis (NCM-2439), Staphylococcus aureus (NCIM-2492), Pseudomonas aeruginosa (NClM-2053) and Escherichia coli (NCIM-2068) organisms, using agar diffusion method. The petroleum ether, chloroform, methanol and aqueous extracts showed significant antibacterial activity. Our findings offer experimental support to the therapeutic claimson this herb as useful against bacterial infections.

Abstract: Background : In our hospital, an abrupt increase in the resistant rate of A. baumannii to imipenem was observed. We evaluated the imipenem minimal inhibitory concentration (MIC) of an automated system that our laboratory is using, by comparing with those of other methods. Methods : During the period from February 2002 to February 2003, the imipenem MICs of the agar dilution method, Etest, and the disk diffusion method, were compared for imipenem-resistant A. baumannii tested by an automated system in 46 samples at Chung-Ang University Phil-Dong Hospital. We tested for susceptibility to imipenem with the Vitek system by using the GNI card, the disk diffusion method by using the imipenem disk (BBL), and the agar dilution method. PCR testing of the isolates for carbapenemase genes (IMP-1 and VIM-2) detected in other hospitals was done using published primers and conditions. Results : By the agar dilution method, 23 (50.0%) isolates were susceptible to imipenem, 14 (30.4%) isolates were intermediate, and 9 (19.6%) isolates were resistant. However, by the Etest, 8 (17.4%) were susceptible to imipenem, and 28 (60.9%) isolates were resistant. By the disk diffusion method, the susceptible isolates were 14 (30.4%) and the resistant isolates were 17 (37.0%). Quantitative agreement between the agar dilution method and the disk diffusion test gave an inverse linear correlation coefficient (r=-0.564). The results of the 13 isolates, whose results of the MIC were below 2 or above 16 in the agar dilution method, corresponded with the Etest and the disk diffusion test. The IMP-1 gene was detected in one isolate. Conclusions : It is recommended that when a gram-negative bacilli isolate including A. baumannii is characterized as resistant to imipenem by the Vitek system, an additional simple test, such as the disk diffusion assay, might be used.

Abstract: This research expresses the potential of the bacterial activity present in the organic extracts obtained from Penicillium sp., isolated from the esponge Irciniafelix. This activity was evaluated through agar diffusion test and Minimal Inhibitory Concentration (MIC). The susceptibility trials of organic fractions were carried out against Staphylococcus aureus, S. epidermidis, Bacillus cereus and B. subtilis. The use of the chromatographic techniques (CLV and TLC), permitted to obtain bioactive organic extracts of different polarities, of which only the EtOAc and MeOH fractions inhibited the growth of the bacteria used. Of the EtOAc fractionation, only fraction number 3 EtOAc/Hex presented greatest activity against the Gram-positive bacteria. Number 1 EtOAc/Hex fraction increased its activity against S. aureus (24 mm) and S. epidermidis (25 mm), which can be explained by the loss of possible antagonistic effect during the fractionation process. The CMI trials were carried out for the EtOAc number I subfraction against S. aureus, S. epidermidis, B. cereus and B. subtilis, wich was clinical interest, and shows the potential of this organic extract as antimicrobial agent.

Abstract: Objective : To investigate the antimicrobial activity of different extracts (chloroform, ethylacetate, methanol, water) of the plant. Heliotropium marifolium Retz. Methods : Antimicrobial activity was assessed by standard dilution test using Mueller Hinton agar (MH) medium. The zone of inhibition of the extracts was compared with that of ciprofloxacin (5 μg / disc) by disc diffusion method. Results : The findings showed potential antimicrobial properties of the extracts against the organisms tested. The minimum inhibitory concentration (MIC) of the extracts was 133.33 μg/ml. The zone of inhibition of all extracts was comparable with that of ciprofloxacin. Conclusions : The study suggests that the plant is promising for development of phytomedicine for antimicrobial properties.

Abstract: The growth responses of Phellodendron amurense root-derived materials against seven intestinal bacteria were examined, using an impregnated paper disk agar diffusion method and spectrometric method under O 2 -free condition. The biologically active constituent of the P. amurense root extract was characterized as berberine chloride (C 20 H 18 NO 41 Cl) using various spectroscopic analyses. The growth responses varied depending on the bacterial strain, chemicals, and dose tested. At 1 mg/disk, berberine chloride strongly inhibited the growth of Clostridium perfringens, and moderately inhibited the growth of Escherichia coli and Streptococcus mutans without any adverse effects on the growth of three lactic acid-bacteria (Bifidobacterium bifidum, B. longum, and Lactobacillus acidophilus). The structure-activity relationship revealed that berberine chloride exhibited more growth-inhibiting activity against C. perfringens, E. coli, and S. mutans than berberine iodide and berberine sulfate. These results, therefore, indicate that the growth-inhibiting activity of the three berberines was much more pronounced as chloridated analogue than iodided and sulphated analogues. As for the morphological effect caused by 1 mg/disk of berberine chloride, most strains of C. perfringens were damaged and killed, indicating that berberine chloride showed a strong inhibition against C. perfringens. As naturally occurring growth-inhibiting agents, the P. amurense root-derived materials described could be useful as a preventive agent against diseases caused by harmful intestinal bacteria such as clostridia.

Abstract: Mini-dissertation submitted in partial compliance with the requirements for the Master's Degree in Technology: Homoeopathy, Durban Institute of Technology, 2003.

Abstract: Inhibitory effect of Lactobacilli strains which were isolated fish on coag. (+) S.aureus, coag. (-) S. aureus, E. coli, E. coli K12, Y. enterocolitica, P. aeroginosa, B. subtilis and, P. fleurescens was determined by the agar diffusion method. Also, bacteriosin produced by the strains and inhibitory activities of these material were examined on the tested bacteria. L. casei HS1 was found to be the highest strain that showed both general inhibition and bacteriosin production on tested bacteria.

Abstract: This study was designed to evaluate "in vitro" the inhibitory effects of spices and essential oils on the growth of psycrotrophic food-borne bacteria: Aeromonas hydrophila, Listeria monocytogenes and Yersinia enterocolitica. The sensitivity to nine spices and their oils (chilli, cinnamon, cloves, ginger, nutmeg, oregano, rosemary, sage, thyme) was studied. Antibacterial activity was evaluated on liquid and solid medium. Spices: 1% concentration of each spice was added separately to Triptic Soy Broth and then inoculated to contain 10(8)/ml organism and held to 4 degrees C for 7 days. Populations of test organism were determined on Triptic Soy Agar. Oils: Inhibition of growth was tested by using the paper disc agar diffusion method (at 35, 20 and 4 degrees C) and measuring their inhibition zone. MIC was determined by the broth microdilution method. Some culinary spices produce antibacterial activity: inhibition of growth ranged from complete (cinnamon and cloves against A. hydrophila) to no inhibition. Antibacterial inhibition zone ranged from 8 mm to 45 mm: thyme essential oil showed the greatest inhibition against A. hydrophila.

Abstract: The objectives of the study were to investigate the antimicrobial efficacy, over time, of combining antibacterial agents with a glass ionomer cement (GIC). This was assessed using an agar diffusion test. Chlorhexidine hydrochloride, cetylpyridinium chloride, cetrimide and benzalkonium chloride were added to Fuji IX GIC at 0, 1, 2 and 4% w/w. Antibacterial-GIC specimens were placed onto agar plates inoculated with one of six bacterial species (Streptococcis, Lactobacillus, and Actinomyces, two each) and the area of inhibition calculated after 24 h incubation. The experiment was repeated weekly and at week 11 the surface of the specimen was abraded prior to replacing on inoculated agar plates. Control specimens of the GIC produced no bacterial inhibition. The antibacterial-GIC combination specimens showed significant inhibition which decreased at different rates over the test period. Resurfacing of the specimens showed a dramatic increase of antibacterial action similar to levels produced on week 1. CT-GIC showed the greatest (p < 0.005) inhibitory effect throughout the experimental period for 4 out of 6 test bacteria. The addition of antibacterial agents to Fuji IX creates a GIC material with significant antimicrobial action in vitro which is dependent on concentration and type of antibacterial agent, and appears to be associated primarily with a release of the antibacterial from the surface layer of the specimen.