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1.  Antimicrobial Activity of Carpolobia Lutea Extracts and Fractions 
Carpolobia lutea (G. Don) (Polygalaceae) is a tropical medicinal plant putative in traditional medicines against gonorrhea, gingivitis, infertility, antiulcer and malaria. The present study evaluated the antimicrobial, antifungal and antihelicobacter effects of extracts C. lutea leaf, stem and root. The extracts were examined using the disc-diffusion and Microplates of 96 wells containing Muller-Hinton methods against some bacterial strains: Eschericia coli (ATCC 25922), E. coli (ATCC10418), Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923), Staphyllococus aureus (ATCC 6571), Enterococcus faecalis (ATCC 29212) and Bacillus subtilis (NCTC 8853) and four clinical isolates: one fungi (Candida albican) and three bacteria (Salmonella, Sheigella and staphylococcus aureus). The Gram-positive bacteria: Staphylococcus aureus (ATCC 25923), Enterococcus faecalis (ATCC 29212), Bacillus subtilis (ATCC 19659) and the Gram-negative bacteria: Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 27853), Cândida albicans (ATCC 18804) and Helicobacter pylori (ATCC 43504). Some of these extracts were found to be active against some tested strains but activity against H. pylori was >1000mg/ml and good fungistatic activity against C. albican. The MIC against C. albican is in the order n-HF > CHF > ETF= EAF.The order of potency of fraction was the ethanol root > n-HF leaf > ethanol fraction stem > chloroform fraction leaf = ethyl acetate fraction leaf. Polyphenols were demonstrated in ethanol fraction, ethyl acetate fraction, crude ethyl acetate extract and ethanol extract, respectively. These polyphenols isolated may partly explain and support the use of C. lutea for the treatment of infectious diseases in traditional Ibibio medicine of Nigeria.
PMCID: PMC3746663  PMID: 23983362
Carpolobia lutea; Polygalaceae; antimicrobial; antifungal; antihelicobacter; Polyphenols
2.  Antibacterial substances from marine algae isolated from Jeddah coast of Red sea, Saudi Arabia 
Marine algae are known to produce a wide variety of bioactive secondary metabolites and several compounds have been derived from them for prospective development of novel drugs by the pharmaceutical industries. However algae of the Red sea have not been adequately explored for their potential as a source of bioactive substances. In this context Ulva reticulata, Caulerpa occidentalis, Cladophora socialis, Dictyota ciliolata, and Gracilaria dendroides isolated from Red sea coastal waters of Jeddah, Saudi Arabia, were evaluated for their potential for bioactivity. Extracts of the algae selected for the study were prepared using ethanol, chloroform, petroleum ether and water, and assayed for antibacterial activity against Escherichia coli ATCC 25322, Pseudomonas aeruginosa ATCC 27853, Stapylococcus aureus ATCC 29213, and Enterococcus faecalis ATCC 29212. It was found that chloroform was most effective followed by ethanol, petroleum ether and water for the preparation of algal extract with significant antibacterial activities, respectively. Results also indicated that the extracts of red alga G. dendroides were more efficient against the tested bacterial strains followed by green alga U. reticulata, and brown algae D. ciliolata. Chemical analyses showed that G. dendroides recorded the highest percentages of the total fats and total proteins, followed by U. reticulata, and D. ciliolate. Among the bioflavonoids determined Rutin, Quercetin and Kaempherol were present in high percentages in G. dendroides, U. reticulata, and D. ciliolate. Estimation of saturated and unsaturated fatty acids revealed that palmitic acid was present in highest percentage in all the algal species analyzed. Amino acid analyses indicated the presence of free amino acids in moderate contents in all the species of algae. The results indicated scope for utilizing these algae as a source of antibacterial substances.
PMCID: PMC3937469  PMID: 24596500
Marine algae; Red sea; Antibacterial activity; Human pathogens; Phyto-chemical analyses
3.  Antimicrobial Potential of Endophytic Fungi Derived from Three Seagrass Species: Cymodocea serrulata, Halophila ovalis and Thalassia hemprichii 
PLoS ONE  2013;8(8):e72520.
Endophytic fungi from three commonly found seagrasses in southern Thailand were explored for their ability to produce antimicrobial metabolites. One hundred and sixty endophytic fungi derived from Cymodoceaserrulata (Family Cymodoceaceae), Halophilaovalis and Thalassiahemprichii (Family Hydrocharitaceae) were screened for production of antimicrobial compounds by a colorimetric broth microdilution test against ten human pathogenic microorganisms including Staphylococcus aureus ATCC 25923, a clinical isolate of methicillin-resistant S. aureus, Escherichia coli ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Candida albicans ATCC 90028 and NCPF 3153, Cryptococcus neoformans ATCC 90112 and ATCC 90113 and clinical isolates of Microsporumgypseum and Penicilliummarneffei. Sixty-nine percent of the isolates exhibited antimicrobial activity against at least one test strain. Antifungal activity was more pronounced than antibacterial activity. Among the active fungi, seven isolates including Hypocreales sp. PSU-ES26 from C. serrulata, Trichoderma spp. PSU-ES8 and PSU-ES38 from H. ovalis, and Penicillium sp. PSU-ES43, Fusarium sp. PSU-ES73, Stephanonectria sp. PSU-ES172 and an unidentified endophyte PSU-ES190 from T. hemprichii exhibited strong antimicrobial activity against human pathogens with minimum inhibitory concentrations (MIC) of less than 10 µg/ml. The inhibitory extracts at concentrations of 4 times their MIC destroyed the targeted cells as observed by scanning electron microscopy. These results showed the antimicrobial potential of extracts from endophytic fungi from seagrasses.
PMCID: PMC3745589  PMID: 23977310
4.  Trimebutine as a potential antimicrobial agent: a preliminary in vitro approach 
Hippokratia  2012;16(4):347-349.
Aim: The aim of this preliminary study was to investigate the in vitro effect of "non-antibiotic" trimebutine against reference strains Staphylococcus aureus ATCC 29213, ATCC 25923, Escherichia coli ATCC 25922, ATCC 35218, Pseudomonas aeruginosa ATCC 27853 and Enterococcus faecalis ATCC 29212; microbiota that are potentially involved in the pathophysiology of post-infectious functional gastrointestinal disorders.
Methods: Trimebutine activity was assessed by the broth microdilution method according to Clinical and Laboratory Standards Institute recommendations against reference strains S. aureus ATCC 29213 and ATCC 25923, E. coli ATCC 25922 and ATCC 35218, P. aeruginosa ATCC 27853 and E. faecalis ATCC 29212. Bactericidal activity of the compound was determined by spreading a 10 μL aliquot on Mueller-Hinton agar from each dilution showing non-visible growth. All tests were carried out in triplicate.
Results: Trimebutine was active against all strains tested presenting with MIC ranging from 1024 to 4000 mg/L. MIC and MBC were similar for E. coli ATCC 25922 and P. aeruginosa ATCC 27853 whereas for Gram-positive isolates and E. coli ATCC 35218 the MBC was higher.
Conclusions: We demonstrated the in vitro bacteriostatic/bactericidal activity of trimebutine against bacteria frequently colonizing the gastrointestinal tract and potentially involved in human gastrointestinal infections that might trigger post-infectious functional gastrointestinal disorders.
PMCID: PMC3738610  PMID: 23935315
trimebutine; antimicrobial effect; post-infectious irritable bowel syndrome; functional dyspepsia; gastroesophageal reflux disease
5.  Antimicrobial activity of green tea extract against isolates of methicillin-resistant Staphylococcus aureus and multi-drug resistant Pseudomonas aeruginosa 
To evaluate antibacterial activity of the Indonesian water soluble green tea extract, Camellia sinensis, against clinical isolates of methicillin-resistant Staphylococcus aureus (S. aureus) (MRSA) and multi-drug resistant Pseudomonas aeruginosa (MDR-P. aeruginosa).
Antimicrobial activity of green tea extract was determined by the disc diffusion method and the minimum inhibitory concentration (MIC) was determined by the twofold serial broth dilutions method. The tested bacteria using in this study were the standard strains and multi-drug resistant clinical isolates of S. aureus and P. aeruginosa, obtained from Laboratory of Clinical Microbiology, Faculty of Medicine, University of Indonesia.
The results showed that the inhibition zone diameter of green tea extracts for S. aureus ATCC 25923 and MRSA were (18.970±0.287) mm, and (19.130±0.250) mm respectively. While the inhibition zone diameter for P. aeruginosa ATCC 27853 and MDR-P. aeruginosa were (17.550±0.393) mm and (17.670±0.398) mm respectively. The MIC of green tea extracts against S. aureus ATCC 25923 and MRSA were 400 µg/mL and 400 µg/mL, respectively, whereas the MIC for P. aeruginosa ATCC 27853 and MDR-P. aeruginosa were 800 µg/mL, and 800 µg/mL, respectively.
Camellia sinensis leaves extract could be useful in combating emerging drug-resistance caused by MRSA and P. aeruginosa.
PMCID: PMC3703562  PMID: 23905026
Camellia sinensis; Green tea; Antibacterial activity; MIC; Multi-drug resistant bacteria
6.  In vitro antimicrobial activity of Medilox® super-oxidized water 
Super-oxidized water is one of the broad spectrum disinfectants, which was introduced recently. There are many researches to find reliable chemicals which are effective, inexpensive, easy to obtain and use, and effective for disinfection of microorganisms leading hospital infections. Antimicrobial activity of super-oxidized water is promising. The aim of this study was to investigate the in-vitro antimicrobial activity of different concentrations of Medilox® super-oxidized water that is approved by the Food and Drug Administration (FDA) as high level disinfectant.
Material and methods
In this study, super-oxidized water obtained from Medilox® [Soosan E & C, Korea] device, which had been already installed in our hospital, was used. Antimicrobial activities of different concentrations of super-oxidized water (1/1, 1/2, 1/5, 1/10, 1/20, 1/50, 1/100) at different exposure times (1, 2, 5, 10, 30 min) against six ATCC strains, eight antibiotic resistant bacteria, yeasts and molds were evaluated using qualitative suspension test. Dey-Engley Neutralizing Broth [Sigma-Aldrich, USA] was used as neutralizing agent.
Medilox® was found to be effective against all standard strains (Acinetobacter baumannii 19606, Escherichia coli 25922, Enterococcus faecalis 29212, Klebsiella pneumoniae 254988, Pseudomonas aeruginosa 27853, Staphylococcus aureus 29213), all clinical isolates (Acinetobacter baumannii, Escherichia coli, vancomycin-resistant Enterococcus faecium, Klebsiella pneumoniae, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, Bacillus subtilis, Myroides spp.), and all yeastsat 1/1 dilution in ≥ 1 minute. It was found to be effective on Aspergillus flavus at 1/1 dilution in ≥ 2 minutes and on certain molds in ≥ 5 minutes.
Medilox® super-oxidized water is a broad spectrum, on-site producible disinfectant, which is effective on bacteria and fungi and can be used for the control of nosocomial infection.
PMCID: PMC4107540  PMID: 25023905
Super-oxidized water; Medilox; Disinfectant; Bacteria; Fungi
7.  Antibacterial and antioxidant activity of methanol extract of Evolvulus nummularius 
Indian Journal of Pharmacology  2009;41(5):233-236.
To evaluate the antibacterial and antioxidant activity of methanol extract of Evolvulus nummularius (L) L.
Materials and Methods:
Disc diffusion and broth serial dilution tests were used to determine the antibacterial activity of the methanol extract against two Gram-positive bacterial strains (Bacillus subtilus NCIM 2718, Staphylococcus aureus ATCC 25923) and three Gram-negative bacterial strains (Pseudomonas aeruginosa ATCC 27853, Klebsiella pneumoniae ATCC 70063 and Escherichia coli ATCC 25922). The methanol extract was subjected to preliminary phytochemical analysis. Free radical scavenging activity of the methanol extract at different concentrations was determined with 2, 2-diphenyl-1picrylhydrazyl (DPPH).
The susceptible organisms to the methanol extract were Escherichia coli (MIC=12.50 mg/ml) and Bacillus subtilus (MIC=3.125 mg/ml) and the most resistant strains were Staphylococcus aureus, Klebsiella pneumoniae and Pseudomonas aeruginosa. The methanol extracts exhibited radical scavenging activity with IC50 of 350 μg/ml.
The results from the study show that methanol extract of E.nummularius has antibacterial activity. The antioxidant activity may be attributed to the presence of tannins, flavonoids and triterpenoids in the methanol extract. The antibacterial and antioxidant activity exhibited by the methanol extract can be corroborated to the usage of this plant in Indian folk medicine.
PMCID: PMC2812784  PMID: 20177496
Antibacterial; DPPH; Evolvulus nummularius
8.  Comparative In Vitro Antimicrobial Activity of Tigecycline, a New Glycylcycline Compound, in Freshly Prepared Medium and Quality Control▿  
Journal of Clinical Microbiology  2007;45(7):2173-2179.
The in vitro spectra of activity of tigecycline and tetracycline were determined for 2,490 bacterial isolates representing 50 different species or phenotypic groups. All isolates were tested simultaneously by broth microdilution using freshly prepared Mueller-Hinton broth and by disk diffusion. Portions of these data were submitted to the Food and Drug Administration (FDA) in support of the sponsor's application for new drug approval. In a separate study, MIC and disk diffusion quality control ranges were determined. The tigecycline MICs at which 50%/90% of bacteria were inhibited were (in μg/ml) as follows: for Streptococcus spp., 0.06/0.12; for Moraxella catarrhalis, 0.06/0.12; for Staphylococcus spp., 0.12/0.25; for Enterococcus spp., 0.12/0.25; for Listeria monocytogenes, 0.12/0.12; for Neisseria meningitidis, 0.12/0.25; for Haemophilus spp., 0.25/0.5; for Enterobacteriaceae, 0.05/2.0; for non-Enterobacteriaceae, 0.5/8.0. Tigecycline was consistently more potent than tetracycline against all species studied. The data from this study confirm the FDA-approved MIC and disk diffusion breakpoints for tigecycline for Streptococcus spp. other than Streptococcus pneumoniae, enterococci, and Enterobacteriaceae. Provisional breakpoints for Haemophilus spp. and S. pneumoniae are proposed based on the data from this study. The following MIC and/or disk diffusion quality control ranges are proposed: Staphylococcus aureus ATCC 29213, 0.03 to 0.25 μg/ml; S. aureus ATCC 25923, 20 to 25 mm; Escherichia coli ATCC 25922, 0.03 to 0.25 μg/ml and 20 to 27 mm; Pseudomonas aeruginosa ATCC 27853, 9 to 13 mm, Enterococcus faecalis ATCC 29212, 0.03 to 0.12 μg/ml; S. pneumoniae ATCC 49619, 0.015 to 0.12 μg/ml and 23 to 29 mm; Haemophilus influenzae ATCC 49247, 0.06 to 0.5 μg/ml and 23 to 31 mm; and Neisseria gonorrhoeae ATCC 49226, 30 to 40 mm.
PMCID: PMC1932989  PMID: 17494717
9.  Phytochemical and Antimicrobial Effects of Chrozophora Senegalensis 
The in vitro antimicrobial activities of the whole plant extract (ethanolic-CEE) of Chrozophora senegalensis and its fractions (ethyl acetate-EAA, n-butanol-NBE, aqueous-AQE) were assayed using the agar plate diffusion and nutrient broth dilution methods. Test microorganisms were Bacillus subtilis (NCTC 8326 B76), Escherichia coli (ATCC 11775), Pseudomonas aeruginosa (ATCC 10145), Staphylococcus aureus (ATCC 021001). Aspergillus flavus, Aspergillus niger, Candida albicans and Salmonella typhi - laboratory isolates. CEE, EAA and NBE inhibited all the test bacterial organisms and a fungus-Aspergillus flavus. AQE inhibited only Salmonella typhi and Bacillus subtilis. None of the extracts had activity on other 3 fungal organisms tested. CEE and EAA showed minimum inhibition concentration (MIC) of 0.390 and 3.125 mg/ml against S. typhi and E. coli, while NBE and AQE had MIC of 3.125 and 1.563 mg/ml against S. typhi respectively. NBE had an MIC of 12.500 mg/ml against E. coli. The minimum bactericidal concentration (MBC) of CEE and EAA was found to be <0.098 against S. typhi. The MBC of AQE was 12.5 mg/ml against E. coli and S. aureus, and 6.25 mg/ml towards P. aeruginosa. CEE and EAA exhibited similar antibacterial activities, followed by AQE. The extracts revealed the presence of carbohydrates, tannins, saponins, sterols determined by utilizing standard methods of analysis.
This study has justified the traditional use of the plant for treating diarrhea, boils and syphilis.
PMCID: PMC2816503  PMID: 20161917
Antimicrobial activity; Chrozophora senegalensis; Extracts; Phytochemical Screening; Euphorbiaceae
10.  Quality Control Guidelines for Disk Diffusion and Broth Microdilution Antimicrobial Susceptibility Tests with Seven Drugs for Veterinary Applications 
Journal of Clinical Microbiology  2000;38(1):453-455.
This multicenter study proposes antimicrobial susceptibility (MIC and disk diffusion methods) quality control (QC) parameters for seven compounds utilized in veterinary health. Alexomycin, apramycin, tiamulin, tilmicosin, and tylosin were tested by broth microdilution against various National Committee for Clinical Laboratory Standards (NCCLS)-recommended QC organisms (Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, Streptococcus pneumoniae ATCC 49619, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853). In addition, disk diffusion zone diameter QC limits were determined for apramycin, enrofloxacin, and premafloxacin by using E. coli ATCC 25922, P. aeruginosa ATCC 27853, and S. aureus ATCC 25923. The results from five or six participating laboratories produced ≥99.0% of MICs and ≥95.0% of the zone diameters within suggested guidelines. The NCCLS Subcommittee for Veterinary Antimicrobial Susceptibility Testing has recently approved these ranges for publication in the next M31 document.
PMCID: PMC88749  PMID: 10618141
11.  Production of microbial medium from defatted brebra (Milletia ferruginea) seed flour to substitute commercial peptone agar 
To investigate and optimize microbial media that substitute peptone agar using brebra seed defatted flour.
'Defatted process, inoculums preparation, evaluation of bacterial growth, preparation of cooked and hydrolyzed media and growth turbidity of tested bacteria were determined.
Two percent defatted flour was found to be suitable concentration for the growth of pathogenic bacteria: Escherichia coli (ATCC 25922) (E. coli), Pseudomonas aeruginosa (ATCC 27853), Salmonella (NCTC 8385) and Shigella flexneri (ATCC 12022) (S. flexneri), while 3% defatted flour was suitable for Staphylococcus aureus (ATCC 25923) (S. aureus). E. coli (93±1) and S. flexneri (524±1) colony count were significantly (P≤0.05) greater in defatted flour without supplement than in supplemented medium. E. coli [(3.72×109±2) CFU/mL], S. aureus [(7.4×109±2) CFU/mL], S. flexneri [(4.03×109±2) CFU/mL] and Salmonella [(2.37×109±1) CFU/mL] in non-hydrolyzed sample were statistically (P≤0.05) greater than hydrolyzed one and commercial peptone agar. Colony count of Salmonella [(4.55×109±3) CFU/mL], S. flexneri [(5.40×109±3) CFU/mL] and Lyesria moncytogenes (ATCC 19116) [(5.4×109±3) CFU/mL] on raw defatted flour agar was significantly (P≤0.05) greater than cooked defatted flour and commercial peptone agar. Biomass of E. coli, S. aureus, Salmonella and Enterococcus faecalis in non-hydrolyzed defatted flour is highly increased over hydrolyzed defatted flour and commercial peptone broth.
The defatted flour agar was found to be better microbial media or comparable with peptone agar. The substances in it can serve as sources of carbon, nitrogen, vitamins and minerals that are essential to support the growth of microorganisms without any supplements. Currently, all supplements of peptone agar are very expensive in the market.
PMCID: PMC3761138  PMID: 24075344
Colony counts; Commercial media; Defatted flour; Microbial media; Pathogenic bacteria; Peptone agar
12.  Synthesis and Antibacterial Activity of Triphenyltinbenzoate 
Triphenyltinbenzoate was synthesized using triphenyltinchloride and silver benzoate prepared from sodium benzoate. The structure of the synthetic compound was elucidated by spectral and C, H analysis. The antibacterial activities of the organotin compound were determined against four bacteria namely Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Streptococcus pyogenes (clinical isolate) and Pseudomonas aeruginosa (ATCC 27853) in vitro experiment. All the bacteria were inhibited at a concentration of 200 μg/ml and 20 μg/ml in dimethylsulphoxide solution and the minimum inhibitory concentration was found to be same, 7.5 μg/ml for Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes and 10 μg/ml for Pseudomonas aeruginosa.
PMCID: PMC3013556  PMID: 21218074
Antibacterial activity; MIC; synthesis; triphenyltinbenzoate; zone of inhibition
13.  In-Vitro Screening of Malaysian Honey from Different Floral Sources for Antibacterial Activity on Human Pathogenic Bacteria 
Different researches on therapeutic effects of honey have been conducted in different regions; however the study on the potential antibacterial activity of Malaysian honey is still limited. In this study, antibacterial activities of different monofloral honey samples were tested against several common human pathogenic bacteria.
Materials and Methods
The well-diffusion method, minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) techniques were employed to investigate the putative antibacterial activity of Malaysian monofloral honey from Koompassia excelsa (Becc.) Taub (Tualang), Melaleuca cajuputi Powell (Gelam) and Durio zibethinus Murr. (Durian). Honey samples were tested against Staphylococcus aureus ATCC6518 and ATCC25923, Staphylococcus epidermidis ATCC12228, Enterococcus faecium LMG16192, Enterococcus faecalis LMG16216 and ATCC29212, Escherichia coli ATCC25922, Salmonella enterica serovar Typhimurium ATCC14028 and Klebsiella pneumoniae ATCC13883.
Marked variations were observed in the antibacterial activity of these honey samples. Durian honey failed to produce substantial antibacterial activity, whereas Tualang and Gelam honey showed a spectrum of antibacterial activity with their growth inhibitory effects against all of the tested bacterial species including vancomycin-resistant enterococci (VRE).
Present findings suggested Gelam honey possesses highest antibacterial effect among the tested Malaysian honey samples.
PMCID: PMC4202638  PMID: 25435614
Honey; monofloral; antibacterial; well-diffusion method; VRE
14.  Analysis of a single reference strain for determination of gentamicin regression line constants and inhibition zone diameter breakpoints in quality control of disk diffusion antibiotic susceptibility testing. 
Journal of Clinical Microbiology  1982;16(5):784-793.
An equation was derived from known formulas to express the size of the inhibition zone diameter in the disk diffusion antibiotic susceptibility test as a function of the disk content of antibiotic. The equation permitted a calculation of regression line constants for the correlation between zone diameter and the minimum inhibitory concentration (MIC) with a single reference strain. Analysis of reference strains Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853, as well as 12 clinical isolates belonging to these species, showed a linearity between zone size squared and the logarithm of disk content in tests with 10-, 30-, and 100-micrograms gentamicin disks. All three species, however, gave regression line constants which were characteristic for the individual bacterial species. Calculations of zone diameter breakpoints corresponding to recommended MIC limits with E. coli and P. aeruginosa reference strains gave an accurate prediction of gentamicin susceptibility. Histogram analysis of 48 strains of Streptococcus faecalis from clinical specimens showed a distribution of zone diameter values which would result in false classification of susceptibility with breakpoints calculated for the other bacterial species studied. Single reference strain analysis of S. faecalis ATCC 29212 (gentamicin MIC, 8 micrograms/ml) permitted the calculation of breakpoints which accurately assigned the strains tested to the intermediate category of susceptibility. Single reference strain analysis offers a quality control method for individual laboratories that allows the determination of inhibition zone diameter breakpoints corresponding to recommended MIC limits with no MIC determinations required.
PMCID: PMC272477  PMID: 6818249
15.  In vitro study of elution kinetics and bio-activity of meropenem-loaded acrylic bone cement 
Use of antibiotic-loaded acrylic bone cement to treat orthopaedic infections continues to remain popular, but resistance to routinely used antibiotics has led to the search for alternative, more effective antibiotics. We studied, in vitro, the elution kinetics and bio-activity of different concentrations of meropenem-loaded acrylic bone cement.
Meropenem-loaded bone cement cylinders of different concentrations were serially immersed in normal saline. Elution kinetics was studied by measuring the drug concentration in the eluate, collected at pre-determined intervals, by high-performance liquid chromatography. Bio-activity of the eluate of two different antibiotic concentrations was tested for a period of 3 weeks against each of the following organisms: Staphylococcus aureus ATCC 2593 (MSSA), Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, S. aureus ATCC 43300 (MRSA) and Klebsiella pneumoniae ATCC 700603 (ESBL).
Meropenem elutes from acrylic bone cement for a period of 3–27 days depending on the concentration of antibiotic. Higher doses of antibiotic concentration resulted in greater elution of the antibiotic. The eluate was found to be biologically active against S. aureus ATCC 2593 (MSSA), P. aeruginosa ATCC 27853, E. coli ATCC 25922 and K. pneumoniae ATCC 700603 (ESBL) for a period of 3 weeks.
The elution of meropenem is in keeping with typical antibiotic-loaded acrylic bone cement elution characteristics. The use of high-dose meropenem-loaded acrylic bone cement seems to be an attractive option for treatment of resistant Gram-negative orthopaedic infections but needs to be tested in vivo.
PMCID: PMC3427696  PMID: 22461001
Local antibiotic delivery; Extended-spectrum beta-lactamase producers; Gram-negative; Orthopaedic infections; Antibiotic bone cement
16.  Evaluation of Antimicrobial and Wound Healing Potential of Justicia flava and Lannea welwitschii 
Microbial infections of various types of wounds are a challenge to the treatment of wounds and wound healing. The aim of the study is to determine the antimicrobial, antioxidant, and in vivo wound healing properties of methanol leaf extracts of Justicia flava and Lannea welwitschii. The antimicrobial activity was investigated using agar well diffusion and microdilution methods. The free radical scavenging activity of the methanol leaf extracts was performed using 1,1-diphenyl-2-picryl-hydrazyl (DPPH). The rate of wound contraction was determined using excision model. The test organisms used were Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 4853, Bacillus subtilis NTCC 10073, Staphylococcus aureus ATCC 25923, and clinical strains of Candida albicans. The MICs of methanol leaf extract of J. flava against test organisms were E. coli (7.5 mg/mL); P. aeruginosa (7.5 mg/mL); S. aureus (5 mg/mL); B. subtilis (7.5 mg/mL); and C. albicans (5 mg/mL). The MICs of methanol leaf extract of L. welwitschii against test organisms were E. coli (5 mg/mL); P. aeruginosa (10 mg/mL); S. aureus (5 mg/mL); B. subtilis (2.5 mg/mL); and C. albicans (2.5 mg/mL). The MBC/MFC of the extract was between 10 and 50 mg/mL. The IC50 of the reference antioxidant, α-tocopherol, was 1.5 μg/mL and the methanol leaf extracts of J. flava and L. welwitschii had IC50 of 65.3 μg/mL and 81.8 μg/mL, respectively. The methanol leaf extracts of J. flava and L. welwitschii gave a significant reduction in wound size as compared to the untreated. The rates of wound closure after the application of the extracts (7.5% w/w) were compared to the untreated wounds. On the 9th day, J. flava extract had a percentage wound closure of 99% (P < 0.01) and that of L. welwitschii exhibited wound closure of 95% (P < 0.05) on the 13th day compared to the untreated wounds. The two extracts significantly (P < 0.01) increased the tensile strength of wounds compared to the untreated wounds. The extracts treated wound tissues showed improved angiogenesis, collagenation, and reepithelialization compared to the untreated wound tissues. The preliminary phytochemical screening of J. flava and L. welwitschii leaf extracts revealed the presence of tannins, alkaloids, flavonoids, and glycosides. The above results indicate that methanol leaf extracts of J. flava and L. welwitschii possess antimicrobial and wound healing properties which may justify the traditional uses of J. flava and L. welwitschii in the treatment of wounds and infections.
PMCID: PMC3789403  PMID: 24159350
17.  Determination of Disk Diffusion and MIC Quality Control Guidelines for JNJ-Q2, a Novel Quinolone▿  
Journal of Clinical Microbiology  2011;49(8):3009-3011.
JNJ-Q2 is a novel fluorinated 4-quinolone in development for treatment of acute bacterial skin and skin structure infection and community-acquired bacterial pneumonia. This quality control (QC) study was performed to establish ranges for control strains: Staphylococcus aureus ATCC 29213 (0.004 to 0.015 μg/ml), Enterococcus faecalis ATCC 29212 (0.015 to 0.06 μg/ml), Pseudomonas aeruginosa ATCC 27853 (0.5 to 2 μg/ml and 17 to 23 mm), Escherichia coli ATCC 25922 (0.008 to 0.03 μg/ml and 30 to 36 mm), Haemophilus influenzae ATCC 49247 (0.002 to 0.015 μg/ml and 31 to 39 mm), Streptococcus pneumoniae ATCC 49619 (0.004 to 0.015 μg/ml and 28 to 35 mm), and S. aureus ATCC 25923 (32 to 38 mm). These ranges will be crucial in evaluating JNJ-Q2 potency as it progresses through clinical trial development.
PMCID: PMC3147733  PMID: 21653768
18.  Antimicrobial activity of the bioactive components of essential oils from Pakistani spices against Salmonella and other multi-drug resistant bacteria 
The main objective of this study was the phytochemical characterization of four indigenous essential oils obtained from spices and their antibacterial activities against the multidrug resistant clinical and soil isolates prevalent in Pakistan, and ATCC reference strains.
Chemical composition of essential oils from four Pakistani spices cumin (Cuminum cyminum), cinnamon (Cinnamomum verum), cardamom (Amomum subulatum) and clove (Syzygium aromaticum) were analyzed on GC/MS. Their antibacterial activities were investigated by minimum inhibitory concentration (MIC) and Thin-Layer Chromatography-Bioautographic (TLC-Bioautographic) assays against pathogenic strains Salmonella typhi (D1 Vi-positive), Salmonella typhi (G7 Vi-negative), Salmonella paratyphi A, Escherichia coli (SS1), Staphylococcus aureus, Pseudomonas fluorescens and Bacillus licheniformis (ATCC 14580). The data were statistically analyzed by using Analysis of Variance (ANOVA) and Least Significant Difference (LSD) method to find out significant relationship of essential oils biological activities at p <0.05.
Among all the tested essential oils, oil from the bark of C. verum showed best antibacterial activities against all selected bacterial strains in the MIC assay, especially with 2.9 mg/ml concentration against S. typhi G7 Vi-negative and P. fluorescens strains. TLC-bioautography confirmed the presence of biologically active anti-microbial components in all tested essential oils. P. fluorescens was found susceptible to C. verum essential oil while E. coli SS1 and S. aureus were resistant to C. verum and A. subulatum essential oils, respectively, as determined in bioautography assay. The GC/MS analysis revealed that essential oils of C. cyminum, C. verum, A. subulatum, and S. aromaticum contain 17.2% cuminaldehyde, 4.3% t-cinnamaldehyde, 5.2% eucalyptol and 0.73% eugenol, respectively.
Most of the essential oils included in this study possessed good antibacterial activities against selected multi drug resistant clinical and soil bacterial strains. Cinnamaldehyde was identified as the most active antimicrobial component present in the cinnamon essential oil which acted as a strong inhibitory agent in MIC assay against the tested bacteria. The results indicate that essential oils from Pakistani spices can be pursued against multidrug resistant bacteria.
PMCID: PMC3853939  PMID: 24119438
Essential oils; Multidrug resistant; Minimum inhibitory concentration; GC/MS; TLC-bioautography
19.  Antibacterial, antifungal and antioxidant activities of the ethanol extract of the stem bark of Clausena heptaphylla 
There is wide spread interest in drugs derived from plants as green medicine is believed to be safe and dependable, compared with costly synthetic drugs that have adverse effects.
We have attempted to evaluate the antioxidant, In vitro thrombolytic, antibacterial, antifungal and cytotoxic effects of Clausena heptaphylla (Rutaceae) stem bark extract ethanol extract.
Ethanolic stem bark extract of Clausena heptaphylla (CHET) contains flavonoids, alkaloids, saponins and steroids but it lacks tannins, anthraquinones and resins. Phenol content of the extract was 13.42 mg/g and flavonoid content was 68.9 mg/g. CHET exhibited significant DPPH free radical scavenging activity with IC50 value of 3.11 μg/ml. Reducing power of CHET was also moderately stronger. In the cytotoxicity assay, LC50 and Chi-square value of the ethanolic extract against brine shrimp nauplii were 144.1461 μg/ml and 0.8533 demonstrating potent cytotoxic effect of the extract. In vitro thrombolytic activity of CHET is significant with 45.38% clot lysis capability compared to that of Streptokinase (65.78%). In antibacterial screening, moderate zone of inhibition (6.5-9.0 mm in diameter) was observed against gram-positive Bacillus subtilis ATCC 11774, Bacillus cereus ATCC 10876, Staphylococcus aureus ATCC 25923, Bacillus polymyxa ATCC 842 and Bacillus megaterium ATCC 13578 and less promising zone of inhibition (3.0-4.5 mm in diameter) against gram-negative Salmonella typhi ATCC 65154, Shigella flexneri ATCC 12022, Proteus vulgaris ATCC 13315 and Escherichia coli ATCC 25922. Shigella sonnei ATCC 8992 did not show any sensitivity. The MIC values against these bacteria were ranged from 2,000 to 3,500 μg/ml. The extract showed significant zone of inhibition against Rhizopus oryzae DSM 2200, Aspergillus niger DSM 737 and Aspergillus ochraceus DSM 824 in antifungal assay.
Further advanced research is necessary to isolate and characterize the chemical components responsible for the therapeutic properties of the plant.
PMCID: PMC3533896  PMID: 23181593
Antioxidant; Antibacterial; Antifungal; Cytotoxic; Clausena heptaphylla
20.  Pseudonocardians A–C, New Diazaanthraquinone Derivatives from a Deap-Sea Actinomycete Pseudonocardia sp. SCSIO 01299 
Marine Drugs  2011;9(8):1428-1439.
Pseudonocardians A–C (2–4), three new diazaanthraquinone derivatives, along with a previously synthesized compound deoxynyboquinone (1), were produced by the strain SCSIO 01299, a marine actinomycete member of the genus Pseudonocardia, isolated from deep-sea sediment of the South China Sea. The structures of compounds 1–4 were determined by mass spectrometry and NMR experiments (1H, 13C, HSQC, and HMBC). The structure of compound 1, which was obtained for the first time from a natural source, was confirmed by X-ray analysis. Compounds 1–3 exhibited potent cytotoxic activities against three tumor cell lines of SF-268, MCF-7 and NCI-H460 with IC50 values between 0.01 and 0.21 μm, and also showed antibacterial activities on Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212 and Bacillus thuringensis SCSIO BT01, with MIC values of 1–4 μg mL−1.
PMCID: PMC3164384  PMID: 21892356
marine actinomycetes; Pseudonocardia; natural products; cytotoxicity; antibacterial; South China Sea
21.  Evidence for different pyoverdine-mediated iron uptake systems among Pseudomonas aeruginosa strains. 
Infection and Immunity  1989;57(11):3491-3497.
Fourteen strains of Pseudomonas aeruginosa (P. aeruginosa ATCC 15692, P. aeruginosa ATCC 27853, and 12 clinical isolates) were checked for the production of pyoverdine and for pyoverdine-mediated iron uptake. Under iron restriction, two isolates produced undetectable amounts of pyoverdine, but all the other strains produced a compound with physicochemical properties identical or close to those of the pyoverdine of P. aeruginosa ATCC 15692 (strain PAO1). The pyoverdines were purified and tested for their growth-promoting activity and for their ability to facilitate 59Fe uptake in homologous experiments involving each pyoverdine and its producing strain, as well as in heterologous systems involving all the other strains. The results of both types of experiments suggested the existence of three specificity groups. This was confirmed by analysis of the amino acid composition of the pyoverdines, which differed for each group. A partially purified polyclonal antiserum raised against a major 80-kilodalton (kDa) iron-regulated outer membrane protein (IROMP) of P. aeruginosa PAO1 recognized the 80-kDa IROMPs from P. aeruginosa PAO1 and the clinical isolates belonging to the same group, whereas the IROMPs from the strains belonging to the two other groups were not detected. A second antiserum raised against the P. aeruginosa ATCC 27853 80-kDa IROMP gave similar results by reacting specifically with the 80-kDa IROMP from the strains belonging to this group. Thus, together with the already known pyoverdine from P. aeruginosa PAO1, two new types of pyoverdines produced by strains belonging to this species were characterized.
PMCID: PMC259858  PMID: 2509364
22.  Interpretive criteria and quality control parameters for testing bacterial susceptibility to the fluoroquinolone PD131628. 
Journal of Clinical Microbiology  1995;33(1):235-238.
For testing bacterial susceptibility to PD131628, a 5-micrograms disk and the following tentative interpretive criteria may be used: > or = 19 mm for susceptible (MIC, < or = 1.0 micrograms/ml), 16 to 18 mm for intermediate (MIC, 2.0 micrograms/ml), and < or = 15 mm for resistant (MIC, > or = 4.0 micrograms/ml). For standard quality control strains, the following limits are proposed: for Escherichia coli ATCC 25922, zones of 31 to 41 mm or a MIC of 0.002 to 0.016 micrograms/ml; for Pseudomonas aeruginosa ATCC 27853, zones of 26 to 34 mm or a MIC of 0.12 to 0.5 micrograms/ml; for Staphylococcus aureus ATCC 25923, zones of 27 to 33 mm; for Staphylococcus aureus ATCC 29213, a MIC of 0.03 to 0.12 micrograms/ml; and for Enterococcus faecalis ATCC 29212, a MIC of 0.12 to 0.5 micrograms/ml.
PMCID: PMC227918  PMID: 7699050
23.  Single-strain regression analysis for determination of interpretive breakpoints for cefoperazone disk diffusion susceptibility testing. 
Journal of Clinical Microbiology  1983;17(6):975-980.
A novel approach for setting interpretive breakpoints in disk diffusion antibiotic susceptibility testing according to determined minimum inhibitory concentration (MIC) limits is described, using the method of single-strain regression analysis. The procedure was tested on reference strains Staphylococcus aureus (ATCC 25923), Streptococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 27853), using published results from cefoperazone disk diffusion experiments. The correlation between logarithm of the disk content and inhibition zone diameter squared was linear, excluding three endpoint values. When constants A and B in the new regression line equation were calculated for the four strains, all four showed different regression lines. Zone diameters corresponding to various MICs were calculated for a disk content of 75 micrograms. The values obtained for the four strains were 20.1, 20.9, 24.9, and 25.8 mm, respectively, for an MIC of 16 micrograms/ml, and 15.7, 15.7, 22.3, and 17.9 mm, respectively, for an MIC of 64 micrograms/ml. The following zone diameter breakpoints were determined for the "I" (intermediate) category, using a 75-micrograms disk: S. aureus, 18 to 15 mm; S. faecalis, 23 to 13 mm; E. coli, 20 to 17 mm; and P. aeruginosa, 20 to 17 mm.
PMCID: PMC272786  PMID: 6223938
24.  Stability of imipenem in Mueller-Hinton agar stored at 4 degrees C. 
The purpose of the present study was to measure the stability of imipenem in Mueller-Hinton agar stored at 4 degrees C over time. MICs for Staphylococcus aureus ATCC 25923, Streptococcus faecalis ATCC 29212, Escherichia coli ATCC 25922, and Pseudomonas aeruginosa ATCC 27853 were determined in triplicate daily for up to 15 days. The calculated mean time to observe a shift of one dilution in MIC endpoints was 4.33 +/- 1.25 days. For routine work, imipenem agar dilution plates should be prepared within 48 to 72 h of the test.
PMCID: PMC176367  PMID: 3867331
25.  Preparation, characterization and in vitro antimicrobial activity of liposomal ceftazidime and cefepime against Pseudomonas aeruginosa strains 
Brazilian Journal of Microbiology  2012;43(3):984-992.
Pseudomonas aeruginosa is an opportunistic microorganism with the ability to respond to a wide variety of environmental changes, exhibiting a high intrinsic resistance to a number of antimicrobial agents. This low susceptibility to antimicrobial substances is primarily due to the low permeability of its outer membrane, efflux mechanisms and the synthesis of enzymes that promote the degradation of these drugs. Cephalosporins, particularty ceftazidime and cefepime are effective against P. aeruginosa, however, its increasing resistance has limited the usage of these antibiotics. Encapsulating antimicrobial drugs into unilamellar liposomes is an approach that has been investigated in order to overcome microorganism resistance. In this study, antimicrobial activity of liposomal ceftazidime and cefepime against P. aeruginosa ATCC 27853 and P. aeruginosa SPM-1 was compared to that of the free drugs. Liposomal characterization included diameter, encapsulation efficiency and stability. Minimum Inhibitory Concentration (MIC) was determined for free and liposomal forms of both drugs. Minimum Bactericidal Concentration (MBC) was determined at concentrations 1, 2 and 4 times MIC. Average diameter of liposomes was 131.88 nm and encapsulation efficiency for cefepime and ceftazidime were 2.29% end 5.77%, respectively. Improved stability was obtained when liposome formulations were prepared with a 50% molar ratio for cholesterol in relation to the phospholipid. MIC for liposomal antibiotics for both drugs were 50% lower than that of the free drug, demonstrating that liposomal drug delivery systems may contribute to increase the antibacterial activity of these drugs.
PMCID: PMC3768856  PMID: 24031917
Pseudomonas aeruginosa; liposomes; cephalosporins

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