American Type Culture Collection Reference Research Articles

A Single-laboratory Performance Evaluation of MALDI-TOF MS in Rapid Identification of Staphylococcus aureus, Cronobacter sakazakii, Vibrio parahaemolyticus, and Some Closely Related Bacterial Species of Public Health Importance.

Staphylococcus is a genus of Gram-positive bacteria, known to cause food poisoning and gastrointestinal illness in humans. Additionally, the emergence of methicillin-resistant S. aureus (MRSA) strains have caused major healthcare burden worldwide. Cronobacter is a group of Gram-negative bacteria, that can survive in extreme dry conditions. Cronobacter sakazakii is known to contaminate the powdered infant formula and cause life-threatening infections in neonates. Vibrio is a genus of human-pathogenic Gram-negative bacteria that can cause foodborne illness by consuming undercooked or raw seafood. Vibrio parahaemolyticus can cause serious gastrointestinal disease in humans. Thus, rapid identification of Staphylococcus spp., Cronobacter spp. and Vibrio spp. is crucial for the source tracking of contaminated food, and to measure the transmission dynamics of these bacterial pathogens causing foodborne diseases and outbreaks. This single-laboratory performance evaluation study used the VITEK MS system to evaluate the potential of MALDI-TOF MS technology for rapid identification of S. aureus-like, C. sakazakii-like, and V. parahaemolyticus-like, isolates of public health importance. A total of 226 isolates recovered from various food, environmental surveillance samples and other sources were identified by bioMérieux VITEK 2 and VITEK MS systems as Staphylococcus spp., Cronobacter spp., and Vibrio spp. Five American Type Culture Collection (ATCC) reference Gram-positive and Gram-negative bacterial isolates were also tested to complete the study. In addition, for some Staphylococcus spp. isolates whole genome sequencing (WGS), and DNA sequencing of 16S rRNA partial region was also performed for species identification. The VITEK MS system was able to provide species identification to all 96 isolates of Staphylococcus spp. and to all 29 isolates of Vibrio spp. examined with a high confidence value (99.9%). Similarly, species identification was observed for the majority of spots (245 of 303) for the 101 Cronobacter spp. isolates (∼82.0%) with a high confidence value (99.9%), and genus level identification was noticed for the rest of the Cronobacter spp. isolates (18.0%; 58 of the 303 spots) analyzed. Species identification data generated by VITEK 2 system was comparable to data obtained by VITEK MS system. The VITEK MS system is a reliable high-throughput platform that can rapidly identify Staphylococcus, Vibrio, and Cronobacter to the genus level, as well as S. aureus, C. sakazakii, V. parahaemolyticus, and other closely related foodborne isolates, and bacterial isolates from additional sources, in most cases. The VITEK MS system can be used in the rapid genus and species identification of human-pathogenic Staphylococcus spp., Cronobacter spp., and Vibrio spp. isolates.

β-TCP/S53P4 Scaffolds Obtained by Gel Casting: Synthesis, Properties, and Biomedical Applications.

The objective of this study was to investigate the osteogenic and antimicrobial effect of bioactive glass S53P4 incorporated into β-tricalcium phosphate (β-TCP) scaffolds in vitro and the bone neoformation in vivo. β-TCP and β-TCP/S53P4 scaffolds were prepared by the gel casting method. Samples were morphologically and physically characterized through X-ray diffraction (XRD) and scanning electron microscope (SEM). In vitro tests were performed using MG63 cells. American Type Culture Collection reference strains were used to determine the scaffold's antimicrobial potential. Defects were created in the tibia of New Zealand rabbits and filled with experimental scaffolds. The incorporation of S53P4 bioglass promotes significant changes in the crystalline phases formed and in the morphology of the surface of the scaffolds. The β-TCP/S53P4 scaffolds did not demonstrate an in vitro cytotoxic effect, presented similar alkaline phosphatase activity, and induced a significantly higher protein amount when compared to β-TCP. The expression of Itg β1 in the β-TCP scaffold was higher than in the β-TCP/S53P4, and there was higher expression of Col-1 in the β-TCP/S53P4 group. Higher bone formation and antimicrobial activity were observed in the β-TCP/S53P4 group. The results confirm the osteogenic capacity of β-TCP ceramics and suggest that, after bioactive glass S53P4 incorporation, it can prevent microbial infections, demonstrating to be an excellent biomaterial for application in bone tissue engineering.

Repositioning of Benzodiazepine Drugs and Synergistic Effect with Ciprofloxacin Against ESKAPE Pathogens.

Infectious diseases are among the leading causes of morbidity and mortality worldwide. Combating them becomes more complex when caused by the pathogens of the ESKAPE group, which are Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp. The purpose of this study was to investigate the repositioning potential of the benzodiazepines clonazepam and diazepam individually and in combination with the antibacterial ciprofloxacin against ESKAPE. The minimum inhibitory concentration and minimum bactericidal concentration against seven American Type Culture Collection (ATCC) reference standard strains and 64 ESKAPE clinical isolates were determined. In addition, the interaction with ciprofloxacin was determined by the checkerboard method and fractional inhibitory concentration index (FICI) of clonazepam against 11 ESKAPE and diazepam against five ESKAPE. We also list the results found and their clinical significance. Benzodiazepines showed similar antibacterial activity against Gram-positive and Gram-negative. The checkerboard and FICI results showed a synergistic effect of these drugs when associated with ciprofloxacin against almost all tested isolates. Viewing the clinical cases studied, benzodiazepines have potential as treatment alternatives. The results allow us to conclude that clonazepam and diazepam, when in combination with ciprofloxacin, have promising activity against ESKAPE, therefore, assuming the position of candidates for repositioning.

In Vitro Studies of Xylitol and Erythritol Inhibition of Streptococcus Mutans and Streptococcus Sobrinus Growth and Biofilm Production.

The aim of this study was to evaluate synergy and inhibitory effects of xylitol and erythritol on Streptococcus mutans and Streptococcus sobrinus growth and biomass production on a polystyrene plastic surface. Study design; S. mutans and sobrinus strains (American Type Culture Collection reference strains 31341, 35668, 25175, sobrinus 33478) were cultivated in media (Todd Hewitt Broth with 1% sucrose or heart-brain infusion broth with 1% sucrose) at differing concentrations of xylitol or erythritol in microtiter assay plates incubated for 48 hours. Bacterial growth was quantified and measured by optical density using a microplate reader. Experiments assessing synergy and biofilm growth were carried out also using microdilution assays. All four strains were inhibited by 30% (w/v) xylitol, and 15% erythritol at 150mg/ml erythritol, 2/4 strains had reduced growth; at 270mg/ml, 4/4 strains were inhibited. Bactericidal effects were not observed at any polyol concentration. Combinations of both polyols in a checker board array were used to determine if there were any benefits of polyol combinations. Results The combination studies yielded mixed outcomes with indifference in growth for strains 68 and 78, potential additive effect for strain 75 and possible antagonism for strain 41. Assessment of biomass formation and polyol interference were also performed post MIC assessment. Strains 41, 68 and 75 produced significant biomass in the absence of either polyol. Both polyols inhibited biomass formation in a dose-dependent fashion. Strain 75 is a poor biomass producer and could not be assessed for polyol effects in our assay. Conclusion: Our results demonstrate significant polyol influence on the oral Streptococcal strains tested in our laboratory.

Synergistic effect of sertraline and disulfiram against multidrug resistant bacteria as a new alternative to drug repositioning

The repositioning of approved drugs is atopic of interest for the academy and the pharmaceutical industry. The synergistic combination of these drugs can be successful in the treatment of infections caused by resistant bacteria. This study aimed to assess the in vitro synergistic antibacterial activity of sertraline and disulfiram and their interaction with ciprofloxacin and sulfamethoxazole/trimethoprim. We determined the minimum inhibitory concentration, the minimum bactericidal concentration and the fractional inhibitory concentration index. Eighteen bacterial strains were used, being nine American Type Culture Collection reference strains and nine multidrug resistant clinical isolates. Synergy was detected between sertraline and disulfiram against a strain of Staphylococcus aureusATCC 25923 and a clinical isolate of S. aureus. When associated to sulfamethoxazole/trimethoprim and ciprofloxacin, sertraline and disulfiram showed eight synergistic events, which occurred against three different standard strains and two multidrug resistant clinical isolates. When the minimum bactericidal concentration was determined, the bactericidal activity of sertraline was enhanced with disulfiram. Our results suggest that these drugs, widely used to treat depression and chronic alcoholism, have antibacterial potential individually, in association, and combined with antimicrobials, what makes their repositioning a promising therapeutic alternative for the effective treatment of infections caused by multidrug resistant bacteria.

The effects of Mn and/or Ni dopants on the in vitro/in vivo performance, structural and magnetic properties of β-tricalcium phosphate bioceramics

The present study describes the effects of dopants of Mn and/or Ni on the structural, magnetic and in vivo/in vitro performances of β-tricalcium phosphate (β-TCP) bioceramics synthesized by a sol-gel method. Both types of dopants caused significant effects on the structure-related parameters of β-TCP, including the crystallite dimensions and lattice parameters. The magnetic properties of as-prepared samples were found to be affected by the type and amount of the dopant(s). The in vitro antimicrobial activities of doped β-TCPs against American Type Culture Collection (ATCC) reference bacterial and fungal strains were evaluated. Among them, only 1.35Ni-TCP displayed an effective antimicrobial activity against Gram-positive bacteria (50 μg/mL Minimum Inhibitory Concentration (MIC) range), while other types of doped β-TCPs did not possess with such effect. The in vivo tests showed that each formulation could be a good nominate for bone healing and bone treatment.

In vitro evaluation of methicillin-resistant and methicillin-sensitive Staphylococcus aureus susceptibility to Saudi honeys

BackgroundHoney has been increasingly recognized as a potential therapeutic agent for treatment of wound infections. There is an urgent need for assessment and evaluation of the antibacterial properties against wound pathogens of honeys that have not yet been tested.MethodsTen Saudi honeys collected from different geographical locations were screened initially for their antibacterial potential against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) by the agar well diffusion method. Manuka honey (UMF-12) was used for comparison. Of the tested honeys, the honey that exhibited the greatest antibacterial activity in the agar well diffusion assay was further evaluated for its minimum inhibitory concentration (MIC) against ten MRSA clinical isolates and three American Type Culture Collection (ATCC) reference strains by the microbroth dilution method.ResultsLocally produced honeys exhibited variable antibacterial activity against the tested isolates in the agar well diffusion assay. They were unable to exhibit antibacterial activity against MSSA and MRSA at 25% dilutions (w/v) in catalase solution. However, Sumra and Talha honeys showed a zone of inhibition at 50% dilutions (w/v) in catalase solution. This finding means that both honeys possess weak non-peroxide-based antibacterial activity. Moreover, Sumra honey showed a larger inhibition zone at 50 and 25% dilutions (w/v) in distilled water than Manuka honey against both MSSA and MRSA. This result demonstrates that Sumra honey has more hydrogen peroxide-related antibacterial activity or total antibacterial activity than Manuka honey. In addition, MIC results obtained through a microbroth dilution assay showed that Sumra honey inhibited the growth of all MRSA clinical isolates (n = 10) and reference strains [MRSA (ATCC 43300) and MSSA (ATCC 29213)] at lower concentrations (12.0% v/v) than those required for Manuka honey-mediated inhibition (14.0% v/v). This result means that Sumra honey has more peroxide or synergistic antibacterial activity than Manuka honey. An equivalent MIC (15.0% v/v) was observed for E. coli (ATCC 25922) between Manuka honey and Sumra honey.ConclusionsSumra honey may be used as an alternative therapeutic agent for infected wounds and burns, where additional hydrogen peroxide-related antibacterial activity is needed. In the future, the physiochemical characteristics of Sumra honey may be evaluated and standardized.

Antimicrobial Susceptibility Testing of Leptospira spp. in the Lao People's Democratic Republic Using Disk Diffusion.

.Leptospirosis is a global zoonotic disease caused by pathogenic bacteria of the Leptospira genus, which are fastidious, slow-growing organisms. Antimicrobial susceptibility data are limited; traditionally, the organisms have not been culturable on solid media. The recent development of Leptospira Vanaporn Wuthiekanun (LVW) agar, which facilitates rapid growth of Leptospira spp., provides the opportunity for antimicrobial susceptibility testing. Eighty-three Leptospira spp. clinical isolates originating from patients in Laos between 2006 and 2016 were tested against six antimicrobials (azithromycin, ceftriaxone, ciprofloxacin, doxycycline, gentamicin, and penicillin G) using disk diffusion on LVW agar. Quality control was undertaken using American Type Culture Collection (ATCC) reference strains with known susceptibilities on both standard media and LVW agar. All Leptospira spp. isolates produced large zones of inhibition around each of the six antimicrobials. All zones were greater than 25 mm: gentamicin produced the smallest zones (median 35 mm; interquartile range 30 mm–37 mm) and azithromycin produced the largest zones (median 85 mm; interquartile range 85 mm–85 mm). Zones produced by non-leptospiral ATCC reference strains on LVW agar were within 2 mm of accepted strain-specific quality control range on standard media. Antimicrobial activity on LVW agar appears to be similar to that on standard media. As there are no published susceptibility guidelines for the Leptospira genus, zone interpretation was subjective. Leptospira Vanaporn Wuthiekanun agar enabled antimicrobial susceptibility testing of multiple Leptospira isolates on solid media; the large zone sizes observed suggest that resistance has not emerged to these six antimicrobials in Lao Leptospira spp.

Antibacterial Efficacy of Commercially Available Essential Oils Tested Against Drug-Resistant Gram-Positive Pathogens

The potential antibacterial activity of basil (Ocimum basilicum), chamomile (Matricaria chamomilla), origanum (Thymus capitatus), tea tree (Melaleuca alternifolia) and thyme (Thymus vulgaris) essential oils, was investigated against 29 Gram-positive bacterial strains isolated from wastewater treatment plants, clinical samples (n = 25) and American Type Culture Collection (ATCC) reference strains (n = 4). Wild bacterial strains included methicillin-resistant Staphylococcus aureus (n = 16) and vancomycin-resistant Enterococcus spp. (n = 9). The antimicrobial activity of the selected oils was studied using the broth macrodilution method. The Minimal Inhibitory Concentration (MIC) values for S. aureus ranged from 0.06 to 0.5% (v/v) for origanum oil, 0.06 to 1% (v/v) for thyme oil, 0.12 to 1% (v/v) for tea tree oil, 0.25 to 4% (v/v) for basil oil and 2 to >4% (v/v) for chamomile oil. For enterococci the MIC values were significantly higher ranging from 0.25 to 1% (v/v), 0.5 to 2% (v/v), 1 to 4% (v/v), 4 to >4% (v/v) and >4% (v/v) for the above-mentioned oils, respectively. The main compounds of the tested essential oils were: estragole (Ocimum basilicum), bisabolol and trans-b-farnesene (Matricaria chamomilla), carvacrol and thymol (Thymus capitatus), terpinen-4-ol and p-cymene (Melaleuca alternifolia), thymol, linalool, and p-cymene (Thymus vulgaris). Origanum essential oil yielded the best antimicrobial results followed by thyme, tea tree, and basil oil, while chamomile oil exhibited weak antibacterial properties.

Application of MALDI-TOF MS Systems in the Rapid Identification of Campylobacter spp. of Public Health Importance

Campylobacteriosis is an infectious gastrointestinal disease caused by Campylobacter spp. In most cases, it is either underdiagnosed or underreported due to poor diagnostics and limited databases. Several DNA-based molecular diagnostic techniques, including 16S ribosomal RNA (rRNA) sequence typing, have been widely used in the species identification of Campylobacter. Nevertheless, these assays are time-consuming and require a high quality of bacterial DNA. Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) MS is an emerging diagnostic technology that can provide the rapid identification of microorganisms by using their intact cells without extraction or purification. In this study, we analyzed 24 American Type Culture Collection reference isolates of 16 Campylobacter spp. and five unknown clinical bacterial isolates for rapid identification utilizing two commercially available MADI-TOF MS platforms, namely the bioMérieux VITEK® MS and Bruker Biotyper systems. In addition, 16S rRNA sequencing was performed to confirm the species-level identification of the unknown clinical isolates. Both MALDI-TOF MS systems identified the isolates of C. jejuni, C. coli, C. lari, and C. fetus. The results of this study suggest that the MALDI-TOF MS technique can be used in the identification of Campylobacter spp. of public health importance.

Evaluation of Two Standard and Two Chromogenic Selective Media for Optimal Growth and Enumeration of Isolates of 16 Unique Bacillus Species

The genus Bacillus is a group of gram-positive endospore-forming bacteria that can cause food poisoning and diarrheal illness in humans. A wide range of food products have been linked to foodborne outbreaks associated with these opportunistic pathogens. The U.S. Food and Drug Administration recommends (in their Bacteriological Analytical Manual) the use of Bacara or mannitol egg yolk polymyxin (MYP) agar plates and the most-probable-number (MPN) method for enumeration and confirmation of Bacillus cereus and related species isolated from foods, sporadic cases, outbreaks, and routine environmental surveillance samples. We performed a comparative analysis of two chromogenic media (Bacara and Brilliance) and two traditional media (MYP and polymyxin egg yolk mannitol bromothymol blue agar [PEMBA]) for the isolation and enumeration of 16 Bacillus species under modified growth conditions that included pH, temperature, and dilution factor. A total of 50 environmental, food, and American Type Culture Collection reference isolates from 16 distinct Bacillus species were evaluated. A food adulteration experiment also was carried out by artificially adulterating two baby food matrices with two isolates each of B. cereus and Bacillus thuringiensis . Our results clearly indicated that chromogenic plating media (Bacara and Brilliance) are better than conventional standard media (MYP and PEMBA) for the detection and enumeration of B. cereus in foods and other official regulatory samples. The comparison of the two chromogenic media also indicated that Brilliance medium to be more efficient and selective for the isolation of Bacillus.

Phytochemical and biological properties of four Astragalus species commonly used in traditional Palestinian medicine

IntroductionSeveral species that belong to the genus Astragalus have been used in Palestinian folk medicine to treat various infectious and non-infectious diseases. This study aimed to assess the phytochemical and biological activities of four Astragalus species growing wild in Jerusalem. MethodsPhytochemical screening, estimation of total phenols, flavonoids and tannin contents, as well as antimicrobial and antioxidant activities were investigated for Astragalus aleppicus, Astragalus angustifolius, Astragalus annularis, and Astragalus boeticus. Phytochemical screening, evaluation of total phenols, flavonoids, tannins contents as well as antioxidant properties were investigated by using standard phytochemical methods. Detection of antimicrobial activities for both the aqueous and the methanolic extracts were performed using the broth micro-dilution method. The antimicrobial activities were examined against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa American Type Culture Collection reference strains, as well as against Methicillin Resistant Staphylococcus aureus and Candida albicans clinical isolates. ResultsA mixture of phytochemical compounds was found in the extracts of all tested Astragalus species. A. boeticus was found to contain high levels of total phenols, flavonoids and tannins with remarkable potential of antioxidant and antimicrobial activities. The current study provides initial data that justify the use and importance of Astragalus species in Palestinian folkloric medicine. ConclusionOur results showed that A. boeticus was superior to the other 3 species in all the conducted tests. In fact, it had the highest polyphenolic content. In addition, its aqueous extract showed the highest antibacterial activity, while the methanolic extract showed the highest antifungal and antioxidant activities. Further studies are needed to identify and characterize these constituents.

Investigating the impacts of various solvents fractions of Bupleurum lancifolium on the antimicrobial and antioxidant potentials

Introduction: In the last thirty years, interests in searching for plants with potential antimicrobial and antioxidant activities have been increased due to their probable health benefits. This study aimed to investigate the antimicrobial and antioxidant effects of various solvents fractions of Bupleurum lancifolium. Methods: The antioxidant activities of four fractions of Bupleurum lancifolium were assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH ) scavenging assay, while the antimicrobial activity was assessed by broth microdilution method. The antimicrobial activity of four plant fractions was examined against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Shigella sonnie and Enterococcus faecium American Type Culture Collection reference strains. Results: The fractionation extraction method succeeds in the assessment of the antibacterial and antioxidant activity of B. lancifolium plant. Hence the methanolic fraction has antioxidant potential with 33.03% of inhibition according to the Trolox antioxidant standard molecule, while n-hexane and methanol fractions showed powerful antibacterial activity against E. faecium and S. sonnie strains with Minimum Inhibitory Concentration (MIC )1.5625 mg/ml for both fractions. Conclusion: All plant fractions have potent antioxidant and antibacterial activities. Further investigations, i.e. isolation, identification, and clinical assessment, of the active compound(s) are needed for the possible formulation of new therapeutic alternatives.

Green Synthesis, Characterization and Antibacterial Activity of Silver Nanoparticles of Endophytic Fungi Aspergillus terreus

In present study, the silver nanoparticles were synthesized using endophytic fungi Aspergillus terreus, isolated from Calotropis procera. Synthesized nanoparticles were characterized using various spectroscopic techniques. The nanoparticles were reported oval to spherical in shape. The average size of silver nanoparticles was 16.54 nm. Gas chromatography-mass spectrometry analysis of Aspergillus terreus showed the presence of 17 compounds. The synthesized silver nanoparticles showed considerable antibacterial activity against tested bacterial strains: 9 American type culture collection reference (ATCC) and 3 multidrug resistance (MDR) strains. Synthesized nanoparticles showed significant antibacterial activity against Salmonella typhi (16.67 ± 0.58 mm), Staphylococcus aureus (15.67 ± 0.58 mm), and Escherichia coli (15.67 ± 0.58 mm). Minimum inhibitory concentration was reported in range of 11.43 μg/ml to 308 μg/ml. Cell leakage analysis reported an increase in protein leakage level and degradation of nucleic acid after treatment with silver nanoparticles. The present study concluded that endophytic fungi Aspergillus terreus isolated from Calotropis procera can be used as a source for synthesis of silver nanoparticles and suggesting as an effective antibacterial agent

Multilocus Sequence Analysis of Phylogroup 1 and 2 Oral Treponeme Strains.

Periodontal diseases are caused by persistent polymicrobial biofilm infections of the gums and underlying tooth-supporting structures and have a complex and variable etiology. Although Treponema denticola is strongly associated with periodontal diseases, the etiological roles of other treponeme species/phylotypes are less well defined. This is due to a paucity of formal species descriptions and a poor understanding of genetic relationships between oral treponeme taxa. Our study directly addresses these issues. It represents one of the most comprehensive analyses of oral treponeme strains performed to date, including isolates from North America, Europe, and Asia. We envisage that our results will greatly facilitate future metagenomic efforts aimed at characterizing the clinical distributions of oral treponeme species/phylotypes, helping investigators to establish a more detailed understanding of their etiological roles in periodontal diseases and other infectious diseases. Our results are also directly relevant to various polymicrobial tissue infections in animals, which also involve treponeme populations.

Rapid infectious disease identification by next-generation DNA sequencing

Currently, there is a critical need to rapidly identify infectious organisms in clinical samples. Next-Generation Sequencing (NGS) could surmount the deficiencies of culture-based methods; however, there are no standardized, automated programs to process NGS data. To address this deficiency, we developed the Rapid Infectious Disease Identification (RIDI™) system. The system requires minimal guidance, which reduces operator errors. The system is compatible with the three major NGS platforms. It automatically interfaces with the sequencing system, detects their data format, configures the analysis type, applies appropriate quality control, and analyzes the results. Sequence information is characterized using both the NCBI database and RIDI™ specific databases. RIDI™ was designed to identify high probability sequence matches and more divergent matches that could represent different or novel species. We challenged the system using defined American Type Culture Collection (ATCC) reference standards of 27 species, both individually and in varying combinations. The system was able to rapidly detect known organisms in <12h with multi-sample throughput. The system accurately identifies 99.5% of the DNA sequence reads at the genus-level and 75.3% at the species-level in reference standards. It has a limit of detection of 146cells/ml in simulated clinical samples, and is also able to identify the components of polymicrobial samples with 16.9% discrepancy at the genus-level and 31.2% at the species-level. Thus, the system's effectiveness may exceed current methods, especially in situations where culture methods could produce false negatives or where rapid results would influence patient outcomes.

Investigation the efficiency of various methods of volatile oil extraction from Trichodesma africanum and their impact on the antioxidant and antimicrobial activities.

Background:Currently, there is an increasing interest in developing more efficient techniques for the extraction of phytochemicals. Microwaves and ultrasonic extraction methods are promising techniques that can be used for this purpose.Objectives:The purpose of this study was to investigate the impact of different extraction methods on yield, antioxidant and antimicrobial activities of volatile oil extracted from Trichodesma africanum.Materials and Methods:Volatile oil was extracted using microwave, ultrasonic, microwave-ultrasonic, and conventional hydrodistillation methods. The extracted oil was evaluated for antioxidant and antimicrobial activities. The antioxidant activity was assessed by 2,2-Diphenyl-1-picrylhydrazyl scavenging assay, whereas the antimicrobial activity was assessed by broth microdilution method. The antimicrobial activity of the volatile oils was examined against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa American type culture collection reference strains, as well as against methicillin-resistant S. aureus (MRSA) and Candida albicans clinical isolates.Results:The volatile oil obtained by the four extraction methods in this study exhibited both antioxidant and antimicrobial activities. Among the four extraction methods used, the microwave-ultrasonic method yielded the largest amount (1.8% v/w) and the yield exhibited the highest antioxidant activity in terms of inhibition (91.83% ± 1.1). The minimum inhibitory concentrations for E. coli, P. aeruginosa, S. aureus, MRSA, and C. albicans were 3, 5, 6, 3, and 9, respectively.Conclusion:Among the extraction techniques used in this study, the microwave-ultrasonic method showed the best results. Moreover, this study suggests that T. africanum volatile oils contain active substances that could potentially be used both as natural preservatives in food and pharmaceutical industries as well as in developing new antimicrobial and antioxidant agents.

Increased Resistance of Contact Lens-Related Bacterial Biofilms to Antimicrobial Activity of Soft Contact Lens Care Solutions

To determine if clinical and reference strains of Pseudomonas aeruginosa, Serratia marcescens, and Staphylococcus aureus form biofilms on silicone hydrogel contact lenses and ascertain antimicrobial activities of contact lens care solutions. Clinical and American Type Culture Collection reference strains of P. aeruginosa, S. marcescens, and S. aureus were incubated with lotrafilcon A lenses under conditions that facilitate biofilm formation. Biofilms were quantified by quantitative culturing (colony-forming units, CFUs), and gross morphology and architecture were evaluated using scanning electron microscopy and confocal microscopy. Susceptibilities of the planktonic and biofilm growth phases of the bacteria to 5 common multipurpose contact lens care solutions and 1 hydrogen peroxide care solution were assessed. Pseudomonas aeruginosa, S. marcescens, and S. aureus reference and clinical strains formed biofilms on lotrafilcon A silicone hydrogel contact lenses, as dense networks of cells arranged in multiple layers with visible extracellular matrix. The biofilms were resistant to commonly used biguanide-preserved multipurpose care solutions. Pseudomonas aeruginosa and S. aureus biofilms were susceptible to a hydrogen peroxide and a polyquaternium-preserved care solution, whereas S. marcescens biofilm was resistant to a polyquaternium-preserved care solution but susceptible to hydrogen peroxide disinfection. In contrast, the planktonic forms were always susceptible. Pseudomonas aeruginosa, S. marcescens, and S. aureus form biofilms on lotrafilcon A contact lenses, which in contrast to planktonic cells are resistant to the antimicrobial activity of several soft contact lens care products.

Genetic Diversity of Trichomonas vaginalis Clinical Isolates Determined by EcoRI Restriction Fragment Length Polymorphism of Heat-Shock Protein 70 Genes

Restriction fragment length polymorphism (RFLP) analysis using a multilocus heat-inducible cytoplasmic heat-shock protein 70 (Hsp70) hybridization probe with EcoRI-digested genomic DNA was used in molecular typing of 129 Trichomonas vaginalis isolates. Results indicate that Trichomonas organisms exhibit considerable polymorphism in their Hsp70 RFLP patterns. Analysis of seven American Type Culture Collection reference strains and 122 clinical isolates, including 84 isolates from Jackson, Mississippi, 18 isolates from Atlanta, Georgia, and 20 isolates from throughout the United States, showed 105 distinct Hsp70 RFLP pattern subtypes for Trichomonas. Phylogenetic analysis of the Hsp70 RFLP data showed that the T. vaginalis isolates were organized into two clonal lineages. These results illustrate the substantial genomic diversity present in T. vaginalis and indicate that a large number of genetically distinct Trichomonas isolates may be responsible for human trichomoniasis in the United States.

Tigecycline MIC Testing by Broth Dilution Requires Use of Fresh Medium or Addition of the Biocatalytic Oxygen-Reducing Reagent Oxyrase To Standardize the Test Method

Tigecycline is a broad-spectrum glycylcycline antibiotic with activity against not only susceptible gram-positive and gram-negative pathogens but also strains that are resistant to many other antibiotics. In the process of determining quality control (QC) limits for the American Type Culture Collection reference strains for tigecycline, a number of inconsistencies in MICs were encountered which appeared to be related to the age of the Mueller-Hinton broth (MHB) medium used in the MIC testing. The objective of this study was to determine the cause of the discrepant MIC results between fresh and aged MHB. The MICs of tigecycline were determined in MHB that was either prepared fresh (<12 h old), prepared and stored at 4 degrees C, stored at room temperature, stored anaerobically, or supplemented with the biocatalytic oxygen-reducing reagent Oxyrase. When tested in fresh media, tigecycline was 2 to 3 dilutions more active against the CLSI-recommended QC strains compared to aged media (MICs of 0.03 to 0.25 and 0.12 to 0.5 mug/ml, respectively). Media aged under anaerobic conditions prior to testing or supplemented with Oxyrase resulted in MICs similar to those obtained in fresh medium (MICs of 0.03 to 0.12 and 0.03 to 0.25 mug/ml, respectively). Time-kill kinetics demonstrated a >3 log(10) difference in viable growth when tigecycline was tested in fresh or Oxyrase-supplemented MHB compared to aged MHB. High-pressure liquid chromatography analysis revealed the accumulation of an early peak (oxidative by-product of tigecycline) to be 3.5% in fresh media and 25.1% in aged media after 24 h and that addition of Oxyrase prevented the accumulation of this oxidized by-product. These results suggested that the activity of tigecycline was affected by the amount of dissolved oxygen in the media. The use of fresh MHB or supplementation with Oxyrase resulted in a more standardized test method for performing MIC tests with tigecycline.