Stability Studies of the Dilution Series of Different Antibiotic Stock Solutions in Culture Medium Incubated at 37 °C.

The long-term stability of antibiotics in culture media remains underexplored in scientific literature. This study evaluated the stability of eight distinct antibiotic stock solutions-amoxicillin, cefotaxime, neomycin, oxytetracycline, florfenicol, enrofloxacin, colistin, and potentiated sulfonamide-and their 10-fold dilution series in tryptone soy broth (TSB) at 37 °C, over 12 days. Samples were collected immediately after preparation and on days 1, 2, 5, 7, 9, and 12, with active substance concentrations measured using ultra-high-performance liquid chromatography (UHPLC) coupled with mass spectrometry. The results indicated that among the ultrapure water stock solutions, neomycin, florfenicol, and potentiated sulfonamide maintained stability (>95%). Within the culture medium, florfenicol showed consistent stability (100%) throughout the study, potentiated sulfonamide experienced minor degradation (>85%), and neomycin underwent significant degradation. Amoxicillin, oxytetracycline, and colistin displayed considerable degradation in both solution types but were more stable in ultrapure water solutions. The stability of cefotaxime and enrofloxacin in ultrapure water solutions and in the medium was very similar when compared; however, 3.6% of the former and 88.7% of the latter remained detectable by day 12. These findings are crucial for minimum inhibitory concentration (MIC) assessments, especially in minimum bactericidal concentration (MBC) studies, and in experiments concerning long-term evolution and co-selection. This study underscores the necessity of stability assessments in culture media to validate future experimental outcomes.

Supplementation with Amino Acid Sources Facilitates Fermentative Growth of Shewanella oneidensis MR-1 in Defined Media.

Shewanella oneidensis MR-1 is a facultative anaerobe that grows by respiration using a variety of electron acceptors. This organism serves as a model to study how bacteria thrive in redox-stratified environments. A glucose-utilizing engineered derivative of MR-1 has been reported to be unable to grow in glucose minimal medium (GMM) in the absence of electron acceptors, despite this strain having a complete set of genes for reconstructing glucose to lactate fermentative pathways. To gain insights into why MR-1 is incapable of fermentative growth, this study examined a hypothesis that this strain is programmed to repress the expression of some carbon metabolic genes in the absence of electron acceptors. Comparative transcriptomic analyses of the MR-1 derivative were conducted in the presence and absence of fumarate as an electron acceptor, and these found that the expression of many genes involved in carbon metabolism required for cell growth, including several tricarboxylic acid (TCA) cycle genes, was significantly downregulated in the absence of fumarate. This finding suggests a possibility that MR-1 is unable to grow fermentatively on glucose in minimal media owing to the shortage of nutrients essential for cell growth, such as amino acids. This idea was demonstrated in subsequent experiments that showed that the MR-1 derivative fermentatively grows in GMM containing tryptone or a defined mixture of amino acids. We suggest that gene regulatory circuits in MR-1 are tuned to minimize energy consumption under electron acceptor-depleted conditions, and that this results in defective fermentative growth in minimal media. IMPORTANCE It is an enigma why S. oneidensis MR-1 is incapable of fermentative growth despite having complete sets of genes for reconstructing fermentative pathways. Understanding the molecular mechanisms behind this defect will facilitate the development of novel fermentation technologies for the production of value-added chemicals from biomass feedstocks, such as electro-fermentation. The information provided in this study will also improve our understanding of the ecological strategies of bacteria living in redox-stratified environments.

Tryptone-stabilized silver nanoparticles' potential to mitigate planktonic and biofilm growth forms of Serratia marcescens.

Several microbial pathogens are capable of forming biofilms. These microbial communities pose a serious challenge to the healthcare sector as they are quite difficult to combat. Given the challenges associated with the antibiotic-based management of biofilms, the research focus has now been shifted towards finding alternate treatment strategies that can replace or complement the antibacterial properties of antibiotics. The field of nanotechnology offers several novel and revolutionary approaches to eradicate biofilm-forming microbes. In this study, we evaluated the antibacterial and antibiofilm efficacy of in-house synthesized, tryptone-stabilized silver nanoparticles (Ts-AgNPs) against the superbug Serratia marcescens. The nanoparticles were of spherical morphology with an average hydrodynamic diameter of 170 nm and considerable colloidal stability with a Zeta potential of - 24 ± 6.15 mV. Ts-AgNPs showed strong antibacterial activities with a minimum inhibitory concentration (MIC50) of 2.5 µg/mL and minimum bactericidal concentration (MBC) of 12.5 µg/mL against S. marcescens. The nanoparticles altered the cell surface hydrophobicity and inhibited biofilm formation. The Ts-AgNPs were also effective in distorting pre-existing biofilms by degrading the extracellular DNA (eDNA) component of the extracellular polymeric substance (EPS) layer. Furthermore, reduction in quorum-sensing (QS)-induced virulence factors produced by S. marcescens indicated that Ts-AgNPs attenuated the QS pathway. Together, these findings suggest that Ts-AgNPs are an important anti-planktonic and antibiofilm agent that can be explored for both the prevention and treatment of infections caused by S. marcescens.

Effect of molecular weight of chitosan on the formation and properties of zein-nisin-chitosan nanocomplexes.

This study evaluated the effect of molecular weight of chitosan (3 kDa,150 kDa, 400 kDa, and 600 kDa) on zein-nisin-chitosan nanocomplexes. The formation mechanism, physicochemical and antibacterial properties of the nanocomplexes (ZNC0.3, ZNC15, ZNC40, and ZNC60) were assessed. The nanocomplexes were characterized by DLS, ζ-potential, atomic force microscopy, scanning electron microscopy, circular dichroism, fourier transform infrared and UV-Vis spectroscopy. The results showed that the lowest molecular weight chitosan (LMWC, 3 kDa) formed nanocomplexes with nisin and zein structurally differed from the higher molecular weights chitosan (HMWC, >3 kDa). LMWC was doped on the surface of the nanocomplexes. HMWC linked and formed a network to adsorb zein and nisin. The antibacterial activity against Staphylococcus aureus showed that the minimum inhibitory concentration of ZNC0.3, ZNC15, ZNC40, and ZNC60 was 7.0625, 14.125, 14.125, and 28.25 µg/mL. ZNC0.3 could be a suitable nisin delivery system for its high encapsulation efficiency (85.38%) and antibacterial properties.

Cytokinins are pervasive among common in vitro culture media: An analysis of their forms, concentrations and potential sources.

The number of organisms that are known to produce cytokinins (CKs) continues to increase. In fact, species from all life kingdoms have now been shown to either produce CKs or at least have the genetic components to make it possible. In vitro growth of microorganisms, plant/animal cells, and tissue cultures often requires nutrient-rich media composed of ingredients with organic origins including: yeast extract, peptone, tryptone, or various plant or animal extracts. These compounds, derived from microbial, plant and animal materials, can be the source of significant levels of exogenous CKs in the culture medium. As CK investigative work continues to expand rapidly, it is of critical importance to draw attention to this complexity; the presence of CKs in growth medium affects CK metabolism of the cultured organism and interferes with the readings of analytical instrumentation used to profile CKs in tested microorganisms or cell cultures.

Amino acids other than glutamate affect the expression of the GAD system in Listeria monocytogenes enhancing acid resistance.

The Glutamate Decarboxylase (GAD) system is important for survival of L. monocytogenes and other microorganisms under acidic conditions. Environmental conditions influence the function of the GAD system. Until now, the only conditions known to lead to increased transcription of the GAD system are the stationary phase in rich media and anoxic conditions. Previously, we showed that transcription of the GAD system requires unidentified compounds other than glutamate present in rich media. Following a test looking at various compounds we identified for first time that peptone, tryptone and casamino acids activate the GAD system under oxic conditions suggesting that amino acid(s) other than glutamate and/or peptides are important for the above process. The defined medium, where the GAD system is inactive, once it is supplemented with the above compounds results in an active intracellular and extracellular GAD system and increased acid resistance. Through functional genomics we show that these compounds are required for GadD2 activity and although we previously showed that GadD3 is active part of the intracellular GAD system, the supplementation did not activate this gene. The above is explained by the fact that only gadD2 transcription was upregulated by these compounds while the transcription of gadD1 and gadD3 remained unaffected. Together our results show that the L. monocytogenes GadD2 decarboxylase is activated in the presence of amino acids or peptides other than glutamate, a finding that has important implications for acid tolerance and food safety.

Biofilm production under different atmospheres and growth media by Streptococcus agalactiae isolated from milk of cows with subclinical mastitis.

Different methods to analyze Streptococcus agalactiae biofilm formation have been investigated, but standardized protocols have not been developed. We compared S. agalactiae biofilm production among different atmospheres and growth media. Biofilm formation was studied in 32 isolates from bovine mastitis cases grown in Tryptone Soy Broth (TSB), Todd Hewitt Broth (THB), Luria Bertani Broth (LB) and Brain Heart Infusion (BHI), under two atmospheres, aerobic and 5% CO2. Regardless of the culture medium, growth under 5% CO2 resulted in a greater proportion of biofilm formation (65.63%), as compared with aerobic conditions (39.84%). Regardless of the atmosphere, the chances of biofilm formation were greater for isolates grown in TSB, as compared with THB [Odds ratio (OR) = 3.02], BHI (OR = 4.57), or LB (OR = 10.20). Thus, we suggest the use of 5% CO2 atmosphere and TSB in biofilm formation assays by Group-B streptococci (GBS) isolated from intramammary infections.

CdSe QD Biosynthesis in Yeast Using Tryptone-Enriched Media and Their Conjugation with a Peptide Hecate for Bacterial Detection and Killing.

The physical and chemical synthesis methods of quantum dots (QDs) are generally unfavorable for biological applications. To overcome this limitation, the development of a novel "green" route to produce highly-fluorescent CdSe QDs constitutes a promising substitute approach. In the present work, CdSe QDs were biosynthesized in yeast Saccharomyces cerevisiae using a novel method, where we showed for the first time that the concentration of tryptone highly affects the synthesis process. The optimum concentration of tryptone was found to be 25 g/L for the highest yield. Different methods were used to optimize the QD extraction from yeast, and the best method was found to be by denaturation at 80 °C along with an ultrasound needle. Multiple physical characterizations including transmission electron microscopy (TEM), dynamic light scattering (DLS), energy-dispersive X-ray spectroscopy (EDX), and spectrophotometry confirmed the optical features size and shape distribution of the QDs. We showed that the novel conjugate of the CdSe QDs and a cell-penetrating peptide (hecate) can detect bacterial cells very efficiently under a fluorescent microscope. The conjugate also showed strong antibacterial activity against vancomycin-resistant Staphylococcus aureus (VRSA), methicillin-resistant Staphylococcus aureus (MRSA), and Escherichia coli, which may help us to cope with the problem of rising antibiotic resistance.

Nitrogen source and pH interact and modulate lipase secretion in a non-clinical strain of Candida parapsilosis

Lipases (E.C. 3.1.1.3) are serine-hydrolases, and act on long chain fatty acid ester bonds. They exhibit specific and enantioselective activities, which are desirable for many industrial applications. This study aimed at screening and optimizing the production of lipases by wild yeast strains from a variety of substrates, as well as characterizing the enzyme. An initial selection was made in oxygenated oil-supplemented minimum medium, and the enzymatic activity of the supernatant was tested over p-nitrophenyl palmitate. One-hundred and twenty-four yeast strains from different substrates were tested, and twenty-three showed significantly higher lipolytic activity (p<0.01). One yeast in particular, QU110, showed best lipase production and therefore was selected for the optimization and characterization processes. This yeast exhibits enzyme secretion in initial pH 6.0, with olive oil and tryptone as carbon and nitrogen sources, respectively. There was a strong interaction between nitrogen source and initial pH, and pH 9.0seems to inhibit enzyme secretion. The crude enzyme (cell-free supernatant) shows stability in surfactants and n-hexane, but not in ethanol or methanol. A Response Surface Model was created and optimal enzyme activity conditions were observed at 36°C and pH 8.0. The lipase is appropriate for transesterification reactions, as the enzyme is more stable in strong apolar solvents than moderately apolar ones. Also, secretion by pH was not reported elsewhere, which should be further investigated and contribute for other yeast bioprocesses as well.

Thermal Inactivation of Listeria monocytogenes in Crab Meat.

Listeria monocytogenes is an important bacterial pathogen in seafood products, but limited information is currently available on the thermal resistance of relevant isolates in seafood. Thermal inactivation studies were undertaken (i) to provide much needed thermal inactivation data for L. monocytogenes in crab meat and (ii) to investigate whether tryptone soya broth (TSB) is representative of crab meat in thermal inactivation studies involving L. monocytogenes. D-values were obtained for a cocktail of two crab isolates (serotypes 1/2a and 4b) at 50, 55, and 60°C. In crab meat, D-values were 174.4, 28.2, and 1.6 min, respectively. Similar D-values of 176.4, 28.8, and 1.4 min were obtained in TSB. The corresponding z-values were 4.9°C (crab meat) and 4.8°C (TSB), respectively. The conclusions were that (i) current pasteurization conditions (e.g., 70°C for 2 min) would achieve complete destruction of any L. monocytogenes present in crab meat and (ii) TSB could be used as a model matrix for assessing the thermal inactivation of L. monocytogenes in crab meat.

Evaluation of an ATP-Bioluminescence Rapid Microbial Screening Method for In-Process Biologics.

This study compared an adenosine triphosphate (ATP)-based bioluminescence rapid microbial method (RMM) with a conventional sterility method for biologics sample testing. The RMM is based on a comparison of ATP levels in inoculated and uninoculated microbiological growth medium samples following growth enrichment incubation. The biologics samples qualified in this study were recombinant monoclonal antibodies and hybridoma cell culture supernatants. Initially, the lot-to-lot variation in background ATP of these samples posed significant challenges. Two strategies to increase the signal-to-noise ratio (positive result/background ATP) were evaluated: enzyme-based signal amplification and reduction of the broth-based noise through broth selection. Following qualification of the RMM for antibody and cell culture samples, the RMM was also utilized for rapid screening of several sources of purified water. This ATP-based RMM has proved invaluable in routine testing of diverse biologics samples at our discovery research site and plays a key role in the investigation of contaminated samples.LAY ABSTRACT: Biologics research laboratories routinely conduct sterility testing of products in development. However, the lengthy turnaround time for detection of microbial contaminants when using a conventional sterility test is a bottleneck in this fast-paced environment. This study investigated an adenosine triphosphate-based bioluminescence rapid microbial method (RMM) for biologics samples, including monoclonal antibodies and hybridoma cell cultures. The results showed that the RMM allowed detection of antibody sample contaminants after only three days of incubation. In addition to being faster than the standard method, the RMM proved more reliable in detecting contaminants in cell culture samples with antibiotics. Since its initial evaluation, this RMM has accelerated biologics sterility testing across multiple projects at our site.

Tryptone-stabilized gold nanoparticles target tubulin and inhibit cell viability by inducing an unusual form of cell cycle arrest.

Gold nanoparticles have been investigated extensively for their molecular mechanisms of action and anticancer potential. We report a novel, tubulin-targeted antiproliferative mechanism of action of tryptone-stabilized gold nanoparticles (TsAuNPs). TsAuNPs, synthesized using HAuCl4·3H2O and tryptone and characterized by a variety of spectroscopic methods and transmission electron microscopy, were found to be inhibitory to viability of human pancreatic (PANC-1), cervical (HeLa), and breast (MDA-MB-231) cancer cell lines in a concentration-dependent manner, with highest efficacy against PANC-1 cells. The particles strongly inhibited the clonogenic propagation of PANC-1 cells. TsAuNPs-mediated inhibition of cell viability involved an unusual mode of cell cycle arrest (arrest at both G0/G1 phase and S-phase) followed by apoptosis. In vitro, TsAuNPs bound purified tubulin, competitively inhibited anilinonaphthalene sulfonate binding to tubulin, and suppressed tubulin assembly. In cells, tubulin-TsAuNPs interactions were manifested as a disrupted microtubule network, defective reassembly of cold-disassembled microtubules, and induction of tubulin acetylation. Our data indicate that TsAuNPs inhibit cell viability by inducing differential cell cycle arrest possibly through disrupted dynamicity of cellular microtubules.

Increasing Growth Yield and Decreasing Acetylation in Escherichia coli by Optimizing the Carbon-to-Magnesium Ratio in Peptide-Based Media.

Complex media are routinely used to cultivate diverse bacteria. However, this complexity can obscure the factors that govern cell growth. While studying protein acetylation in buffered tryptone broth supplemented with glucose (TB7-glucose), we observed that Escherichia coli did not fully consume glucose prior to stationary phase. However, when we supplemented this medium with magnesium, the glucose was completely consumed during exponential growth, with concomitant increases in cell number and biomass but reduced cell size. Similar results were observed with other sugars and other peptide-based media, including lysogeny broth. Magnesium also limited cell growth for Vibrio fischeri and Bacillus subtilis in TB7-glucose. Finally, magnesium supplementation reduced protein acetylation. Based on these results, we conclude that growth in peptide-based media is magnesium limited. We further conclude that magnesium supplementation can be used to tune protein acetylation without genetic manipulation. These results have the potential to reduce potentially deleterious acetylated isoforms of recombinant proteins without negatively affecting cell growth.IMPORTANCE Bacteria are often grown in complex media. These media are thought to provide the nutrients necessary to grow bacteria to high cell densities. In this work, we found that peptide-based media containing a sugar are magnesium limited for bacterial growth. In particular, magnesium supplementation is necessary for the bacteria to use the sugar for cell growth. Interestingly, in the absence of magnesium supplementation, the bacteria still consume the sugar. However, rather than use it for cell growth, the bacteria instead use the sugar to acetylate lysines on proteins. As lysine acetylation may alter the activity of proteins, this work demonstrates how lysine acetylation can be tuned through magnesium supplementation. These findings may be useful for recombinant protein production, when acetylated isoforms are to be avoided. They also demonstrate how to increase bacterial growth in complex media.

Impact of phosphate and other medium components on physiological regulation of bacterial laccase production.

Laccases are multicopper oxidases known to catalyze the transformation of a wide range of phenolic and non-phenolic substrates using oxygen as electron acceptor and forming water as the only by product. Their potential relevance in several industries requires the constant search for novel laccases. Positive outcome of the isolation of laccase producing bacteria depends on the nature and concentration of media constituents. Several attempts to isolate laccase producing bacteria failed when the phosphate-containing M9 minimal medium was used. Shift to phosphate-less M162 medium led to successful isolations. Seven bacterial isolates belonging to genera Bacillus, Lysinibacillus, Bhargavaea and Rheinheimera were used to study the effect of medium constituents on laccase production. Inorganic phosphate (≥50 mM) was found to regulate laccase synthesis negatively though no inhibitory effect of phosphate (10-500 mM) was seen on laccase activity. All isolates ceased laccase synthesis when grown in the presence of tryptone (0.2-1%), with R. tangshanensis as an exception, or yeast extract (1.5-2%) as the only C/N source in M162 medium. Supplementation upto 0.1% of glucose in basal M162 medium increased laccase production in five isolates but decreased at higher concentrations. The influence of medium components on laccase synthesis was further affirmed by zymographic studies. These observations offer possibilities of isolating promising laccase producers from diverse environmental sources. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:541-548, 2017.

Photoactivated disinfection (PAD) of dental root canal system - An ex-vivo study.

AIM: To investigate the efficacy of photo activated disinfection (PAD) in reducing colony-forming unit (CFU) counts of Enterococcus faecalis (E. faecalis) in infected dental root canals. The study compared the efficacy of PAD with conventional endodontic treatment (CET) and also a combination of CET along with PAD. MATERIAL AND METHODS: 53 maxillary incisors were taken for the study. Teeth were divided into 3 groups, CET (Group I) (n = 11), PAD (Group II) (n = 21), and a combination of CET and PAD (Group III) which consisted of (n = 21) samples, Group II and Group III were further divided into 2 subgroups, Group IIa, IIb and Group IIIa, IIIb. Strains of E. faecalis were inoculated in all the root canals. CET group samples were treated by chemo-mechanical preparation (CMP) alone, PAD samples were treated with laser alone at 2 different exposure time (4 min and 2 min). In the combination treatment, samples were treated initially by CET and then by PAD for a time period of 4 min and 2 min. Contents of the root canal were aspirated, diluted and plated in Tryptone Soya Broth (TSB) and plates were incubated for 24 h to observe the bacterial regrowth. RESULTS: Showed PAD used along with CMP reduced the bacterial load of E. faecalis by 99.5% at 4 min and 98.89% at 2 min. CONCLUSION: PAD may be an adjunctive procedure to kill residual bacteria in the dental root canal systems after standard endodontic root canal preparation.

Employing Tryptone as a General Phase Transfer Agent to Produce Renal Clearable Nanodots for Bioimaging.

Hydrophobic ultrasmall nanoparticles synthesized in nonpolar solvents exhibit great potential in biomedical applications. However, a major challenge when applying these nanomaterials in biomedical research is the lack of a versatile strategy to render them water dispersible while preserving the hydrodynamic diameter (HD) to be less than 8 nm for efficient renal clearance. To address this problem, tryptone is employed as the novel ligand to fabricate a simple, versatile, and inexpensive strategy for transferring hydrophobic NaGdF(4) nanodots (3 nm in diameter) from organic phase into aqueous phase without any complicated organic synthesis. The paramagnetic properties of NaGdF(4) nanodots are well retained after the phase transfer process. In particular, the tryptone-NaGdF(4) nanodots have ultrasmall HD (ca., 7.5 nm), which greatly improves their tumor accumulation and facilitates renal clearance within 24 h postinjection. The as-prepared tryptone-NaGdF(4) nanodots can also be further functionalized with other molecules for extensively biomedical and bioanalytical applications. Furthermore, the proposed strategy can easily be extended to transfer other types of inorganic nanoparticles from hydrophobic to hydrophilic for facilitating biomedical applications.

Development and validation of a liquid chromatography-mass spectrometry assay for polymyxin B in bacterial growth media.

There is increasing interest in the optimization of polymyxin B dosing regimens to treat infections caused by multidrug-resistant Gram-negative bacteria. We aimed to develop and validate a liquid chromatography-single quadrupole mass spectrometry (LC-MS) method to quantify polymyxin B in two growth media commonly used in in vitro pharmacodynamic studies, cation-adjusted Mueller-Hinton and tryptone soya broth. Samples were pre-treated with sodium hydroxide (1.0M) and formic acid in acetonitrile (1:100, v/v) before analysis. The summed peak areas of polymyxin B1 and B2 relative to the summed peak areas of colistin A and B (internal standard) were used to quantify polymyxin B. Quality control samples were prepared and analyzed to assess the intra- and inter-day accuracy and precision. The robustness of the assay in the presence of bacteria and commonly co-administered antibiotics (rifampicin, doripenem, imipenem, cefepime and tigecycline) was also examined. Chromatographic separation was achieved with retention times of approximately 9.7min for polymyxin B2 and 10.4min for polymyxin B1. Calibration curves were linear between 0.103 and 6.60mg/L. Accuracy (% relative error) and precision (% coefficient of variation), pooled for all assay days and matrices (n=84), were -6.85% (8.17%) at 0.248mg/L, 1.73% (6.15%) at 2.48mg/L and 1.54% (5.49%) at 4.95mg/L, and within acceptable ranges at all concentrations examined. Further, the presence of high bacterial concentrations or of commonly co-administered antibiotics in the samples did not affect the assay. The accuracy, precision and cost-efficiency of the assay make it ideally suited to quantifying polymyxin B in samples from in vitro pharmacodynamic models.

Differential gene expression in Giardia lamblia under oxidative stress: significance in eukaryotic evolution.

Giardia lamblia is a unicellular, early branching eukaryote causing giardiasis, one of the most common human enteric diseases. Giardia, a microaerophilic protozoan parasite has to build up mechanisms to protect themselves against oxidative stress within the human gut (oxygen concentration 60 µM) to establish its pathogenesis. G. lamblia is devoid of the conventional mechanisms of the oxidative stress management system, including superoxide dismutase, catalase, peroxidase, and glutathione cycling, which are present in most eukaryotes. NADH oxidase is a major component of the electron transport chain of G. lamblia, which in concurrence with disulfide reductase, protects oxygen-labile proteins such as pyruvate: ferredoxin oxidoreductase against oxidative stress by sustaining a reduced intracellular environment. It also contains the arginine dihydrolase pathway, which occurs in a number of anaerobic prokaryotes, includes substrate level phosphorylation and adequately active to make a major contribution to ATP production. To study differential gene expression under three types of oxidative stress, a Giardia genomic DNA array was constructed and hybridized with labeled cDNA of cells with or without stress. The transcriptomic data has been analyzed and further validated using real time PCR. We identified that out of 9216 genes represented on the array, more than 200 genes encoded proteins with functions in metabolism, oxidative stress management, signaling, reproduction and cell division, programmed cell death and cytoskeleton. We recognized genes modulated by at least ≥ 2 fold at a significant time point in response to oxidative stress. The study has highlighted the genes that are differentially expressed during the three experimental conditions which regulate the stress management pathway differently to achieve redox homeostasis. Identification of some unique genes in oxidative stress regulation may help in new drug designing for this common enteric parasite prone to drug resistance. Additionally, these data suggest the major role of this early divergent ancient eukaryote in anaerobic to aerobic organism evolution.

Helicobacter pylori inactivation and virulence gene damage using a supported sensitiser for photodynamic therapy.

About half of the world's population is currently infected with Helicobacter pylori, which is involved in the development of several gastro-duodenal pathologies. The increasing number of antibiotic resistance reduces the effectiveness of the first-line therapy, so new strategies to improve the H. pylori eradication rates are needed. Antimicrobial Photodynamic Therapy (APDT) benefits from photogenerated reactive oxygen species, such as singlet oxygen, which inactivate microorganisms by means of photosensitising dyes and visible light. Therefore, it could be a suitable alternative for H. pylori eradication in the gastro-duodenal tract, particularly in patients infected with antibiotic resistant strains. We evaluated APDT against H. pylori, in vitro, using a new photosensitising material (PSM) based on a ruthenium(II) complex covalently bound to micrometric glass beads. Five H. pylori isolates (classified according to cagA genotype, and metronidazole-clarithromycin resistance) were used. Bacteria were mixed with the PSM and incubated in the dark or illuminated by blue light. Aliquots (min 1', 2', 5', 15' and 30') were cultured and colonies were counted after 2-3 days. A 99.99999% decrease was detected in the number of colonies in the irradiated wells where the bacterium was mixed with the PSM, compared to non-illuminated wells or with irradiated wells without PSM. It was also confirmed that DNA is a molecular target for oxidant species released during APDT (evaluated by alkaline gel electrophoresis after endonuclease III incubation, ureC and cagA RT-PCR, and bacterial fingerprint). Results were independent of cagA gene and antibiotic resistances.

Plants traditionally used individually and in combination to treat sexually transmitted infections in northern Maputaland, South Africa: antimicrobial activity and cytotoxicity.

ETHNOPHARMACOLOGICAL RELEVANCE: Although medicinal plants are used extensively to treat sexually transmitted infections (STIs) in rural northern Maputaland, KwaZulu-Natal, the efficacy and safety of these plants have not previously been evaluated. AIM OF STUDY: A study was designed to investigate the in vitro antimicrobial activity and cytotoxicity profiles of a selection (individual plants and selected combinations) of traditionally used plants in this study area. MATERIALS AND METHODS: Aqueous and organic (dichloromethane: methanol, 1:1) extracts were prepared. Antimicrobial activity was assessed using the minimum inhibitory concentration (MIC) assay against the STI associated pathogens; Candida albicans ATCC 10231, Ureaplasma urealyticum clinical strain, Oligella ureolytica ATCC 43534, Trichomonas vaginalis clinical strain, Gardnerella vaginalis ATCC 14018 and Neisseria gonorrhoeae ATCC 19424. For the combination study, interactions were assessed using the fractional inhibitory concentration (ΣFIC). The plant species were assessed for safety using the 3-[4,5-dimethyl-2-thiazol-yl]-2,5-diphenyl-2H-tetrazolium bromide (MTT) cellular viability assay on the human embryonic kidney epithelial (Graham, HEK-293) cell line. RESULTS: For the antimicrobial studies, U. urealyticum was the most sensitive of the six test organisms, with the aqueous extract of Ranunculus multifidus (0.02mg/ml) and the organic extract of Peltophorum africanum (0.04mg/ml) being the most antimicrobially active plant species studied. Sclerocarya birrea was found to have the broadest spectrum of activity (mean MIC of 0.89mg/ml). The only plant species to exhibit some degree of cytotoxicity against the kidney epithelial cell line was Kigelia africana (100µg/ml), with 22% and 16% cell death for the aqueous and organic extracts, respectively. Of the 13 combinations studied, several synergistic combinations were evident, the most prominent being the combination of Albizia adianthifolia and Trichilia dregeana (aqueous extract) with an ΣFIC value of 0.15 against O. ureolytica. Synergistic interactions were observed regardless of the ratio of the aqueous mixtures of the two plants. Syzygium cordatum and S. birrea (aqueous extract) was also a combination of interest, demonstrating synergistic (ΣFIC=0.42) interactions against O. ureolytica. This combination, however, also displayed some cytotoxicity towards the human epithelial cell line. CONCLUSION: This study demonstrated that anecdotal evidence of plant use does not always correlate with in vitro activity. Furthermore, the toxicological profiling is of utmost importance as if not combined in its correct ratio can lead to potential adverse effects.