Multiple antibiotic resistances in metal tolerant E. coli from hospital waste water

Study of antibiotic resistance was done among the metal tolerant E. coli isolates from hospital wastewater at Lucknow city. Metal tolerance was determined in terms of visible growth on metal amended plates at their varying concentrations. MICs were also determined among all metal tolerant E. coli isolates. All the isolates showed their MIC in between 100-2000 µg/ml while maximum isolates demonstrated their MICs at 400, 800 and 1600 µg/ml against all the metal tested. 23.07% of the isolates showed their MIC at 2000 µg/ml against Ni³⁺. Multiple antibiotic resistances were recorded among all the metal resistant E.coli isolates. A high level of resistance was observed against Methicillin (86.53%) followed by penicillin (73.07%), Cephradin (57.69%), Rifampicin (34.61%), Erythromycin (26.92%), Nalidixic acids (25%), Chloramphenicol (3.84%) and least to Gentamycine (1.92%). Streptomycin was recorded most effective against E.coli isolates among the entire antibiotic tested. Antimicrobial resistance observed among the bacteria from the aquatic system contaminated with hospital wastes may be threatful for the environment and public health both.     

The Rationale for Using Rifabutin in the Treatment of MDR and XDR Tuberculosis Outbreaks

Genetically related Mycobacterium tuberculosis strains with alterations at codon 516 in the rpoB gene were observed amongst a substantial number of patients with drug resistant tuberculosis in the Eastern Cape Province (ECP) of South Africa. Mutations at codon 516 are usually associated with lower level rifampicin (RIF) resistance, while susceptibility to rifabutin (RFB) remains intact. This study was conducted to assess the rationale for using RFB as a substitution for RIF in the treatment of MDR and XDR tuberculosis outbreaks. Minimum inhibitory concentrations (MICs) of 34 drug resistant clinical isolates of M tuberculosis were determined by MGIT 960 and correlated with rpoB mutations. RFB MICs ranged from 0.125 to 0.25 µg/ml in the 34 test isolates thereby confirming phenotypic susceptibility as per critical concentration (CC) of 0.5 µg/ml. The corresponding RIF MICs ranged between 5 and 15 µg/ml, which is well above the CC of 1.0 µg/ml. Molecular-based drug susceptibility testing provides important pharmacogenetic insight by demonstrating a direct correlation between defined rpoB mutation and the level of RFB susceptibility. We suggest that isolates with marginally reduced susceptibility as compared to the epidemiological cut-off for wild-type strains (0.064 µg/ml), but lower than the current CC (≤0.5 µg/ml), are categorised as intermediate. Two breakpoints (0.064 µg/ml and 0.5 µg/ml) are recommended to distinguish between susceptible, intermediate and RFB resistant strains. This concept may assist clinicians and policy makers to make objective therapeutic decisions, especially in situations where therapeutic options are limited. The use of RFB in the ECP may improve therapeutic success and consequently minimise the risk of ongoing transmission of drug resistant M. tuberculosis strains.     

How do combinations of rpsL- and miaA- generate streptomycin dependence?

Summary Petrullo et al. (1983) have studied the consequences of combining a mutation (rpsL ^–) that normally generates streptomycin resistant (Sm^r) ribosomes with a mutation (miaA ^–) that leads to loss of a tRNA hypermodification. They found surprisingly that such doubly mutant bacteria become streptomycin dependent (Sm^d). Here, we show in vitro that ribosomes purified from an Sm^r mutant behave very like Sm^d ribosomes when they are combined with tRNA from an miaA ^– mutant. Our analysis suggests that proofreading becomes excessively intense when the mutant components are combined, and that this reduces the efficiency of translation to the very low levels characteristic of Sm^d ribosomes. We show that Sm increases the efficiency of translation in vitro by suppressing the proofreading flows. We suggest that this will explain the growth stimulatory effect of Sm on the rpsL ^–, miaA ^–double mutants.     

Bacterioplankton in Antarctic Ocean Waters During Late Austral Winter: Abundance, Frequency of Dividing Cells, and Estimates of Production

Bacterioplankton productivity in Antarctic waters of the eastern South Pacific Ocean and Drake Passage was estimated by direct counts and frequency of dividing cells (FDC). Total bacterioplankton assemblages were enumerated by epifluorescent microscopy. The experimentally determined relationship between in situ FDC and the potential instantaneous growth rate constant (μ) is best described by the regression equation ln μ = 0.081 FDC − 3.73. In the eastern South Pacific Ocean, bacterioplankton abundance (2 × 10⁵ to 3.5 × 10⁵ cells per ml) and FDC (11%) were highest at the Polar Front (Antarctic Convergence). North of the Subantarctic Front, abundance and FDC were between 1 × 10⁵ to 2 × 10⁵ cells per ml and 3 to 5%, respectively, and were vertically homogeneous to a depth of 600 m. In Drake Passage, abundance (10 × 10⁵ cells per ml) and FDC (16%) were highest in waters south of the Polar Front and near the sea ice. Subantarctic waters in Drake Passage contained 4 × 10⁵ cells per ml with 4 to 5% FDC. Instantaneous growth rate constants ranged between 0.029 and 0.088 h⁻¹. Using estimates of potential μ and measured standing stocks, we estimated productivity to range from 0.62 μg of C per liter · day in the eastern South Pacific Ocean to 17.1 μg of C per liter · day in the Drake Passage near the sea ice.     

Chromosomal Gene Inactivation in the Green Sulfur Bacterium Chlorobium tepidum by Natural Transformation

Conditions for inactivating chromosomal genes of Chlorobium tepidum by natural transformation and homologous recombination were established. As a model, mutants unable to perform nitrogen fixation were constructed by interrupting nifD with various antibiotic resistance markers. Growth of wild-type C. tepidum at 40°C on agar plates could be completely inhibited by 100 μg of gentamicin ml⁻¹, 2 μg of erythromycin ml⁻¹, 30 μg of chloramphenicol ml⁻¹, or 1 μg of tetracycline ml⁻¹ or a combination of 300 μg of streptomycin ml⁻¹ and 150 μg of spectinomycin ml⁻¹. Transformation was performed by spotting cells and DNA on an agar plate for 10 to 20 h. Transformation frequencies on the order of 10⁻⁷ were observed with gentamicin and erythromycin markers, and transformation frequencies on the order of 10⁻³ were observed with a streptomycin-spectinomycin marker. The frequency of spontaneous mutants resistant to gentamicin, erythromycin, or spectinomycin-streptomycin was undetectable or significantly lower than the transformation frequency. Transformation with the gentamicin marker was observed when the transforming DNA contained 1 or 3 kb of total homologous flanking sequence but not when the transforming DNA contained only 0.3 kb of homologous sequence. Linearized plasmids transformed at least an order of magnitude better than circular plasmids. This work forms a foundation for the systematic targeted inactivation of genes in C. tepidum, whose 2.15-Mb genome has recently been completely sequenced.     

Conformation dependent binding of netropsin and distamycin to DNA and DNA model polymers

The binding of the antibiotics netropsin and distamycin A to DNA has been studied by thermal melting, CD and sedimentation analysis. Netropsin binds strongly at antibiotic/nucleotide ratios up to at least 0.05. CD spectra obtained using DNA model polymers reveal that netropsin binds tightly to poly (dA) · poly (dT), poly (dA-dT) · poly(dA-dT) and poly (dI-dC) · poly (dI-dC) but poorly, if at all, to poly (dG) · poly (dC). Binding curves obtained with calf thymus DNA reveal one netropsin-binding site per 6.0 nucleotides (K_a=2.9 · 10⁵ M⁻¹); corresponding values for distamycin A are one site per 6.1 nucleotides with K_a= 11.6 · 10⁵ M⁻¹. Binding sites apparently involve predominantly A·T-rich sequences whose specific conformation determines their high affinity for the two antibiotics. It is suggested that the binding is stabilized primarily by hydrogen bonding and electrostatic interactions probably in the narrow groove of the DNA helix, but without intercalation. Any local structural deformation of the helix does not involve unwinding greater than approximately 3° per bound antibiotic molecule.     

Production and Characterization of Amylase from Calvatia gigantea

α-Amylase (EC 3.2.1.1) was excreted by Calvatia gigantea in liquid growth media containing different sources of starch. Among the factors affecting enzyme production in shake flasks were the type and the concentration of starch and the nitrogen source supplied. Optimum cultural conditions for maximum enzyme production were: soluble starch concentration, 5%; inoculum size, 3.75 × 10⁵ conidia per ml; 5-day cultivation time at 28 to 30°C. The observed maximum yield of 81.3 U of saccharifying enzyme activity per ml of growth medium was the highest ever reported in the literature for submerged cultures. Partially purified enzyme functioned optimally at pH 4.5 to 5.5 and 53 to 58°C. The activation energy of enzymic hydrolysis of starch in the range of 20 to 40°C was 8,125 cal/mol (ca. 3.41 × 10⁴ J). The apparent K_m value of the enzyme at 25°C was 7.68 × 10⁻⁴ g/ml. Some of the properties of the enzyme under investigation were similar to those of α-amylases excreted from molds producing large amounts of the enzyme.     

Streptomycin resistance in Rhizobium japonicum

Mutants resistant to varying concentrations of streptomycin were recovered from two streptomycin-sensitive, effective nitrogen-fixing strains of Rhizobium japonicum. To determine if there were an upper limit of resistible antibiotic concentration, 3 mutants which were resistant to 10000 μg/ml were challenged by higher concentrations of streptomycin. Only one grew well at 25000 μg/ml, and none grew at 50000 μg/ml. All mutants maintained a smooth colonial morphology, and none exhibited streptomycin-dependence. Streptomycin-resistant mutants of both strains were examined for properties of infectivity and effectiveness. All mutants tested retained the symbiotic properties of the parental strains. The retention of these parental properties by the streptomycin-resistant mutants of R. japonicum is different from the properties described for phenotypically similar mutants in certain other rhizobial species.     

Alteration of ribosomal protein L6 in mutants of Escherichia coli resistant to gentamicin.

Summary Spontaneous and ethylmethane-sulfonate induced mutants of Escherichia coli resistant to gentamicin sulfate were isolated and investigated for alterations in the ribosomal protein pattern. It was found by two-dimensional polyacrylamide gel electrophoresis that three independently isolated strains did not show any spot for ribosomal protein L6. On cochromatography of radioactively labelled mutant and wild-type ribosomal proteins on carboxymethyl-cellulose columns a shift of the elution position of protein L6 was observed, the new elution positions being characteristic for the individual mutants analyzed which indicates that they possess different alterations in the L6 primary structure.Genetic analysis showed that the gentamicin resistant strains contain at least two mutations. One of them correlates with the altered L6 protein and causes an increased minimal inhibitory concentration of the drug by about 5 to 10-fold. The other mutation is not yet biochemically characterized. Its presence is connected with an about 10 to 20-fold increase in the resistance. Both mutations, when put together, confer resistance to 50 to 100 g/ml of the antibiotic in a low salt rich medium and to 1 mg/ml in a defined medium with a high concentration of phosphate. Cross-resistance analysis demonstrated that the three gentamicin-resistant (double-mutant) strains with the altered L6 protein are resistant to 50–100 g per ml of all other aminoglycoside antibioties tested. This forms a sharp contrast to the streptomycin resistance mutations present in strA1, strA40 or strA60 mutants which do not confer markedly increased levels of resistance to most of the other aminoglycosides.     

In vitro antioxidant capacity and free radical scavenging evaluation of active metabolite constituents of Newbouldia laevis ethanolic leaf extract

Background

The aim of the present study was to evaluate the in vitro antioxidant and free radical scavenging capacity of bioactive metabolites present in Newbouldia laevis leaf extract.

Results

Chromatographic and spectrophotometric methods were used in the study and modified where necessary in the study. Bioactivity of the extract was determined at 10 μg/ml, 50 μg/ml, 100 μg/ml, 200 μg/ml and 400 μg/ml concentrations expressed in % inhibition. The yield of the ethanolic leaf extract of N.laevis was 30.3 g (9.93%). Evaluation of bioactive metabolic constituents gave high levels of ascorbic acid (515.53 ± 12 IU/100 g [25.7 mg/100 g]), vitamin E (26.46 ± 1.08 IU/100 g), saponins (6.2 ± 0.10), alkaloids (2.20 ± 0.03), cardiac glycosides(1.48 ± 0.22), amino acids and steroids (8.01 ± 0.04) measured in mg/100 g dry weight; moderate levels of vitamin A (188.28 ± 6.19 IU/100 g), tannins (0.09 ± 0.30), terpenoids (3.42 ± 0.67); low level of flavonoids (1.01 ± 0.34 mg/100 g) and absence of cyanogenic glycosides, carboxylic acids and aldehydes/ketones. The extracts percentage inhibition of DPPH, hydroxyl radical (OH^.), superoxide anion (O₂^.-), iron chelating, nitric oxide radical (NO), peroxynitrite (ONOO⁻), singlet oxygen (¹O₂), hypochlorous acid (HOCl), lipid peroxidation (LPO) and FRAP showed a concentration-dependent antioxidant activity with no significant difference with the controls. Though, IC₅₀ of the extract showed significant difference only in singlet oxygen (¹O₂) and iron chelating activity when compared with the controls.

Conclusions

The extract is a potential source of antioxidants/free radical scavengers having important metabolites which maybe linked to its ethno-medicinal use.     

Studies on the congenitally goitrous sheep. The iodinated compounds of serum, and circulating thyroid-stimulating hormone

1. A group of normal and congenitally goitrous Merino sheep were investigated to identify the metabolic defect present in the abnormal animals. 2. Protein-bound iodine concentrations of serum from goitrous animals (average 5·7μg./100ml.) were higher than normal (average 4·2μg./100ml.; P 0·001), but the hormonal iodine measured as butanol-extractable ¹³¹I was low in the serum of goitrous (average 40·3% of protein-bound ¹³¹I) compared with that of normal (84·2%; P 0·02) sheep. The non-hormonal iodine of the serum of goitrous sheep appeared to include iodotyrosines and iodinated protein. 3. Starch-gel-electrophoretic separations of sera from normal and goitrous sheep after ¹³¹I injection (100–500μc) showed no qualitative differences in the radioactivity of protein components. No significant differences in thyroxine-binding in vitro by serum proteins of normal and goitrous sheep were observed. 4. The clearance rates of ¹³¹I-labelled iodotyrosines (t_½ 1·2–2·9hr.) and iodothyronines (t_½ 33·5–47·4hr.) were similar in normal and goitrous sheep. 5. The concentration of circulating thyroid-stimulating hormone was significantly higher (P<0·01 in three sheep, P<0·05 in one sheep) in goitrous sheep. 6. The congenital goitre appears to be due to compensatory hypertrophy of the gland resulting from an inability to synthesize an adequate supply of thyroid hormone.     

ELECTRON MICROSCOPIC EXAMINATION OF SUBCELLULAR FRACTIONS : II. Quantitative Analysis of the Mitochondrial Population Isolated from Rat Liver

Large granule fractions, containing about 80% of the cytochrome oxidase of the tissue, were isolated from rat liver and used to prepare thin pellicles of packed particles which were submitted to quantitative electron microscopic examination. Various parameters describing the mitochondrial population were determined by measuring the size and number of mitochondrial profiles in sections, and the ratio of the inner to the outer membrane area. The mean particle radius and volume were found to be respectively 0.38 µ and 0.29 µ³; the average areas per mitochondrion were 2 and 5 µ² for the outer and inner membranes respectively. On the basis of the cytochrome oxidase activity recovered in the particulate fractions, the results were extrapolated to the whole liver, and it was concluded that rat liver contains about 5.10¹¹ mitochondria per gram; this corresponds to a volume of 0.14 ml/g and to an area of 2.5 and 1 m²/g for the inner and outer membranes respectively. The validity and the accuracy of these determinations is discussed and the results are compared to the information which has been obtained by independent methods or by other investigators.     

Streptomycin Induced Stress Response in Salmonella enterica Serovar Typhimurium Shows Distinct Colony Scatter Signature

We investigated the streptomycin-induced stress response in Salmonella enterica serovars with a laser optical sensor, BARDOT (bacterial rapid detection using optical scattering technology). Initially, the top 20 S. enterica serovars were screened for their response to streptomycin at 100 μg/mL. All, but four S. enterica serovars were resistant to streptomycin. The MIC of streptomycin-sensitive serovars (Enteritidis, Muenchen, Mississippi, and Schwarzengrund) varied from 12.5 to 50 μg/mL, while streptomycin-resistant serovar (Typhimurium) from 125–250 μg/mL. Two streptomycin-sensitive serovars (Enteritidis and Mississippi) were grown on brain heart infusion (BHI) agar plates containing sub-inhibitory concentration of streptomycin (1.25–5 μg/mL) and a streptomycin-resistant serovar (Typhimurium) was grown on BHI containing 25–50 μg/mL of streptomycin and the colonies (1.2 ± 0.1 mm diameter) were scanned using BARDOT. Data show substantial qualitative and quantitative differences in the colony scatter patterns of Salmonella grown in the presence of streptomycin than the colonies grown in absence of antibiotic. Mass-spectrometry identified overexpression of chaperonin GroEL, which possibly contributed to the observed differences in the colony scatter patterns. Quantitative RT-PCR and immunoassay confirmed streptomycin-induced GroEL expression while, aminoglycoside adenylyltransferase (aadA), aminoglycoside efflux pump (aep), multidrug resistance subunit acrA, and ribosomal protein S12 (rpsL), involved in streptomycin resistance, were unaltered. The study highlights suitability of the BARDOT as a non-invasive, label-free tool for investigating stress response in Salmonella in conjunction with the molecular and immunoassay methods.     

The Sonic Hedgehog Signaling Pathway Induces Myopic Development by Activating Matrix Metalloproteinase (MMP)-2 in Guinea Pigs

Purpose

To investigate whether the Sonic hedgehog (Shh) signaling induces myopic development by increasing the expression of matrix metalloproteinase (MMP)-2 in guinea pigs.

Methods

A translucent diffuser was glued onto the right eye to induce form-deprivation myopia (FDM) in 10 guinea pigs. Four guinea pigs were served as a control group. The other 100 guinea pigs were subdivided into 5 groups (20 per group) and received a 10 µl intravitreal injection every 2 days for 4 times. Two groups were injected with 20 or 50 µg/ml Shh amino-terminal peptide (Shh-N) into the right eye and 0.1% bovine serum albumin into the other. FDM was induced in the right eyes of the three cyclopamine-treated groups and both eyes were injected with 50, 100, or 200 µg/ml cyclopamine. Retinoscopic refraction and eye dimensions were assessed on Day 14 of treatment. MMP-2 protein expression was determined in both scleras by western blotting.

Results

Both concentrations of Shh-N stimulated myopic development and axial growth as compared with control eyes. Myopia and axial elongation were significantly greater in the 50 µg/ml than in the 20 µg/ml Shh-N group (P<0.001 and P = 0.0019, respectively). All three doses of cyclopamine significantly attenuated myopic development compared with the FDM group (P<0.0001). Cyclopamine at 100 or 200 µg/ml significantly reduced axial elongation compared with the FDM group (P = 0.044 and P = 0.001, respectively). FDM-induced myopia and axial elongation were significantly greater in the 50 µg/ml than in the 200 µg/ml cyclopamine group (P<0.0001 and P = 0.008, respectively). MMP-2 expression was significantly greater in Shh-N–treated eyes than in the control eyes, and was lower in the cyclopamine plus FDM groups than in the FDM group.

Conclusions

The Shh signaling pathway induces myopic development by activating MMP-2 in guinea pigs.     

Requirement for Cell Dispersion Prior to Selection of Induced Azaguanine-Resistant Colonies of Chinese Hamster Cells

With V79 Chinese hamster cell cultures treated with a mutagen, the maximum frequency of colonies resistant to 8-azaguanine (AZG) was attained when the cells were dispersed after a suitable expression time before adding the selection medium. V79–4 cells were exposed to 500 µM MMS, 7 µM AFAA, or 10 µM MNNG and allowed to multiply before being reseeded at 4 x 10⁴ cells/60 mm dish and selected with 10 µg/ml AZG. Maximum frequencies of 4 x 10^-5, 4 x 10^-4, and 2.4 x 10^-3 were obtained about 100, 130, and 200 hrs after exposure to MMS, AFAA, and MNNG, respectively. The maximum frequencies following MMS or MNNG treatments were about 10-fold greater than those obtained when induction and selection of AZG-resistant colonies were performed in the same culture dish. The reseeding of treated cells eliminated the possibility of metabolic cooperation within mosaic colonies of wild-type and mutant cells and achieved expression of the induced changes before intercolony crossfeeding reduced the frequency of resistant colonies.—AZG-resistant colonies were selected in medium containing dialyzed fetal bovine serum, and the selection medium was replaced at least twice. Both serum dialysis and selection medium replacement were necessary for consistent achievement of background frequencies of resistant colonies near 10^-6. Reconstruction experiments with AZG-resistant V79 lines showed that the efficiency of recovery of resistant cells in the selection medium was constant over a range of 0–20 colonies observed/dish. A mixed population of V79 and AZG-resistant cells was also correctly analyzed by the procedure used in mutagenesis studies.     

220D-F2 from Rubus ulmifolius Kills Streptococcus pneumoniae Planktonic Cells and Pneumococcal Biofilms

Streptococcus pneumoniae (pneumococcus) forms organized biofilms to persist in the human nasopharynx. This persistence allows the pneumococcus to produce severe diseases such as pneumonia, otitis media, bacteremia and meningitis that kill nearly a million children every year. While bacteremia and meningitis are mediated by planktonic pneumococci, biofilm structures are present during pneumonia and otitis media. The global emergence of S. pneumoniae strains resistant to most commonly prescribed antibiotics warrants further discovery of alternative therapeutics. The present study assessed the antimicrobial potential of a plant extract, 220D-F2, rich in ellagic acid, and ellagic acid derivatives, against S. pneumoniae planktonic cells and biofilm structures. Our studies first demonstrate that, when inoculated together with planktonic cultures, 220D-F2 inhibited the formation of pneumococcal biofilms in a dose-dependent manner. As measured by bacterial counts and a LIVE/DEAD bacterial viability assay, 100 and 200 µg/ml of 220D-F2 had significant bactericidal activity against pneumococcal planktonic cultures as early as 3 h post-inoculation. Quantitative MIC’s, whether quantified by qPCR or dilution and plating, showed that 80 µg/ml of 220D-F2 completely eradicated overnight cultures of planktonic pneumococci, including antibiotic resistant strains. When preformed pneumococcal biofilms were challenged with 220D-F2, it significantly reduced the population of biofilms 3 h post-inoculation. Minimum biofilm inhibitory concentration (MBIC)₅₀ was obtained incubating biofilms with 100 µg/ml of 220D-F2 for 3 h and 6 h of incubation. 220D-F2 also significantly reduced the population of pneumococcal biofilms formed on human pharyngeal cells. Our results demonstrate potential therapeutic applications of 220D-F2 to both kill planktonic pneumococcal cells and disrupt pneumococcal biofilms.     

Analysis of Triclosan-Selected Salmonella enterica Mutants of Eight Serovars Revealed Increased Aminoglycoside Susceptibility and Reduced Growth Rates

The biocide triclosan (TRC) is used in a wide range of household, personal care, veterinary, industrial and medical products to control microbial growth. This extended use raises concerns about a possible association between the application of triclosan and the development of antibiotic resistance. In the present study we determined triclosan mutant prevention concentrations (MPC) for Salmonella enterica isolates of eight serovars and investigated selected mutants for their mechanisms mediating decreased susceptibility to triclosan. MPC_TRC values were 8 - 64-fold higher than MIC values and ranged between 1 - 16 µg/ml. The frequencies at which mutants were selected varied between 1.3 x 10^-10 - 9.9 x 10^-11. Even if MIC values of mutants decreased by 3-7 dilution steps in the presence of the efflux pump inhibitor Phe-Arg-β-naphtylamide, only minor changes were observed in the expression of genes encoding efflux components or regulators, indicating that neither the major multidrug efflux pump AcrAB-TolC nor AcrEF are up-regulated in triclosan-selected mutants. Nucleotide sequence comparisons confirmed the absence of alterations in the regulatory regions acrRA, soxRS, marORAB, acrSE and ramRA of selected mutants. Single bp and deduced Gly₉₃→Val amino acid exchanges were present in fabI, the target gene of triclosan, starting from a concentration of 1 µg/ml TRC used for MPC determinations. The fabI genes were up to 12.4-fold up-regulated. Complementation experiments confirmed the contribution of Gly₉₃→Val exchanges and fabI overexpression to decreased triclosan susceptibility. MIC values of mutants compared to parent strains were even equal or resulted in a more susceptible phenotype (1-2 dilution steps) for the aminoglycoside antibiotics kanamycin and gentamicin as well as for the biocide chlorhexidine. Growth rates of selected mutants were significantly lower and hence, might partly explain the rare occurrence of Salmonella field isolates exhibiting decreased susceptibility to triclosan.     

Production of Aseptic Spores of Vesicular-Arbuscular Endophytes and Their Viability After Chemical and Physical Stress

The survival of germinating spores of vesicular-arbuscular endophytes after treatments with oxidizing agents, antibiotics, moist heat, ultrasonic radiation, and ultraviolet radiation was compared with that of their contaminating microbes. Spores of three species were rapidly decontaminated by treatment with 0.42% (wt/vol) chlorine available from 5.0% (wt/vol) chloramine-T at 30°C for 20 to 40 min depending on the species and the soil from which they were extracted. This treatment did not change spore viability. The survival of spores was reduced by exposure for 20 min to 1.11% chlorine at 30°C for Glomus caledonius or at 35°C for Acaulospora laevis. Growth of any bacteria surviving treatment with oxidizing agents was inhibited by 100 μg of chloramphenicol per ml in agar; however, spore germination and germ tube growth were reduced only by concentrations greater than 200 μg/ml in agar. Spore germination was decreased by concentration of pimaracin, which controlled fungal growth. The spores survived moist heat at 40°C for 80 min, 55°C for 10 min, and 60°C for less than 1 min. The viability of spores was unaffected by ultrasonic irradiation for up to 4 min. Spores of G. caledonius and A. laevis were extremely resistant to ultraviolet radiation. Their viability was unaffected by exposure to 5 × 10⁸ ergs cm⁻² from an ultraviolet source of 253.7nm. The spores had very thick, pigmented walls, and the possibility that these provided some protection against the physical and chemical treatments is discussed. The degree of physiological damage to the spores caused by the treatments demonstrated some adverse effects of basic laboratory procedures. This information, together with that on the comparative sensitivity of contaminating microbes to the treatments, was used in the development of protocol for producing large numbers of uncontaminated spores.     

Reverse regulation of soluble receptor for advanced glycation end products and proinflammatory factor resistin and S100A12 in Kawasaki disease

Introduction

Kawasaki disease (KD), an acute febrile disease, characterized by systemic vasculitis, predominantly affects infants and children under 5 years of age. Coronary artery lesions (CALs) are its most critical complication, and the etiology remains unknown yet. In order to explore the value of resistin, S100A12 and soluble receptor for advanced glycation end products (sRAGE) in the pathophysiology of KD, we studied the serum levels of resistin, S100A12 and sRAGE in different stages of KD.

Methods

Serum levels of resistin, S100A12 and sRAGE were measured by enzyme-linked immunosorbent assay (ELISA) method in 15 healthy children and 40 KD patients at acute, afebrile and subacute stage.

Results

The resistin and S100A12 levels, including the ratio of resistin to sRAGE and S100A12 to sRAGE increased significantly in the acute stage, and decreased progressively in the afebrile and subacute stage. However, the sRAGE levels decreased significantly in the acute stage, and increased progressively in the afebrile and subacute stage. In the acute, afebrile and subacute stage, the resistin levels were higher in intravenous immunoglobulin (IVIG) non-responders (0.64 ± 0.30, 0.48 ± 0.35, 0.28 ± 0.19, × 10² ng/ml) than in IVIG responders (0.35 ± 0.24, 0.21 ± 0.19, 0.12 ± 0.05, × 10² ng/ml). In the acute and subacute stage, the S100A12 levels were higher in IVIG non-responders (7.92 ± 2.61, 4.98 ± 4.75, × 10² ng/ml) than in IVIG responders (5.05 ± 3.22, 2.35 ± 2.26, × 10² ng/ml). In the afebrile and subacute stage, the sRAGE levels were lower in IVIG non-responders (3.51 ± 2.64, 3.65 ± 3.27, × 10² pg/ml) than in IVIG responders (6.00 ± 2.78, 7.19 ± 2.88, × 10² pg/ml). The resistin levels were positively correlated with S100A12 levels. The sRAGE levels were negatively related with S100A12 and resistin levels.

Conclusions

Resistin, S100A12 and sRAGE are involved in the pathophysiology of KD.     

Probability Distribution of Drug-Resistant Mutants in Unselected Populations of Mycobacterium tuberculosis

The fluctuation test shows that Mycobacterium tuberculosis mutates to resistance to isoniazid, streptomycin, ethambutol and rifampin spontaneously and at random. The average mutation rates for the drugs, in the same order, were calculated to be 2.56 × 10⁻⁸, 2.95 × 10⁻⁸, 10⁻⁷, and 2.25 × 10⁻¹⁰ mutation per bacterium per generation. The relatively high mutation rate to ethambutol resistance and the low mutation rate to rifampin resistance were confirmed by analyzing the increase in the proportion of mutants with time in a growing population of the tubercle bacilli. The highest proportions of mutants to be expected in unselected populations of the tubercle bacilli were calculated from the results of fluctuation tests.