Bacterial Contamination of Tissue Allografts – Experiences of the Donor Tissue Bank of Victoria

The aim of this study is to report the experience of the Donor Tissue Bank of Victoria with bacteria isolated from musculoskeletal, skin and cardiac allografts retrieved from cadaveric donors. The results of all quality control samples for bacterial culture, taken during retrieval and processing of allografts at the DTBV for a 12 month period, were extracted and analysed. It was found that 15.7% of skin, 15.1% of heart valves and 5.8% of musculoskeletal samples had positive culture results. The number and types of organisms isolated varied with tissue type. The most commonly isolated organisms were Staphylococcus species (including S. aureus). The identity of the isolate and the number of positive specimens from the same donor were considerations in the decision concerning the suitability of tissue for subsequent implantation.     

Isolation and preservation of the living embryo sac ofCrinum asiaticum L. var.japonicum baker

The enzymatic maceration method was used to isolate an intact embryo sac ofCrinum asiaticum and its component cells. Best results were obtained when using enzyme solutions that contained pectinase hemicellulase, cellulase and pectolyase. Aseptic ovules were incubated in the enzyme solution for 1.5 hr at 25 C. This allowed the isolation of embryo sacs to yield up to 20% of the amount present. An isolated embryo sac usually consists of an egg cell, synergids, antipodals and a central cell. Some embryo sacs can be digested as gametophytic protoplast. The size, shape and position of the isolated embryo sac seemingly possessed similarities with those of the fixed embryo sac in the ovary. An isolated embryo sac can be in a living state when the result of the fluorochromatic reaction (FCR) and protoplasmic streaming is positive. When cultured in proper media, 68% of the isolated gametophytic protoplasts were observed to have sustained their positive FCR for more than 1 month.     

Mevinolin production by some fungi

The potentiality of twenty-five fungal species belonging to 14 genera isolated from Egyptian soils to produce mevinolin, a hypocholesterolemic agent, when grown on selected substrates was tested. For the first screening samples of culture filtrates were tested by TLC and the positive results were further estimated by HPLC analysis. It was found that nearly one-third of the tested fungi showed positive results as to production of mevinolin.Aspergillus terreus was distinguished by its capacity to produce mevinolin when cultivated on a selected medium. The maximum mevinolin yields were achieved after on 8-d incubation at 30°C. An initial pH value of 5–6 was found to be the optimum for growth ofA. terreus and mevinolin production.     

白及内生真菌多样性研究

白及( Bletilla striata)是兰科地生型多年生植物,也是我国传统中药材之一。利用菌根技术进行白及的保护和人工栽培,需要获得白及可培养的内生真菌。该研究以广西野生的白及根和叶为材料,采用分离培养法分离内生真菌,并结合真菌形态特征,及其核糖体的转录间隔区( ITS)序列分析,确定内生真菌的分类地位。结果表明：从2株白及植物90块组织中分离获得37株内生真菌,鉴定为15个分类单元,由9个属组成,分属于2门4纲7目8科,包括锤舌菌纲( Leotiomycetes)、座囊菌纲( Dothideomycetes)和粪壳菌纲( Sordariomy-cetes),伞菌纲( Agaricomycetes)。从根中分离获得内生真菌12种,蜡壳菌属为优势属;从叶中分离获得内生真菌3种,刺盘孢属为优势属;刺盘孢菌属( Colletotrichum)和蜡壳菌属( Sebacina)真菌的相对多度值均达到20％;4株担子菌均分布于根中,叶组织中未有分布。根组织中内生真菌的多样性指数(H＝1.863)高于叶组织(1.098)。该研究结果及其所分离培养的担子菌类真菌,为更好地利用菌根技术进行白及等兰科植物资源的保护与可持续利用奠定了基础。     

Application of biotinylated and32P probes for detection of P-fimbriae in urinaryE. coli

Escherichia coli is the common causative agent of urinary tract infections. Twenty-six strains ofEscherichia coli were isolated from children with pyelonephritis, symptomatic urinary tract infections and asymptomatic bacteriuria. Biotinylated and³²P-DNA probes were prepared for detection of P-fimbriae in the isolates. Of the 13 strains isolated from patients with pyelonephritis 11 were positive for the presence of the P gene by both probes. Strains isolated from cases of symptomatic urinary tract infections revealed the presence of P gene only in three samples of the total of nine isolated. None of the isolatedE. coli strains from asymptomatic bacteriuria was found positive for the presence of the P gene. The biotynylated probe was simple and easily applicable in standard laboratory conditions and therefore the authors recommend it for use in diagnostic laboratories.     

Spirochetal non-borrelia microorganism isolated fromIxodes ricinus

Spirochetal microorganisms were isolated from femaleIxodes ricinus in Slovakia. Morphological, immunochemical and molecular biological analysis showed that the microorganism shared several common antigens withBorrelia species while other genetic traits were distinct and not related toBorrelia burgdorferi sensu lato. Lyme disease patient’s serum contained antibodies reacting with antigens of this microorganism. On the one hand the cross-reacting antigens represent a risk of false positive results in laboratory diagnostics, while on the other hand they have a certain potential for vaccine development against Lyme disease.     

Survey of Campylobacter jejuni in stray cats in southern Italy

Aim: To evaluate the presence of thermotolerant Campylobacter spp. in stray cats in southern Italy. Methods and Results: One hundred and thirteen animals were trapped in two different environments (urban area, harbour area) of the city of Naples. From each cat, rectal swabs were collected. The samples were processed in order to detect thermotolerant Campylobacter spp. by culture methods. The positive samples were then confirmed by multiplex polymerase chain reaction. Campylobacter jejuni was isolated from 19/113 (16·8%) stray cats, whereas Campylobacter coli was not isolated. The cat data (age, environment and sex) were analysed by two statistical analyses using the C. jejuni status (positive/negative) as a dependent variable. As regards statistical regression model results, age and environment were risk factors for C. jejuni positivity. In particular, cats older than 1 year had a significantly higher risk of being positive for C. jejuni than cats aged up to 1 year (OR = 10·440; P = 0·000). Moreover, cats living in the harbour area had a significantly higher risk to be a carrier of C. jejuni than cats living in urban area (OR = 17·911; P = 0·008). Conclusion: The findings of the present survey confirm stray cats as potential carriers of C. jejuni. Significance and Impact of the Study: This is the first study on the prevalence of C. jejuni in stray cats in Europe.     

利用酵母双杂交技术筛选与生殖支原体黏附蛋白(MgPa)相互作用的宿主蛋白

从利用基于分离的泛素介导膜蛋白酵母双杂交系统构建的人尿道上皮细胞cDNA文库中筛选生殖支原体(Mg)黏附蛋白(MgPa)的互作蛋白。以pET30a-MgPa为模板,经PCR扩增MgPa基因,将其分别连接到pBT3STE和pBT3SUC载体以构建诱饵质粒pBT3STE-MgPa和pBT3SUC-MgPa;将诱饵质粒分别转化到酵母菌株NMY32内,检测其毒性和自激活活性;将人尿道上皮细胞cDNA文库质粒pPR3-N-207-Zeng转入到含pBT3SUC-MgPa诱饵质粒的酵母菌株中,筛选阳性克隆。检测阳性克隆LacZ报告基因的活性。提取阳性克隆质粒进行DNA测序与BLAST分析。结果显示,成功构建的诱饵质粒pBT3STE-MgPa和pBT3SUC-MgPa两者都没有自激活功能,且pBT3SUC-MgPa的活性强于pBT3STE-MgPa。用pBT3SUC-MgPa转化NMY32酵母作为受体酵母细胞。经DNA测序与BLAST分析结果表明筛选到的28个阳性克隆归属于23个不同的蛋白。从利用分离的泛素介导膜蛋白酵母双杂交系统构建的人尿道上皮细胞cDNA文库中筛选到23个与MgPa相互作用的蛋白,为阐明MgPa的功能及Mg可能的致病机制提供参考。     

Rapid Identification by Polymerase Chain Reaction of Staphylococcal Exfoliative Toxin Serotype A and B Genes

A new system was designed to detect staphylococcal exfoliative toxin A (ETA) and B (ETB) genes by the polymerase chain reaction (PCR). The primer pairs for the ETA gene (eta) were 20 and 20-mer, and its PCR product was a 741-bp eta fragment, while the primer pairs for the ETB gene (etb) were also 20 and 20-mer, and its PCR product was a 629-bp etb fragment. When these primers were simultaneously used in the PCR, the two types of ET were clearly detected as two bands in an ETA and ETB double-producer using only one colony within 3 hr. We examined 66 strains of Staphylococcus aureus isolated from patients with staphylococcal scalded skin syndrome (SSSS) and compared the results obtained by ELISA and PCR. The same results were obtained for 56 of the strains, i.e., 30 strains were ETA producers, 20 strains were ETB producers, and 6 strains were double-producers. However, positive results were obtained for 5 of the 10 non-ET-producing strains. Two of these strains were judged by PCR as ETA producers and three as ETB producers. Thus, PCR is very sensitive and rapid in detecting ETA and ETB gene fragments in colonies isolated from patients with SSSS.     

Serratia species isolated from plants

Using the selective caprylate-thallous agar medium, the presence ofSerratia species was systematically examined in 623 plant samples. A total of 167Serratia strains was isolated from these plant samples and identified to species and biogroups. Uniform and characteristicSerratia populations were found in figs and coconuts: (i)Serratia ficaria was recovered from most figs collected in California, Tunisia, and France; various biotypes ofS. marcescens also were found in figs; (ii) onlyS. marinorubra was recovered from coconuts bought on two continents. From plants other than figs and coconuts, representatives were isolated of all eightSerratia species we presently recognize—with a large preponderance ofS. liquefaciens andS. proteamaculans. These other plant samples fell into threeSerratia-prevalence groups: (i) vegetables-mushrooms-mosses-decaying plant material (53.8% of these samples were positive forSerratia); (ii) grasses (23.7% positive); and (iii) trees and shrubs-small plants (8.4% positive). PigmentedS. marcescens biotypes were rarely isolated from plants (except from figs). Of theS. marcescens biogroups most frequently encountered in nosocomial and iatrogenic infections of man, A3 and A4 were isolated from plants in this study, but A5/8 and TCT were not.     

Evaluation of chemical constituents and antinociceptive properties of Myricaria elegans Royle

The phytochemical screening of Myricaria elegans Royle (Tamaricaceae) gave strongly positive results for terpenes. A total of six triterpenes were isolated from the CHCl₃ fraction, including eleganene‐A, eleganene‐B, corsolic acid, betulin, ursolic acid, and erythrodiol. The in vivo antinociceptive investigation of the plant showed a significant increase in the tail‐flick latency, accompanied by mild sedation and severe ataxia. Considering the known activities of some of the compounds isolated from the plant, it may be hypothesized that the increase in the tail‐flick latency may be the combined effect of analgesia, ataxia, and sedation, rather than analgesia alone. These findings suggest M. elegans to be a potential source for activity‐guided isolation of important natural compounds with a variety of effects.     

PCR-based assay for the rapid detection of fumonisin-producing Fusarium species in maize-based animal and poultry feeds in Karnataka,India

Abstract

One hundred and fourteen animal feedstuffs and eighty poultry feed mixtures commonly used for animal and poultry nutrition in Karnataka, India, were analysed for Fusarium contamination. The total counts of fusaria in animal feeds and poultry feed mixtures revealed a high incidence of F. verticillioides, being isolated from all positive samples. Most contaminated samples were maize pellets (71.4%), cotton seed (66.6%), maize powder (60%) and fine wheat bran (50%), respectively, while no Fusarium species was isolated from Bengal gram husk and wheat flakes. All the Fusarium species were identified by the PCR method using genus specific ITS and group specific FUM 1 primers. Of the 374 Fusarium isolates tested with ITS set of primers, all fusaria scored positive, whereas only 244 (65%) isolates tested positive with the FUM 1 set of primers. The specificity of the primers provides the basis for a simple, accurate and precise detection of Fusarium species that represents fumonisin producers, which are a considerable risk for animal, poultry and human health.     

Diazotrophic bacilli isolated from the sunflower rhizosphere and the potential of Bacillus mycoides B38V as biofertiliser

The nitrogen fixation by strains belonging to the Bacillus genus remains poorly explored. In this work, the diversity of endospore‐forming bacilli isolated from the rhizosphere of sunflower was evaluated. A total of 101 strains were identified based on the V1‐V2 variable regions of the 16S rRNA gene. Strains belonging to the genera Bacillus and Paenibacillus represented 41.6% and 58.4%, respectively, of total isolates. The production of indolic compounds was a common trait among the isolates, and approximately 75% of them exhibited positive nitrogenase activity; but only 9.2% displayed activities higher than 1 nmol C₂H₄ mg protein h^?1. Within the genus Bacillus, the isolates related to the B. cereus group displayed the highest nitrogenase activity and were the second most frequent group of Bacillus sp. isolated. Plants inoculated with the isolate B38V showed the highest N content, and their shoot dry weights were significantly increased compared with positive control. Our results indicated that B38V, which belongs to the B. mycoides species, has the potential to promote sunflower growth. The data obtained in this study provide additional information concerning the diversity of Gram‐positive diazotrophic within the genera Bacillus and Paenibacillus and their potential for the biofertilisation of sunflower crops.     

Miconazole in the treatment of superficial mycoses

Miconazole nitrate (2%) cream was evaluated in the treatment of superficial mycoses. Out of 116 patients having multiple clinical diagnoses, 66 cases were found to be positive by culture. Species of Trichophyton were the predominant etiological agents (in over 60%) followed by Candida species (20%) and Epidermophyton floccosum (15%). All the cases selected for study were followed up to a period of 4–18 months. A cure rate of 94.6 per cent was observed in all the cases where causal organisms were isolated. Significantly high cure rate (66%) was also seen in cases where causal organisms could not be isolated, including cases of tinea versicolor. Results of mycological examination were in confirmity with the clinical results. Read at the Symposium on Human Mycoses and Miconazole held in New Delhi, India, February, 1975.     

Production of enterotoxin,verotoxin, hemolysin and cytotoxic necrotizing factor byEscherichia coli of intestinal and extraintestinal origin

Seventy-sixEscherichia coli strains were examined for heat-labile enterotoxin (LT), verotoxin (VT), hemolysin (HLy) and cytotoxic necrotizing factor (CNF). Thirty-six strains were isolated from patients suffering from diarrhea and forty from different extraintestinal infections. The number of LT-producing strains was low (2.6%) (one of intestinal and one of extraintestinal origin). Verotoxin was produced only by one extraintestinal strain. Four intestinal strains were hemolytic (11.2%) and also positive for CNF. From 24 hemolytic strains of extraintestinal origin (60%), 17 produced also CNF. Most of the hemolytic (30%) as well as CNF-producing strains (22.5%) were isolated from urine. Our results are similar to those of other studies confirming the close association between hemolysin and CNF production as well as a possible role of these toxic factors in pathogenesis of extraintestinal, infections caused byE. coli.     

Chemical and Immunological Characterization of a Low Molecular Weight Outer Membrane Protein of Salmonella typhi

A new immunogenic outer membrane protein, Omp-28 (MW 28,000 and pI 4.6), was isolated from smooth Salmonella typhi cells by the use of an extracting medium containing 6 m urea, 1% deoxycholate and 5 mM EDTA. The purification of Omp-28 was performed by gel filtration and fast ion exchange chromatography. This protein showed to be the prevalent component isolated by the latter methodology. Omp-28 is formed by three identical subunits (MW 9,000), not linked by disulfide bonds. The partial N-terminal amino acid sequence of Omp-28 presented great homology with part of the sequence of an Escherichia coli protein found in a precursor whose sequence was predicted by c-DNA. ELISA and Western blotting identified Omp-28 as the major antigenic protein present in the outer membrane protein fraction, isolated by gel filtration. Antibodies against Omp-28 were detected by ELISA in 43% of 28 sera from typhoid fever convalescent patients. The antisera from mice immunized with Omp-28 and the highest positive typhoid fever convalescent serum gave a positive bactericidal test, killing 50% of Salmonella typhi cells in serum dilutions of 1/80 and 1/320, respectively. These results indicate the immunogenic importance of Omp-28 isolated from Salmonella typhi outer membrane and strongly suggest it should be used in further studies of animal protection against the disease caused by this pathogenic bacteria.     

Chemical Composition,Antibacterial, Antioxidant and in Vitro Antidiabetic Activities of Essential Oils from Eruca vesicaria

This work describes the study of the chemical composition and bioactivity of the essential oils (EOs) of the different organs (leaves, flowers, stems and roots) from Eruca vesicaria. According to the GC and GC/MS analysis, all the EOs were dominated by erucin (4‐methylthiobutyl isothiocyanate) with a percentage ranging from 17.9 % (leaves) to 98.5 % (roots). The isolated EOs were evaluated for their antioxidant (DPPH, ABTS and β‐carotene/linoleic acid), antibacterial and inhibitory property against α‐amylase and α‐glucosidase. Most EOs exhibited an interesting α‐glucosidase and α‐amylase inhibitory potential. The roots essential oil was found to be the most active with IC₅₀ values of 0.80±0.06 and 0.11±0.01 μg mL^?1, respectively. The essential oil of roots exhibited the highest antioxidant activity (DPPH, PI=92.76±0.01 %; ABTS, PI=78.87±0.19; and β‐carotene, PI=56.1±0.01 %). The isolated oils were also tested for their antibacterial activity against two Gram‐positive and three Gram‐negative bacteria. Moderate results have been noted by comparison with Gentamicin used as positive control.     

Spatial Associations, Size–Distance Relationships and Population Structure of Two Dominant Life Forms in a Semiarid Enclave of the Venezuelan Andes

The role of three thorny legume species as nurse plants and competitive relationships with columnar cacti were evaluated in a semiarid enclave of the Venezuelan Andes. Abundance and size of three columnar cacti species (Stenocereus griseus, Cereus repandus and Pilosocereus tillianus) under isolated shrubs of three thorny legumes species (Prosopis juliflora, Acacia farnesiana and A. macracantha) were recorded and compared with open areas. Using size–distance data we inferred the intensity of intra- and interspecific competition between both life forms in a “cardonal” (xeric zone) and “espinar” (mesic zone) of the enclave. Sixty-one columnar cacti were found beneath the three thorny legume species (92%), while only 5 cactus individuals were found in open areas (8%). Comparison of observed and expected number of cactus individuals shows that S. griseus and C. repandus are significantly associated with isolated shrubs of A. farnesiana and A. macracantha. Although P. juliflora contributed more than 40% of the total legume plant cover, the number of columnar cacti under its isolated shrubs was significantly lower than would be expected by chance. In all, 19 of 21 possible plant–plant combinations between and within columnar cacti and legume shrubs were recorded (espinar: 18, cardonal: 13, common combinations: 12). In both zones, intra- and interspecific combinations among columnar cacti species were relatively high (positive correlation between the sum of neighbour plant sizes and the distances separating them). Our results strongly suggest, at least for case of S. griseus and C. repandus, that these columnar cacti species require nurse plants for their establishment. The results on interference need still support from further research. We discuss the effects of plant–plant positive interactions on natural regeneration of these columnar cacti.     

Molecular characterization of Vibrio parahaemolyticus of similar serovars isolated from sewage and clinical cases of diarrhoea in Calcutta,India

Vibrio parahaemolyticus is a seafood-borne halophilic pathogen that causes acute gastroenteritis in humans. During the course of an investigation on the incidence of V. parahaemolyticus in sewage water samples of Calcutta, India, we isolated eight (26.7%) strains of V. parahaemolyticus from 30 samples. Among these strains, five (62.5%) carried the thermostable direct hemolysin (tdh) gene, a major virulence marker of V. parahaemolyticus. Two strains belonged to serovar O5:K3 and the remaining three to O5:KUT, which is common among clinical strains of V. parahaemolyticus isolated from hospitalized patients of Calcutta with acute diarrhoea. The tdh positive sewage strains of V. parahaemolyticus were compared by randomly amplified polymorphic DNA (RAPD)-PCR and pulsed-field gel electrophoresis (PFGE) with strains of similar serovars selected from our culture collection to determine the genetic relatedness. Our results showed that except for sharing the similar serovar, sewage and clinical strains of V. parahaemolyticus were genetically different. In addition, toxRS-targeted group-specific (GS) PCR and open reading frame 8 (ORF-8) PCR showed that the sewage strains did not belong to the pandemic genotype. Since the sewage in Calcutta is directly used for cultivation of vegetables and for pisciculture, the presence of tdh positive V. parahaemolyticus in the sewage highlights the need for constant monitoring of the environment.