Comparison of coagulase-negative staphylococci by pulsed-field gel electrophoresis

Five pathogenic strains each of Staphylococcus epidermidis, S. haemolyticus, S. lugdunensis and S. schleiferi were analysed by conventional electrophoresis and field inversion gel electrophoresis. For these coagulase-negative staphylococci, the restriction endonuclease SmaI emerged as the most suitable enzyme for pulsed-field electrophoresis by providing an adequate number of clearly separated DNA fragments. Field inversion gel electrophoresis confirmed the differences among strains already discriminated by conventional electrophoresis, and furthermore, differentiated strains which had previously appeared identical. Among the species that were studied, S. epidermidis showed great genomic diversity with a few common bands. On the contrary, S. haemolyticus, S. lugdunensis and S. schleiferi showed less diversity. Although these minor variations may be epidemiologically significant, this question has to be investigated on a larger number of strains.     

2D-immunoblotting analysis of Sporothrix schenckii cell wall

We utilized two-dimensional gel electrophoresis and immunoblotting (2D-immunoblotting) with anti-Sporothrix schenckii antibodies to identify antigenic proteins in cell wall preparations obtained from the mycelial and yeast-like morphologies of the fungus. Results showed that a 70-kDa glycoprotein (Gp70) was the major antigen detected in the cell wall of both morphologies and that a 60-kDa glycoprotein was present only in yeast-like cells. In addition to the Gp70, the wall from filament cells showed four proteins with molecular weights of 48, 55, 66 and 67 kDa, some of which exhibited several isoforms. To our knowledge, this is the first 2D-immunoblotting analysis of the S. schenckii cell wall.     

Genotypic analysis of strains of mutans streptococci by pulsed-field gel electrophoresis

The species and serotypes of various strains of S. mutans and S. sobrinus were characterized by pulsed-field gel electrophoresis after the genomic DNA from the various strains had been digested with five restriction enzymes (EcoR I, Xba I, Hind III, Sfi I and BssH II) separately. Among these restriction enzymes, BssH II was very useful for the characterization of species and serotypes and, in particular, digestion discriminated between serotypes d and g. The restriction patterns obtained from the genomic DNA of isolates isolated from children's saliva were essentially identical to those from the genomic DNA of the standard laboratory strains. Patterns of BssH II digests of the genomic DNA of 10 isolates identified as S. sobrinus were characteristic of serotype g of the standard laboratory strains. Our results indicate that digestion with BssH II and subsequence analysis by pulsed-field gel electrophoresis should be useful for the characterization of species and serotypes and for epidemiological studies of mutans streptococci.     

Variants of Sporothrix schenckii with attenuated virulence for mice

Strains of Sporothrix schenckii preserved under mineral oil were examined for virulence in BALB/c mice. The mice were inoculated with S. schenckii conidia and development of cutaneous lesions, signs of inactivity, weight loss, survival rates, number of viable yeast cells in lung and spleen, splenomegaly and organ lesions were evaluated. After intravenous injection of 7.5 x 10(6) conidia, two of five S. schenckii strains were unable to induce systemic disease and to kill the mice, only producing cord-like lesions on the tail that regressed with mouse maturation. Very small numbers of viable cells isolated from the spleen confirmed the lower invasive ability of these strains when compared with other strains studied here. These results suggest a relationship between the attenuation of virulence and the storage method under mineral oil after long periods of time.     

Molecular heterogeneity of heat-stable toxin-producing and non-producing Escherichia coli O25 strains isolated in Japan

Escherichia coli O25 strains that produce heat-stable toxin (ST) have been recently isolated in Japan, and epidemiological study of this type of enterotoxigenic E. coli is required. In this study the heterogeneity of 16 ST-producing and non-producing strains of E. coli O25 was investigated. All eight ST-producing strains were shown to have STIb gene, and seven of them had similar profiles of plasmids, ladder-banding of LPS in SDS-polyacrylamide gel electrophoresis, and chromosomal DNA digestions in pulsed-field gel electrophoresis (PFGE). In contrast, ST-non-producing strains were more heterogeneous in all parameters examined. PFGE of the digested chromosomal DNA with several restriction enzymes was proved to be an effective procedure to compare the closely related strains of E. coli O25.     

Pulsed-field gel electrophoretic analysis and some characteristics of Staphylococcus aureus isolated from retail foods and human hands

This study investigates whether there is a predominant Staphylococcus aureus strain in retail foods and healthy human hands, and examines the relationship between pulsed-field gel electrophoresis (PFGE) banding patterns and the S. aureus characteristics of staphylococcal enterotoxin (SE) type, coagulase type, and β-lactamase activity. Ninety-four strains of S. aureus isolated from retail foods and healthy human hands were analyzed by PFGE. Several strains isolated from the same shop or a chain store showed identical patterns, indicating that the origins of these strains were identical. After excluding these strains showing identical patterns, 54 strains were used for the PFGE analysis. No spread of a particular clone in the environment surrounding the food was apparent. The PFGE analysis of these 54 strains was classified in 6 lineages (L1-L6). There was no relationship between the PFGE banding pattern and coagulase type or SE type. Eleven (84.6%) of the 13 isolates in PFGE banding pattern L5 did not produce β-lactamase, suggesting that the production of β-lactamase influenced a specific PFGE banding pattern.     

Genetic diversity of human isolates of Salmonella enterica serovar Enteritidis in Malaysia

AIMS: The study was undertaken to determine clonal relationship and genetic diversity of the human strains of Salmonella enterica serovar Enteritidis isolated from 1995 to 2002 from different parts of Malaysia. METHODS AND RESULTS: Antimicrobial susceptibility test, plasmid profiling and pulsed-field gel electrophoresis were applied to analyse 65 human isolates of S. Enteritidis obtained over an eight year period from different parts of Malaysia. Four nonhuman isolates were included for comparison. A total of 14 distinct XbaI-pulsed-field profiles (PFPs) were observed, although a single PFP X1 was predominant and this particular clone was found to be endemic in Malaysia. The incidence of drug resistant S. Enteritidis remained relatively low with only 37% of the strains analysed being resistant to one or more antimicrobial agents. All except one resistant strain carried at least one plasmid ranging in size from 3.7 to 62 MDa giving nine plasmid profiles. The three isolates from raw milk and one from well-water had similar PFPs to that of the human isolates. CONCLUSIONS: Salmonella Enteritidis strains were more diverse than was previously thought. Fourteen subtypes were noted although one predominant clone persisted in Malaysia. The combination of pulsed-field gel electrophoresis, plasmid profiling and antibiograms provided additional discrimination to the highly clonal strains of S. Enteritidis. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report to assess the genotypes of the predominant clinical S. Enteritidis in different parts of the country. As S. Enteritidis is highly endemic in Malaysia, the data generated would be useful for tracing the source during outbreaks of gastroenteritis in the study area.     

儿童孢子丝菌病的临床分析及致病菌的基因型检测

目的 对吉林省扶余县35例儿童孢子丝菌病进行临床分析并对部分菌株进行基因测定,了解该地区孢子丝菌病的致病菌菌株基因是否发生变异而致致病力增强及发病率增高.方法 收集2011年1月1日～4月30日来自扶余县就诊于我科并确诊为孢子丝菌病的患儿35例,进行临床分析;从中选取8株菌,对其核糖体保守区及内转录间隔区(ITS)、非转录间隔区(NTS)区进行基因测定,了解基因水平上的异同.结果 8株孢子丝菌菌株中5株NTS区可见较大范围的碱基缺失.结论 吉林省扶余县孢子丝菌菌株可能由于NTS区碱基较大范围缺失而致致病力增强,导致该地区短时间内发病率增高.     

Identification of lactoferrin-binding proteins in Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus agalactiae isolated from cows with mastitis

Three strains of Streptococcus dysgalactiae subsp. dysgalactiae (S. dysgalactiae) and five strains of Streptococcus agalactiae were used to identify lactoferrin-binding proteins (LBPs). LBPs from extracted surface proteins were detected by polyacrylamide gel electrophoresis and Western blotting. All strains of S. dysgalactiae evaluated had 52- and 74-kDa protein bands. All strains of S. agalactiae evaluated had 52-, 70- and 110-kDa protein bands. In addition, a 45-kDa band was detected in two of five S. agalactiae strains evaluated. This study demonstrated that S. dysgalactiae and S. agalactiae of bovine origin contain two and three major LBPs, respectively.     

Chromosomal Polymorphism in the Sporothrix schenckii Complex

Sporotrichosis is a polymorphic disease caused by a complex of thermodimorphic fungi including S. brasiliensis, S. schenckii sensu stricto (s. str.), S. globosa and S. luriei. Humans and animals can acquire the disease through traumatic inoculation of propagules into the subcutaneous tissue. Despite the importance of sporotrichosis as a disease that can take epidemic proportions there are just a few studies dealing with genetic polymorphisms and genomic architecture of these pathogens. The main objective of this study was to investigate chromosomal polymorphisms and genomic organization among different isolates in the S. schenckii complex. We used pulsed field gel electrophoresis (PFGE) to separate chromosomal fragments of isolated DNA, followed by probe hybridization. Nine loci (β-tubulin, calmodulin, catalase, chitin synthase 1, Internal Transcribed Spacer, Pho85 cyclin-dependent kinase, protein kinase C Ss-2, G protein α subunit and topoisomerase II) were mapped onto chromosomal bands of Brazilian isolates of S. schenckii s. str. and S. brasiliensis. Our results revealed the presence of intra and interspecies polymorphisms in chromosome number and size. The gene hybridization analysis showed that closely related species in phylogenetic analysis had similar genetic organizations, mostly due to identification of synteny groups in chromosomal bands of similar sizes. Our results bring new insights into the genetic diversity and genome organization among pathogenic species in the Sporothrix schenckii complex.     

Ecology of Sporothrix schenckii and of Ceratocystis stenoceras in Corsica and Alsace, French provinces free of sporotrichosis]

The study was conducted in 2 French Provinces free of sporotrichosis. Of 40 samples (soil, plant fragments, . . .) taken from Corsica, 59 isolations were obtained. All of these contained S. schenckii type of growth. Of 30 retained for further study, 14 were later identified as Ceratocystis stenoceras. All 14 needed pyrimidine as a growth factor. They developed well at 37 degrees and gave a yeast-like growth in shaken medium at 35 degrees and 25 degrees. One of the strains was pathogenic to mice. From 422 small mammals trapped in Alsace 37 isolations were made which morphologically were identified as S. schenckii. From these 31 were identified as C. stenoceras. Six isolations were considered as S. schenckii. The presence of nature of C. stenoceras and S. scheckii is discussed. Both fungi were isolated from various materials collected in tropical areas where sporotrichosis is endemic but also in such temperate areas as Corsica and Alsace where the mycosis is non-existent.     

西藏仲巴五彩沙漠放线菌资源勘探及生物活性筛选

【背景】细菌耐药性问题日益严峻,新抗生素的研发速度远远落后于临床需要,从特殊生境中挖掘微生物药物资源有望解决以上问题。【目的】勘探西藏仲巴五彩沙漠土壤放线菌多样性并进行生物活性筛选,为发现药用放线菌资源、开发新型抗生素奠定基础。【方法】采用8种分离培养基,通过平板稀释涂布法分离放线菌;根据分离菌株的16S r RNA基因序列同源性分析放线菌多样性;采用PCR技术对分离的放线菌菌株进行II型聚酮合酶(PKS-II)酮缩酶结构域KS、非核糖体多肽合成酶(NRPS)腺苷酸化结构域A、安莎类抗生素生物合成前体3-氨基-5-羟基-苯甲酸合酶(AHBA)保守区、黄素腺嘌呤二核苷酸卤化酶(Halo)保守区抗生素生物合成基因检测;对生物合成基因检测阳性的菌株进行液体发酵,发酵液经乙酸乙酯萃取、菌体经丙酮浸提,获得提取浓缩物样品进行抑菌活性和抗氧化活性筛选。【结果】从4份土样中分离纯化到231株放线菌,分布于7个属,其中链霉菌为优势菌属。68株放线菌的生物合成基因分析显示至少具有1种生物合成基因簇,其中6株同时具有4种生物合成基因簇;进一步的抑菌活性检测显示所有检测的菌株至少表现为对1株检定菌具有抑菌活性,其中8株具有广谱抗菌活性;抗氧化活性筛选结果为13株显示总抗氧化能力阳性,10株具有较好的羟自由基清除能力,3株显示较强的氧自由基清除能力。【结论】西藏仲巴五彩沙漠土壤中含有较丰富的放线菌药用资源,具有从中发现放线菌新菌种和开发新抗生素的潜力。     

Fatty acid composition of polar and neutral lipids of Sporothrix schenckii and Ceratocystis stenoceras]

The fatty acids contained in the neutral and polar lipids from Sprorthrix schenckii and Ceratocystis stenoceras and 3 mutants of the latter fungus were found to be identical. The major fatty acids were palmitic, oleic and linoleic. The fungi had similar quantitative composition especially the mutant strains and S. schenckii. This observation provides more data regarding the possible relationship between Ceratocystis and sporothrix.     

Polymorphism of Sporothrix schenckii surface polysaccharides as a function of morphological differentiation.

The alkali-extractable polysaccharides from different morphological types of two Sporothrix schenckii strains (1099.12 and 1099.18) were investigated. Dissociation of morphological phase transition and temperature effects was possible in a synthetic medium which produced cultures with 100% yeast forms either at 25 or at 37 degrees C. Only rhamnomannans with single-unit alpha-L-rhamnopyranosyl side chains were formed by the yeast forms irrespective of the incubation temperature. The higher temperature inhibited formation of 4-O- and 2,4-di-O-substituted alpha-D-mannopyranose units in the rhamnomannan. An apparently unsporulated mycelium culture of one S. schenckii strain (1099.12) synthesized a galactomannan whose structure was partially determined by methylation analysis and by proton and 13C nuclear magnetic resonance spectroscopy. In another strain (1099.18), a mannan was excreted in the medium of an apparently conidia-less mycelial form at 25 degrees C with short incubation. Its structure was also partially determined. An apparent mixture of this mannan and a rhamnomannan rich in alpha-L-rhamnopyranosyl-(1 leads to 2)-alpha-L-rhamnopyranose side chains formed in these cultures on prolonged incubation. The proportion of the excreted rhamnomannan increased as the mycelium sporulated and conidia were more numerous. Mannans or galactomannans may be transient polysaccharides in the young mycelium of S. schenckii. As the culture develops, rhamnomannans are formed in amounts usually masking the presence of other mannose-containing polysaccharides. It is suggested that in S. schenckii different polysaccharides are formed with side chains containing different proportions of rhamnose, mannose, or galactose, as a function of morphological differentiation.     

Decrease in ribosomal proteins 1, 2/3, L4, and L7 in Drosophila melanogaster in the absence of X rDNA

Summary The rRNA genes (rDNA) in Drosophila melanogaster are found in two clusters, one on the X and one on the Y chromosome. We have compared the ribosomal protein composition of wild-type Oregon-R flies containing both X-linked and Y-linked rDNA with that of flies containing only the Y-linked rDNA by two-dimensional polyacrylamide gel electrophoresis. Four basic proteins (1, 2/3, L4, and L7) normally present in wild-type flies were either electrophoretically not detectable (1, 2/3, and L4) or marginally detectable (L7) in flies with only Y-linked rDNA. No additional proteins were observed in these flies. However, immunodiffusion assays using specific antibodies raised against purified protein L4 confirmed that L4 was present but in relatively lower amounts in these Y-linked rDNA flies. An investigation was carried out to determine whether these electrophoretically undetectable proteins were more readily lost during ribosome preparation and hence were not readily detectable in the 80S particles by gel electrophoresis or whether they had been modified. Thus the proteins in the post-ribosomal cell supernatant and the high salt sucrose gradient were analyzed by two-dimensional gel electrophoresis and immunochemical assays with antibodies raised against protein L4 and total 80S ribosomal proteins. The experimental evidence indicates that there is a small amount of protein L4 and probably proteins 1, 2/3, and L7 in flies with only Y-linked rDNA but significantly less of these proteins than in wild-type flies.     

The expression of lipopolysaccharide by strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii and their cross-reacting strains of Escherichia coli

Strains of Shigella dysenteriae, Shigella flexneri and Shigella boydii express lipopolysaccharides, that enable the serotyping of strains based on their antigenic structures. Certain strains of S. dysenteriae, S. flexneri and S. boydii are known to share epitopes with strains of Escherichia coli ; however, the lipopolysaccharide profiles of the cross-reacting organisms have not been compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) lipopolysaccharides profiling. In the present study, type strains of these bacteria were examined using SDS-PAGE/silver staining to compare their respective lipopolysaccharide profiles. Strains of S. dysenteriae, S. boydii and S. flexneri all expressed long-chain lipopolysaccharide, with distinct profile patterns. The majority of strains of Shigella spp., known to cross-react with strains of E. coli , had lipopolysaccharide profiles quite distinct from the respective strain of E. coli . It was concluded that while cross-reacting strains of Shigella spp. and E. coli may express shared lipopolysaccharide epitopes, their lipopolysaccharide structures are not identical.     

Heterogeneity of the genes coding for 5 S RNA in three related strains of the genus Bacillus.

Summary Three related strains of the genus Bacillus, viz. B. licheniformis, B. subtilis and Bacillus Q were all found to contain a minor species of 5 S RNA in an amount indicating that it is transcribed from only one of the multiple 5 S RNA cistrons known to be present in these strains. The major and minor types of 5 S RNA from each of the three strains could be separated from each other by polyacrylamide gel electrophoresis in the presence of urea. The complete nucleotide sequences of the minor B. subtilis and Bacillus Q 5 S RNAs have been determined. Comparison of these sequences to the previously determined sequence of the minor 5 S RNA from B. licheniformis (Raué et al., 1976) showed the three minor types of 5 S RNA to be identical except for the residues at positions 79, 85 and 95. Moreover, seven out of the eight sequence differences between the major and the minor 5 S RNA are the same in all three strains. Thus, the single cistron coding for minor 5 S RNA has been strongly conserved in all three strains, which may indicate a biological significance for the minor 5 S RNA species.     

Chemical alterations in cell envelopes of polymyxin-resistant Pseudomonas aeruginosa isolates.

Cell envelopes from Pseudomonas aeruginosa strains resistant to polymyxin were compared with cell envelopes from polymyxin-sensitive strains as to their content of total protein, carbohydrate, and 2-keto-3-deoxyoctonate and as to their protein composition as determined by slab polyacrylamide gel electrophoresis. The cell envelopes of the polymyxin-resistant strains had reduced amounts of lipopolysaccharide, as indicated a reduction in both carbohydrate and 2-keto-3-deoxyoctonate concentrations, and a greatly altered protein composition as shown by polyacrylamide gel electrophoresis. There was a quantitative increase in total cell envelop protein in these strains. However, those protein bands identified as being major outer membrane proteins upon polyacrylamide gel electrophoresis of separated outer and cytoplasmic membranes were reduced greatly in concentration in the polymyxin-resistant cell envelopes. Thus, it appears that polymyxin resistance in these strains is associated with the alteration of the outer membrane through a loss of lipopolysaccharide and outer membrane proteins.     

Protease inhibitor system in horses: classification and detection of a new allele

A method of horizontal thin layer polyacrylamide gel electrophoresis at acid pH has been developed for the separation of the prealbumins in equine plasma. Using this method, it has been possible to split the S allele into two, S₁ and S₂, bringing the total number of prealbumin alleles in Thoroughbred horses to eight. The gene frequencies of these eight alleles in Australian Thoroughbreds are presented. All eight prealbumin types exhibit antiprotease activity and therefore, it is suggested that the name prealbumin (Pr) should be abandoned in favour of protease inhibitor (Pi) although at this stage it is not known whether this incorporates the Pi1 and Pi2 described by Juneja et al. (1979).