Molecular Typing of Environmental and Clinical Strains of Vibrio vulnificus Isolated in the Northeastern USA

Vibrio vulnificus is a ubiquitous marine bacterium that is responsible for infections and some seafood-related illnesses and deaths in the United States, mainly in individuals with compromised health status in the Gulf of Mexico region. Most phylogenetic studies focus on V. vulnificus strains isolated in the southern United States, but almost no genetic data are available on northeastern bacterial isolates of clinical or environmental origin. Our goal in this study was to examine the genetic diversity of environmental strains isolated from commercially-produced oysters and in clinical strains of known pathogenicity in northeastern United States. We conducted analyses of a total of eighty-three strains of V. vulnificus, including 18 clinical strains known to be pathogenic. A polyphasic, molecular-typing approach was carried out, based upon established biotypes, vcg, CPS, 16S rRNA types and three other genes possibly associated with virulence (arylsulfatase A, mtlABC, and nanA). An established Multi Locus Sequence Typing (MLST) method was also performed. Phylogenetic analyses of these markers and MLST results produced similar patterns of clustering of strains into two main lineages (we categorized as ‘LI’ and ‘LII’), with clinical and environmental strains clustering together in both lineages. Lineage LII was comprised primarily but not entirely of clinical bacterial isolates. Putative virulence markers were present in both clinical and environmental strains. These results suggest that some northeastern environmental strains of V. vulnificus are phylogenetically close to clinical strains and probably are capable of virulence. Further studies are necessary to assess the risk of human illness from consuming raw oysters harvested in the northeastern US.     

Molecular Typing of Candida glabrata

Mycopathologia - The yeast Candida glabrata has emerged, second only to Candida albicans, to be one of the most frequently isolated fungi in clinical specimen from human. Its frequent resistance...     

乳腺癌分子分型的研究进展

乳腺癌是目前世界上女性最为常见的恶性肿瘤之一。随着个体化的乳腺癌的化疗、内分泌治疗和靶向治疗的广泛使用,乳腺癌的分子分型检测也因此得到高度重视和广泛使用。2013年St.Gallen国际乳腺癌会议上重新定义了乳腺癌的分子分型标准,使Luminal A型和Luminal B型乳腺癌的比例有所改变,导致原本分类为Luminal A型的部分患者重新分类为Luminal B型而使Luminal B型患者数量有所增加。人上皮生长因子受体-2(human epidermalgrowth factor receptor-2,HER2)型乳腺癌的靶向药物曲妥珠单抗与其他化疗药物的联合应用及不同时间应用的治疗效果有了进一步研究。雄激素受体(Androgen Receptor,AR)认为可用于常规评估三阴性乳腺癌,除此之外,三阴性乳腺癌的小亚型的分型也为三阴性乳腺癌的个体化治疗提供了新的思路。尽管乳腺癌分子分型方面已取得一定进展,并获得国际同行间的认可,给乳腺癌患者的治疗和预后复发预测提供了重要依据,但目前所有的分子标记物仍不能够满足治疗需求使其临床实用性仍旧具有局限性,因此还期待更多更深入的研究。     

Detection and Strain Typing of Ancient Mycobacterium leprae from a Medieval Leprosy Hospital

Nine burials excavated from the Magdalen Hill Archaeological Research Project (MHARP) in Winchester, UK, showing skeletal signs of lepromatous leprosy (LL) have been studied using a multidisciplinary approach including osteological, geochemical and biomolecular techniques. DNA from Mycobacterium leprae was amplified from all nine skeletons but not from control skeletons devoid of indicative pathology. In several specimens we corroborated the identification of M. leprae with detection of mycolic acids specific to the cell wall of M. leprae and persistent in the skeletal samples. In five cases, the preservation of the material allowed detailed genotyping using single-nucleotide polymorphism (SNP) and multiple locus variable number tandem repeat analysis (MLVA). Three of the five cases proved to be infected with SNP type 3I-1, ancestral to contemporary M. leprae isolates found in southern states of America and likely carried by European migrants. From the remaining two burials we identified, for the first time in the British Isles, the occurrence of SNP type 2F. Stable isotope analysis conducted on tooth enamel taken from two of the type 3I-1 and one of the type 2F remains revealed that all three individuals had probably spent their formative years in the Winchester area. Previously, type 2F has been implicated as the precursor strain that migrated from the Middle East to India and South-East Asia, subsequently evolving to type 1 strains. Thus we show that type 2F had also spread westwards to Britain by the early medieval period.     

Multilocus Sequence Typing of Pathogenic Candida albicans Isolates Collected from a Teaching Hospital in Shanghai,China: A Molecular Epidemiology Study

Molecular typing of Candida albicans is important for studying the population structure and epidemiology of this opportunistic yeast, such as population dynamics, nosocomial infections, multiple infections and microevolution. The genetic diversity of C. albicans has been rarely studied in China. In the present study, multilocus sequence typing (MLST) was used to characterize the genetic diversity and population structure of 62 C. albicans isolates collected from 40 patients from Huashan Hospital in Shanghai, China. A total of 50 diploid sequence types (DSTs) were identified in the 62 C. albicans isolates, with 41 newly identified DSTs. Based on cluster analysis, the 62 isolates were classified into nine existing clades and two new clades (namely clades New 1 and New 2). The majority of the isolates were clustered into three clades, clade 6 (37.5%), clade 1 (15.0%) and clade 17 (15.0%). Isolates of clade New 2 were specifically identified in East Asia. We identified three cases of potential nosocomial transmission based on association analysis between patients’ clinical data and the genotypes of corresponding isolates. Finally, by analyzing the genotypes of serial isolates we further demonstrated that the microevolution of C. albicans was due to loss of heterozygosity. Our study represents the first molecular typing of C. albicans in eastern China, and we confirmed that MLST is a useful tool for studying the epidemiology and evolution of C. albicans.     

Epidemiology of Pseudomonas aeruginosa in a Burns Hospital: Surveillance by a Combined Typing System

For 3 months, 259 cultures of Pseudomonas aeruginosa isolated from nonpatient environmental sources and 262 cultures from 16 infected patients in the Intensive Care Unit (ICU) of Shriners Burns Hospital were typed by a combined system with a high degree of reliability. Sinks were major sources of environmental contamination. Serotypes 1 and 2 were the predominant types found in patients, and they were most prevalent among typable strains from sinks. Strain designations were made on the basis of similarities in data from serological and phage typing. All nontypable strains were typed by pyocin production. Two infected patients carried different strains of P. aeruginosa that remained the same type for 45 days, even though their beds in ICU were approximately 6 feet apart. Cross-contamination from patient to patient and spread of infection by nursing personnel were eliminated as major modes of transmission because nasopharyngeal swabs, hair samples, and hands of nursing staff were consistently negative. Splashing of water from contaminated sinks to fomites was suggested as a possible mode of transfer for this infectious agent.     

Molecular genetics in Aspergillus fumigatus

Manipulation of the genome of the human pathogen Aspergillus fumigatus is not well developed. Approaches and data from related model organisms are being used to develop molecular genetic systems in A. fumigatus; for example, the molecular typing of strains during infection. A genome-sequencing programme has begun and will form the basis for future development.     

Differentiation of Lactobacillus Species by Molecular Typing

A total of 64 type, reference, clinical, health food, and stock isolates of microaerophilic Lactobacillus species were examined by restriction fragment length polymorphisms. Of particular interest were members of six of the eight species most commonly recovered from the vaginas of healthy premenopausal women, namely, Lactobacillus jensenii, L. casei, L. rhamnosus, L. acidophilus, L. plantarum, and L. fermentum. Six main groupings were identified on the basis of ribotyping. This technique was able to classify fresh isolates to the species level. In the case of the ribotype A grouping for L. rhamnosus, differences between strains were evident by chromosome typing (chromotyping). Many isolates did not possess plasmids. Six L. rhamnosus strains isolated from four different health food products appeared to be identical to L. rhamnosus ATCC 21052. The molecular typing system is useful for identifying and differentiating Lactobacillus isolates. Studies of strains of potential importance to the urogenital flora should include molecular characterization as a means of comparing genetic traits with those of strains whose characteristics associated with colonization and antagonism against pathogens have been defined.Lactobacilli colonizing human tissues have long been considered important for the maintenance of a healthy gastrointestinal tract (4) and urogenital tract (20, 21, 22, 25). The disruption of the lactobacillus flora has been associated with many urogenital infections, and as these afflict over 150 million women worldwide each year, this area of study is an important one. Indeed, many patients resort to taking health food products containing lactobacilli as a means of trying to maintain a healthy intestinal (and in some cases vaginal) flora.The typing of lactobacilli has generally been conducted by cell and colony morphology and biochemical tests. These techniques type bacteria based on their ability to ferment sugars and produce acids such as lactic acid and acetic acid (9). Unfortunately, these typing methods are not completely accurate, and strains which show intermediate characteristics are frequently encountered (9, 33).Many studies emphasize that the classification of lactobacilli is unsatisfactory and does not reflect the real phylogenetic relatedness of different strains and species (6, 19, 30). Several new genetic and chemotaxonomic approaches have been used during the last 14 years with an aim of improving the classification and identification of lactobacilli: for example, analysis of plasmid content (18), sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of whole-cell protein (19) and of total soluble cell protein (8), sequencing of rRNA (5, 6, 19), restriction endonuclease fingerprinting (14, 30), and DNA-DNA hybridization (6, 17, 19, 27). All of these approaches have improved the taxonomic knowledge of the generic and suprageneric relationships of lactobacilli. However, no analysis of urogenital or health food isolates of lactobacilli by molecular typing has been reported.Plasmid typing of Lactobacillus strains has been suggested as a taxonomic tool in a number of reports (27, 31), but there is evidence to suggest that it is not very effective (2, 12, 15, 28), since plasmids can be absent or unstable.Chromosome typing (restriction endonuclease fingerprinting of chromosomal DNA) (chromotyping) has been applied to the discrimination of strains of lactobacilli and has been found more specific and reproducible than plasmid content analysis (14). However, one of the disadvantages of chromotyping is that comparing electrophoretic patterns consisting of up to 100 bands is difficult. Hence, chromotyping alone may not be widely used for typing large numbers of strains.A sensitive but rapid method for species differentiation involves the use of ribotyping (1, 27), which combines Southern hybridization of chromosomal DNA fingerprints with the use of Escherichia coli rRNA probes, thereby discriminating between various species and individual strains of lactobacilli. The method involves the separation and identification of the rRNA genes present within the bacterial genome, which exist in various copy numbers (10, 13), making it possible to delineate species based on differences in the restriction fragment length polymorphisms of the rRNA genes. Of particular importance is the fact that specific regions of the rRNA genes have remained well conserved because of their functional importance, thus allowing the detection of a broad range of bacteria with 16S and 23S rRNA of E. coli as probes.The aim of this study was to develop a method to test the efficacy of ribotyping of Lactobacillus type and reference strains and to use this method to characterize a number of clinical, health food, and laboratory isolates.     

Molecular Typing of Aeromonas Isolates in Natural Mineral Waters

A total of 103 isolates of Aeromonas spp. were obtained over a 3-year period from a natural mineral water and from surface streams located within the boundaries of the watershed of the natural mineral water wells and were typed by macrorestriction analysis of genomic DNA with XbaI and by pulsed-field gel electrophoresis. All Aeromonas caviae isolates from the natural mineral water belonged to the same clone, and an analogous clonal identity was found among Aeromonas hydrophila isolates. These two clones expressed no hemolytic or cytotoxic activities. Aeromonas isolates from surface waters showed high molecular heterogeneity and were not related to the clones found in the natural mineral water. The presence of aeromonads chronically found in the natural mineral water was a likely consequence of a localized development of a biofilm, with no exogenous contamination of the aquifer. Molecular fingerprinting of drinking water isolates is a useful tool in explaining possible reasons for bacterial occurrences.     

绿脓杆菌分子分型方法研究进展

绿脓杆菌是一种常见的人畜共患机会致病菌,广泛存在于自然界,是造成实验动物污染和医院内感染的重要病原菌之一。分子分型方法是病原菌流行病学分析的重要手段,对于确定感染来源和途径,检测交叉污染和流行菌株方面非常有效。本文主要对绿脓杆菌分子分型方法的研究进展进行综述。     

Vancomycin-Resistant Enterococci in a Chinese Hospital

In investigating the prevalence of vancomycin-resistant enterococci (VREs) in a hospital in China, 338 enterococci were detected by using multiplex polymerase chain reaction. Eleven VREs were found, including one VanA E. faecalis, one VanB E. faecium, three VanB E. faecalis, five VanC₁ E. gallinarum, and one VanC₂ E. flavescens. VITEK 2, microbroth dilution, and E-test were used to determine the susceptibilities of the VREs to certain antimicrobial agents; multiple-drug resistance of the 11 VREs was distinct. Compared with phenotypic methods, multiplex PCR rapidly detected 11 VREs and provided van genotype information. This is the first study that sequenced van genes of VRE isolates in China to date and found type VanB in China. Because of the relatively low isolating rate, early detection of VRE is vital.     

Molecular polymorphism and phenotypic variation in Aspergillus carbonarius

Thirteen collection strains and field isolates of Aspergillus carbonarius were examined by using various genotypic and phenotypic approaches. Restriction fragment length polymorphism analysis of the ribosomal RNA gene cluster and the mitochondrial DNA of the strains revealed only slight variations, except for one field isolate (IN7), which exhibited completely different ribosomal RNA gene cluster and mitochondrial DNA patterns. The mitochondrial DNAs of these strains were found to be much larger (45 to 57 kb) than those found earlier in the A. niger aggregate. Strain-specific characters could be detected by the random amplified polymorphic DNA technique. Isoenzyme analysis and examination of carbon source utilisation patterns of the strains also revealed some intraspecific variability, though much smaller than that observed by using DNA-based techniques. The dendrograms constructed based on genotypic and phenotypic data suggest that strain IN7 might represent a new subspecies of A. carbonarius.Abbreviations kb kilobase pair - mtDNA mitochondrial DNA - RAPD random amplified polymorphic DNA - rDNA ribosomal RNA gene cluster - RFLP restriction fragment length polymorphisms     

Prevalence and Molecular Genetic Typing of Nonsyndromic Sensorineural Deafness in Chuvash Republic

Summarized genetic epidemiological characteristics of nonsyndromic sensorineural deafness in six raions of Chuvash Republic (Cheboksary, Kanash, Morgaushi, Tsivil'sk, Mariinski Posad, and Alatyr') are presented. A total of 264419 individuals were examined. Forty-five families (60 affected individuals) with autosomal recessive (AR) and 8 families (18 affected individuals) with autosomal dominant (AD) nonsyndromic sensorineural deafness (NSSD) were identified. The load of AD and AR NSSD in the raions examined was estimated. A correlation between the distribution of AR NSSD and genetic drift was demonstrated. Furthermore, the load of AR NSSD was substantially higher in the regions with higher differentiation level. The Spearman's correlation coefficient value was 0.87. Typing of the 35delG mutation in the gene for connexion 26 was carried out in 34 patients from 26 families with AR NSSD. Comparative estimates of the NSSD prevalence in a number of Russian populations were performed.     

无乳链球菌耐药性及分子分型方法的研究进展

无乳链球菌(Streptococcus agalactiae)是链球菌属最主要的致病菌之一,又被称为B群链球菌(group B Streptococcus,GBS)。S. agalactiae致病性主要由毒力因子和表面蛋白引起,毒力因子包括荚膜多糖、溶血素、菌毛岛屿、透明质酸酶、磷酸甘油激酶和CAMP因子,表面蛋白是αC蛋白、表面免疫相关蛋白、黏附蛋白、纤维蛋白原结合蛋白、层黏连蛋白结合蛋白和纤溶酶受体蛋白。近8年的S. agalactiae耐药情况统计数据发现,S.agalactiae已对19种抗菌抗药物产生耐药,检出20个耐药基因和12种毒力因子。国内外S. agalactiae分子分型方法主要致力于血清型、多位点序列、脉冲场凝胶电泳、菌毛岛屿和细菌前噬菌体基因分型。本文阐述了S.agalactiae生物学特性、流行性致病信息、耐药性研究现状和分子分型方法研究进展,以期为进一步探明S.agalactiae耐药机制、开发治疗S. agalactiae的新型药物提供参考。     

Molecular Investigations into the Mechanics of a Muscle Anchoring Complex

The titin-telethonin complex, essential for anchoring filaments in the Z-disk of the sarcomere, is composed of immunoglobulin domains. Surprisingly, atomic force microscopy experiments showed that it resists forces much higher than the typical immunoglobulin domain and that the force distribution is unusually broad. To investigate the origin of this behavior, we developed a multiscale simulation approach, combining minimalist and atomistic models (SOP-AT). By following the mechanical response of the complex on experimental timescales, we found that the mechanical stability of titin-telethonin is modulated primarily by the strength of contacts between telethonin and the two titin chains, and secondarily by the timescales of conformational excursions inside telethonin and the pulled titin domains. Importantly, the conformational transitions executed by telethonin in simulations support its proposed role in mechanosensing. Our SOP-AT computational approach thus provides a powerful tool for the exploration of the link between conformational diversity and the broadness of the mechanical response, which can be applied to other multidomain complexes.     

Monitoring Environmental Aspergillus spp. Contamination and Meteorological Factors in a Haematological Unit

The opportunistic pathogens belonging to the Aspergillus genus are present in almost all seasons of the year, and their concentration is related to meteorological conditions. The high density of Aspergillus spp. conidia in a haematological hospital ward may be a significant risk factor for developing invasive fungal diseases in immunocompromised patients. Aim of the present study was to evaluate the variability of airborne Aspergillus spp. conidia contamination in a Haematological Unit (HU) within a period of 16 months in relation with some meteorological parameters. An environmental Aspergillus surveillance was conducted in the HU in four rooms and their bathrooms, in the corridor and in three external sites using an agar impact sampler. During each sampling, temperature and relative humidity at each site were recorded and current wind speed and rainfall events were taken from the official weather service. Aspergillus spp. conidia concentration differed significantly across the sampling sites. Internal Aspergillus spp. loads were significantly dependent on temperature, internal relative humidity and rain. External conidia concentrations were significantly influenced by outdoor temperature and relative humidity. A suitable indicator was introduced to evaluate the seasonal distribution of Aspergillus spp. conidia in the sampling sites, and a significant dependence on this indicator was observed inside the HU. Seventeen different fungal species belonging to the Aspergillus genus were detected during the sampling period. Aspergillus fumigatus was the most frequently isolated species and its distribution depended significantly on the seasonal indicator both inside and outside the hospital ward.     

A Review Molecular Typing Methods for <Emphasis Type="Italic">Aspergillus flavus</Emphasis> Isolates

Aspergillus flavus is the second most important Aspergillus species causing human infections. The importance of this fungus increases in regions with a dry and hot climate. Small phylogenetic studies in Aspergillus flavus indicate that the morphological species contains several genetically isolated species. Different genotyping methods have been developed and employed in order to better understand the genetic and epidemiological relationships between environmental and clinical isolates. Understanding pathogen distribution and relatedness is essential for determining the epidemiology of nosocomial infections and aiding in the design of rational pathogen control methods. Typing techniques can also give us a deeper understanding of the colonization pattern in patients. Most of these studies focused on Aspergillus fumigatus because it is medically the most isolated species. To date, there has not been any publication exclusively reviewing the molecular typing techniques for Aspergillus flavus in the literature. This article reviews all these different available methods for this organism.