Development of Multiple-Locus Variable-Number Tandem-Repeat Analysis for the Molecular Subtyping of Enterobacter sakazakii

The genomic content of Enterobacter sakazakii strain ATCC BAA-894 was analyzed for variable-number tandem repeats (VNTRs). In this study we report the development of a multiple-locus VNTR analysis (MLVA) strategy for the subtyping of E. sakazakii. The method is based on a GeneScan analysis of four VNTR loci labeled with multiple fluorescent dyes. This approach was applied to a collection of 112 isolates representing all 16 of the currently defined E. sakazakii biogroups. MLVA successfully discriminated among these isolates and compared favorably with pulsed-field gel electrophoresis. The method was relatively fast and easy to perform. The potential value of MLVA as an epidemiological tool is discussed.     

Multiple-Locus Variable-Number Tandem-Repeat Analysis for Clonal Identification of Vibrio parahaemolyticus Isolates by Using Capillary Electrophoresis

Epidemics of Vibrio parahaemolyticus in Chile have occurred since 1998. Direct genome restriction enzyme analysis (DGREA) using conventional gel electrophoresis permitted discrimination of different V. parahaemolyticus isolates obtained from these outbreaks and showed that this species consists of a highly diverse population. A multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) approach was developed and applied to 22 clinical and 91 environmental V. parahaemolyticus isolates from Chile to understand their clonal structures. To this end, an advanced molecular technique was developed by applying multiplex PCR, fluorescent primers, and capillary electrophoresis, resulting in a high-resolution and high-throughput (HRHT) genotyping method. The genomic basis of this HRHT method was eight VNTR loci described previously by Kimura et al. (J. Microbiol. Methods 72:313-320, 2008) and two new loci which were identified by a detailed molecular study of 24 potential VNTR loci on both chromosomes. The isolates of V. parahaemolyticus belonging to the same DGREA pattern were distinguishable by the size variations in the indicative 10 VNTRs. This assay showed that these 10 VNTR loci were useful for distinguishing isolates of V. parahaemolyticus that had different DGREA patterns and also isolates that belong to the same group. Isolates that differed in their DGREA patterns showed polymorphism in their VNTR profiles. A total of 81 isolates was associated with 59 MLVA groups, providing fine-scale differentiation, even among very closely related isolates. The developed approach enables rapid and high-resolution analysis of V. parahaemolyticus with pandemic potential and provides a new surveillance tool for food-borne pathogens.Food-borne infections by Vibrio parahaemolyticus cause gastroenteritis, which is the most common clinical manifestation (38). An increasing number of V. parahaemolyticus infections and outbreaks caused by strains belonging to a pandemic clonal complex have been observed throughout the world since 1996 (2, 6, 9, 12, 13, 31, 32, 36, 40). Epidemics of Vibrio parahaemolyticus in Chile have occurred since the summer of 1998 and were caused by the pandemic clone O3:K6 that had emerged in Southeast Asia in 1996 (12, 13, 15). However, this strain was only a minor component of a highly diverse V. parahaemolyticus population in shellfish, as demonstrated by an improved method for restriction enzyme analysis, using total bacterial DNA, named direct genome restriction enzyme analysis (DGREA), in combination with conventional gel electrophoresis (12). This method has a discrimination index similar to that of restriction fragment length polymorphism-pulsed-field gel electrophoresis (PFGE) (12, 13, 19).A variety of molecular typing methods have been applied to V. parahaemolyticus, such as ribotyping (3, 10, 14), PFGE (3, 30), group-specific PCR (32), arbitrarily primed PCR (18, 32, 36), and multilocus sequence typing (7, 16). The use of DGREA permitted discrimination of different V. parahaemolyticus Chilean isolates and showed that these bacteria consist of a highly diverse population comprising at least 23 different genotypic groups among the environmental isolates obtained from shellfish and 5 different groups of clinical isolates (19).Epidemiological analyses of infections caused by pathogenic bacteria depend on the accurate identification of strains, preferably at the clonal level. Variable-number tandem repeats (VNTRs) comprising short sequence repeats constitute a rich source of genetic polymorphism and have been used extensively as markers for discrimination between strains of many different bacterial genera (27, 46). VNTRs have been used to discriminate among individual strains within several food- or waterborne pathogens with little genetic variation, including Escherichia coli O157:H7 (25, 35), Pseudomonas aeruginosa (37), Staphylococcus aureus (41), and Salmonella enterica subsp. enterica serovar Typhimurium (26), and to characterize other important human pathogens, such as Neisseria meningitidis (42), Listeria monocytogenes (28), Legionella pneumophila (34, 39), Leptospira interrogans (43), and Mycobacterium tuberculosis (45). VNTR loci have even been found in genetically highly homogenous pathogens, such as Bacillus anthracis (1, 21, 29). Multiple-locus VNTR analysis (MLVA) is defined as the analysis of a set of loci spread throughout the bacterial genome (23). Individual strains within a bacterial species often maintain the same sequence elements but with different copy numbers due to variations introduced by slipped-strand mispairing during DNA replication (33).Recently, a study of the polymorphism of tandem repeats in V. parahaemolyticus showed the utility of the MLVA approach for characterizing recently emerged and highly homogeneous pandemic strains of serotype O3:K6 (22). These authors reported a scheme of eight genomic VNTR loci, comparing PFGE results for clinical strains of V. parahaemolyticus serotype O3:K6. The study by Kimura et al. (22) comprised only strains of serogroup O3:K6 and used conventional gel electrophoresis to evaluate VNTRs. In epidemiological studies, a more rapid technique is needed for mass application of MLVA that also provides improved resolution and has been validated for nonserogroup O3:K6 isolates. Capillary electrophoresis has become the preferred technology to improve resolution and accuracy in bacterial VNTR analysis due to the availability of multiple fluorescent labels and better accuracy and reproducibility (27).In our study we describe the use of an improved MLVA for discriminating genotypically a diverse collection of clinical and environmental V. parahaemolyticus isolates from Chile. These very closely related isolates have been analyzed and grouped by DGREA previously (12). To this end, we developed and applied multiplex PCR of 10 VNTR loci, tagged with multiple fluorescent dyes, and analyzed the amplicons by capillary electrophoresis. The results demonstrated that MLVA typing is able to distinguish between V. parahaemolyticus isolates that have different DGREA patterns and isolates that belong to the same group, allowing accurate sizing of amplicons by assignment of the fragment size. Validation of this typing method with 113 Chilean isolates demonstrated the utility of this technique also for nonserogroup O3:K6 clinical isolates, thereby providing a new tool for the study of the molecular epidemiology of V. parahaemolyticus.     

Differentiation of Clostridium botulinum Serotype A Strains by Multiple-Locus Variable-Number Tandem-Repeat Analysis

Ten variable-number tandem-repeat (VNTR) regions identified within the complete genomic sequence of Clostridium botulinum strain ATCC 3502 were used to characterize 59 C. botulinum strains of the botulism neurotoxin A1 (BoNT/A1) to BoNT/A4 (BoNT/A1-A4) subtypes to determine their ability to discriminate among the serotype A strains. Two strains representing each of the C. botulinum serotypes B to G, including five bivalent strains, and two strains of the closely related species Clostridium sporogenes were also tested. Amplified fragment length polymorphism analyses revealed the genetic diversity among the serotypes and the high degree of similarity among many of the BoNT/A1 strains. The 10 VNTR markers amplified fragments within all of the serotype A strains but were less successful with strains of other serotypes. The composite multiple-locus VNTR analysis of the 59 BoNT/A1-A4 strains and 3 bivalent B strains identified 38 different genotypes. Thirty genotypes were identified among the 53 BoNT/A1 and BoNT/A1(B) strains, demonstrating discrimination below the subtype level. Contaminating DNA within crude toxin preparations of three BoNT/A subtypes (BoNT/A1 to BoNT/A3) also supported amplification of all of the VNTR regions. These markers provide clinical and forensics laboratories with a rapid, highly discriminatory tool to distinguish among C. botulinum BoNT/A1 strains for investigations of botulism outbreaks.     

High-Resolution In Situ Genotyping of Legionella pneumophila Populations in Drinking Water by Multiple-Locus Variable-Number Tandem-Repeat Analysis Using Environmental DNA

Central to the understanding of infections by the waterborne pathogen Legionella pneumophila is its detection at the clonal level. Currently, multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) of L. pneumophila isolates can be used as a tool for high-resolution genotyping. Since L. pneumophila is difficult to isolate, the isolation of outbreak strains often fails due to a viable but nonculturable (VBNC) state of the respective environmental population. Therefore, we developed a cultivation-independent approach to detect single clones in drinking water. This approach is based on the extraction of DNA from drinking water followed by PCR using a set of eight VNTR primer pairs necessary for MLVA genotyping of L. pneumophila. The PCR amplicons were analyzed by single-strand conformation polymorphism (SSCP) and capillary electrophoresis to obtain the respective MLVA profiles. Parallel to the high-resolution analysis, we used the same environmental DNA to quantify the number of L. pneumophila cells in drinking water using real-time PCR with 16S rRNA gene-targeted primers. We used a set of drinking water samples from a small-scale drinking water network to test our approach. With these samples we demonstrated that the developed approach was directly applicable to DNA obtained from drinking water. We were able to detect more L. pneumophila MLVA genotypes in drinking water than we could detect by isolation. Our approach could be a valuable tool to identify outbreak strains even after the outbreak has occurred and has the potential to be applied directly to clinical material.Legionella pneumophila is a Gram-negative, facultative, intracellular pathogen that accounts for the majority of cases of Legionnaires'' disease in Europe (16). It is also the causative agent of a milder form of infection, Pontiac fever (14). Legionellae are ubiquitous inhabitants of natural and human-made aquatic environments. They occur in bulk water and biofilms, where they replicate within protozoa, which can serve as a transmission vehicle and as a protective shell against disinfection or heat treatment (2, 6, 7). In drinking water supply systems (DWSSs), legionellae can survive in dead-end tubings, stagnated water in plumbings, or seldom-used facilities (2). The pathogen is transmitted via small droplets of water, e.g., aerosols from cooling towers, showerheads, or air conditioners. In the human lung, it is able to enter and replicate within alveolar macrophages, causing a severe pneumonia. Among the 48 species of the genus Legionella (1, 4, 21, 22), L. pneumophila is responsible for approximately 91% of all reported community-acquired cases of legionellosis. Among the 15 serogroups of L. pneumophila, serogroup 1 accounts for 84% of confirmed cases, as assessed by an international collaborative survey (40). Even among serogroup 1 isolates, a high level of genetic diversity was observed by several studies (12, 30, 35).Epidemiological analyses of infections caused by L. pneumophila depend on the accurate identification of strains, preferably at the clonal level. Therefore, several typing methods have been implemented in the last years, e.g., MLST (multiple-locus sequence typing), which is based on DNA sequencing of multiple polymorphic DNA segments (13, 33). Recently, a multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) was implemented by Pourcel et al. and approved by Eurosurveillance (30, 31). MLVA typing is used to determine the allele-related repeat size variation on different VNTR loci of L. pneumophila isolates. The method was further improved by adapting the eight-locus-comprising MLVA (MLVA-8) to capillary electrophoresis (CE) with the use of fluorescently labeled primers, thus providing a fast, reproducible, and low-cost genotyping method for L. pneumophila isolates (26). Upstream isolation procedures face several problems: especially in hot water with temperatures above 37°C, legionellae can lose culturability and enter a viable but nonculturable (VBNC) state (28). This VBNC state is mostly the reason why L. pneumophila cannot be isolated from aquatic environments that are suspected to be the source of infection (37). Additionally, cultivation of this fastidious bacterium is difficult due to its slow growth and overgrowing by competing bacteria on agar plates.The aim of this study was to evaluate the utility of MLVAs directly on environmental DNA obtained from finished drinking water. Using DNA-based single-strand conformation polymorphism (SSCP) analysis, this method should (i) allow the identification of strains causing outbreaks, (ii) allow the monitoring of L. pneumophila strains present in a given sample at the clonal level without cultivation, and (iii) provide sequence information on VNTR markers obtained directly from the sequencing of SSCP gel bands, i.e., environmental DNA. To this end, we tested our approach with DNA from a set of drinking water samples from a small-scale drinking water network. We demonstrated that the method provides a reliable tool for the analysis of drinking water samples where the number of L. pneumophila cells present is relatively low and where isolation procedures did not succeed. Additionally, complete sequence information on the VNTR locus could be obtained from PCR amplicons separated on SSCP gels to identify the specific MLVA genotype.     

Subtyping of a Large Collection of Historical Listeria monocytogenes Strains from Ontario,Canada, by an Improved Multilocus Variable-Number Tandem-Repeat Analysis (MLVA)

Listeria monocytogenes is responsible for severe and often fatal food-borne infections in humans. A collection of 2,421 L. monocytogenes isolates originating from Ontario''s food chain between 1993 and 2010, along with Ontario clinical isolates collected from 2004 to 2010, was characterized using an improved multilocus variable-number tandem-repeat analysis (MLVA). The MLVA method was established based on eight primer pairs targeting seven variable-number tandem-repeat (VNTR) loci in two 4-plex fluorescent PCRs. Diversity indices and amplification rates of the individual VNTR loci ranged from 0.38 to 0.92 and from 0.64 to 0.99, respectively. MLVA types and pulsed-field gel electrophoresis (PFGE) patterns were compared using Comparative Partitions analysis involving 336 clinical and 99 food and environmental isolates. The analysis yielded Simpson''s diversity index values of 0.998 and 0.992 for MLVA and PFGE, respectively, and adjusted Wallace coefficients of 0.318 when MLVA was used as a primary subtyping method and 0.088 when PFGE was a primary typing method. Statistical data analysis using BioNumerics allowed for identification of at least 8 predominant and persistent L. monocytogenes MLVA types in Ontario''s food chain. The MLVA method correctly clustered epidemiologically related outbreak strains and separated unrelated strains in a subset analysis. An MLVA database was established for the 2,421 L. monocytogenes isolates, which allows for comparison of data among historical and new isolates of different sources. The subtyping method coupled with the MLVA database will help in effective monitoring/prevention approaches to identify environmental contamination by pathogenic strains of L. monocytogenes and investigation of outbreaks.     

Molecular responses of Campylobacter jejuni to cadmium stress

Cadmium ions are a potent carcinogen in animals, and cadmium is a toxic metal of significant environmental importance for humans. Response curves were used to investigate the effects of cadmium chloride on the growth of Camplyobacter jejuni. In vitro, the bacterium showed reduced growth in the presence of 0.1 mm cadmium chloride, and the metal ions were lethal at 1 mm concentration. Two-dimensional gel electrophoresis combined with tandem mass spectrometry analysis enabled identification of 67 proteins differentially expressed in cells grown without and with 0.1 mm cadmium chloride. Cellular processes and pathways regulated under cadmium stress included fatty acid biosynthesis, protein biosynthesis, chemotaxis and mobility, the tricarboxylic acid cycle, protein modification, redox processes and the heat-shock response. Disulfide reductases and their substrates play many roles in cellular processes, including protection against reactive oxygen species and detoxification of xenobiotics, such as cadmium. The effects of cadmium on thioredoxin reductase and disulfide reductases using glutathione as a substrate were studied in bacterial lysates by spectrophotometry and nuclear magnetic resonance spectroscopy, respectively. The presence of 0.1 mm cadmium ions modulated the activities of both enzymes. The interactions of cadmium ions with oxidized glutathione and reduced glutathione were investigated using nuclear magnetic resonance spectroscopy. The data suggested that, unlike other organisms, C. jejuni downregulates thioredoxin reductase and upregulates other disulfide reductases involved in metal detoxification in the presence of cadmium.     

Differentiation of Campylobacter jejuni and Campylobacter coli Using Multiplex-PCR and High Resolution Melt Curve Analysis

Campylobacter spp. are important causes of bacterial gastroenteritis in humans in developed countries. Among Campylobacter spp. Campylobacter jejuni (C. jejuni) and C. coli are the most common causes of human infection. In this study, a multiplex PCR (mPCR) and high resolution melt (HRM) curve analysis were optimized for simultaneous detection and differentiation of C. jejuni and C. coli isolates. A segment of the hippuricase gene (hipO) of C. jejuni and putative aspartokinase (asp) gene of C. coli were amplified from 26 Campylobacter isolates and amplicons were subjected to HRM curve analysis. The mPCR-HRM was able to differentiate between C. jejuni and C. coli species. All DNA amplicons generated by mPCR were sequenced. Analysis of the nucleotide sequences from each isolate revealed that the HRM curves were correlated with the nucleotide sequences of the amplicons. Minor variation in melting point temperatures of C. coli or C. jejuni isolates was also observed and enabled some intraspecies differentiation between C. coli and/or C. jejuni isolates. The potential of PCR-HRM curve analysis for the detection and speciation of Campylobacter in additional human clinical specimens and chicken swab samples was also confirmed. The sensitivity and specificity of the test were found to be 100% and 92%, respectively. The results indicated that mPCR followed by HRM curve analysis provides a rapid (8 hours) technique for differentiation between C. jejuni and C. coli isolates.     

Molecular origin of two polysaccharides of Campylobacter jejuni 81116

The nature of the polysaccharide molecules of the human enteric pathogen Campylobacter jejuni has been the subject of debate. Previously, C. jejuni 81116 was shown to contain two different polysaccharides, one acidic (polysaccharide A) and the other neutral (polysaccharide B), occurring in a 3 : 1 ratio, respectively. The aim of this study was to determine the molecular origin of these polysaccharides. Using a combination of centrifugation, gel permeation chromatography, chemical assays, and (1)H-NMR analysis, polysaccharide B was shown to be derived from lipopolysaccharide and polysaccharide A from capsular polysaccharide. Thus, C. jejuni 81116 produces both lipopolysaccharide-like molecules and capsular polysaccharide.     

Multiple-Locus Variable-Number Tandem-Repeats Analysis of Escherichia coli O157 using PCR multiplexing and multi-colored capillary electrophoresis

The Multiple-Locus Variable-Number Tandem-Repeats Analysis (MLVA) method is currently being used as the primary typing tool for Shiga-toxin-producing Escherichia coli (STEC) O157 isolates in our laboratory. The initial assay was performed using a single fluorescent dye and the different patterns were assigned using a gel image. Here, we present a significantly improved assay using multiple dye colors and enhanced PCR multiplexing to increase speed, and ease the interpretation of the results. The different MLVA patterns are now based on allele sizes entered as character values, thus removing the uncertainties introduced when analyzing band patterns from the gel image. We additionally propose an easy numbering scheme for the identification of separate isolates that will facilitate exchange of typing data. Seventy-two human and animal strains of Shiga-toxin-producing E. coli O157 were used for the development of the improved MLVA assay. The method is based on capillary separation of multiplexed PCR products of VNTR loci in the E. coli O157 genome labeled with multiple fluorescent dyes. The different alleles at each locus were then assigned to allele numbers, which were used for strain comparison.     

Molecular characterization of a strain-specific sequence in Campylobacter jejuni

Campylobacter jejuni strains 81116 and NCTC 11392 were shown to contain a region of DNA that was not present in other strains. This region was cloned from the chromosome of strain 81116 and the nucleotide sequence determined. It was found to contain an insert of 742 bp which was flanked by direct repeats of 45 bp. Although the nature of this sequence is unknown at present, it is clear that it only presents in certain strains of Camp. jejuni and it may therefore be useful as an epidemiological tool.     

Molecular characterization of a cryptic plasmid from Campylobacter jejuni

Campylobacter jejuni is a leading bacterial cause of human enterocolitis. Molecular genetic characterization of this pathogen has been hampered by the lack of genetic tools that are functional in this organism. Cloning vectors commonly used in other organisms usually do not replicate within C. jejuni. To develop a system for functional analysis of C. jejuni genes, a small plasmid (pCJ01) identified in a poultry isolate of C. jejuni was sequenced and characterized in this study. By using inverse PCR, the full sequence of pCJ01 was amplified and subsequently determined. Results indicate that pCJ01 is a circular molecule of 3212 bp, with a G + C content of 33.5%. A typical plasmid replication origin with iteron sequences is identified upstream of the DNA sequences encoding replication initiation proteins. Four open reading frames (ORFs) are present in pCJ01. ORF1 and ORF2 share high homology with the putative RepA and RepB proteins, respectively, of known C. coli plasmids. ORF3 and ORF4, of unknown function, do not exhibit homology with any sequences deposited in the GenBank database. Hydropathy analysis predicts that ORF3 and ORF4 contain multiple stretches of hydrophobic amino acids, suggesting that they may encode transmembrane proteins. Since pCJ01 is a small plasmid and can be readily prepared from C. jejuni, it may be modified for use in molecular characterization of C. jejuni virulence genes.     

毛细管电泳在细菌分离分析中的应用

介绍了近年来毛细管电泳技术在细菌分离分析方面的研究进展。毛细管电泳以细菌表面的特征信息为分离的基础,可以快速鉴定相应的菌株,可以对微生物进行快速定量,可以反映细菌特殊时期的生理特征,也可以研究微生物与分子之间的相互作用。同时应用该技术可分离分析自然界不能纯培养的微生物。因而毛细管电泳分离与检测细菌方法的建立及其应用在分离科学和微生物学方面都有很大的实际意义。     

氨基酸的毛细管电泳检测研究新进展

氨基酸是生命有机体的重要组成部分,参与人体各项生理活动和新陈代谢,在人体中发挥着特殊的作用,是人体必不可少的营养成分之一。近年来,生命科学的研究与食品、药品和保健品的开发,以及麻醉药品、精神药品对兴奋性神经递质的影响的研究,促进了对存在于各种检验材料中的氨基酸的分析方法的研究,探索高灵敏度和高选择性的氨基酸分析检测方法引起了人们极大的兴趣,所以建立快速、准确的氨基酸的检测方法越来越重要。毛细管电泳(capillary electrophoresis,CE)是继高效液相色谱之后发展起来的一种新的分离和分析方法,具有分离效率高、分析速度快、样品及试剂用量少等特点,本文参考近几年的相关文献,对氨基酸进行毛细管电泳检测使用紫外检测器、荧光检测器、电化学检测器和质谱检测器的方法作一综述。     

Molecular Evidence for the Thriving of Campylobacter jejuni ST-4526 in Japan

Campylobacter jejuni is a leading cause of human gastroenteritis worldwide. This study aimed at a better understanding of the genetic diversity of this pathogen disseminated in Japan. We performed multilocus sequence typing (MLST) of Campylobacter jejuni isolated from different sources (100 human, 61 poultry, and 51 cattle isolates) in Japan between 2005 and 2006. This approach identified 62 sequence types (STs) and 19 clonal complexes (CCs), including 11 novel STs. These 62 STs were phylogenetically divided into 6 clusters, partially exhibiting host association. We identified a novel ST (ST-4526) that has never been reported in other countries; a phylogenetic analysis showed that ST-4526 and related STs showed distant lineage from the founder ST, ST-21 within CC-21. Comparative genome analysis was performed to investigate which properties could be responsible for the successful dissemination of ST-4526 in Japan. Results revealed that three representative ST-4526 isolates contained a putative island comprising the region from Cj0737 to Cj0744, which differed between the ST-4526 isolates and the reference strain NCTC11168 (ST-43/CC-21). Amino acid sequence alignment analyses showed that two of three ST-4526 isolates expressed 693aa- filamentous hemagglutination domain protein (FHA), while most of other C. jejuni strains whose genome were sequenced exhibited its truncation. Correspondingly, host cell binding of FHA-positive C. jejuni was greater than that of FHA-truncated strains, and exogenous administration of rFHA protein reduced cell adhesion of FHA-positive bacteria. Biochemical assays showed that this putative protein exhibited a dose-dependent binding affinity to heparan sulfate, indicating its adhesin activity. Moreover, ST-4526 showed increased antibiotic-resistance (nalidixic acid and fluoroquinolones) and a reduced ability for DNA uptake. Taken together, our data suggested that these combined features contributed to the clonal thriving of ST-4526 in Japan.     

Colonization of cattle intestines by Campylobacter jejuni and Campylobacter lanienae

The location and abundance of Campylobacter jejuni and Campylobacter lanienae in the intestines of beef cattle were investigated using real-time quantitative PCR in two studies. In an initial study, digesta and tissue samples were obtained along the digestive tract of two beef steers known to shed C. jejuni and C. lanienae (steers A and B). At the time of slaughter, steer B weighed 540 kg, compared to 600 kg for steer A, yet the intestine of steer B (40.5 m) was 36% longer than the intestine of steer A (26.1 m). In total, 323 digesta samples (20-cm intervals) and 998 tissue samples (3.3- to 6.7-cm intervals) were processed. Campylobacter DNA was detected in the digesta and in association with tissues throughout the small and large intestines of both animals. Although C. jejuni and C. lanienae DNA were detected in both animals, only steer A contained substantial quantities of C. jejuni DNA. In both digesta and tissues of steer A, C. jejuni was present in the duodenum and jejunum. Considerable quantities of C. jejuni DNA also were observed in the digesta obtained from the cecum and ascending colon, but minimal DNA was associated with tissues of these regions. In contrast, steer B contained substantial quantities of C. lanienae DNA, and DNA of this bacterium was limited to the large intestine (i.e., the cecum, proximal ascending colon, descending colon, and rectum); the majority of tissue-associated C. lanienae DNA was present in the cecum, descending colon, and rectum. In a second study, the location and abundance of C. jejuni and C. lanienae DNA were confirmed in the intestines of 20 arbitrarily selected beef cattle. DNA of C. jejuni and C. lanienae were detected in the digesta of 57% and 95% of the animals, respectively. C. jejuni associated with intestinal tissues was most abundant in the duodenum, ileum, and rectum. However, one animal contributed disproportionately to the abundance of C. jejuni DNA in the ileum and rectum. C. lanienae was most abundant in the large intestine, and the highest density of DNA of this bacterium was found in the cecum. Therefore, C. jejuni colonized the proximal small intestine of asymptomatic beef cattle, whereas C. lanienae primarily resided in the cecum, descending colon, and rectum. This information could be instrumental in developing efficacious strategies to manage the release of these bacteria from the gastrointestinal tracts of cattle.     

Specific Multilocus Variable-Number Tandem-Repeat Analysis Genotypes of Mycoplasma pneumoniae Are Associated with Diseases Severity and Macrolide Susceptibility

Clinical relevance of multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) in patients with community-acquired pneumonia (CAP) by Mycoplasma pneumoniae (M. pneumoniae) is unknown. A multi-center, prospective study was conducted from November 2010 to April 2012. Nine hundred and fifty-four CAP patients were consecutively enrolled. M. pneumoniae clinical isolates were obtained from throat swabs. MLVA typing was applied to all isolates. Comparison of pneumonia severity index (PSI) and clinical features among patients infected with different MLVA types of M. pneumoniae were conducted. One hundred and thirty-six patients were positive with M. pneumoniae culture. The clinical isolates were clustered into 18 MLVA types. One hundred and fourteen (88.3%) isolates were resistant to macrolide, covering major MLVA types. The macrolide non-resistant rate of M. pneumoniae isolates with Mpn13-14-15-16 profile of 3-5-6-2 was significantly higher than that of other types (p≤0.001). Patients infected with types U (5-4-5-7-2) and J (3-4-5-7-2) had significantly higher PSI scores (p<0.001) and longer total duration of cough (p = 0.011). Therefore it seems that there is a correlation between certain MLVA types and clinical severity of disease and the presence of macrolide resistance.     

Colonization of Cattle Intestines by Campylobacter jejuni and Campylobacter lanienae

The location and abundance of Campylobacter jejuni and Campylobacter lanienae in the intestines of beef cattle were investigated using real-time quantitative PCR in two studies. In an initial study, digesta and tissue samples were obtained along the digestive tract of two beef steers known to shed C. jejuni and C. lanienae (steers A and B). At the time of slaughter, steer B weighed 540 kg, compared to 600 kg for steer A, yet the intestine of steer B (40.5 m) was 36% longer than the intestine of steer A (26.1 m). In total, 323 digesta samples (20-cm intervals) and 998 tissue samples (3.3- to 6.7-cm intervals) were processed. Campylobacter DNA was detected in the digesta and in association with tissues throughout the small and large intestines of both animals. Although C. jejuni and C. lanienae DNA were detected in both animals, only steer A contained substantial quantities of C. jejuni DNA. In both digesta and tissues of steer A, C. jejuni was present in the duodenum and jejunum. Considerable quantities of C. jejuni DNA also were observed in the digesta obtained from the cecum and ascending colon, but minimal DNA was associated with tissues of these regions. In contrast, steer B contained substantial quantities of C. lanienae DNA, and DNA of this bacterium was limited to the large intestine (i.e., the cecum, proximal ascending colon, descending colon, and rectum); the majority of tissue-associated C. lanienae DNA was present in the cecum, descending colon, and rectum. In a second study, the location and abundance of C. jejuni and C. lanienae DNA were confirmed in the intestines of 20 arbitrarily selected beef cattle. DNA of C. jejuni and C. lanienae were detected in the digesta of 57% and 95% of the animals, respectively. C. jejuni associated with intestinal tissues was most abundant in the duodenum, ileum, and rectum. However, one animal contributed disproportionately to the abundance of C. jejuni DNA in the ileum and rectum. C. lanienae was most abundant in the large intestine, and the highest density of DNA of this bacterium was found in the cecum. Therefore, C. jejuni colonized the proximal small intestine of asymptomatic beef cattle, whereas C. lanienae primarily resided in the cecum, descending colon, and rectum. This information could be instrumental in developing efficacious strategies to manage the release of these bacteria from the gastrointestinal tracts of cattle.     

单链构象多态性毛细管电泳分析研究进展

基因突变的检测在临床疾病诊断中起十分重要的作用.单链构象多态性(Single Strand Conform ationPolym orphism,SSCP)分析是检测突变最流行的方法之一.SSCP分析与毛细管电泳(Capillary Electrophoresis,CE)相结合的技术更是具有灵敏度高、花费低、简单、快速的优点.目前,这项技术已应用于人类原癌基因、抑癌基因以及其它致病基因的突变检测.主要综述了各种参数对CES-SCP分析的影响以及CES-SCP分析技术将来的发展方向.     

毛细管电泳测定蛋白激酶A活力的新方法

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