Bacterial Typing Techniques ; methods ; Brucella ; classification

Bacterial Typing Techniques ; Leptospira interrogans ; classification ; genetics ; Minisatellite Repeats ; Multilocus Sequence Typing ; methods

OBJECTIVETo evaluate the feasibility of the application of variable-number tandem repeat (VNTR) loci of Salmonella Enteritidis (S. enteritidis) in subtyping mutiple-locus variable-number tandem repeat analysis (MLVA).

METHODSA total of 16 isolates of S.enteritidis from different place and time in China were preliminarily assessed by choosing 11 reported VNTR loci, the loci with single amplified bands were picked to subtype all 104 S. enteritidis isolates. The isolates were also analyzed by pulse field gel electrophoresis (PFGE) to compare the superiority or inferiority of MLVA method and PFGE method.

RESULTSSeven VNTR loci were selected from the preliminary screening to expand the analysis, and the 7 VNTR loci had grouped 104 of S.enteritidis isolates into either 16 MLVA subtypes or 22 PFGE subtypes, with the D value at 0.7222 and 0.7974, respectively. Comparing with the isolates in MLVA subtypes, the isolates in PFGE showed a stronger resolving power. Meanwhile the results in PFGE showed a more disperse frequency distribution than those in MLVA.

CONCLUSIONThese results indicate that some VNTR locus which have shown a good polymorphism internationally, may fail to show polymorphism in China, thereby, more VNTR loci should be included in MLVA and the wide screening may benefit the unity of global laboratorial methods.

Bacterial Typing Techniques ; methods ; Electrophoresis, Gel, Pulsed-Field ; Minisatellite Repeats ; Multilocus Sequence Typing ; methods ; Salmonella enteritidis ; classification ; genetics

OBJECTIVETo type the Chinese Anaplasma phagocytophilum isolates by Multispacer typing (MST).

METHODSBased on the genomes of the 4 published Anaplasma strains, 4 genomic sequences were analyzed by Mauve 2.3.1 software and variable spacer sequences were selected for designing primers with the bio-software Primer Premier 5.0. A total of 11 Chinese A. phagocytophilum isolates, obtained from different areas of China during 2009-2012 were assayed by the MST. Twenty two intergenic sequences for each isolate tested and the reference A. phagocytophilum strain Webster and A. phagocytophilum strain HZ were concatenated in the order of HGA-mst 1F/1R-mst 2F/2R, HGA-mst 22F/22R.

RESULTSTwenty two pairs of primers were successfully used for typing the Human granulocytic anaplasmosis (HGA) strains in the study. Those 22 intergenic sequences exhibited a great diversity among the strains tested and each of the strain tested was identified as unique genotype, according to the alignment analysis of the 22 concatenated intergenic sequences. Of these single nucleotide polymorphism (SNPs) identified in the study, the nucleotide transitions shared the highest percentage (60.2%, 251/417) and then the nucleotide transversion, accounted for 23.0% (96/417) and the indel events (insertion/deletion) were observed of 16.7% (70/417)SNPs. Phylogenetic analysis indicated that the 5 strains from patients (LZ-H1, LZ-H2, LZ-H3, LZ-H4, LZ-H5) from Laizhou areas, Shandong province and 1 tick strain (LZ-T1) from Haemaphysalis longicornis collected from the same areas where the patients lived were grouped in the same clan with the reference A. phagocytophilum strain Webster and strain HZ. Beijing isolates (BJ-H1) grouped with Xinjiang isolates (XJ-H1 and XJ-H3) while another tick isolates from Laizhou areas (LZ-T2) and another Xinjiang human isolate(XJ-H2)were in the same clan, which was closely related to the isolates from severe patients in Laizhou.

CONCLUSIONChinese HGA isolates exhibited a great diversity of intergenic regions. MST seemed a valuable tool for the detection and tracing for any endemic strains of Anaplasma during the outbreak investigations in the public health events.

Anaplasma phagocytophilum ; classification ; genetics ; Bacterial Typing Techniques ; methods ; China ; DNA, Ribosomal Spacer ; genetics ; Polymorphism, Single Nucleotide

OBJECTIVETo analyze the genetic relations of Shigella isolated from Shenzhen in 2001-2006 and develop primary molecular subtyping surveillance network of Shigella.

METHODSChromosomal DNAs from 55 isolated in agarose were digested with the restriction enzyme Xba I, and then were analyzed by pulsed-field gel electrophoresis. Pulsed-field gel electrophoresis (PFGE) patterns were clustered using BioNumerics software.

RESULTSAll 41 distinctive PFGE patterns were identified among 55 strains. 32 strains belonged to one cluster. Differences were observed in other strains.

CONCLUSIONBoth genetic-related clones and non-related clones of Shigella existed in Shenzhen. The development of PFGE molecular subtyping surveillance network would contribute to the active surveillance, outbreak investigation and source tracking for Shigellosis.

Bacterial Typing Techniques ; China ; Electrophoresis, Gel, Pulsed-Field ; methods ; Feces ; microbiology ; Humans ; Shigella ; classification ; isolation & purification

OBJECTIVESelecting variable-number tandem-repeat (VNTR) loci for different serogroups of Shigella spp to explore and establish multilocus variable-number tandem-repeat analysis (MLVA) method, in order to study the molecular characteristic of the isolated strains.

METHODSOf the Shigella strains found by dysentery surveillance in Beijing from 2001 to 2009, 180 strains were selected for this study, according to the number and serotypes of the surveillant strains, at the ratio of 15%; including 50 strains of Shigella sonnei and 130 strains of Shigella flexneri. After screening the polymorphism of the 18 VNTR loci, 10 VNTR loci (sh1-sh10) were retained and constructed three groups of multi-PCR methods to detect all he 180 strains and analyze MLVA molecular subtypes using capillary segments.

RESULTSA range of 2 to 11 alleles were found on the 10 VNTR loci among the 180 Shigella strains, with a diversity index value between 0.158 and 0.766. The 10 loci showed diversity in different serogroups, such as only one allele found in sh6 of Shigella flexneri, sh2 and sh3 of Shigella sonnei individually. The isolated 180 strains were divided into 84 MLVA subtypes, with a resolution ratio D value at 0.967 (95%CI: 0.956 - 0.978). The 130 strains of Shigella flexneri were divided into 63 subtypes, named as TF001-TF063; among which TF001, TF002 and TF 005 were the dominant subtypes, accounting to 17, 16 and 15 strains respectively. The 50 strains of Shigella sonnei were divided into 21 subtypes, named as TS001-TS021; among which TS002 (14 strains) and TS001 (7 strains) were the dominant subtypes.

CONCLUSIONMLVA subtyping method including 10 VNTR loci was preliminarily developed. The MLVA cluster analysis revealed that the subtypes of Shigella strains isolated in Beijing were diverse, and suggested the possibility of multiple-clone source.

Alleles ; Bacterial Typing Techniques ; methods ; China ; DNA, Bacterial ; genetics ; Genotype ; Minisatellite Repeats ; Polymorphism, Genetic ; Shigella ; classification ; genetics ; isolation & purification

Using three Austrian case studies, the variegated applications of molecular typing in today's public health laboratories are discussed to help illustrate preventive management strategies relying on DNA subtyping. DNA macrorestriction analysis by pulsed field gel electrophoresis has become the gold standard for subtyping of food borne pathogens like listeria, salmonella, campylobacter and Bacillus cereus. Using a Salmonella Mbandaka outbreak from the year 2010 as example, it is shown how the comparison of patterns from human isolates, food isolates, animal isolates and feed isolates can allow to identify and confirm a source of disease. An epidemiological connection between the simultaneous occurrence of tuberculosis in cattle and deer with cases of human tuberculosis due to Mycobacterium caprae in 2010 was excluded using mycobacterial interspersed repetitive units variable-number tandem repeats subtyping. Also in 2010, multilocus sequence typing with nonselective housekeeping genes, the so-called sequence based typing protocol, was used to elucidate connections between an environmental source (a hospital drinking water system) and a case of legionellosis. During the last decades, molecular typing has evolved to become a routine tool in the daily work of public health laboratories. The challenge is now no longer to simply type microorganisms, but to type them in a way that allows for data exchange between public health laboratories all over the world.
Bacterial Typing Techniques/*methods ; Clinical Laboratory Techniques/*methods ; DNA Fingerprinting ; DNA, Bacterial/*analysis ; Disease Outbreaks ; Electrophoresis, Gel, Pulsed-Field/methods ; Food Microbiology ; Humans ; Laboratories ; Molecular Typing/*methods ; Preventive Medicine ; *Public Health

OBJECTIVETo develop a pulsed field gel electrophoresis (PFGE) method for molecular typing of Lactobacillus and Streptococcus thermophilus (S. thermophilus) and to apply it in identification and characterization of both bacteria isolated from yoghurt collected from Beijing supermarket.

METHODSThe five most useful restriction enzymes including Apa I, Not I, Sfi I, Xba I and Sma I were chosen to cut DNA of 52 strains of Lactobacillus, S. thermophilus as well as associated standard bacteria strains. The endonucleases and electrophoresis conditions for PFGE analysis were optimized and applied in molecular typing of Lactobacillus and S.thermophilus isolates. Cluster analysis based on the PFGE data was conducted. The identification results of PFGE were compared with those obtained in biochemical and 16s ribosomal RNA PCR identification tests.

RESULTSNot I was suitable for L. bulgaricus, L. fermentum and L. delbrueckii digestion. While Apa I was an appropriate endonuclease for S. thermophilus, L. acidophilus and L. casei digestion. The results of molecular typing indicated that 24 strains of L.bulgaricus and 15 strains of S. thermophilus were grouped into 8 types by PFGE method, respectively. While 7 strains of L.acidophilus were grouped into 3 types and 2 strains of L. delbrueckii were grouped into 2 different PFGE types.

CONCLUSIONThe results of PFGE analysis are in compliance with those of 16s rRNA PCR and biochemical identification. The PFGE method developed in this study is suitable for molecular characterization of both Lactobacillus and S. thermophilus.

Bacterial Typing Techniques ; methods ; Electrophoresis, Gel, Pulsed-Field ; methods ; Lactobacillus ; classification ; isolation & purification ; Streptococcus thermophilus ; classification ; isolation & purification

OBJECTIVETo apply pulse-field gel electrophoresis analysis(PFGE) in analysing a case of food poisoning caused by Vibrio parahaemolyticus.

METHODSPFGE using restriction enzyme Not I was employed in molecular subtyping of thirty strains of V. parahaemolyticus isolated from a case of food poisoning in Guangzhou city and PFGE patterns were analyzed by using BioNumerics Version 4.0 software to perform cluster analysis. Pattern profiles were compared by using the Dice coefficient and unweighted pair group method with arithmetic averages (UPGMA).

RESULTSThirty strains were of the same type of pulsotype.

CONCLUSIONSMolecular subtyping by PFGE might disclose the epidemiological relationships of the strains from humans, food and the environment, giving a strong molecular epidemiological evidence and a support for the source-tracking of outbreak events.

Bacterial Typing Techniques ; methods ; China ; Electrophoresis, Gel, Pulsed-Field ; methods ; Foodborne Diseases ; microbiology ; Humans ; Vibrio parahaemolyticus ; classification ; genetics ; isolation & purification

Bacterial Typing Techniques ; methods ; Communicable Diseases ; diagnosis ; microbiology ; virology ; Epidemiologic Methods ; Humans ; Molecular Diagnostic Techniques ; methods ; Oligonucleotide Array Sequence Analysis ; methods