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

BACKGROUNDMulti-locus sequence typing (MLST) is widely used to explore the population structure of numerous bacterial pathogens. However, for genotypically-restricted pathogens, the sensitivity of MLST is limited by a paucity of variation within selected loci. For Bartonella henselae (B. henselae), although the MLST scheme currently used has been proven useful in defining the overall population structure of the species, its reliability for the accurate delineation of closely-related sequence types, between which allelic variation is usually limited to, at most, one or two nucleotide polymorphisms. Exploitation of high-throughput sequencing data allows a more informed selection of MLST loci and thus, potentially, a means of enhancing the sensitivity of the schemes they comprise.

METHODSWe carried out SOLiD resequencing on 12 representative B. henselae isolates and explored these data using single nucleotide polymorphism (SNP) analysis. We determined the number and distribution of SNPs in the genes targeted by the established MLST scheme and modified the position of loci within these genes to capture as much genetic variation as possible.

RESULTSUsing genome-wide SNP data, we found the distribution of SNPs within each open reading frame (ORF) of MLST loci, which were not represented by the established B. henselae MLST scheme. We then modified the position of loci in the MLST scheme to better reflect the polymorphism in the ORF as a whole. The use of amended loci in this scheme allowed previously indistinguishable ST1 strains to be differentiated. However, the diversity of B. henselae was still rare in China.

CONCLUSIONSOur study demonstrates the use of SNP analysis to facilitate the selection of MLST loci to augment the currently-described scheme for B. henselae. And the diversity among B. henselae strains in China is markedly less than that observed in B. henselae populations elsewhere in the world.

Bartonella henselae ; genetics ; Molecular Sequence Data ; Multilocus Sequence Typing ; methods ; Open Reading Frames ; genetics ; Polymorphism, Single Nucleotide ; genetics

OBJECTIVETo establish and compare the pulsed-field gel electrophoresis (PFGE), multiple-locus variable number tandem repeat analysis (MLVA) and automated ribotyping for subtyping of Citrobacter strains.

METHODSPFGE protocol was optimized in terms of plug preparation procedure, restriction enzymes and configuration of electrophoretic parameters. MLVA method was evaluated by finding variable number tandem repeats in two genomes of Citrobacter strains. The ribotyping was performed by using the automated RiboPrinter system.

RESULTSWe optimized the plug preparation procedure, focused on the cell suspension concentration (turbidity of 2.5 to 3.5), SDS addition (no SDS needed) and lysis time (1 h), and selected the appropriate restriction enzyme (XbaI) and the electrophoretic parameters (1.0 s-20.0 s for 19 h) of PFGE. There was nearly no discriminatory power of MLVA between Citrobacter strains. For 51 Citrobacter strains, automated ribotyping gave a D-value of 0.9945, while PFGE gave a D-value of 0.9969. Both PFGE and automated ribotyping clustered strains from the same sources (with the same species from the same place at the same time identified as the same source) and divided strains from different sources (from different years, places and hosts) into different subtypes.

CONCLUSIONPFGE protocol established in this paper and automated ribotyping are suitable for application in Citrobacter subtyping.

Automation ; Citrobacter ; classification ; genetics ; Electrophoresis, Gel, Pulsed-Field ; methods ; Minisatellite Repeats ; genetics ; Multilocus Sequence Typing ; methods ; Phylogeny ; Ribotyping ; methods

OBJECTIVETuberculosis remains one of the most serious infectious diseases in the world. In this study, a scheme of Mycobacterium tuberculosis (M. tuberculosis) multilocus sequence analysis (MLSA) was established for the phylogenetic and epidemiology analysis.

METHODSTo establish the scheme of M. tuberculosis MLSA, the genome of H37Rv, CCDC5079 and CCDC5180 were compared, and some variable genes were chosen to be the MLSA typing scheme. 44 M. tuberculosis clinical isolates were typed by MLSA, IS6110-RFLP, and soligotyping, to evaluate the MLSA methods.

RESULTSAfter comparison of the genome, seven high discrimination gene loci (recX, rpsL, rmlC, rpmG1, mprA, gcvH, ideR) were chosen to be the MLSA typing scheme finally. 11 variable SNP sites of those seven genes were found among the 44 M. tuberculosis isolate strains and 11 sequence types (STs) were identified. Based on the Hunter-Gaston Index (HGI), MLSA typing was not as good for discrimination at the strain level as IS6110-RFLP, but the HGI was much better than that of spoligotyping. In addition, the MEGA analysis result of MLSA data was similar to spoligotyping/PGG lineage, showing a strong phylogenetic signal in the modern strains of M. tuberculosis. The MLSA data analysis by eBURST revealed that 4 sequence types (ST) came into a main cluster, showing the major clonal complexes in those 44 strains.

CONCLUSIONMLSA genotyping not only can be used for molecular typing, but also is an ideal method for the phylogenetic analysis for M. tuberculosis.

Chromosome Mapping ; Chromosomes, Bacterial ; Gene Expression Regulation, Bacterial ; Genome, Bacterial ; Genotype ; Multilocus Sequence Typing ; methods ; Mycobacterium tuberculosis ; genetics ; metabolism

OBJECTIVETo analyze molecular and evolution characteristics of Salmonella Paratyphi A isolates from 2000 to 2008, China.

METHODSUsing pulsed-field gel electrophoresis (PFGE) method with SpeI restriction enzyme, and multilocus sequence typing (MLST) method based on housekeeping genes (aroC, thrA, hisD, purE, sucA, dnaN, hemD, adk, and purA), the genomic variations of 118 Salmonella Paratyphi A isolates from 10 regions during 2000 to 2008 were analyzed.

RESULTSUsing PFGE method, 118 Salmonella Paratyphi A isolates were clustered into 32 PFGE patterns, and 5 patterns were predominant (5 isolates or above). However, only 2 MLST types were identified for all isolates with MLST method. Among all Salmonella Paratyphi A isolates, the sequences of housekeeping genes were highly conservative and showed a high degree of cloning.

CONCLUSIONFor Chinese epidemic Salmonella Paratyphi A isolates during 2000 - 2008, MLST method showed low discrimination power and the MLST method should not be applied to outbreak and epidemiological surveillance of Salmonella Paratyphi A. Currently, nationwide paratyphoid fever epidemics is caused by highly clonal isolates in China. As the time changes, these isolates also accumulate sporadic mutations.

Bacterial Typing Techniques ; China ; DNA, Bacterial ; genetics ; Electrophoresis, Gel, Pulsed-Field ; methods ; Humans ; Multilocus Sequence Typing ; Paratyphoid Fever ; epidemiology ; microbiology ; Salmonella paratyphi A ; classification ; genetics ; isolation & purification ; Sequence Analysis, DNA ; Serotyping

BACKGROUND: We evaluated the efficacy of multilocus sequence typing (MLST) for assessing the genetic relationship among Candida albicans isolates from patients with candidemia in a hospital setting. METHODS: A total of 45 C. albicans isolates from 21 patients with candidemia were analyzed. The MLST results were compared with results obtained by Southern blot hybridization (C1 fingerprinting) and pulsed-field gel electrophoresis (PFGE). PFGE analysis included karyotyping and restriction endonuclease analysis of genomic DNAs using BssHII (REAG-B) and SfiI (REAG-S). RESULTS: The 45 isolates yielded 20 unique diploid sequence types (DSTs) by MLST, as well as 12 karyotypes, 15 REAG-B patterns, 13 REAG-S patterns, and 14 C1 fingerprinting types. Microevolution among intra-individual isolates was detected in 6, 5, 3, 5, and 7 sets of isolates by MLST (1 or 2 allelic differences), REAG-B, REAG-S, C1 fingerprinting, and a combination of all methods, respectively. Among 20 DSTs, 17 were unique, and 3 were found in more than 1 patient. The results of 2 DSTs obtained from 9 patient isolates were in agreement with REAG and C1 fingerprinting patterns. However, the remaining DST, which was shared by 2 patient isolates, showed 2 different PFGE and C1 fingerprinting patterns. In addition, 3 sets of isolates from different patients, which differed in only 1 or 2 alleles by MLST, also exhibited different PFGE or C1 fingerprinting patterns. CONCLUSIONS: MLST is highly discriminating among C. albicans isolates, but it may have some limitations in typing isolates from different patients, which may necessitate additional analysis using other techniques.
Alleles ; Blotting, Southern ; Candida albicans/*classification/genetics/isolation & purification ; Candidemia/*microbiology ; DNA, Fungal/*analysis ; Electrophoresis, Gel, Pulsed-Field ; Genotype ; Humans ; Karyotyping ; Multilocus Sequence Typing/*methods

Propionibacterium acnes, a gram-positive, anaerobic, and aerotolerant bacterium that is found frequently in the skin as part of the human microbiome causes inflammatory acne, shoulder infection, and the contamination of medical devices. The study goals were the antibiotic resistant and molecular epidemiological characterization of the P. acnes isolates in Korea. A total of 22 P. acnes isolates originated from diverse patients were obtained from three National Culture Collections for Pathogens in South Korea. The hemolytic properties and minimum inhibitory concentrations (MIC) of five antibiotics (tetracycline, doxycycline, clindamycin, erythromycin, and minocycline) were determined. Only one isolate showed high MIC values and resistance to all five antibiotics. Genotypic characterization was achieved by multilocus sequence typing (MLST) for eight loci (aroE, guaA, tly, camp2, atpD, gmk, lepA, and sodA) and repetitive-sequence-based PCR (rep-PCR) analysis using the DiversiLab kit. MLST revealed four phylogroups that were type IA₁ (27.3%), type IA₂ (18.2%), type IB (13.6%), and type II (40.9%). Rep-PCR results demonstrated three clusters that were cluster I (39.1%), cluster II (45.5%), and cluster III (13.6%). The isolates of cluster I were part of phylogroup type IA (both IA₁ and IA₂), and the isolates of cluster II belonged to phylogroup type II. All isolates of phylogroup type IB were hemolytic and belonged to cluster III. The results of rep-PCR clustering analysis showed a good correlation with those of MLST phylogroups, suggesting that rep-PCR could be an alternative method to track P. acnes subtype lineages.
Acne Vulgaris ; Anti-Bacterial Agents ; Clindamycin ; Doxycycline ; Erythromycin ; Humans ; Korea ; Methods ; Microbial Sensitivity Tests ; Microbiota ; Multilocus Sequence Typing* ; Polymerase Chain Reaction* ; Propionibacterium acnes* ; Propionibacterium* ; Shoulder ; Skin

BACKGROUND: From January 2014 to December 2015, 69 clones of Enterobacter cloacae showing multidrug resistance to six classes of antimicrobial agents were collected from two medical centers in Korea. METHODS: Minimum inhibitory concentrations were determined using the E-test method, and 17 genes were detected using polymerase chain reaction (PCR). The epidemiological relatedness of the strains was identified using repetitive element sequence-based PCR and multilocus sequence typing. RESULTS: The 69 E. cloacae clones produced extended spectrum β lactamase (ESBL) and AmpC and showed multidrug resistance to cefotaxime, ceftazidime, and aztreonam. We identified the following sequence types: ST56 of type VI for ESBL SHV (N=12, 17.4%); ST53, ST114, ST113, and ST550 of types I, IV, VI, and VII, respectively, for CTX-M (N=11, 15.9%); and ST668 of type III for the carbapenemase NDM gene (N=1, 1.5%). The AmpC DHA gene (N=2, 2.89%) was confirmed as ST134, although its type was not identified, whereas EBC (MIR/ACT; N=18, 26.1%) was identified as ST53, ST24, ST41, ST114, ST442, ST446, ST484, and ST550 of types V, I, III, IV, VII, and VI, respectively. The formed subclasses were bla CTX-M-3 and bla CTX-M-22 by CTX-M-1, bla CTX-M-9 and bla CTX-M-125 by CTX-M-9, bla DHA-1 by DHA, and bla MIR-7 and bla ACT-15,17,18,25,27,28 by EBC (MIR/ACT). CONCLUSIONS: There were no epidemiological relationships between the gene products and the occurrence of resistance among the strains.
Anti-Infective Agents ; Aztreonam ; Cefotaxime ; Ceftazidime ; Cloaca ; Clone Cells ; Drug Resistance, Multiple* ; Enterobacter cloacae* ; Enterobacter* ; Korea ; Methods ; Microbial Sensitivity Tests ; Multilocus Sequence Typing ; Polymerase Chain Reaction

BACKGROUNDStreptococcus pneumoniae (S. pneumoniae) is a major causative agent of severe infections, including sepsis, pneumonia, meningitis, and otitis media, and has become a major public health concern. We report the pneumococcal serotype and sequence type (ST) distribution, and antimicrobial resistance of 39 S. pneumoniae strains from seven hospitals in China.

METHODSBlood/cerebrospinal fluid (CSF) and sputum isolates from patients were analyzed to determine S. pneumoniae serotypes by polymerase chain reaction (PCR) and the Neufeld Quellung reaction, the multilocus sequence types (MLST) by PCR and sequencing, and susceptibility to antimicrobial agents by the VITEK Gram Positive Susceptibility Card.

RESULTSA total of 39 isolates were collected including 21 blood/CSF and 18 sputum isolates. Conventional serotyping by the Quellung reaction required 749 reactions. In contrast, PCR based typing needed only 106 PCR reactions. The most frequent serotypes from the blood/CSF isolates were 14 (38.1%), 19A (14.3%), 23F (9.5%), and 18C (9.5%). In the sputum isolates the most frequent serotypes were 19F (33.3%), 23F (16.7%), 19A (11.1%), and 3 (11.1%). The incidence of penicillin resistance in the blood/CSF and sputum isolates was 66.7% and 55.6%, respectively. Statistical analysis showed that patients = 5 years old had a higher resistance to penicillin when they compared with the patients = 65 years old (P = 0.011). Serotypes 14, 19A and 19F were significantly associated with penicillin resistance (P < 0.001). ST320, ST271, and ST876 isolates showed high resistant rates to several antibiotics including penicillin (P = 0.006). All of the isolates of serotype 19A were resistant to both penicillin and erythromycin, and they were all multi-drug resistant (MDR) isolates.

CONCLUSIONSThe specificity and sensitivity of multiplex-PCR are good, and this method represents a substantial savings of time and money, and can be widely used in the laboratory and clinical practice. Data from this research showed an extremely high prevalence of penicillin resistance and an increasing prevalence of multi-drug resistant (MDR) rate in S. pneumoniae. A distinctive emergence of serotype 19A was observed which was also associated with the increasing prevalence of antimicrobial resistance. Therefore, nationwide surveillance of pneumococcal resistance and serotypes is strongly warranted.

Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; Drug Resistance, Multiple, Bacterial ; Humans ; Infant ; Microbial Sensitivity Tests ; Middle Aged ; Molecular Typing ; methods ; Multilocus Sequence Typing ; methods ; Pneumococcal Infections ; microbiology ; Serotyping ; Streptococcus pneumoniae ; classification ; drug effects