No Abstract Available.
Clostridium* ; Molecular Typing*

OBJECTIVE: A core genome multilocus sequence typing (cgMLST) scheme to genotype and identify potential risk clonal groups (CGs) in Proteus mirabilis. METHODS: In this work, we propose a publicly available cgMLST scheme for P. mirabilis using chewBBACA. In total 72 complete P. mirabilis genomes, representing the diversity of this species, were used to set up a cgMLST scheme targeting 1,842 genes, 635 unfinished (contig, chromosome, and scaffold) genomes were used for its validation. RESULTS: We identified a total of 205 CGs from 695 P. mirabilis strains with regional distribution characteristics. Of these, 159 unique CGs were distributed in 16 countries. CG20 and CG3 carried large numbers of shared and unique antibiotic resistance genes. Nine virulence genes ( papC, papD, papE, papF, papG, papH, papI, papJ, and papK) related to the P fimbrial operon that cause severe urinary tract infections were only found in CG20. These CGs require attention due to potential risks. CONCLUSION This research innovatively performs high-resolution molecular typing of P. mirabilis using whole-genome sequencing technology combined with a bioinformatics pipeline (chewBBACA). We found that the CGs of P. mirabilis showed regional distribution differences. We expect that our research will contribute to the establishment of cgMLST for P. mirabilis.
Genome, Bacterial ; Proteus mirabilis/genetics* ; Multilocus Sequence Typing ; Molecular Epidemiology ; Genotype

PURPOSE: We investigated the molecular types of uropathogenic Escherichia coli (UPEC) by using conventional phylogrouping, multilocus sequence typing (MLST), and fimH genotyping. METHODS: Samples of patients younger than 18 years of age were collected from the Chung-Ang University Hospital over 2 years. Conventional phylogenetic grouping for UPEC strains was performed by polymerase chain reaction (PCR). Bacterial strain sequence types (STs) were classified on the basis of the results of partial sequencing of seven housekeeping genes. In addition, we analyzed nucleotide variations in a 424-base pair fragment of fimH, a major virulence factor in UPEC. RESULTS: Sixty-four UPEC isolates were analyzed in this study. Phylogenetic grouping revealed that group B2 was the most common type (n=54, 84%). We identified 16 distinctive STs using MLST. The most common STs were ST95 (35.9%), ST73 (15.6%), ST131 (12.5%), ST69 (7.8%), and ST14 (6.3%). Fourteen fimH allele types were identified, of which 11 had been previously reported, and the remaining three were identified in this study. f1 (n=28, 45.2%) was found to be the most common allele type, followed by f6 and f9 (n=7, 11.3% each). Comparative analysis of the results from the three different molecular typing techniques revealed that both MLST and fimH typing generated more discriminatory UPEC types than did PCR-based phylogrouping. CONCLUSION: We characterized UPEC molecular types isolated from Korean children by MLST and fimH genotyping. fimH genotyping might serve as a useful molecular test for large epidemiologic studies of UPEC isolates.
Alleles ; Child* ; Epidemiologic Studies ; Genes, Essential ; Humans ; Molecular Typing* ; Multilocus Sequence Typing ; Phylogeny ; Polymerase Chain Reaction ; Urinary Tract Infections* ; Uropathogenic Escherichia coli* ; Virulence

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

BACKGROUND: Multilocus sequence typing (MLST) is useful in determining the long-term evolutionary process and minimizes differences in experimental results across individuals and laboratories. It is also useful in determining evolutionary origins and backgrounds of bacterial species. This study carries out MLST analysis on VanA-type vancomycin-resistant Enterococcus faecium isolated from patient specimens in a single university hospital over nine years in order to observe changes in genetic evolution over time. METHODS: During the years from 2007 to 2015, 44 clinical isolates of vanA-containing E. faecium were collected from Ajou University Hospital in Korea. Species were identified by the VitekII system (bio-Merieux, USA), and antibiotic susceptibility testing was performed by disk diffusion and E-test according to Clinical and Laboratory Standards Institute (CLSI) guidelines. To determine genetic relatedness, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF M/S) was employed. To characterize clonal diversity, MLST analysis was used. RESULTS: All isolates were highly resistant to ampicillin, ciprofloxacin, and vancomycin but showed variable levels of resistance to teicoplanin. The 44 clinical isolates were genetically unrelated according to MALDI-TOF M/S analysis. MLST showed that the clinical isolates harbored 6 sequence types (ST), with ST17 (n=19) being the most common, followed by ST78 (n=13), ST192 (n=6), ST64 (n=4), ST262 (n=1), and ST414 (n=1). CONCLUSION: The MLST analysis showed that the sequence types of most isolates belonged to clonal complex 17 This is consistent with outbreaks in hospitals. We had single observations for ST262 and ST414, suggesting that they were random occurrences. MLST can be useful for speculating the genetic evolution of VanA-containing E. faecium isolates.
Ampicillin ; Ciprofloxacin ; Diffusion ; Disease Outbreaks ; Enterococcus faecium* ; Enterococcus* ; Evolution, Molecular ; Humans ; Korea ; Mass Spectrometry ; Multilocus Sequence Typing* ; Teicoplanin ; Vancomycin

PURPOSE: Bacillus cereus has been reported as the cause of nosocomial infections in cancer patients. In our pediatric cancer ward, a sudden rise in the number of patients with B. cereus bacteremia was observed in 2013 to 2014. This study was performed to investigate the molecular epidemiology of increased B. cereus bacteremia cases in our center. METHODS: Pediatric cancer patients who developed B. cereus bacteremia were identified from January 2001 to June 2014. The B. cereus bacteremia in this study was defined as a case in which at least one B. cereus identified in blood cultures, regardless of true bacteremia. Available isolates were further tested by multilocus sequence typing (MLST) analysis. A retrospective chart review was performed. RESULTS: Nineteen patients developed B. cereus bacteremia during the study period. However, in 2013, a sudden increase in the number of patients with B. cereus bacteremia was observed. In addition, three patients developed B. cereus bacteremia within 1 week in July and the other three patients within 1 week in October, respectively, during emergency room renovation. However, MLST analysis revealed different sequence types without consistent patterns. Before 2013, five tested isolates were ST18, ST26, ST177, and ST147-like type, and ST219-like type. Isolates from 2013 were ST18, ST73, ST90, ST427, ST784, ST34-like type, and ST130-like type. CONCLUSIONS: MLST analyses showed variable ST distribution of B. cereus isolates. Based on this study, there was no significant evidence suggesting a true outbreak caused by a single ST among patients who developed B. cereus bacteremia.
Bacillus cereus* ; Bacillus* ; Bacteremia ; Cross Infection ; Disease Outbreaks ; Emergency Service, Hospital ; Humans ; Molecular Epidemiology* ; Multilocus Sequence Typing ; Pediatrics ; Retrospective Studies

OBJECTIVETo characterize the genetic background of multidrug-resistant Acinetobacter baumannii (A. baumannii) from China, and the population structure of this pathogen.

METHODSA previously reported MLST scheme was applied to a collection of 33 multidrug-resistant strains of A. baumannii from China, and the data of all the strains in the A. baumannii MLST database were downloaded for the population structure analysis. The sequence types and clonal complexes were identified, the presence or absence of recombination was analyzed for each MLST locus, and the values of I(A)(S), and recombination/mutation ratio were calculated for the whole strain collection. A phylogenetic tree was constructed using all the allelic profiles in the database.

RESULTSA total of six sequence types were identified from the 33 Chinese strains tested, and 29 of these strains belonged to the CC92 clonal complex. Three (gdhB, gpi, and rpoD) of the seven MLST loci (gltA, gyrB, recA, cpn60, gdhB, gpi, rpoD) had undergone recombination with statistical evidence. For all allele profiles in the MLST database, the I(A)(S) value was 0.155 and the recombination/mutation ratio was 6.083. Sequence types from each clonal complex were grouped closely in the phylogenetic tree, which gave an overview of the microevolution of this pathogen.

CONCLUSIONThe spread of multidrug-resistant A. baumannii in China was closely related to the CC92 clonal complex. A. baumannii had an 'epidemic' population structure, i.e., a superficially clonal structure with high levels of recombination, in which successful epidemic clones arise especially including worldwide dissemination of the CC92 clonal complex to cause a widespread occurrence of multidrug-resistant infections.

Acinetobacter baumannii ; classification ; genetics ; isolation & purification ; Bacterial Typing Techniques ; China ; Cluster Analysis ; DNA, Bacterial ; genetics ; Drug Resistance, Multiple, Bacterial ; Genetic Variation ; Genetics, Population ; Molecular Epidemiology ; Molecular Sequence Data ; Multilocus Sequence Typing ; Phylogeny

BACKGROUND: To investigate the national molecular epidemiology and resistance profiles of vancomycin-intermediate Staphylococcus aureus (VISA), we analyzed the characteristics of methicillin-resistant Staphylococcus aureus (MRSA) collected from clinical samples at tertiary or general hospitals participating in a nationwide surveillance program for VISA and vancomycin-resistant Staphylococcus aureus (VRSA) in Korea during an 12-week period in each year from 2007 to 2013. METHODS: VISA was defined by agar dilution, broth dilution and E-test methods with vancomycin minimum inhibitory concentrations of >2 μg/mL. All VISA isolates were characterized by multilocus sequence typing, staphylococcal cassette chromosome mec typing, spa typing, accessory gene regulator typing, Diversilab analysis, and antibiogram analysis. RESULTS: Of 109,345 MRSA isolates, 87,354 were screened and 426 isolates were identified as positive on brain heart infusion agar containing 4 μg/mL vancomycin (BHI-V4). Of 426 isolates, 76 isolates were identified as VISA. No VRSA isolates were detected among the isolates. Overall, a total of 6 genotypes were identified among VISA strains and the predominant clones were ST5-II-t2460, ST72-IV-t324, and ST239-III-t037 (44.7%, 15.8%, and 10.5%, respectively). Of note, ST72-IV-t324 clones are known to be a typical community-associated MRSA. ST239-III-t037 strains were more resistant to trimethoprim-sulfamethoxazole than any other type of strain. ST72-IV-t324 strains were susceptible to all of the antimicrobial agents tested except erythromycin and daptomycin. All of the VISA isolates were susceptible to linezolid and quinupristin-dalfopristin. CONCLUSION: Although VRSA is still rare, continuous monitoring of VRSA occurrence is needed, as well as VISA prevalence, epidemic clonal shift, and antimicrobial resistance.
Agar ; Anti-Infective Agents ; Brain ; Clone Cells ; Daptomycin ; Erythromycin ; Genotype ; Heart ; Hospitals, General ; Korea* ; Linezolid ; Methicillin-Resistant Staphylococcus aureus ; Microbial Sensitivity Tests ; Molecular Epidemiology ; Molecular Typing* ; Multilocus Sequence Typing ; Prevalence ; Staphylococcus aureus* ; Staphylococcus* ; Trimethoprim, Sulfamethoxazole Drug Combination ; Vancomycin

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

BACKGROUND: We investigated the usefulness of infrequent-restriction-site polymerase chain reaction (IRS-PCR) compared with pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) on the molecular epidemiologic analysis of methicillin-resistant Staphylococcus aureus (MRSA). MATERIALS AND METHODS: We used fifty clinical isolates of MRSA collected from 10 university hospitals located in Seoul. We performed three procedures on these isolates: PFGE using SmaI, IRS-PCR using XbaI-Hha I or EagI-Hha I, and MLST using seven house-keeping genes. We determined the clusters of molecular types by dendrogram using the unweighted pair group method with arithmetic mean (UPGMA) and Dice coefficients. RESULTS: MLST analysis showed that isolates exhibited ST1, ST5, ST72, ST89, and ST239. In PFGE, the isolates clustered into 5 major groups with 80% similarity, which subsequently became classified into 18 subgroups with 95% similarity. In IRS-PCR using EagI-HhaI restriction enzymes, there was little resolution among the patterns of isolates. However, XbaI-HhaI IRS-PCR showed 5 groups with a 90% similarity. These groups were then classified into 9 subgroups with a 95% similarity. There were no significant differences among the isolates from different hospitals. CONCLUSIONS: The XbaI-HhaI IRS-PCR method could be a useful tool in the molecular epidemiology of MRSA. Its resolution power was good enough to analyze isolates, because the patterns of IRS-PCR were closely correlated with those of MLST and showed diverse groups.
Electrophoresis, Gel, Pulsed-Field ; Genes, Essential ; Hospitals, University ; Methicillin ; Methicillin Resistance ; Methicillin-Resistant Staphylococcus aureus ; Molecular Epidemiology ; Multilocus Sequence Typing ; Polymerase Chain Reaction ; Staphylococcus ; Staphylococcus aureus