Comparison of four methods, including semi-automated rep-PCR, for the typing of vancomycin-resistant Enterococcus faecium

We have assessed the performance of semi-automated rep-PCR (Diversilab®) and multilocus sequence typing (MLST) in comparison to pulsed-field gel electrophoresis (PFGE) for typing a collection of 29 epidemiologically characterized vancomycin-resistant Enterococcus faecium (VRE). Sixteen strains that harbored the Tn1546 element were typed by PCR mapping. The discriminative power of the typing methods was calculated by the Simpson's index of diversity, and the concordance between methods was evaluated by the Kendall's coefficient of concordance. Semi-automated rep-PCR appeared as discriminative as PFGE and was further compared with PFGE for typing 67 VRE isolated during a hospital outbreak. Rep-PCR appeared to be more discriminative than PFGE for this second set of strains. Reproducibility of DiversiLab® was also tested against 35 selected isolates. Only three showed less than 97% similarity, indicating high reproducibility at this level of discrimination. In conclusion, semi-automated rep-PCR is a useful tool for rapid screening of VRE isolates during an outbreak, although cost of the system may be limiting for routine implementation. PFGE, which remains the reference method, should be used for confirmation and evaluation of the genetic relatedness of epidemic isolates.     

Clonal spread of carbapenem resistant Acinetobacter baumannii ST92 in a Chinese Hospital during a 6-year period

The carbapenem resistance rate of Acinetobacter baumannii in our hospital has increased steadily since 2004. The molecular epidemiology of carbapenem resistant A. baumannii (CRAB) clinical isolates was characterized by multilocus sequence typing (MLST) and rep-PCR in parallel, with pan-drug susceptible A. baumannii (PSAB) used as control. MLST was performed to determine the sequence types (STs), and eBURST algorithm was used to analyze their relatedness. Carbapenem resistance related genes (oxa-23, oxa-24, oxa-51, oxa-58, imp, vim, and adeB) were screened using Polymerase Chain Reaction (PCR) method. 23 STs were identified in the 65 included isolates, with ST92 being the predominant clone. PSAB clustered into more singletons than CRAB. The positivity of oxa-23 and adeB correlated with high level carbapenem resistance (MIC_IPM>32 mg/L, MIC_MEM >32 mg/L) of CRAB ST92 isolates in 2009, which was different from the resistance pattern (MIC_IPM≤4 mg/L, 8 mg/L ≤MIC_MEM≤16 mg/L) of CRAB ST92 isolates in 2004. These observations suggest that clonal spread of CRAB ST92 isolates longitudinally is the possible reason for carbapenem resistance rate increase and correlate with high level carbapenem resistance in our hospital.     

Characterization of Staphylococcus aureus isolates from buffalo, bovine, ovine, and caprine milk samples collected in Rio de Janeiro State, Brazil

Eighty-four staphylococcal isolates were obtained from milk samples from cows, sheep, goats, and buffalo with subclinical mastitis and from colonization samples from ostriches. The animals were hosted in 18 small dairy herds and an ostrich breeding located in 10 municipalities of the state of Rio de Janeiro, Brazil. Thirty isolates were identified as Staphylococcus aureus by biochemical and molecular techniques and were comparatively characterized by phenotypic and genotypic methods. The molecular characterization by pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST) revealed five clonal types (PFGE A, spa type t359, sequence type 747 [ST747]; PFGE B, spa type t1180, ST750; PFGE C, spa type t605, ST126; PFGE D, spa type t127, ST751; and PFGE F, spa type t002, ST5). None of the isolates harbored the Panton-Valentine leukocidin or exfoliative toxin D gene. The detection of major clone A (in 63% of the isolates) in different herds, among all animal species studied, and in infection and colonization samples evidenced its geographical spread among Rio de Janeiro State and no host preference among the animal species. Comparison with S. aureus from a human origin suggested that all but one clone found in the present study might be animal specific.     

Analysis of the Pan Genome of <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> Isolates Recovered from Poultry by Pulsed-Field Gel Electrophoresis,Multilocus Sequence Typing (MLST), and Repetitive Sequence Polymerase Chain Reaction (rep-PCR) Reveals Different Discriminatory Capabilities

Campylobacter jejuni is one of the leading bacterial causes of food-borne illness in the USA. Molecular typing methods are often used in food safety for identifying sources of infection and pathways of transmission. Moreover, the identification of genetically related isolates (i.e., clades) may facilitate the development of intervention strategies for control and prevention of food-borne diseases. We analyzed the pan genome (i.e., core and variable genes) of 63 C. jejuni isolates recovered from chickens raised in conventional, organic, and free-range poultry flocks to gain insight into the genetic diversity of C. jejuni isolates recovered from different environments. We assessed the discriminatory power of three genotyping methods [i.e., pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and repetitive extragenic palindromic polymerase chain reaction (rep-PCR)]. The rep-PCR fingerprint was generated by determining the presence of repetitive sequences that are interspersed throughout the genome via repetitive extragenic palindromic PCR, enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), and BOX element PCR (BOX-PCR) and combining the data to form a composite fingerprint. The genetic fingerprints were subjected to computer-assisted pattern analysis. Comparison of the three genotypic methods revealed that repREB-PCR showed greater discriminatory power than PFGE and MLST. ERIC-PCR and BOX-PCR yielded the highest number of PCR products and greatest reproducibility. Regardless of the genotyping method, C. jejuni isolates recovered from chickens reared in conventional, organic, and free-range environments all exhibit a high level of genotypic diversity.     

Optimization of analytical parameters for inferring relationships among Escherichia coli isolates from repetitive-element PCR by maximizing correspondence with multilocus sequence typing data

Repetitive-element PCR (rep-PCR) is a method for genotyping bacteria based on the selective amplification of repetitive genetic elements dispersed throughout bacterial chromosomes. The method has great potential for large-scale epidemiological studies because of its speed and simplicity; however, objective guidelines for inferring relationships among bacterial isolates from rep-PCR data are lacking. We used multilocus sequence typing (MLST) as a "gold standard" to optimize the analytical parameters for inferring relationships among Escherichia coli isolates from rep-PCR data. We chose 12 isolates from a large database to represent a wide range of pairwise genetic distances, based on the initial evaluation of their rep-PCR fingerprints. We conducted MLST with these same isolates and systematically varied the analytical parameters to maximize the correspondence between the relationships inferred from rep-PCR and those inferred from MLST. Methods that compared the shapes of densitometric profiles ("curve-based" methods) yielded consistently higher correspondence values between data types than did methods that calculated indices of similarity based on shared and different bands (maximum correspondences of 84.5% and 80.3%, respectively). Curve-based methods were also markedly more robust in accommodating variations in user-specified analytical parameter values than were "band-sharing coefficient" methods, and they enhanced the reproducibility of rep-PCR. Phylogenetic analyses of rep-PCR data yielded trees with high topological correspondence to trees based on MLST and high statistical support for major clades. These results indicate that rep-PCR yields accurate information for inferring relationships among E. coli isolates and that accuracy can be enhanced with the use of analytical methods that consider the shapes of densitometric profiles.     

Characterization of Staphylococcus aureus Isolates from Buffalo, Bovine, Ovine, and Caprine Milk Samples Collected in Rio de Janeiro State, Brazil

Eighty-four staphylococcal isolates were obtained from milk samples from cows, sheep, goats, and buffalo with subclinical mastitis and from colonization samples from ostriches. The animals were hosted in 18 small dairy herds and an ostrich breeding located in 10 municipalities of the state of Rio de Janeiro, Brazil. Thirty isolates were identified as Staphylococcus aureus by biochemical and molecular techniques and were comparatively characterized by phenotypic and genotypic methods. The molecular characterization by pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST) revealed five clonal types (PFGE A, spa type t359, sequence type 747 [ST747]; PFGE B, spa type t1180, ST750; PFGE C, spa type t605, ST126; PFGE D, spa type t127, ST751; and PFGE F, spa type t002, ST5). None of the isolates harbored the Panton-Valentine leukocidin or exfoliative toxin D gene. The detection of major clone A (in 63% of the isolates) in different herds, among all animal species studied, and in infection and colonization samples evidenced its geographical spread among Rio de Janeiro State and no host preference among the animal species. Comparison with S. aureus from a human origin suggested that all but one clone found in the present study might be animal specific.     

A comparative evaluation of five typing techniques for determining the diversity of fluorescent pseudomonads

Five typing methods were evaluated, utilising 63 strains of fluorescent pseudomonads, to assess their usefulness as tools to study the bacterial diversity within this complex group. The methods used were Biolog metabolic profiling, restriction fragment length polymorphism ribotyping, PCR ribotyping, and repetitive element sequence-based PCR (rep-PCR) utilising BOX and enterobacterial repetitive intergenic consensus (ERIC) primers. Cluster analysis of the results clearly demonstrated the considerable homogeneity of Pseudomonas aeruginosa isolates and, conversely, the heterogeneity within the other species, in particular P. putida and P. fluorescens, which need further taxonomic investigation. Biolog metabolic profiling enabled the best differentiation among the species. Rep-PCR proved to be highly discriminatory, more so than the other DNA fingerprinting techniques, demonstrating its suitability for the analysis of highly clonal isolates. RFLP ribotyping, PCR ribotyping, and rep-PCR produced specific clusters of P. aeruginosa isolates, which corresponded to their origins of isolation, hence we recommend these methods for intraspecific typing of bacteria.     

Optimization of Analytical Parameters for Inferring Relationships among Escherichia coli Isolates from Repetitive-Element PCR by Maximizing Correspondence with Multilocus Sequence Typing Data

Repetitive-element PCR (rep-PCR) is a method for genotyping bacteria based on the selective amplification of repetitive genetic elements dispersed throughout bacterial chromosomes. The method has great potential for large-scale epidemiological studies because of its speed and simplicity; however, objective guidelines for inferring relationships among bacterial isolates from rep-PCR data are lacking. We used multilocus sequence typing (MLST) as a “gold standard” to optimize the analytical parameters for inferring relationships among Escherichia coli isolates from rep-PCR data. We chose 12 isolates from a large database to represent a wide range of pairwise genetic distances, based on the initial evaluation of their rep-PCR fingerprints. We conducted MLST with these same isolates and systematically varied the analytical parameters to maximize the correspondence between the relationships inferred from rep-PCR and those inferred from MLST. Methods that compared the shapes of densitometric profiles (“curve-based” methods) yielded consistently higher correspondence values between data types than did methods that calculated indices of similarity based on shared and different bands (maximum correspondences of 84.5% and 80.3%, respectively). Curve-based methods were also markedly more robust in accommodating variations in user-specified analytical parameter values than were “band-sharing coefficient” methods, and they enhanced the reproducibility of rep-PCR. Phylogenetic analyses of rep-PCR data yielded trees with high topological correspondence to trees based on MLST and high statistical support for major clades. These results indicate that rep-PCR yields accurate information for inferring relationships among E. coli isolates and that accuracy can be enhanced with the use of analytical methods that consider the shapes of densitometric profiles.     

Molecular typing of Vibrio parahaemolyticus isolated from seafood harvested along the south-west coast of India

Aims: Evaluation of protein profiling for typing Vibrio parahaemolyticus using 71 strains isolated from different seafood and comparison with other molecular typing techniques such as random amplified polymorphic DNA analysis (RAPD) and enterobacterial repetitive intergenic consensus sequence (ERIC)‐PCR. Methods and Results: Three molecular typing methods were used for the typing of 71 V. parahaemolyticus isolates from seafood. RAPD had a discriminatory index (DI) of 0·95, while ERIC‐PCR showed a DI of 0·94. Though protein profiling had less discriminatory power, use of this method can be helpful in identifying new proteins which might have a role in establishment in the host or virulence of the organism. Conclusions: The use of protein profiling in combination with other established typing methods such as RAPD and ERIC‐PCR generates useful information in the case of V. parahaemolyticus associated with seafood. Significance and Impact of the Study: The study demonstrates the usefulness of nucleic acid and protein‐based studies in understanding the relationship between various isolates from seafood.     

Rep-PCR应用于快速鉴定啤酒污染菌的研究

为评价rep-PCR在快速鉴定啤酒污染菌中的应用,首先比较了DNA提取方法,确定CTAB法作为制备rep-PCR的DNA模板的方法。并通过PCR产物直接测序的方法,从分离菌中鉴定得到11种常见的啤酒污染菌。用BOXA1R和(GTG)_5引物扩增分离菌,采用Gel ComparⅡ软件处理电泳图,构建污染菌的标准指纹图库。经过聚类分析表明,BOXA1R和(GTG)5对Lactobacillusbrevis、L.buchneri、L.casei/paracasei、L.plantarum和L.fermentum的聚类效果具有互补性,并首次提出指纹比对快速鉴定的相似系数阈值的概念。对来自三个不同来源的9株乳酸菌的快速鉴定结果表明,rep-PCR鉴定技术简单、快速、可靠,在快速鉴定方面将具有重要的应用价值。     

The Molecular Epidemiology of the Highly Virulent ST93 Australian Community Staphylococcus aureus Strain

In Australia the PVL - positive ST93-IV [2B], colloquially known as "Queensland CA-MRSA" has become the dominant CA-MRSA clone. First described in the early 2000s, ST93-IV [2B] is associated with skin and severe invasive infections including necrotizing pneumonia. A singleton by multilocus sequence typing (MLST) eBURST analysis ST93 is distinct from other S aureus clones. To determine if the increased prevalence of ST93-IV [2B] is due to the widespread transmission of a single strain of ST93-IV [2B] the genetic relatedness of 58 S. aureus ST93 isolated throughout Australia over an extended period were studied in detail using a variety of molecular methods including pulsed-field gel electrophoresis, spa typing, MLST, microarray DNA, SCCmec typing and dru typing. Identification of the phage harbouring the lukS-PV/lukF-PV Panton Valentine leucocidin genes, detection of allelic variations in lukS-PV/lukF-PV, and quantification of LukF-PV expression was also performed. Although ST93-IV [2B] is known to have an apparent enhanced clinical virulence, the isolates harboured few known virulence determinants. All PVL-positive isolates carried the PVL-encoding phage ΦSa2USA and the lukS-PV/lukF-PV genes had the same R variant SNP profile. The isolates produced similar expression levels of LukF-PV. Although multiple rearrangements of the spa sequence have occurred, the core genome in ST93 is very stable. The emergence of ST93-MRSA is due to independent acquisitions of different dru-defined type IV and type V SCCmec elements in several spa-defined ST93-MSSA backgrounds. Rearrangement of the spa sequence in ST93-MRSA has subsequently occurred in some of these strains. Although multiple ST93-MRSA strains were characterised, little genetic diversity was identified for most isolates, with PVL-positive ST93-IVa [2B]-t202-dt10 predominant across Australia. Whether ST93-IVa [2B] t202-dt10 arose from one PVL-positive ST93-MSSA-t202, or by independent acquisitions of SCCmec-IVa [2B]-dt10 into multiple PVL-positive ST93-MSSA-t202 strains is not known.     

Bacteriocin Typing of Burkholderia (Pseudomonas) solanacearum Race 1 of the French West Indies and Correlation with Genomic Variation of the Pathogen

Burkholderia solanacearum race 1 isolates indigenous to the French West Indies were characterized by bacteriocin typing and two genomic fingerprinting methods: pulsed-field gel electrophoresis of genomic DNA digested by rare-cutting restriction endonucleases (RC-PFGE) and PCR with primers corresponding to repetitive extragenic palindromic (REP), enterobacterial repetitive intergenic consensus (ERIC), and BOX elements (collectively known as rep-PCR). The survey comprised 24 reference strains and 65 isolates obtained from a field trial in Guadeloupe in 1993. Comparison of the data identified RC-PFGE as the most discriminatory method, delineating 17 pulsed-field gel profile types. rep-PCR and bacteriocin typing identified nine rep-PCR profile types and nine bacteriocin groups. Independent determination of similarity coefficients and clustering of RC-PFGE and rep-PCR data identified six groups common to both sets of data that correlated to biovar and bacteriocin groups. Further study of bacteriocin production in planta gave results consistent with in vitro bacteriocin typing. It was observed that spontaneous bacteriocin-resistant mutants exhibited a cross-resistance to other bacteriocins as identified by the typing scheme and that such mutants possessed a selective advantage for growth over isogenic nonmutants in the presence of a bacteriocin. The results are significant in the search for biological control of disease by nonpathogenic mutants of the wild-type organism.     

Genome sequences of three Acinetobacter baumannii strains assigned to the multilocus sequence typing genotypes ST2, ST25, and ST78

Acinetobacter baumannii is an emerging opportunistic gram-negative pathogen responsible for hospital-acquired infections. A. baumannii epidemics described in Europe and worldwide were caused by a limited number of genotypic clusters of multidrug-resistant strains. Here, we report the availability of draft genome sequences for three multidrug-resistant A. baumannii strains assigned to multilocus sequence typing genotypes ST2, ST25, and ST78 that were more frequently isolated during outbreaks occurred in Greece, Italy, Lebanon, and Turkey.     

A practical method for subtyping of Streptococcus agalactiae serotype III, of human origin, using rolling circle amplification

Group B streptococcus (GBS; Streptococcus agalactiae) serotype III is one of the most common and virulent serotypes of the species. It can be divided into several subtypes, which vary in their distribution among invasive isolates from different patient groups. In this study, we used 91 well-characterized GBS serotype III isolates to compare three subtyping methods, and developed a novel padlock probe and rolling circle amplification (RCA) method to identify informative single nucleotide polymorphisms (SNPs) that define the major subtypes. There was good agreement between partial sequencing of the capsule polysaccharide synthesis (cps) gene cluster, a 3-set genotyping system and multilocus sequence typing (MLST). Serosubtype III-2/multilocus sequence type (ST)-17 represents a virulent clone which is significantly associated with late onset GBS neonatal infections. RCA provides a simple, reproducible method for rapid identification of the two most common GBS serotype III subtypes (III-1/ST-19 and III-2/ST-17).     

Application of Rep-PCR fingerprint in rapid identification of beer-spoilage bacteria

The application potential of rep-PCR in typing beer-spoilage isolates was studied. The effects of different factors, including DNA templates and primers, on the quality and reproducibility of fingerprints were investigated. The CATB protocol was shown to be the feasible method for DNA extraction. Primers BOXA1R and (GTG)₅ were used in rep-PCR, and the PCR products were sequenced to identify strains isolated from two breweries. Rep-PCR fingerprint profiles were obtained using GelCompar II software. Cluster analysis showed that the isolates belonging to Lactobacillus brevis, L. buchneri, L. casei/paracasei, and L. plantarum are divided into 2 or 3 subgroups. In addition, the two rep-PCR fingerprint profiles complemented each other in typing these isolates. By combining the similarity coefficient cut-off (SCC) of species, 9 unknown isolates were rapidly identified using both fingerprint databases. The results indicate that rep-PCR is a simple, reliable, and promising method for the rapid identification of beer-spoilage bacteria.     

The emerging methicillin-resistant Staphylococcus aureus ST398 clone can easily be typed using the Cfr9I SmaI-neoschizomer

Aim:  To establish a PFGE protocol using Cfr9I, neoschizomer of SmaI, for typing of Staphylococcus aureus isolates belonging to the emerging MRSA ST398 clone.
Methods and Results:  Staphylococcus aureus ST398 and non-ST398 isolates were analysed using the PFGE conditions recommended by the HARMONY consensus protocol. Genomic DNA of non-ST398 isolates could be digested with SmaI, XmaI (also a SmaI-neoschizomer) and Cfr9I. The DNA of SmaI-nontypeable ST398 isolates was partially resistant to XmaI, but could be digested with Cfr9I. By PCR-amplification/sequencing, the presence of a novel C5-cytosine methyltransferase gene ( sauST398M ) was detected in the ST398 isolates. The encoded enzyme, which shows high similarity with C5-cytosine methyltransferases that modify the CCCGGG recognition sequence, could be responsible for the different restriction results.
Conclusion:  SmaI-PFGE is regarded as the 'gold standard' for typing S. aureus . Because of different susceptibility of the GGGCCC recognition sites of the ST398 DNA against SmaI, XmaI and Cfr9I, the proposed protocol is a valuable tool for ST398 typing.
Significance and Impact of the Study:  The use of this protocol allows the comparison of results from SmaI-nontypeable isolates with S. aureus SmaI-PFGE databases and can be applied for outbreak investigations and traceability studies of this emerging MRSA clone.     

Comparison of a semi-automated rep-PCR system and multilocus sequence typing for differentiation of Salmonella enterica isolates

The accurate sub-typing of Salmonella enterica isolates is essential for epidemiological investigations and surveillance of Salmonella infections. Salmonella isolates are currently identified using the Kauffman-White serotyping scheme. Multilocus sequence typing (MLST) schemes have been developed for the major bacterial pathogens including Salmonella and have assisted in understanding the molecular epidemiology and population biology of these organisms. Recently, the DiversiLab rep-PCR system has been developed using micro-fluidic chips to provide standardized, semi-automated fingerprinting for pathogens including S. enterica. In the current study, 71 isolates of S. enterica, representing 21 serovars, were analyzed using MLST and the DiversiLab rep-PCR system. MLST was able to identify 31 sequence types (STs), while the DiversiLab system revealed 38 DiversiLab types (DTs). The rep-PCR distinguished isolates of different serovars and showed greater discriminatory power (0.95) than MLST typing (0.89). Rep-PCR exhibited 92% concordance with MLST and 90% with serotyping, while the concordance level of MLST typing with serotyping was 96%, representing a strong association. Comparison of rep-PCR profiles with those held in an online library database led to the accurate prediction of serovar in 63% of cases and resulted in inaccurate predictions for 10% of profiles. MLST and the rep-PCR system may provide useful additional informative techniques for the molecular identification of S. enterica. We conclude that the DiversiLab rep-PCR system may provide a rapid (less than 4 h) and standardized method for sub-typing isolates of S. enterica.     

Genetic and virulence characterization of <Emphasis Type="Italic">Streptococcus suis</Emphasis> type 2 isolates from swine in the provinces of Zhejiang and Henan,China

This study was aimed to examine the genetic characteristics of 44 Streptococcus suis type 2 (SS2) isolates and the virulence attributes of 23 representative isolates. Multilocus sequence typing revealed five sequence types (ST1, ST7, ST28, ST86, and ST162) with 19 isolates assigned to ST7 (43.2%), 14 to ST1 (31.8%), and 9 to ST28 (20.5%). PCR typing of the pilus gene clusters classified the isolates into three types: A (72.7%), B (22.7%), and N (4.5%). All isolates of pilus types A and N were assigned to the ST1 complex containing ST1, ST7, and ST86, while those of type B belonged to the ST27 complex comprising ST28 and ST162. Only two strains had the putative pathogenicity island 89-kb cluster (89K) and were of type N. The type B strains had a significantly lower adhesion, were more readily killed by macrophage, and had lower virulence to mice than those of types A and N. We conclude that SS2 strains of both ST1 and ST27 complexes, parallel to pilus types A and B, were prevalent in the pig populations in Zhejiang Province, and ST7 and ST1 strains were the predominant genotypes in the diseased pigs with pneumonia.     

Analysis of genomic diversity among photosynthetic stem-nodulating rhizobial strains from northeast Argentina

The genomic diversity among photosynthetic rhizobia from northeast Argentina was assessed. Forty six isolates obtained from naturally occurring stem and root nodules of Aeschynomene rudis plants were analyzed by three molecular typing methods with different levels of taxonomic resolution: repetitive sequence-based PCR (rep-PCR) genomic fingerprinting with BOX and REP primers, amplified 16S rDNA restriction analysis (ARDRA), and 16S-23S rDNA intergenic spacer-restriction fragment length polymorphism (IGS-RFLP) analysis. The in vivo absorption spectra of membranes of strains were similar in the near infrared region with peaks at 870 and 800 nm revealing the presence of light harvesting complex I, bacteriochlorophyll-binding polypeptides (LHI-Bchl complex). After extraction with acetone-methanol the spectra differed in the visible part displaying peaks belonging to canthaxanthin or spirilloxanthin as the main carotenoid complement. The genotypic characterization by rep-PCR revealed a high level of genomic diversity among the isolates and almost all the photosynthetic ones have identical ARDRA patterns and fell into one cluster different from Bradyrhizobium japonicum and Bradyrhizobium elkanii. In the combined analysis of ARDRA and rep-PCR fingerprints, 7 clusters were found including most of the isolates. Five of those contained only photosynthetic isolates; all canthaxanthin-containing strains grouped in one cluster, most of the other photosynthetic isolates were grouped in a second large cluster, while the remaining three clusters contained a few strains. The other two clusters comprising reference strains of B. japonicum and B. elkanii, respectively. The IGS-RFLP analysis produced similar clustering for almost all the strains. The 16S rRNA gene sequence of one representative isolate was determined and the DNA sequence analysis confirmed the position of photosynthetic rhizobia in a distinct phylogenetic group within the Bradyrhizobium rDNA cluster.     

Molecular characterization of Enterococcus faecium isolated from hospitalized patients in Korea

AIMS: Multilocus sequence typing (MLST) was performed for vancomycin-resistant Enterococcus faecium (VREF) from diverse geographical areas in Korea to obtain insights into the genetic relationships with other molecular profiles. To understand the diversity of lineages, vancomycin-susceptible E. faecium (VSEF) were included. METHODS AND RESULTS: A total of 60 E. faecium isolates were analysed by MLST and esp profile. Molecular typing of Tn1546 of 30 VREF strains was evaluated by overlapping PCR of Tn1546 and DNA sequencing. Seven sequence types (ST) were found among 30 VSEF isolates, and four STs were found among 30 VREF isolates. The types most frequently encountered were ST 78 (26 isolates) and ST 203 (16 isolates). Of the 60 E. faecium isolates, 35 isolates were positive for the esp gene. On molecular typing of Tn1546, all VREF isolates were divided into four main types. Strains with the same ST showed divergence in Tn1546 types and strains with the same Tn1546 type represented different STs. CONCLUSIONS: An association between Tn1546 typing and MLST was not found. SIGNIFICANCE AND IMPACT OF THE STUDY: These results suggest that the horizontal spread of Tn1546 between strains plays a major role in the dissemination of vancomycin resistance in Korea.