Brevundimonas diminuta is a lactose non-fermenting Gram-negative rod associated with infection in immunocompromised patients. In three patients from two general wards, B. diminuta was isolated in blood culture sample. The clinical features of the patients did not coincide with the blood culture result, and pseudo-outbreak was suspected. These isolated were biochemically identified as Brevundimonas diminuta, and 16S rRNA sequencing confirmed their identification. The PFGE result showed a single pattern, and their clonality was assumed.
Electrophoresis, Gel, Pulsed-Field ; Humans ; Immunocompromised Host ; Lactose ; Patients' Rooms

BACKGROUND: It is important to define a source of infection when outbreak of Legionella infections has occurred. The performance of a molecular strain typing method was evaluated for environmental and clinical isolates of Legionella pneumophila. METHODS: Thirteen environmental strains, eleven clinical isolates and one type strain (ATCC 33152) of Legionella pneumophila were used for the analysis of pulsed field gel electrophoresis. RESULTS: All 25 strains were discriminated into 21 types. Two strains isolated from different locations in a same building showed different types. Each two, four, and two strains were shown as the same PFGE patterns. CONCLUSIONS: Even though PFGE typing of Legionella pneumophila is excellent for strain differentiation, the same pattern does not necessarily indicate the same source of isolates.
Busan* ; Electrophoresis, Gel, Pulsed-Field ; Korea* ; Legionella pneumophila ; Legionella* ; Water*

Objective: To identify a suspected clustered Typhoid fever by whole genome sequencing(WGS) and pulsed field gel electrophoresis (PFGE) subtyping. Methods: The nature of the epidemic was determined by combination of subtyping results of isolates and epidemiological information. Results: Five S. typhimurium isolates showed identical PFGE patterns and almost the same whole genome sequence. Epidemiological survey showed that five cases had dined in the same restaurant on the same day. Conclusion: Combined with the longest incubation period of typhoid fever, molecular subtyping of pathogenic bacteria and the field epidemiological survey, it can be preliminarily determined that the five cases have common infection sources.
Electrophoresis, Gel, Pulsed-Field ; Epidemics ; Humans ; Typhoid Fever/microbiology*

OBJECTIVETo investigate the epidemiological and molecular typing features of the pathogenic Yersinia enterocolitica strains isolated in China,using pulsed field gel electrophoresis(PFGE) and standardized PFGE method as well as typing database of Yersinia enterocolitica.

METHODSPFGE analysis was performed as Laboratory Directions for molecular subtyping of Salmonella by PFGE (PulseNet,USA) with some modifications and the results of PFGE were analyzed by BioNumerics soft (Version 4.0, Applied Maths BVBA, Belium).

RESULTS114 O:3 Yersinia enterocolitica strains were typed by 25 patterns to have found that K6GN11C30012 (50 strains), K6GN11C30015(19 strains) and K6GN11C30016(10 strains) were the major patterns. K6GNllC30012 had 92.2% cluster similarity with K6GN11C30009-K6GN11C30023. This clone included 91.23% strains of 114 0:3 Yersinia enterocolitica strains. 51 0:9 Yersinia enterocolitica strains were typed by 14 patterns; K6GN11C90004 (22 strains) and K6GN11C90010 (13 strains)were the major patterns. K6GN11C90004 had 81.8% cluster similarity with K6GN11C90010 patterns. The major patterns of 0:3 and 0:9 serotypes were quite different.

CONCLUSIONO:3 Yersinia enterocolitica strains might originate from the same clone and had very few variation in different years and provinces but O:9 Yersinia enterocolitica strains from two different clones with some changes.

In order to develop a rapid and reliable method for B. cereus genotyping, factors influencing PFGE results, including preparation of bacterial cells embedded in agarose, lysis of embedded cells, enzymatic digestion of intact genomic DNA, and electrophoresis parameters allowing for reproducible and meaningful DNA fragment separation, were controlled. Optimal cellular growth (Luria-Bertani agar plates for 12-18 h) and lysis conditions (4 h incubation with 500 µg/mL lysozyme) produced sharp bands on the gel. Restriction enzyme NotI was chosen as the most suitable. Twenty-two isolates were analyzed by NotI digestion, using three electrophoretic parameters (EPs). The EP-a was optimal for distinguishing between isolates. The optimized protocol could be completed within 40 h which is a significant improvement over the previous methods.
Bacillus cereus ; genetics ; isolation & purification ; Bacteriological Techniques ; DNA, Bacterial ; chemistry ; genetics ; Electrophoresis, Gel, Pulsed-Field ; methods

OBJECTIVETo determine Cronobacter spp. contamination in infant and follow-up powdered formula in China.

METHODSAll of 2282 samples were collected from the retail markets in China from January 2012 to December 2012, and analyzed for Cronobacter spp. by the Chinese National Food Safety Standard. Characterization of the isolates was analyzed by pulsed-field gel electrophoresis (PFGE) with XbaI and SpeI restriction enzymes.

RESULTSCronobacter spp. strains were isolated from 25 samples, and the positive rates in infant powdered formulas and follow-up powdered formulas were 0.90% (10/1011) and 1.18% (15/1271), respectively. Analysis of variable data regarding different purchasing store formats, seasonality, and production locations as well as comparison of infant versus follow-up formulas did not reveal statistically significant factors. During the sampling period, one of six surveillance zones did exhibit a statistically significant trend towards higher positive rate. PFGE characterization of Cronobacter spp. to elucidate genetic diversity revealed only three pairs of Cronobacter spp. out of 25 having the same PFGE patterns.

CONCLUSIONThe current investigation indicated a lower positive rate of Cronobacter spp. in the powdered formula in China. This evidence suggested contamination originating from multiple different sources during the manufacturing process.

OBJECTIVETo develop a multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) assay for Acinetobacter pittii typing.

METHODSPolymorphic VNTRs were searched by Tandem Repeats Finder. The distribution and polymorphism of each VNTR locus were analyzed in all the A. pittii genomes deposited in the NCBI genome database by BLAST and were evaluated with a collection of 20 well-characterized clinical A. pittii strains and one reference strain. The MLVA assay was compared with pulsed-field gel electrophoresis (PFGE) for discriminating A. pittii isolates.

RESULTSTen VNTR loci were identified upon bioinformatic screening of A. pittii genomes, but only five of them showed full amplifiability and good polymorphism. Therefore, an MLVA assay composed of five VNTR loci was developed. The typeability, reproducibility, stability, discriminatory power, and epidemiological concordance were excellent. Compared with PFGE, the new optimized MLVA typing scheme provided the same and even greater discrimination.

CONCLUSIONCompared with PFGE, MLVA typing is a faster and more standardized alternative for studying the genetic relatedness of A. pittii isolates in disease surveillance and outbreak investigation.

OBJECTIVETo analyze the molecular types of Salmonella paratyphi A strains isolated in the recent years, and to construct the standard S. paratyphi A databank in the laboratory surveillance network PulseNet China.

METHODSS. paratyphi A isolates from 4 provinces were analyzed with the standard pulsed-field gel electrophoresis (PFGE) protocol used in PulseNet and their patterns compared. The databank was constructed with BioNumerics.

RESULTSEleven PFGE patterns were obtained, in which 3 predominant patterns were identifies with a similarity coefficient of 96.3%. The strains of these patterns, accounted for 86.5% of the analyzed strains, appeared in different provinces and years.

CONCLUSIONThe databank of S. paratyphi A was constructed and could be used in laboratory surveillance of S. paratyphi A in PulseNet China. From the analyses on molecular typing of the isolates, data suggested that the predominant strains might cause the epidemics in different regions.

BACKGROUND: Pulsed-field gel electrophoresis (PFGE) has been regarded a standard method for genotyping in epidemiologic studies. However, it is tedious and time-consuming to perform. Two alternative genotyping methods have recently been developed using the polymerase chain reaction (PCR):amplified fragment length polymorphism (AFLP) and infrequent restriction site-polymerase chain reaction (IRS-PCR). These methods have not yet been applied yet to common pathogens such as Staphylococcus aureus. The purpose of this study was to determine the applicability of AFLP and IRS-PCR for the genotyping of E. coli and S. aureus isolates. METHODS: We performed PFGE, AFLP, and IRS-PCR on clinical isolates of E. coli (n=27) and S. aureus (n=30). We assessed each method in terms of discriminatory power, quality, and efficiency. RESULTS: In E. coli, the discriminatory powers of IRS-PCR and AFLP were comparable to that of PFGE. PFGE discerned 24 (88.8%) out of 27 strains, IRS-PCR discerned 22 (81.5%) out of 27, and AFLP discerned 25 (92.6%) out of 27. In the case of S. aureus, PFGE discerned 27 (90%) out of 30 strains, while both IRA-PCR and AFLP discerned 12 (40%) out of 30. The test-ing took four days to complete with PFGE, two days with AFLP, and was completed within one day with IRS-PCR. IRS-PCR showed better resolution than both PFGE and AFLP. CONCLUSION: In cases of E. coli, AFLP and IRS-PCR could be good alternatives for epidemiologic typing, as they offer better efficiency and comparable discriminatory power to PFGE. On the other hand, IRS-PCR and AFLP do not seem to be suitable for the strain-to-strain differentiation of S. aureus.
Electrophoresis, Gel, Pulsed-Field* ; Escherichia coli* ; Escherichia* ; Hand ; Molecular Typing* ; Polymerase Chain Reaction ; Staphylococcus aureus* ; Staphylococcus*

BACKGROUND: No outbreak of Enterohemorrhagic Escherichia coli (EHEC) infection has occurred as a group in Korea. On July 2004, an outbreak of EHEC infection occurred in an elementary school in Gwangju metropolitan city. Epidemic investigation was undertaken to track the source of infection and the mode of transmission of EHEC. METHODS: All students and staffs of the elementary school were interviewed and completed questionnaires. We surveyed their clinical symptoms and the foods that they ate. Microbiologic examinations were also carried out on the above school-related persons and many environmental specimens. We also investigated the facilities of the school, some suppliers of food materials, and other associated institutions. All the EHEC-positive persons were isolated in 5 hospitals and tested everyday for verotoxin until they turned out to be negative twice in succession, and their family were also interviewed and tested for EHEC. Pulsed-field gel electrophoresis (PFGE) was performed to find out the genetic relationship between isolates. RESULTS: Of the 1,643 school-related persons, 77 persons (4.7%) were positive for EHEC. Most of them were asymptomatic. All the isolated strains were non-O157 EHEC. Serotype O91 was the most frequent serotype (68 isolates), and the isolates revealing O91 serotypes showed identical PFGE patterns. The school meal was significantly associated with this outbreak (relative risk=13.29, p=0.00). CONCLUSIONS: This is the first EHEC outbreak occurred as a group in Korea, All the isolated strains were non-O157 serotypes and the mode of transmission was most likely by school meal.
Electrophoresis, Gel, Pulsed-Field ; Enterohemorrhagic Escherichia coli* ; Gwangju ; Humans ; Korea ; Meals ; Shiga Toxins ; Surveys and Questionnaires