DNA fingerprinting of isolates of Streptococcus mutans by pulsed-field gel electrophoresis

Forty isolates and five standard laboratory strains, representing serotypes c, e and f of Streptococcus mutans were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion of the genomic DNA with BssH II. The digestion patterns of standard laboratory strains were characteristic of serotypes c, e and f. Serotypes c and f generated diagnostic DNA fragments of approximately 145 kbp and of approximately 130-175 kbp in length, respectively. Serotype e generated a ladder of at least 14 fragments of 15-155 kbp in length. The digestion patterns of isolates were essentially similar to those of the standard laboratory strains. The patterns of almost all isolates obtained from a single individual were identical, but patterns of a few different types were also observed among isolates obtained from two individuals. Digestion with BssH II revealed differences among isolates obtained from different individuals. We used differences in banding patterns among isolates to construct a dendrogram. The dendrogram included two major clusters, one that consisted of isolates of serotypes c and f, and an other that consisted of isolates of serotype e. Our results indicate that BssH II is a useful enzyme for distinguishing among isolates of S. mutans and that digestion patterns obtained by PFGE can be used for chromosomal DNA fingerprinting.     

Multiphasic approach reveals genetic diversity of environmental and patient isolates of Mycobacterium mucogenicum and Mycobacterium phocaicum associated with an outbreak of bacteremias at a Texas hospital

Between March and May 2006, a Texas hospital identified five Mycobacterium mucogenicum bloodstream infections among hospitalized oncology patients using fluorescence high-performance liquid chromatography analysis of mycolic acids. Isolates from blood cultures were compared to 16 isolates from environmental sites or water associated with this ward. These isolates were further characterized by hsp65, 16S rRNA, and rpoB gene sequencing, hsp65 PCR restriction analysis, and molecular typing methods, including repetitive element PCR, random amplified polymorphic DNA PCR, and pulsed-field gel electrophoresis (PFGE) of large restriction fragments. Three of five patient isolates were confirmed as M. mucogenicum and were in a single cluster as determined by all identification and typing methods. The remaining two patient isolates were identified as different strains of Mycobacterium phocaicum by rpoB sequence analysis. One of these matched an environmental isolate from a swab of a hand shower in the patient's room, while none of the clinical isolates of M. mucogenicum matched environmental strains. Among the other 15 environmental isolates, 11 were identified as M. mucogenicum and 4 as M. phocaicum strains, all of which were unrelated by typing methods. Although the 16S rRNA gene sequences matched for all 14 M. mucogenicum isolates, there were two each of the hsp65 and rpoB sequevars, seven PCR typing patterns, and 12 PFGE patterns. Among the seven M. phocaicum isolates were three 16S rRNA sequevars, two hsp65 sequevars, two rpoB sequevars, six PCR typing patterns, and six PFGE patterns. This outbreak represents the first case of catheter-associated bacteremia caused by M. phocaicum and the first report of clinical isolates from a U.S. hospital. The investigation highlights important differences in the available typing methods for mycobacteria and demonstrates the genetic diversity of these organisms even within narrow confines of time and space.     

Molecular analysis of environmental and human isolates of Salmonella typhi.

Molecular characterization of a total of 54 isolates of Salmonella typhi from Santiago, Chile, was performed by pulsed-field gel electrophoresis (PFGE) after digestion of chromosomal DNA with three restriction endonucleases: XbaI (5'-TCTAGA-3'), AvrII (5'-CCTAGG-3'), and SpeI (5'-ACTAGT-3'). Thirteen of the 54 isolates were obtained from environmental sources (sewage and river water), and the rest were isolates from clinical cases of typhoid fever. Considerable genetic diversity was detected among the human isolates obtained in 1994, as evidenced by the presence of 14 to 19 different PFGE patterns among 20 human isolates, with F (coefficient of similarity) values ranging from 0.69 to 1.0 (XbaI), 0.61 to 1.0 (AvrII), and 0.70 to 1.0 (SpeI). A total of eight phage types were detected among these 20 isolates, with 50% possessing the E1 or 46 phage type. There was no correlation between PFGE pattern and phage types. Similar diversity was seen among 21 isolates obtained in 1983, with 17 to 19 PFGE patterns detected and F values of 0.56 to 1.0 (XbaI), 0.55 to 1.0 (AvrII), and 0.67 to 1.0 (SpeI). Comparison of these two groups of human isolates obtained 11 years apart indicated that certain molecular types of S. typhi are shared and are able to persist for considerable periods. A similar degree of genetic diversity was also detected among the environmental isolates of S. typhi, for which 10 to 12 different PFGE patterns were detected among the 13 isolates analyzed, with F values ranging from 0.56 to 1.0 (XbaI), 0.52 to 1.0 (AvrII), and 0.69 to 1.0 (SpeI). Certain molecular types present among the environmental isolates of S. typhi were also found among the human isolates from the same time period, providing evidence for the epidemiological link between environmental reservoirs and human infection.     

Typings of Campylobacter jejuni isolated from patients of 2 outbreak cases by genotypic and phenotypic methods

We compared Campylobacter jejuni strains isolated from the patient stools associated with two food-borne diarrheal outbreak cases by the serotypic methods (Lior and Penner systems) and the genotypic methods (restriction fragment length polymorphism (RFLP) of flaA gene and pulsed-field gel electrophoresis (PFGE)). Fla-RFLP was based on the digestion of 410 bp DNA fragment by MboI restriction enzyme amplified from a 5' portion of C. jejuni flaA gene. Six distinctive fla-RFLP patterns were identified by examining 29 serotype reference strains and 58 strains isolated from the patients infected with C. jejuni independently. In the first outbreak case, 4 isolates were shown to be the same patterns each other by the fla-RFLP and PFGE, and by the Lior serotyping, except the Penner system that serotyped into 2 distinct types. On the other hand, in the second case, out of 10 isolates, 5 isolates were identical by the both genotypic and the both serotypic methods, and 4 isolates were not differentiated by the fla-RFLP and Penner system, but were separated into 4 types by PFGE in a little difference. The rest isolate was completely different from the other isolates by the all of methods used now. The findings suggest that the second case occurred by the infection of at least 3 different strains of C. jejuni.     

Construction of a Helicobacter pylori genome map and demonstration of diversity at the genome level.

Genomic DNA from 30 strains of Helicobacter pylori was subjected to pulsed-field gel electrophoresis (PFGE) after digestion with NotI and NruI. The genome sizes of the strains ranged from 1.6 to 1.73 Mb, with an average size of 1.67 Mb. By using NotI and NruI, a circular map of H. pylori UA802 (1.7 Mb) which contained three copies of 16S and 23S rRNA genes was constructed. An unusual feature of the H. pylori genome was the separate location of at least two copies of 16S and 23S rRNA genes. Almost all strains had different PFGE patterns after NotI and NruI digestion, suggesting that the H. pylori genome possesses a considerable degree of genetic variability. However, three strains from different sites (the fundus, antrum, and body of the stomach) within the same patient gave identical PFGE patterns. The genomic pattern of individual isolates remained constant during multiple subcultures in vitro. The reason for the genetic diversity observed among H. pylori strains remains to be explained.     

Species distribution and antifungal susceptibility profile of Candida spp. bloodstream isolates from Latin American hospitals

From March 1999 to March 2000, we conducted a prospective multicenter study of candidemia involving five tertiary care hospitals from four countries in Latin America. Yeast isolates were identified by classical methods and the antifungal susceptibility profile was determined according to the National Committee for Clinical Laboratory Standards microbroth assay method. During a 12 month-period we were able to collect a total of 103 bloodstream isolates of Candida spp. C. albicans was the most frequently isolated species accounting for 42% of all isolates. Non-albicans Candida species strains accounted for 58% of all episodes of candidemia and were mostly represented by C. tropicalis (24.2%) and C. parapsilosis (21.3%). It is noteworthy that we were able to identify two cases of C. lusitaniae from different institutions. In our casuistic, non-albicans Candida species isolates related to candidemic episodes were susceptible to fluconazole. Continuously surveillance programs are needed in order to identify possible changes in the species distribution and antifungal susceptibility patterns of yeasts that may occurs after increasing the use of azoles in Latin American hospitals.     

Molecular investigation of two consecutive nosocomial clusters of Candida tropicalis candiduria using pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) typing was applied to the epidemiological investigation of 21 Candida tropicalis isolates collected from urine specimens of 11 patients and one healthcare worker, in an intensive care unit (ICU) over a 4-month period. Seventeen epidemiologically unrelated strains from 14 patients were also tested to determine the discriminatory power of PFGE. PFGE typing consisted of electrophoretic karyotyping (EK) and restriction endonuclease analysis of genomic DNA (REAG), using two restriction enzymes (BssHII and SfiI). The EK pattern was the same in all 38 isolates, while REAG using SfiI separated the isolates into nine types. However, 16 different PFGE types were identified by REAG with BssHII, and the same results were obtained when the results of both REAG tests were combined. In serial urinary isolates from 10 patients, all strains from each patient had the same PFGE pattern. While the epidemiologically unrelated strains from 14 patients consisted of 13 different PFGE types, the 20 isolates from the 11 ICU patients fell into only two PFGE types (types C1 and C2), and these apparently originated from the two different outbreaks. All strains of type C1 (n = 12) were isolated from six patients, between November 1999 and January 2000, and all of the type C2 strains (n=8) were isolated from five patients, during January and February 2000. This study shows two consecutive clusters of C. tropicalis candiduria in an ICU, defined by PFGE typing, and also demonstrates that a PFGE typing method using BssHII is perhaps the most useful method for investigating C. tropicalis candiduria.     

Use of pulsed field gel electrophoresis as an epidemiological tool for analysis of sporadic associated strains of Salmonella typhi isolated in Taiwan

In order to characterize the subtypes of Salmonella typhi which cause sporadic disease in Taiwan, 55 isolates of Salm. typhi obtained from unrelated patients of sporadic cases during 1992-96 were subjected to chromosomal DNA digestion and pulsed field gel electrophoresis (PFGE). When DNAs of these 55 Salm. typhi strains were digested with XbaI, 41 PFGE patterns were observed. Strains sharing the same XbaI digestion pattern could not be further discriminated by PFGE analysis using SpeI and NotI as digestion enzymes. Thus, considerable genetic diversity exists among the Salm. typhi isolates. Although strains of the same patterns were mainly isolated during the same time, recirculation of certain infectious strains could be possible. When 12 antibiotics, i.e. ampicillin, trimethoprim/sulfamethoxazole, erythromycin, norfloxacin, tetracycline, sulphonamide, streptomycin, neomycin, chloramphenicol, kanamycin, cefoperazone and gentamycin were used to test the antibiotic susceptibility for these Salmonella isolates, only three antibiogram patterns were obtained and 49 of the 55 Salm. typhi isolates were found to belong to one pattern. Phage typing and plasmid profiles were also poor in discriminating these strains. Thus, PFGE alone may be used as a powerful tool for analysis of sporadic associated Salm. typhi strains.     

121株甲型副伤寒病原菌的噬菌体型和脉冲场凝胶电泳型

目的认识甲型副伤寒疫病区甲型副伤寒沙门菌的噬菌体型和脉冲场凝胶电泳（PFGE）型,确定噬菌体型和PFGE型之间的关系以及菌型的分布和流行率。方法采用沙门菌组合噬菌体和SpeI、XbaI消化染色体DNA的PFGE对来自玉溪市7县（区）的121株甲型副伤寒菌进行分型。结果121株菌存在4个完全噬菌体型或1个噬菌体型;用SpeI或XbaI消化产物分别得出以SpeI01、SpeI02或XbaI01占优势的5种或4种PFGE型,SpeI01型和SpeI02型分别占37．2％和57．9％,XbaI01型占95．1％。结论121株菌的噬菌体型与PFGE型之间无一致性联系,PFGE型的SpeI01和SpeI02或XbaI01是玉溪地区的主要流行型,采用SpeI和XbaI的PFGE是鉴别甲型副伤寒菌流行克隆的一项有用技术。     

Pulsed-field gel electrophoresis as an epidemiological tool for clonal identification of Aeromonas hydrophila

Pulsedfield gel electrophoresis (PFGE) was used to characterize Aeromonas hydrophila strains isolated from a cluster of hospital-acquired infections that occurred over approximately 1 month in a French hospital. Five isolates from patients and 10 isolates from the water supply were characterized by biotyping and antibiotic susceptibility patterns and compared with 10 epidemiologically unrelated strains isolated from patients and rivers, by PFGE of digests of chromosomal DNA. Five environmental and four clinical isolates belonged to the same biotype and antibiotic susceptibility pattern type. The endonucleases XbaI, SpeI and SwaI gave satisfactory profiles whereas DraI did not. The profiles were stable, reproducible and discriminatory. The 10 epidemiologically unrelated strains exhibited 10 different patterns after digestion with XbaI , the least expensive, suitable endonuclease. PFGE is a rapid and discriminatory technique for the typing of Aeromonas hydrophila where a common origin of infection is suspected.     

Possible relationship of PFGE patterns of Moraxella catarrhalis between hospital- and community-acquired respiratory infections in a community hospital

We describe a prospective study of molecular analysis of Moraxella catarrhalis isolated from a community hospital. Our study was designed to investigate the possible relationship of pulsed-field gel electrophoresis (PFGE) patterns of M. catarrhalis between hospital- and community-acquired respiratory infections. A nosocomial outbreak of M. catarrhalis was observed between September 2000 and September 2001. During the study period, 40 strains of M. catarrhalis were isolated from a total of 32 patients with respiratory infections (26 strains from 18 inpatients, and 14 strains from 14 outpatients). We compared the PFGE patterns in 40 strains of M. catarrhalis isolated from the respiratory tract of the study patients. The genomic types of M. catarrhalis were classified into three PFGE patterns (A, B, and C). Interestingly, the nosocomial outbreak of M. catarrhalis included two patterns (A and B). Of the three patterns, two patterns (A and B) were found in both inpatients and outpatients. More interestingly, two subtypes of pattern B (B1 and B4) were simultaneously found in both inpatients and outpatients. Our results indicated that PFGE with SmaI chromosomal digestion is a suitable technique to establish the inter-strain genetic relatedness of M. catarrhalis, and suggested that the outbreak of M. catarrhalis occasionally included miscellaneous PFGE patterns. The results also showed that PFGE patterns of M. catarrhalis isolates were similar between hospital- and community-acquired respiratory infections. Analysis of the subtypes suggested that there might be some association between hospital- and community-acquired respiratory infections caused by M. catarrhalis.     

Genomic relatedness within five common Finnish Campylobacter jejuni pulsed-field gel electrophoresis genotypes studied by amplified fragment length polymorphism analysis, ribotyping, and serotyping

Thirty-five Finnish Campylobacter jejuni strains with five SmaI/SacII pulsed-field gel electrophoresis (PFGE) genotypes selected among human and chicken isolates from 1997 and 1998 were used for comparison of their PFGE patterns, amplified fragment length polymorphism (AFLP) patterns, HaeIII ribotypes, and heat-stable (HS) serotypes. The discriminatory power of PFGE, AFLP, and ribotyping with HaeIII were shown to be at the same level for this selected set of strains, and these methods assigned the strains into the same groups. The PFGE and AFLP patterns within a genotype were highly similar, indicating genetic relatedness. The same HS serotypes were distributed among different genotypes, and different serotypes were identified within one genotype. HS serotype 12 was only associated with the combined genotype G1 (PFGE-AFLP-ribotype). These studies using polyphasic genotyping methods suggested that common Finnish C. jejuni genotypes form genetic lineages which colonize both humans and chickens.     

Demonstration of genome DNA diversity of strains of urease-positive thermophilic Campylobacter isolated from the natural environment by pulsed-field gel electrophoresis analysis

Pulsed-field gel electrophoresis (PFGE) analysis was carried out after separate digestion with Apa I, Sal I and Sma I of the genomic DNA from sixteen isolates of urease-positive thermophilic Campylobacter (UPTC) obtained from the natural environment, namely from oysters and mussels, in Northern Ireland. Five NCTC strains previously isolated in England were used for the analysis. Although the eight isolates of UPTC in Northern Ireland and a strain of UPTC in England showed that one or no fragments appeared after digestion with Apa I around 1,900 to 1,640 kb region of the gel, Apa I was shown to cut the genomic DNA from all of the other twelve strains of UPTC and Sal I and Sma I from all of the 21 strains in a distinctly different and distinguishable manner. Consequently, the present study clearly demonstrated that the sixteen isolates of UPTC in Northern Ireland and the five strains in England gave the diversity of the genomic DNA by using PFGE. Some strains of UPTC examined were shown to carry genomes from 1.6 to 1.9 Mb in length, thus the heterogeneous profiles of PFGE and the length of the genomes are thought to occur among the isolates of UPTC in Northern Ireland, as well as among the five representative strains of UPTC from NCTC.     

Differentiation of Citrobacter strains using chromosomal DNA restriction patterns

The aim of this study was checking of the usefulness of chromosomal DNA restriction patterns in differentiation of Citrobacter strains. Molecular characterization of total 56 isolates of Citrobacter from Poland and Czech Republic, was performed by pulsed-field gel electrophoresis after digestion of chromosomal DNA with restriction endonuclease Xba I (5'-TCTAGA-3'). Chromosomal DNA of all tested Citrobacter strains gave after electrophoresis 12 to 21 bands and patterns consisting of 12 to 21 fragments ranging in size from 790 kb to 48.5 kb and smaller, which where not distinguishable. Pulsed-field gel electrophoresis patterns were useful for comparing Citrobacter strains. Identical restriction patterns generated by PFGE were observed in the case of selected strains e.g. strains C. sedlakii studied in this study, coming from an outbreak, having the some phenotype. In addition, PFGE patterns can be used to evaluate the clonal relatedness among bacterial isolates. PFGE can be helpful for assessing genetic relatedness among strains epidemiologicaly unrelated e.g. C. werkmanii strains tested in this study. The sum of DNA fragments after Xba I digestion indicates the genome size of Citrobacter strains. This suggests that PFGE should be useful for epidemiological investigations of Citrobacter strains.     

Comparison of pulsed-field gel electrophoresis patterns of antimicrobial-resistant Escherichia coli and enterococci isolates from the feces of livestock and livestock farmers in Japan

Seven hundred thirty-nine animal strains and 662 livestock-farmer strains, consisting of Escherichia coli and enterococci, were examined for their pulsed-field gel electrophoresis (PFGE) and antimicrobial-resistance patterns. Two hundred fifty-eight and 203 PFGE patterns were found among 739 animal strains isolated from animals comprising broilers, pigs and cattle, and 662 human strains isolated from livestock farmers, respectively, from 27 farms in Japan. These results demonstrated that the PFGE patterns found among E. coli and enterococci strains from animals and livestock-farmers were heterogeneous and considerably diverse. The strains having both the identical PFGE pattern and the same drug-resistance pattern were defined as a single clone in this study. Seven types of E. coli and enterococci clones were shared among animals within the same farms and between the different farms housing the same animal species. The 25 strains (3.4%) of 739 E. coli and enterococci animal strains belonged to these seven types of clones. Only three types of E. coli clones were shared among animals between the different farms housing different animal species, but no identical E. faecalis or E. faecium clones were found between different animal species farms. The 15 strains (2.0%) of 739 E. coli and enterococci animal strains belonged to these three types of clones. Additionally, the 11 strains (1.5%) of 739 E. coli and enterococci strains isolated from animals were identical clones to strains isolated from livestock farmers of the same farm. These results suggest that the transmission of animal clones to livestock farmers or vice versa is less common.     

Pulsed-field gel electrophoresis typing of Hafnia alvei isolated from chub-packed and retail ground beef

Thirty-eight isolates of Hafnia alvei were characterized by biochemical profiles, ribotyping and pulsed-field gel electrophoresis (PFGE) patterns. The isolates were recovered from chub-packed (19 isolates) or retail (nine isolates) ground beef, or were obtained from culture repositories (10 isolates). Biochemical profiling differentiated the 38 isolates into five groups and a commercial ribotyping method recognized 11 groups, whereas PFGE differentiated the same 38 isolates into 19 groups. These data substantiate that PFGE is a highly discriminatory tool for establishing the relatedness among Hafnia alvei strains.     

Analysis of the clonal relationships between strains of Neisseria meningitidis by pulsed field gel electrophoresis.

Fingerprint patterns were generated from strains of Neisseria meningitidis by digestion of chromosomal DNA samples with 'rare-site' restriction endonucleases and resolution of the resultant fragments by pulsed field gel electrophoresis (PFGE). The potential of this technique for the rapid establishment of the clonal relationships between different isolates of the meningococcus was investigated. The fingerprint patterns from various serogroup A strains, previously assigned to clonal subgroups on the basis of their electrophoretic types (ETs), were compared. Fingerprints generated with the endonucleases SfiI, SpeI and NheI each gave distinctive patterns for the clonal subgroups I-IV of serogroup A. Further, the endonucleases SpeI and, particularly, NheI were capable of resolving differences between various subgroup III strains isolated at different times and geographical locations. Strains isolated during the 'new wave' pandemic, which was associated with the Haj, from Europe, America, and Africa, had a characteristic fingerprint pattern and appeared to be distinct from 'old wave' pandemic strains. The PFGE technique is a relatively rapid and sensitive method for establishing clonal relationships among epidemic strains of N. meningitidis.     

Discrimination of Staphylococcus aureus biotypes by pulsed-field gel electrophoresis of DNA macro-restriction fragments

AIMS: To examine whether pulsed-field gel electrophoresis (PFGE) of DNA macro-restriction fragments could provide better discrimination among the different biotypes previously described within the species Staphylococcus aureus than the traditional biochemical approach. METHODS AND RESULTS: Seventy three Staph. aureus strains from various sources (human, animal or food origin) and belonging to eight biotypes, including the poultry-like biotype, tentatively designated as an 'abattoir' biotype, were genotyped by PFGE after SmaI digestion of DNA. The PFGE patterns were compared using the average linkage matching method (UPGMA) with the Dice coefficient. A total of 61 PFGE patterns were observed, showing between 31 and 100% similarity. In most cases, strains with the same biotype were grouped specifically into one, two or three separate sub-clusters. Strains from the 'abattoir' biotype were clustered in one separate sub-cluster. CONCLUSIONS: The PFGE typing is useful to distinguish the traditional biotypes of Staph. aureus and has a more discriminatory power than the biochemical typing. SIGNIFICANCE AND IMPACT OF THE STUDY: The PFGE typing confirms the 'abattoir' biotype as a separate group on a genetic level and is well suited to investigate modes of staphylococcal contamination of food.     

Characterization of Listeria monocytogenes isolates from food animal clinical cases: PFGE pattern similarity to strains from human listeriosis cases

Twenty-one isolates of Listeria monocytogenes from food animal clinical cases that involved meningitis or meningoencephalitis, encephalitis, mastitis and abortion were characterized by serotyping and pulsed-field gel electrophoresis (PFGE) in order to improve our understanding of the genetic links between individual strains and strains recovered from human listeriosis cases. Results showed that five of the isolates were serotype 1/2a, six were 1/2b, nine were 4b, and one was untypeable. A caprine, two bovine and an ovine brain isolate shared identical PFGE patterns indicating that strains of L. monocytogenes are not host specific. Other isolates exhibited distinct patterns that were not shared, indicating a genetic diversity. Dendrogram analysis revealed that PFGE patterns of the isolates clustered primarily according to serotype. We compared the PFGE types obtained for these isolates with PFGE types for human clinical isolates present in the CDC national PulseNet database. Six (29%) of the twenty-one strains had patterns that were indistinguishable from pathogenic human isolates in the database. Our observations offer preliminary evidence that food animals could be significant reservoirs of L. monocytogenes that lead to human infections and support the inclusion of PFGE patterns of veterinary clinical isolates in the national PulseNet database for increased surveillance.     

An Epidemiological Study of Salmonella enteritidis by Pulsed-Field Gel Electrophoresis (PFGE): Several PFGE Patterns Observed in Isolates from a Food Poisoning Outbreak

An epidemiological analysis of Salmonella enteritidis from a food poisoning was done using pulsed-field gel electrophoresis (PFGE) of BlnI- or XbaI-digested fragments of chromosomal DNA of isolates. S. enteritidis isolates obtained from 19 patients had identical PFGE patterns. Therefore, a strain giving the same pattern was considered to be the causative agent of this outbreak. In addition, four isolates that had different BlnI-digested PFGE patterns were obtained from three patients, suggesting that the observed variations in PFGE patterns might occur as the result of some point mutations of chromosomal DNA during growth or from the existence of several S. enteritidis strains from various sources. Subsequent PFGE analysis of continuously subcultured strains supported the former possibility. These observations indicate that PFGE analysis on multiple numbers of colonies from each patient are necessary for the epidemiologic investigation of S. enteritidis.