Isolation and characterization by immunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western blot, restriction fragment length polymorphism-PCR, 16S rRNA gene sequencing, and pulsed-field gel electrophoresis of Rochalimaea quintana from a patient with bacillary angiomatosis.

Rochalimaea quintana was isolated from the blood of a French human immunodeficiency virus-infected patient with bacillary angiomatosis. The isolate showed the typical growth characteristics of Rochalimaea species and was inert when typical biochemical testing was used. The purpose of the present work was to characterize and compare this new isolate with reference strains of R. quintana, Rochalimaea vinsonii, and Rochalimaea henselae by using immunofluorescence, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot (immunoblot), restriction fragment length polymorphism-PCR of the citrate synthase gene, 16S rRNA gene sequencing, and pulsed-field gel electrophoresis. SDS-PAGE, Western blot, restriction fragment length polymorphism-PCR with TaqI enzyme, and 16S rRNA gene sequencing could differentiate the three Rochalimaea species and allowed characterization of the French isolate as R. quintana. However, identification of the Rochalimaea isolate to the species level was more easily obtained by immunofluorescence with specific murine antisera. Pulsed-field gel electrophoresis allowed differentiation of the French R. quintana isolate from R. quintana Fuller and may serve as an epidemiological tool.     

Comparison of pulsed-field gel electrophoresis and amplified fragment length polymorphism techniques for investigating outbreaks of enteritis due to campylobacters

Campylobacters are the most commonly reported cause of acute bacterial enteritis in the United Kingdom and United States, with poultry, milk, and water implicated as sources or vehicles of infection. The majority of campylobacter infections are sporadic, although outbreaks may occur, and these provide an opportunity to evaluate genotypic fingerprinting techniques. In this study, pulsed-field gel electrophoresis (PFGE) was compared with single-enzyme-amplified fragment length polymorphism (SAFLP). The results for the three separate episodes indicated that SAFLP and PFGE both clustered the strains from the first incident as 100% homologous. The strains from the second and third incidents clustered as distinct from both the first incident and from each other. PFGE is well recognized as a discriminatory fingerprinting technique for campylobacters; however, SAFLP has proven to be equally discriminatory, but far less labor intensive and with the added advantages of less "hands-on" time and inexpensive equipment, it is an excellent alternative to PFGE for investigation of outbreaks.     

DNA fingerprinting of Streptococcus pneumoniae strains by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis of genomic DNA was carried out on Streptococcus pneumoniae strains to determine its value in the epidemiological survey of pneumococcal infections. Twenty-one clinical strains were chosen to cover a broad range of diversity according to geographic location, penicillin susceptibility, serotype, and multilocus enzyme electrophoresis (MLEE) pattern. The restriction endonucleases ApaI and SmaI were used to digest intact chromosomes, and the fragments were resolved by field inversion gel electrophoresis (FIGE). Each digest produced 10 to 19 fragments for comparison between strains. All the strains, including strains of the same serotype and strains with the same MLEE profile, had different FIGE patterns. In some cases, the restriction patterns differed by only a few fragment bands, and two isolates differed only in the location of a single DNA fragment. The polymorphism obtained with FIGE was greater than those obtained with serotyping and MLEE analysis. The stability of the FIGE profiles was established by testing of two independent clones derived from pneumococcus strain R36A. These results indicated that pulsed-field gel electrophoresis should be an effective tool for the typing of S. pneumoniae strains, capable of subdividing serotypes or MLEE types and of tracing the origin of pneumococcal strains.     

Molecular typing of a suspected cluster of Nocardia farcinica infections by use of randomly amplified polymorphic DNA, pulsed-field gel electrophoresis, and amplified fragment length polymorphism analyses

Randomly amplified polymorphic DNA (RAPD), pulsed-field gelelectrophoresis (PFGE), and amplified fragment length polymorphism (AFLP) analyses were used to investigate a possible outbreak of Nocardia farcinica. RAPD and PFGE analyses yielded irreproducible and unsatisfactory results, respectively. AFLP analysis seem to be a promising and welcome addition for molecular analysis of Nocardia isolates.     

Discrimination of human pathogenic subspecies of Francisella tularensis by using restriction fragment length polymorphism

We describe the use of two insertion sequence elements (ISFtu1 and ISFtu2) in Francisella tularensis to type strains by restriction fragment length polymorphism (RFLP). The RFLP profiles of 17 epidemiologically unrelated isolates were determined and compared. Our results showed that RFLP profiles can be used to assign F. tularensis strains into five main groups corresponding to strains of F. tularensis subsp. tularensis, F. tularensis strain ATCC 6223, strains of F. tularensis subsp. holarctica, strains of F. tularensis subsp. holarctica from Japan, and F. tularensis subsp. mediaasiatica. The results confirm the genetic identities of these subspecies and also support the suggestion that strains of F. tularensis subsp. holarctica from Japan should be considered members of a separate biovar. These findings should support future studies to determine the genetic differences between strains of F. tularensis at the whole-genome level.     

Determination of genome size and restriction fragment length polymorphism of four Chinese rickettsial isolates by pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) was used to determine the genome size and the restriction fragment length polymorphism (RFLP) of four new Chinese isolates of spotted fever group (SFG) rickettsiae. The genome size of the isolates Ha-91, BJ-90, 054 and 036 was 1,253 kb, 1,236 kb, 1,272 kb, and 1,272 kb, respectively. The isolates 054 and 036 had identical RFLP profiles. All the isolates differed in the properties under study from the so far known SFG rickettsiae. The unique RFLP profiles of the isolates supported our opinion that they are new strains of SFG rickettsiae or even new species of SFG rickettsiae.     

Evaluation of fluorescence-based amplified fragment length polymorphism analysis for molecular typing in hospital epidemiology: comparison with pulsed-field gel electrophoresis for typing strains of vancomycin-resistant Enterococcus faecium

Fluorescence-based amplified fragment length polymorphism (fbAFLP) is a novel assay based on the fluorescent analysis of an amplified subset of restriction fragments. The fbAFLP assay involves the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The ligation of adapters with primer-specific sites coupled with primers containing selective nucleotides allowed the full potential of PCR to be realized while maintaining the advantages of restriction endonuclease analysis. Fluorescence-based fragment analysis with polyacrylamide gel electrophoresis provides the accurate band sizing required for homology assessment. The large number of phylogenetically informative characters obtained by fbAFLP is well suited for cluster analysis and database development. The method demonstrated excellent reproducibility and ease of performance and interpretation. We typed 30 epidemiologically well-characterized isolates of vancomycin-resistant enterococci from an outbreak in a university hospital by fbAFLP. Clustering of fbAFLP data matched epidemiological, microbiological, and pulsed-field gel electrophoresis data. This study demonstrates the unprecedented utility of fbAFLP for epidemiological investigation. Future developments in standardization and automation will set fbAFLP as the "gold standard" for molecular typing in epidemiology.     

Identification of Bartonella species directly in clinical specimens by PCR-restriction fragment length polymorphism analysis of a 16S rRNA gene fragment

It is now established that two species of Bartonella, namely, Bartonella henselae and B. quintana, cause bacillary angiomatosis in human immunodeficiency virus-infected patients. In addition, B. henselae causes cat scratch disease and B. quintana, B. henselae, and B. elizabethae can cause bacteremia and endocarditis in immunocompetent persons. We have developed a PCR-restriction fragment length polymorphism-based assay for direct detection and identification to species level of Bartonella in clinical specimens. This is accomplished by PCR amplification of Bartonella DNA using primers derived from conserved regions of the gene carrying the 16S ribosomal DNA, followed by restriction analysis using DdeI and MseI restriction endonucleases. We amplified a Bartonella genus-specific 296-bp fragment from 25 clinical samples obtained from 25 different individuals. Restriction analysis of amplicons showed that identical patterns were seen from digestion of B. henselae and B. quintana amplicons with DdeI, whereas a different unique pattern was seen by using the same enzyme with B. vinsonii and B. elizabethae. With MseI digestion, B. henselae and B. vinsonii gave nearly identical patterns while B. quintana and B. elizabethae gave a different pattern. By combining the restriction analysis data generated with MseI and DdeI, unique "signature" restriction patterns characteristic for each species were obtained. These patterns were useful in identifying the Bartonella species associated with each tissue specimen.     

Rapid identification of Campylobacter, Arcobacter, and Helicobacter isolates by PCR-restriction fragment length polymorphism analysis of the 16S rRNA gene

A rapid two-step identification scheme based on PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 16S rRNA gene was developed in order to differentiate isolates belonging to the Campylobacter, Arcobacter, and Helicobacter genera. For 158 isolates (26 reference cultures and 132 clinical isolates), specific RFLP patterns were obtained and species were successfully identified by this assay.     

Genome fingerprinting by pulsed-field gel electrophoresis and ribotyping to differentiatePseudomonas aeruginosa serotype O11 strains

The performance of ribotyping and pulsed-field gel electrophoresis was compared in the differentiation of a collection of 44Pseudomonas aeruginosa serotype O11 strains isolated in seven hospitals in Singapore. Digestion of genomic DNA byEcoRI andSacI followed by Southern hybridization with thePseudomonas aeruginosa 16S and 23S rRNA gene revealed seven distinct ribotypes. Ribotyping using a combination of both enzymes revealed 11 ribotypes. In contrast, electrophoretic analysis differentiated 41 different strain types among the 44 clinical isolates using eitherSpeI orDraI. Pulsed-field gel electrophoresis demonstrated greater sensitivity than ribotyping in the differentiation ofPseudomonas aeruginosa strains of the same ribotype and could thus be used alone in epidemiological investigations of hospital outbreaks ofPseudomonas aeruginosa serotype O11 infection.     

Identification of Abiotrophia adiacens and Abiotrophia defectiva by 16S rRNA gene PCR and restriction fragment length polymorphism analysis.

Abiotrophia adiacens and Abiotrophia defectiva, previously referred to as nutritionally variant streptococci, Streptococcus adjacens and Streptococcus defectivus, respectively, are causes of infective endocarditis. We describe a method of identifying these two species and also of distinguishing them from 15 other major etiological pathogens of infective endocarditis by means of 16S rRNA gene PCR amplification followed by restriction fragment length polymorphism analysis (PCR-RFLP). The 16S rRNA genes were successfully amplified with a set of universal primers from all 17 species of bacteria examined, including viridans group streptococci. The RFLP patterns of A. adiacens and A. defectiva obtained by HaeIII or MspI digestion were readily distinguished from each other and from those of other bacteria. When PCR analysis was performed with the supernatant of a suspension of a boiled colony, the 16S rRNA genes of 80 of 82 isolates (97%) of A. adiacens and all isolates (11 of 11) of A. defectiva were amplified. The HaeIII RFLP patterns of the isolates were the same as those of the corresponding type strains, although 28% of A. adiacens isolates revealed intraspecies polymorphism. The detection limit of this method was 0.1 pg of genomic DNA, as assessed by using the digoxigenin-labeling DNA detection system. Thus, the PCR-RFLP analysis that we developed is applicable for the routine detection of Abiotrophia from clinical specimens.     

Combination of pulsed-field gel electrophoresis (PFGE) and single-enzyme amplified fragment length polymorphism (SAFLP) for differentiation of multiresistant Salmonella enterica serotype typhimurium

Objective   To assess the combined application of plasmid profile typing, pulsed-field gel electrophoresis (PFGE) and PCR-based single-enzyme amplified fragment length polymorphism ( S AFLP) for the differentiation of 18 multiresistant (MR) and one drug-sensitive strain of Salmonella enterica serotype typhimurium from humans and food animals.
Methods   Strains were phage typed and tested for resistance to a panel of antimicrobial agents. Strains were also tested for the ability to transfer resistance either directly or by mobilization to standard strains of Escherichia coli K12. Plasmid DNA was extracted from both drug-resistant donor strains and from drug-resistant exconjugants. Total genomic DNA was characterized by PFGE following digestion with the restriction endonuclease Xba I. The resultant patterns were categorized and analyzed by dendrogram analysis using the Dice coefficient and by data clustering using unweighted pair-group arithmetic averaging (UPGMA). Isolates were also characterized and categorized by SAFLP. The levels of discrimination achieved by each method were assessed individually and in combination.
Results   Plasmid DNA was detected in all of the 18 MR isolates but, not in the drug-sensitive isolate. Using PFGE, 19 different profiles were identified, falling into eight major categories. However, by SAFLP, only eight profiles were observed. Subsequent investigations have demonstrated epidemiologic relationships within at least one of these SAFLP profile groupings.
Conclusions   These studies have demonstrated that PFGE and SAFLP can be used independently for the differentiation of MR S. Typhimurium from humans and food animals. However, when used in combination, SAFLP can provide a format for broad epidemiologic groupings. These groupings can be further subdivided by PFGE to provide detailed information on putative strain relationships at the genotypic level.     

Identification of oral peptostreptococcus isolates by PCR-restriction fragment length polymorphism analysis of 16S rRNA genes

Oral Peptostreptococcus isolates tentatively identified by conventional microbiological culture methods were identified to the species level by a combination of PCR amplification of 16S rRNA genes and restriction enzyme analysis of the amplified products. This method is a reliable and rapid alternative to conventional methods for identification of these bacterial species.     

Identification of Clostridium species and DNA fingerprinting of Clostridium perfringens by amplified fragment length polymorphism analysis

An amplified fragment length polymorphism (AFLP) method was applied to 129 strains representing 24 different Clostridium species, with special emphasis on pathogenic clostridia of medical or veterinary interest, to assess the potential of AFLP for identification of clostridia. In addition, the ability of the same AFLP protocol to type clostridia at the strain level was assessed by focusing on Clostridium perfringens strains. All strains were typeable by AFLP, so the method seemed to overcome the problem of extracellular DNase production. AFLP differentiated all Clostridium species tested, except for Clostridium ramosum and Clostridium limosum, which clustered together with a 45% similarity level. Other Clostridium species were divided into species-specific clusters or occupied separate positions. Wide genetic diversity was observed among Clostridium botulinum strains, which were divided into seven species-specific clusters. The same AFLP protocol was also suitable for typing C. perfringens at the strain level. A total of 29 different AFLP types were identified for 37 strains of C. perfringens; strains initially originating from the same isolate showed identical fingerprinting patterns and were distinguished from unrelated strains. AFLP proved to be a highly reproducible, easy-to-perform, and relatively fast method which enables high throughput of samples and can serve in the generation of identification libraries. These results indicate that the AFLP method provides a promising tool for the identification and characterization of Clostridium species.     

DNA fingerprinting of Escherichia coli O157:H7 strains by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis of genomic DNA was carried out on Escherichia coli O157:H7 strains from different geographic locations to determine its value in an epidemiological survey of O157 infections. Pulsed-field gel electrophoresis of XbaI-digested DNA fragments clearly separated E. coli O157:H7 strains from nontoxigenic E. coli O157:H19, O157:H43, and O157:H45 strains and from Shiga-like-toxin-producing E. coli strains of other serogroups. However, among the E. coli O157:H7 strains, the restriction patterns either were identical or differed only by a few fragment bands. In some cases, it was therefore impossible to distinguish among epidemiologically unrelated strains. Hybridization experiments with a DNA probe complementary to Shiga-like toxin II sequences revealed that the Shiga-like toxin II genes were located on DNA fragments of different lengths. Our data show that for a single highly conserved clone, such as E. coli O157:H7, other typing techniques may need to be performed in addition to DNA fingerprinting in epidemiological surveys.     

Identification of Aeromonas clinical isolates by restriction fragment length polymorphism of PCR-amplified 16S rRNA genes.

Identification of Aeromonas species, emergent pathogens for humans, has long been controversial due to their phenotypic and genomic heterogeneities. Computer analysis of the published 16S rRNA gene sequences revealed that restriction fragment length polymorphism of the PCR-amplified 16S rRNA gene is a good and rapid way of assessing the identities of all known species of Aeromonas. The method was evaluated with the reference strains of all species (or DNA homology groups) and 76 clinical isolates of diverse origin. Most results from the two approaches were in agreement, but some discrepancies were discerned. Advantages over previous phenotypic and genetic methods are discussed.     

Comparative study using amplified fragment length polymorphism fingerprinting, PCR genotyping, and phenotyping to differentiate Campylobacter fetus strains isolated from animals

A collection of Campylobacter fetus strains, including both C. fetus subsp. fetus and C. fetus subsp. venerealis, were phenotypically identified to the subspecies level and genotypically typed by PCR and amplified fragment length polymorphism (AFLP) analysis. Phenotypic subspecies determination methods were unreliable. Genotyping of the strains by PCR and AFLP showed a clear discrimination between the two subspecies.     

Molecular typing and epidemiological study of Salmonella enterica serotype Typhimurium isolates from cattle by fluorescent amplified-fragment length polymorphism fingerprinting and pulsed-field gel electrophoresis

One hundred twenty Salmonella enterica serotype Typhimurium strains, including 103 isolates from cattle gathered between 1977 and 1999 in the prefecture located on the northern-most island of Japan, were analyzed by using fluorescent amplified-fragment length polymorphism (FAFLP) and pulsed-field gel electrophoresis (PFGE) to examine the genotypic basis of the epidemic. Among these strains, there were 17 FAFLP profiles that formed four distinct clusters (A, B, C, and D). Isolates that belonged to cluster A have become increasingly common since 1992 with the increase of bovine salmonellosis caused by serotype Typhimurium. PFGE resolved 25 banding patterns that formed three distinct clusters (I, II, and III). All the isolates that belonged to FAFLP cluster A, in which all the strains of definitive phage type 104 examined were included, were grouped into PFGE cluster I. Taken together, these results indicate that clonal exchange of serotype Typhimurium has taken place since 1992, and they show a remarkable degree of homogeneity at a molecular level among contemporary isolates from cattle in this region. Moreover, we have sequenced two kinds of FAFLP markers, 142-bp and 132-bp fragments, which were identified as a polymorphic marker of strains that belonged to clusters A and C, respectively. The sequence of the 142-bp fragment shows homology with a segment of P22 phage, and that of the 132-bp fragment shows homology with a segment of traG, which is an F plasmid conjugation gene. FAFLP is apparently as well suited for epidemiological typing of serotype Typhimurium as is PFGE, and FAFLP can provide a source of molecular markers useful for studies of genetic variation in natural populations of serotype Typhimurium.     

Rapid identification of Campylobacter species by restriction fragment length polymorphism analysis of a PCR-amplified fragment of the gene coding for 16S rRNA.

Restriction fragment length polymorphism analysis of a PCR-amplified DNA fragment of the gene coding for 16S rRNA was performed on 148 previously characterized strains of Campylobacter, Helicobacter, Arcobacter, and Wolinella succinogenes and 13 Campylobacter-like isolates. These strains included clinical, animal, and environmental isolates. PCR amplification generated a 283-bp fragment from all species. The amplicon from each strain was digested with six restriction endonucleases (AccI, AvaI, DdeI, HaeIII, HpaII, XhoI). DdeI was useful for the initial grouping of the strains. Additional discrimination within the different DdeI groups was obtained with AccI, HaeIII, HpaII, and XhoI digestions. The PCR-restriction fragment length polymorphism analysis allowed for the discrimination of members of the genus Campylobacter from members of closely related genera and discrimination between Campylobacter species. The proposed method is simple and rapid and can be useful for the routine identification of Campylobacter-like organisms in clinical or epidemiologic studies.