Identification of three clinically relevant Borrelia burgdorferi sensu lato genospecies by PCR-restriction fragment length polymorphism analysis of 16S-23S ribosomal DNA spacer amplicons

We report the results of a study of the prevalences of three clinically relevant Borrelia burgdorferi sensu lato genospecies (Borrelia burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii) in 1,040 questing Ixodes ticks from all regions of Latvia, where Lyme borreliosis is endemic. The prevalences of Borrelia in Ixodes ricinus and Ixodes persulcatus were 22.6 and 27.9%, respectively. Molecular typing of B. burgdorferi from infected ticks was performed by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified fragments of the 16S-23S (rrs-rrlA) rRNA intergenic spacer by using species-specific primers and subsequent sequencing. The dominant Borrelia species in both Ixodes species was B. afzelii. In addition, different restriction patterns of B. garinii and B. afzelii were also identified. This study demonstrates that the 16S-23S rRNA PCR-RFLP typing method is simple, sensitive, and fast and that it allows one to differentiate among B. burgdorferi species and subspecies with various degrees of pathogenic potential directly in ticks. These features are important in monitoring Lyme disease.     

Novel PCR-restriction fragment length polymorphism analysis for rapid typing of staphylococcal cassette chromosome mec elements

We developed a novel PCR-restriction fragment length polymorphism test for the ccrB gene by using HinfI and BsmI for rapid typing of staphylococcal cassette chromosome mec (SCCmec). When tested with reference strains and methicillin-resistant Staphylococcus aureus isolates, the method proved to be valid and useful for rapid identification of four SCCmec types, especially type IV.     

Development of a PCR-restriction fragment length polymorphism assay for the epidemiological analysis of Shiga toxin-producing Escherichia coli

Six characteristic regions (I to VI) were identified in Shiga toxin 2 (Stx2) phages (T. Sato, T. Shimizu, M. Watarai, M. Kobayashi, S. Kano, T. Hamabata, Y. Takeda, and S. Yamasaki, Gene 309:35-48, 2003). Region V, which is ca. 10 kb in size and is located in the upstream region of the Stx operons, includes the most distinctive region among six Stx phages whose genome sequences have been determined. In this study, we developed a PCR-restriction fragment length polymorphism (RFLP) assay for the epidemiological analysis of Shiga toxin-producing Escherichia coli (STEC) on the basis of the diversity of region V. When region V was amplified by long and accurate-PCR (LA-PCR) with five control E. coli strains carrying six different Stx phages such as E. coli strains C600 (Stx1 phage), C600 (933W phage), C600 (Stx2 phage-I), C600 (Stx2 phage-II), and O157:H7 Sakai strain RIMD0509952 (VT1-Sakai phage and VT2-Sakai phage), an expected size of the band was obtained. Restriction digest of each PCR product with BglI or EcoRV also gave the expected sizes of banding patterns and discriminated the RFLPs of five control strains. When a total of 204 STEC O157 strains were analyzed by LA-PCR, one to three bands whose sizes ranged from 8.2 to 14 kb were obtained. Two STEC O157 strains, however, did not produce any bands. Subsequent restriction digest of the PCR products with BglI or EcoRV differentiated the RFLPs of 202 STEC O157 strains into 24 groups. The RFLP patterns of pulsed-field gel electrophoresis (PFGE) of representative strains of STEC O157 divided into 24 groups were well correlated with those of PCR-RFLP when STEC O157 strains were isolated in the same time period and in the close geographic area. To evaluate the PCR-RFLP assay developed here, ten strains, each isolated from four different outbreaks in different areas in Japan (Tochigi, Hyogo, Aichi, and Fukuoka prefecture), were examined to determine whether the strains in each group showed the same RFLP patterns in the PCR-RFLP assay. In accordance with the results of PFGE except for strains isolated in an area (Fukuoka), which did not produce any amplicon, ten strains in each group demonstrated the same RFLP pattern. Taken together, these data suggest that the PCR-RFLP based on region V is as useful as PFGE but perhaps more simple and rapid than PFGE for the molecular epidemiological analysis of STEC strains during sporadic and common source outbreaks.     

Identification of Staphylococcus spp. by PCR-restriction fragment length polymorphism analysis of dnaJ gene

A PCR-restriction fragment length polymorphism (RFLP) analysis method that analyzes a part of the dnaJ gene was designed for the rapid and accurate identification of Staphylococcus spp. XapI or Bsp143I digestion of the PCR-generated products rendered distinctive RFLP patterns that allowed 41 reference species and subspecies to be identified with a high degree of specificity. The novel method was validated by the identification of 23 clinical staphylococcal strains, and the results were compared with those obtained by other genotypic identification methods. A 100% concordance of the results was shown. Therefore, PCR-RFLP analysis of the dnaJ gene is proposed as a reliable and reproducible method for the identification of Staphylococcus spp.     

Evaluation of PCR-restriction profile analysis and IS2404 restriction fragment length polymorphism and amplified fragment length polymorphism fingerprinting for identification and typing of Mycobacterium ulcerans and M. marinum

Mycobacterium ulcerans and M. marinum are emerging necrotizing mycobacterial pathogens that reside in common reservoirs of infection and exhibit striking pathophysiological similarities. Furthermore, the interspecific taxonomic relationship between the two species is not clear as a result of the very high phylogenetic relatedness (i.e., >99.8% 16S rRNA sequence similarity), in contrast to only 25 to 47% DNA relatedness. To help understand the genotypic affiliation between these two closely related species, we performed a comparative analysis including PCR restriction profile analysis (PRPA), IS2404 restriction fragment length polymorphism (RFLP), and amplified fragment length polymorphism (AFLP) on a set of M. ulcerans (n = 29) and M. marinum (n = 28) strains recovered from different geographic origins. PRPA was based on a triple restriction of the 3' end region of 16S rRNA, which differentiated M. ulcerans into three types; however, the technique could not distinguish M. marinum from M. ulcerans isolates originating from South America and Southeast Asia. RFLP based on IS2404 produced six M. ulcerans types related to six geographic regions and did not produce any band with M. marinum, confirming the previous findings of Chemlal et al. (K. Chemlal, K. DeRidder, P. A. Fonteyne, W. M. Meyers, J. Swings, and F. Portaels, Am. J. Trop. Med. Hyg. 64:270-273, 2001). AFLP analysis resulted in profiles which grouped M. ulcerans and M. marinum into two separate clusters. The numerical analysis also revealed subgroups among the M. marinum and M. ulcerans isolates. In conclusion, PRPA appears to provide a rapid method for differentiating the African M. ulcerans type from other geographical types but is unsuitable for interspecific differentiation of M. marinum and M. ulcerans. In comparison, whole- genome techniques such as IS 2404-RFLP and AFLP appear to be far more useful in discriminating between M. marinum and M. ulcerans, and may thus be promising molecular tools for the differential diagnosis of infections caused by these two species.     

Usefulness of PCR-restriction fragment length polymorphism typing of the coagulase gene to discriminate arbekacin-resistant methicillin-resistant Staphylococcus aureus strains

We compared the results of two typing methods for 678 strains of methicillin-resistant Staphylococcus aureus and methicillin-susceptible S. aureus. PCR-restriction fragment length polymorphism typing of the coagulase gene was a more reliable method than coagulase serotyping from the viewpoint of arbekacin resistance.     

Flagellin gene typing of Campylobacter jejuni by restriction fragment length polymorphism analysis.

We developed and studied a molecular typing approach for Campylobacter spp. with restriction fragment length polymorphism (RFLP) analysis of the flagellin gene flaA in C. jejuni. Using polymerase chain reaction, we amplified the flaA gene from strains comprising different HL:O serotypes by using a primer set directed at the conserved 5' and 3' flaA gene sequence to generate a 1.7-kb amplicon. The amplicon was further digested with the restriction enzyme DdeI, and the fragments generated were analyzed by agarose gel electrophoresis. In 43 non-outbreak strains of six common HL serotypes (HL 1, 2, 4, 5, 9, and 36) in the United States, 18 RFLP patterns were observed. In U.S. outbreak strains previously studied by 10 other typing methods, flaA typing correlated with the HL serotype within each outbreak, and six additional flaA types were identified. Our results suggest that RFLP analysis of the flaA gene from Campylobacter spp. has sufficient discrimination to be useful as a practical typing method for clinical and epidemiologic investigations.     

Identification of Staphylococcus spp. by PCR-restriction fragment length polymorphism of gap gene

Oligonucleotide primers specific for the Staphylococcus aureus gap gene were previously designed to identify 12 Staphylococcus spp. by PCR. In the present study, AluI digestion of PCR-generated products rendered distinctive restriction fragment length polymorphism patterns that allowed 24 Staphylococcus spp. to be identified with high specificity.     

Evaluation of groEL gene analysis for identification of Borrelia burgdorferi sensu lato

The nucleotide sequences of the groEL genes, the flagellin genes, and the 16S rRNA genes from 22 reference strains of Borrelia were compared. groEL sequence analysis is useful not only in interspecies differentiation but also in intraspecies differentiation of Borrelia afzelii and Borrelia garinii isolates.     

Correlation of plasmids with infectivity of Borrelia burgdorferi sensu stricto type strain B31.

The correlation of plasmid profiles with infectivity was investigated by using five clones of Borrelia burgdorferi sensu stricto strain B31 (ATCC 35210). Plasmid profiles were determined by pulsed-field and two-dimensional gel electrophoresis. The 50% infectious dose (ID50) in hamsters was determined. The ID50 of the clone that possessed a full complement of eight linear and three circular plasmids was 10(3) cells. The loss of the 27.5- and 40-kb linear plasmids did not decrease the infectivity of these cells. Rather, the loss of the 27.5-kb linear plasmid was associated with a more disseminated infection. A moderate decrease of the ID50 from 10(3) to 10(5) cells correlated with the loss of the 9.0-kb circular plasmid and the 27.5-kb linear plasmid. A major loss of infectivity (ID50 > 10(3) cells) occurred with cells that lost the 24.7- and 27.5-kb linear plasmids and the 9.0-kb circular plasmid. A 3.0-kb HindIII fragment of the 24.7-kb linear plasmid was used as a probe to determine the presence of the homologous sequences in the three genospecies of Lyme disease spirochetes. An analysis of 21 infectious strains of B. burgdorferi sensu stricto, B. garinii, and B. afzelii revealed a consistent association of infectivity with strains possessing a linear plasmid (size range, 24 to 36 kb) that hybridized with the HindIII fragment. Western immunoblotting with hamster antisera against infectious B31 clone C-3 revealed two proteins with molecular masses of 28 and 43 kDa that were absent in the noninfectious B31 clone C-1. Additionally, a 14-kDa protein was absent in C-1 but present in infectious clone C-9 as shown by two-dimensional polyacrylamide gel electrophoresis.     

Demonstration of Borrelia burgdorferi sensu stricto infection in ticks from the northeast of Mexico

Borrelia burgdorferi sensu lato infection has been confirmed in clinical cases in the northeast of Mexico; however, the bacterium has not been identified as infecting the tick vector Ixodes , Amblyomma and Dermacentor ticks were collected from mammals and plants in northeastern Mexico and examined for Borrelia . Eighteen of 214 ticks were PCR-positive for the fla and 16S rRNA genes and 15 for the ospA gene. Southern blotting with a fla probe and sequencing of ospA genes confirmed infection with B. burgdorferi sensu stricto . These findings, together with reports of indigenous cases, fulfil the criteria that allow northeastern Mexico to be considered as a zone endemic for Lyme disease.     

Rapid differentiation of Borrelia garinii from Borrelia afzelii and Borrelia burgdorferi sensu stricto by LightCycler fluorescence melting curve analysis of a PCR product of the recA gene

To differentiate the Borrelia burgdorferi sensu lato genospecies, LightCycler real-time PCR was used for the fluorescence (SYBR Green I) melting curve analysis of borrelial recA gene PCR products. The specific melting temperature analyzed is a function of the GC/AT ratio, length, and nucleotide sequence of the amplified product. A total of 32 DNA samples were tested. Of them three were isolated from B. burgdorferi reference strains and 16 were isolated from B. burgdorferi strains cultured from Ixodes ricinus ticks; 13 were directly isolated from nine human biopsy specimens and four I. ricinus tick midguts. The melting temperature of B. garinii was 2 degrees C lower than that of B. burgdorferi sensu stricto and B. afzelii. Melting curve analysis offers a rapid alternative for identification and detection of B. burgdorferi sensu lato genospecies.     

Identification of 54 mycobacterial species by PCR-restriction fragment length polymorphism analysis of the hsp65 gene

A total of 121 reference and clinical strains of both slowly and rapidly growing mycobacteria belonging to 54 species were studied for restriction fragment length polymorphism of a PCR-amplified 439-bp segment of the gene encoding the 65-kDa heat shock protein. Restriction digests were separated by 10% polyacrylamide gel electrophoresis (PAGE). By including a size standard in each sample, the restriction fragment profile was calculated using a computer-aided comparison program. An algorithm describing these 54 species (including 22 species not previously described) is proposed. We found that this assay based on 10% PAGE provided a more precise estimate than that based on agarose gel electrophoresis of the real size of restriction fragments as deduced from the sequence analysis and allowed identification of mycobacteria whose PCR-restriction fragment length polymorphism analysis patterns were unequivocally identified by fragments shorter than 60 bp.     

Species identification of mycobacteria by PCR-restriction fragment length polymorphism of the rpoB gene

PCR-restriction fragment length polymorphism analysis (PRA) using the novel region of the rpoB gene was developed for rapid and precise identification of mycobacteria to the species level. A total of 50 mycobacterial reference strains and 3 related bacterial strains were used to amplify the 360-bp region of rpoB, and the amplified DNAs were subsequently digested with restriction enzymes such as MspI and HaeIII. The results from this study clearly show that most of the mycobacterial species were easily differentiated at the species level by this PRA method. In addition, species with several subtypes, such as Mycobacterium gordonae, M. kansasii, M. celatum, and M. fortuitum, were also differentiated by this PRA method. Subsequently, an algorithm was constructed based on the results, and a blinded test was carried out with more than 260 clinical isolates that had been identified on the basis of conventional tests. Comparison of these two sets of results clearly indicates that this new PRA method based on the rpoB gene is more simple, more rapid, and more accurate than conventional procedures for differentiating mycobacterial species.     

In vitro susceptibility testing of four antibiotics against Borrelia burgdorferi: a comparison of results for the three genospecies Borrelia afzelii,Borrelia garinii,and Borrelia burgdorferi sensu stricto

MICs and minimal bactericidal concentrations (MBCs) were evaluated for the four antibiotics azithromycin, amoxicillin, ceftriaxone, and doxycycline against the three main genospecies of Borrelia burgdorferi sensu lato. In MBC testing, statistically significant differences between the genospecies could be found in 7 out of 12 comparative evaluations (P < 0.05).     

Sequence analysis of ospA genes shows homogeneity within Borrelia burgdorferi sensu stricto and Borrelia afzelii strains but reveals major subgroups within the Borrelia garinii species

The genes coding for the outer surface protein A (OspA) of 19 different Borrelia burgdorferi strains belonging to the seven OspA-serotypes 1–7, previously described [Wilske et al. (1993) J Clin Microbiol, 31: 340–350], have been investigated. B. burgdorferi sensu lato strains were chosen from various biological sources (ticks, human skin and cerebrospinal fluid) as well as different geographical origins (Germany, Slovenia, Austria, United States). The open reading frames of all ospA genes consist of 819–825 nucleotides corresponding to proteins of approximately 30 kDa. The ospA sequences obtained in this study and previous published studies were compared with the results from OspA serotyping with monoclonal antibodies. The classification into the seven OspA serotypes could be confirmed on a genetic basis (ospA genotypes 1–7) for all strains analyzed so far (n=29). In addition, one strain without OspA expression could be assigned to ospA genotype 2. Genetic stability could be proven for the ospA gene of B. burgdorferi strain PWudI after inocculation and reisolation from a gerbil. However, we found evidence for intragenic recombination by cluster analysis of ospA sequence data. Accordance of ospA genotype 1 strains with B. burgdorferi sensu stricto and ospA genotype 2 strains with B. afzelii, as well as the ospA genotype strains 3–7 with B. garinii was confirmed by pulsed-field gel electrophoresis of MluI-digested genomic DNA. B. garinii is not only more heterogenous in respect to the OspA-encoding genes, but shows moreover major subgroups formed by genotypes 4, 5 and 6 and genotypes 3 and 7, respectively. The latter group has not been described previously and is specifically recognized by an OspA-specific monoclonal antibody L32 1F7.     

Comparison of PCR-restriction fragment length polymorphism analysis and PCR-direct sequencing methods for differentiating Helicobacter pylori ureB gene variants

A method utilizing PCR-restriction fragment length polymorphism (RFLP) in the Helicobacter pylori genes is widely used to differentiate strains. However, with this typing method only a single base change at a specific restriction site can be detected. In addition, it is unclear whether the nucleotide base change recognized by RFLP is related to a substitution of encoded amino acid. To examine the validity of the PCR-RFLP method, 933-bp PCR products were obtained from 41 different clinical H. pylori isolates and were digested with Sau3A restriction endonuclease. Furthermore, the nucleotides of the same region in the ureB gene were directly sequenced and compared. PCR-RFLP confirmed that there was genetic diversity within the ureB gene with three distinct types, one being well conserved and the other two being variations. However, the direct sequencing method revealed that there was no difference at the nucleotide level among these RFLP types. Base substitutions recognized by Sau3A occurred in the third-base position and did not change the encoded amino acid. In addition, many nucleotide mutations, which could not be recognized by Sau3A, were frequently found. These results suggest that the PCR-RFLP method provides for an easy typing scheme of isolates, but does not reveal the true extent of genetic diversity. It is proposed that careful observation is required for the interpretation of results when clinical isolates are differentiated.     

PCR-restriction fragment length polymorphism analysis for identification of Bacteroides spp. and characterization of nitroimidazole resistance genes

Bacteroides spp. are opportunist pathogens that cause blood and soft tissue infections and are often resistant to antimicrobial agents. We have developed a combined PCR-restriction fragment length polymorphism (RFLP) technique to characterize the 16S rRNA gene for identification purposes and the nitroimidazole resistance (nim) gene for detection of resistance to the major antimicrobial agent used to treat Bacteroides infections: metronidazole (MTZ). PCR-RFLP analysis of 16S ribosomal (rDNA) with HpaII and TaqI produced profiles that enabled discrimination of type strains and identification of 70 test strains to the species level. The 16S rDNA PCR-RFLP identification results agreed with routine phenotypic testing for 62 of the strains. The discrepancies between phenotypic and PCR-RFLP methods for eight strains were resolved by 16S rDNA sequencing in three cases, but five strains remain unidentified. The presence of nim genes was indicated by PCR in 25 of 28 strains that exhibited reduced sensitivity to MTZ. PCR-RFLP of the nim gene products identified the four reported genes (nimA, -B, -C, and -D) and indicated the presence of a previously unreported nim gene in 5 strains. This novel nim gene exhibited 75% DNA sequence similarity with nimB. These rapid, accurate, and inexpensive methods should enable improved identification of Bacteroides spp. and the detection of MTZ resistance determinants.     

Sensitive PCR-restriction fragment length polymorphism assay for detection and genotyping of Giardia duodenalis in human feces

An assay that uses heminested PCR-restriction fragment length polymorphism analysis for the detection and genotyping of Giardia duodenalis on the basis of polymorphism in the triose phosphate isomerase (tpi) gene was developed. This assay was evaluated with DNA extracted from purified parasite material, bacterial cultures, whole human feces containing G. duodenalis and other parasites, and their corresponding immunofluorescence-stained fecal smears on glass microscope slides. The assay was specific and discriminated between G. duodenalis assemblages A and B. RFLP analysis further distinguished two groups (designated groups I and II) within assemblage A. Among 35 DNA samples extracted from whole feces from patients with confirmed sporadic giardiasis, the tpi gene was amplified from 33 (94%). Of these, nine (27%) samples contained assemblage A group II, 21 (64%) contained assemblage B, and 3 (9%) contained a mixture of assemblage A group II and assemblage B. The tpi gene of G. duodenalis assemblage B was amplified from 21 of 24 (88%) DNA samples extracted from whole feces from patients with confirmed cases of infection in a nursery outbreak. No amplification was detected from the remaining three DNA samples. Overall, analysis of DNA extracted from material recovered from stained microscope slides identified identical G. duodenalis genotypes in 35 (65%) of the 54 samples for which a genotype was established with DNA from whole feces. The heminested PCR method developed is sensitive, simple, and rapid to perform and is applicable for the analysis of other intestinal pathogens.     

Proximal region of the gene encoding cytadherence-related protein permits molecular typing of Mycoplasma genitalium clinical strains by PCR-restriction fragment length polymorphism

Restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified proximal region of the gene encoding cytadherence accessory protein P110 (MG192) revealed DNA sequence divergences among 54 Mycoplasma genitalium clinical strains isolated from the genitourinary tracts of women attending a sexually transmitted disease-related health clinic, plus one from the respiratory tract and one from synovial fluid. Seven of 56 (12.5%) strains exhibited RFLPs following digestion of the proximal region with restriction endonuclease MboI or RsaI, or both. No sequence variability was detected in the distal portion of the gene.