PCR-restriction Fragment Length Polymorphism Patterns Research Articles

Discrimination of Relapsing FeverBorrelia persicaandBorrelia microttiby Diagnostic Species-Specific Primers and Polymerase Chain Reaction–Restriction Fragment Length Polymorphism

Tick-borne relapsing fever (TBRF) is endemic in Africa and Eurasia and attributed to different Borrelia species. In the Central Asia and Middle Eastern countries, TBRF is caused mainly by Borrelia persica; however, other Borrelia species such B. microtti, B. latyschewii, B. baltazardi, and B. caucasica have also been described. The classic taxonomy of Borrelia spp. is based on the cospeciation concept that is very complex and rather confusing. In this study, we report two DNA-based methods to discriminate B. persica and B. microtti, the two main prevalent species in the region. Molecular typing of the species was performed using (i) restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified fragments of either 16S-rRNA or glpQ genes, and (ii) species-specific PCR of glpQ gene. Sequence analyses of the data obtained in this study indicate that the glpQ gene is more variable than 16S-rRNA (6.9% vs. 1.2%); thus glpQ is a more useful marker for discrimination of B. persica from B. microtti. The 16S-rRNA fragment comprises only one useful species-specific restriction site (TaqI), whereas the glpQ fragment includes several species-specific restriction sites and its digestion by DraI, TaqI, EcoRV, HinfI, or SspI results in distinctively different PCR-restriction fragment length polymorphism patterns for the two species. Further, the species-specific primers amplified a 253-bp fragment for B. persica and a 451-bp one for B. microtti. Phylogenetic analysis of the data revealed that B. microtti and B. persica are associated to the African and new world RF agents, respectively. This study demonstrates that both typing methods are simple, sensitive, and fast, and that they allow one to differentiate between B. persica and B. microtti. This could prove that both methods are important and useful in monitoring of TBRF disease in endemic areas.

Characterization of widespread canine leishmaniasis among wild carnivores from Spain

Visceral Leishmaniasis (VL) is an emerging zoonotic parasitic disease caused by Leishmania infantum in Mediterranean countries, with sand flies ( Phlebotomus spp.) as vectors and dogs as the main domestic reservoir. The role of wild carnivores in the epidemiology of leishmaniasis is still controversial. In order to determine the prevalence of natural infection with L. infantum in wild carnivores from Spain, we analyzed 217 samples by PCR and western blotting and used restriction fragment length polymorphism (RFLP) to compare the patterns present in wild carnivores with those of domestic dogs from the same areas. DNA of the parasite was detected in spleen or blood samples from 35 (16.12%) analyzed wild carnivores, including 8 of 39 (20.5%) wolves ( Canis lupus), 23 of 162 (14.1%) foxes ( Vulpes vulpes), 2 of 7 (28.6%) Egyptian mongooses ( Herpestes ichneumon), 1 of 4 genets ( Geneta geneta), and 1 of 4 Iberian lynxes ( Lynx pardinus). No significant sex or age differences in prevalence were observed in wolves and foxes ( P > 0.05), but there was a significant difference among regions in foxes ( P < 0.05). A total of 12 PCR–RFLP patterns were found in foxes, 6 in wolves, 4 in dogs, 2 in Egyptian mongooses and 1 in lynx and genet. RFLP patterns differed between dogs and foxes in the two areas where they could be compared. This is the first study of canine leishmaniasis in wild canids and other carnivores from different regions of Spain by PCR. The prevalence of infection indicates the existence of natural infection in apparently healthy wild carnivore populations, and our results are suggestive of a sylvatic cycle independent of dogs.

Environmental Occurrence ofMadurella mycetomatis, the Major Agent of Human Eumycetoma in Sudan

Madurella mycetomatis is the main causative agent of human eumycetoma, a severe debilitating disease endemic in Sudan. It has been suggested that eumycetoma has a soil-borne or thorn prick-mediated origin. For this reason, efforts were undertaken to culture M. mycetomatis from soil samples (n = 43) and thorn collections (n = 35) derived from areas in which it is endemic. However, ribosomal sequencing data revealed that the black fungi obtained all belonged to other fungal species. In addition, we performed PCR-mediated detection followed by restriction fragment length polymorphism (RFLP) analysis for the identification of M. mycetomatis DNA from the environmental samples as well as biopsies from patients with mycetoma. In the case of the Sudanese soil samples, 17 out of 74 (23%) samples were positive for M. mycetomatis DNA. Among the thorn collections, 1 out of 22 (5%) was positive in the PCR. All PCR RFLP patterns clearly indicated the presence of M. mycetomatis. In contrast, 15 Dutch and English control soil samples were all negative. Clinically and environmentally obtained fungal PCR products share the same PCR RFLP patterns, suggesting identity, at least at the species level. These observations support the hypothesis that eumycetoma is primarily environmentally acquired and suggest that M. mycetomatis needs special conditions for growth, as direct isolation from the environment seems to be impossible.

Genetic and enzymatic analyses of metalloprotease (aureolysin) from Staphylococcus aureus isolated from domestic animals

The gene ( aur) encoding the metalloprotease (aureolysin) of Staphylococcus aureus from domestic animals was analyzed by polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (PCR-RFLP) and sequencing. The aur gene was detected in all 74 isolates from cows, pigs and chickens by PCR amplification and was classified into types I and II by PCR-RFLP patterns. The type II aur gene was contained in 36 (94.7%) of 38 protease-positive isolates as judged by skim milk agar plate culture, while type I was contained in 28 (77.8%) of 36 protease-negative isolates. The deduced amino acid sequences of aureolysins from type I and II isolates were almost identical with those of the published data. Subsequently, the two aureolysins were purified from the culture supernatants of type I and II isolates. The molecular weights of purified type I and II aureolysins were both estimated at 34 kDa by SDS-polyacrylamide gel electrophoresis. These aureolysins had maximum proteolytic activity at 30–50 °C and pH 7.0–8.0. Their activity was inhibited by metal- and zinc-specific inhibitors, such as EDTA, EGTA and 1,10-phenanthroline. Specific activity (activity/protein) of type II aureolysin was two times higher than that of type I. These results indicated that the aur gene is highly conserved with two allelic forms (types I and II) among bovine, porcine and avian isolates of S. aureus.

Isolation and epidemiological study of vancomycin-resistant Enterococcus faecium from patients of a haematological unit in Poland

Enterococcus faecium has recently emerged as a serious nosocomial pathogen. Vancomycin resistant enterococci (VRE) have been isolated in Europe and the USA since 1988. This is the first report on isolation of vancomycin resistant E. faecium (VREM) strains in Poland, from Haematological Unit patients in the Clinical Hospital in Gdańsk. In total, 6412 samples were examined between December 1996 and October 1997. Five hundred and five isolates of Enterococcus spp. were collected. One hundred and one were classified as Enterococcus faecium of which 49 were resistant to vancomycin (MIC>256 mg/L) and teicoplanin (MIC>256 mg/L), characteristic of the VanA phenotype. Twenty-nine patients were infected or colonized. A PCR-based specific diagnostic assay confirmed the phenotype. The multiplex PCR-restriction fragment length polymorphism patterns were consistent with VanA-type of vancomycin-resistant E. faecium for all isolates examined. These isolates were epidemiologically-related as shown by PCR-fingerprinting.

Phylogenetic Analysis of Borrelia Species Based on Flagellin Gene Sequences and Its Application for Molecular Typing of Lyme Disease Borreliae

We determined almost complete flagellin gene sequences of various Borrelia species and aligned them with previously published sequences. A neighbor-joining phylogenetic analysis showed that the genus Borrelia was divided into the following three major clusters: New World relapsing fever borreliae (Borrelia turicatae, Borrelia parkeri, and Borrelia hermsii), Old World relapsing fever borreliae (Borrelia crocidurae, Borrelia duttonii, and Borrelia hispanica), and Lyme disease borreliae (Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii). Agents of animal spirochetosis (Borrelia coriaceae and Borrelia anserina) and species of unknown pathogenicity (Borrelia miyamotoi and Borrelia lonestari) were related to relapsing fever borreliae. Although the Lyme disease borreliae, two related species (Borrelia japonica and Borrelia andersonii), and some newly described genomic groups (groups PotiB2, VS116, DN127, Hk501, and Ya501) were closely related to each other, each taxon formed an independent branch on the phylogenetic tree. The data obtained in this study indicate that the flagellin genes are useful in Borrelia taxonomy. To distinguish the Lyme disease borreliae from related organisms easily, we designed an oligonucleotide primer set for the flagellin gene and performed a PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. The primer set amplified an approximately 580-bp DNA fragment that included species-specific restriction sites, and HapII, HhaI, CelII, HincII, or DdeI digestion of the product resulted in distinctively different PCR-RFLP patterns. The PCR-RFLP typing method which we developed should facilitate rapid identification of Lyme disease borreliae and related organisms obtained from biological and clinical specimens.

Characterization of Erwinia chrysanthemi by pectinolytic isozyme polymorphism and restriction fragment length polymorphism analysis of PCR-amplified fragments of pel genes.

Conserved regions about 420 bp long of the pelADE cluster specific to Erwinia chrysanthemi were amplified by PCR and used to differentiate 78 strains of E. chrysanthemi that were obtained from different hosts and geographical areas. No PCR products were obtained from DNA samples extracted from other pectinolytic and nonpectinolytic species and genera. The pel fragments amplified from the E. chrysanthemi strains studied were compared by performing a restriction fragment length polymorphism (RFLP) analysis. On the basis of similarity coefficients derived from the RFLP analysis, the strains were separated into 16 PCR RFLP patterns grouped in six clusters, These clusters appeared to be correlated with other infraspecific levels of E. chrysanthemi classification, such as pathovar and biovar, and occasionally with geographical origin. Moreover, the clusters correlated well with the polymorphism of pectate lyase and pectin methylesterase isoenzymes. While the pectin methylesterase profiles correlated with host monocot-dicot classification, the pectate lyase polymorphism might reflect the cell wall microdomains of the plants belonging to these classes.

Epidemiological study of Mycoplasma pneumoniae infections in japan based on PCR-restriction fragment length polymorphism of the P1 cytadhesin gene.

Two hundred fifty strains of Mycoplasma pneumoniae isolated during the past 20 years in Japan were classified into two groups (I and II) based upon different PCR-restriction fragment length polymorphism patterns of their P1 cytadhesin genes. Clear shifts between the M. pneumoniae groups were observed but did not appear to be correlated with M. pneumoniae epidemic cycles. Patients' sera showed relatively higher levels of antiadhesin antibodies to M. pneumoniae strains homologous with the infecting strain.