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.     

Differentiation of Helicobacter pylori strains directly from gastric biopsy specimens by PCR-based restriction fragment length polymorphism analysis without culture.

Recent studies have shown the usefulness of PCR-based restriction fragment length polymorphism (RFLP) analysis for differentiating Helicobacter pylori strains isolated by culture. For this study, a PCR-based RFLP assay was developed for directly typing H. pylori strains from gastric biopsy specimens. Nineteen gastric biopsy specimens obtained from patients undergoing endoscopy for gastrointestinal complaints were cultured for isolation of H. pylori. Genomic DNA preparations from these gastric biopsy specimens and the corresponding H. pylori isolates were tested by our PCR-based RFLP assay. The 1,179-bp H. pylori DNA fragments amplified by the PCR assay were digested with the restriction enzymes HhaI, MboI, and AluI and analyzed by agarose gel electrophoresis. HhaI, MboI, and AluI digestion produced 11, 10, and 6 distinguishable digestion patterns, respectively, from the 19 H. pylori isolates tested and generated 13, 11, and 6 different patterns, respectively, from the 19 gastric biopsy specimens. The patterns from 13 of the 19 gastric biopsy specimens matched those of the H. pylori isolates from the corresponding patients. The patterns from the remaining six biopsy specimens appeared to represent infection by two strains of H. pylori; the pattern of one strain was identical to that of the isolate from the corresponding patient. By combining all the restriction enzyme digestion patterns obtained by using HhaI, MboI, and AluI, we observed 19 distinct RFLP patterns from the 19 specimens. The results suggest that the PCR-based RFLP analysis method may be useful as a primary technique to identify and distinguish H. pylori strains directly from gastric biopsy specimens without culture of the organisms.     

Detection of oxazolidinone-resistant Enterococcus faecalis and Enterococcus faecium strains by real-time PCR and PCR-restriction fragment length polymorphism analysis

A real-time PCR assay identified linezolid-resistant Enterococcus faecalis and Enterococcus faecium isolates with a G2576U rRNA mutation. PCR-restriction fragment length polymorphism analysis of ribosomal DNA amplicons with NheI also detected this mutation. Both assays detected isolates heterozygous at this position. Recognition of isolates with what is presently the most frequent oxazolidinone resistance mutation may aid surveillance and individual case management.     

Rapid identification of mycobacteria from smear-positive sputum samples by nested PCR-restriction fragment length polymorphism analysis

The rapid identification of mycobacteria from smear-positive sputum samples is an important clinical issue. Furthermore, the availability of a cheap, technically simple, and accurate method also would benefit mycobacterial laboratories in developing countries. In the present study, we aimed to develop an assay allowing the identification of the Mycobacterium tuberculosis complex (MTBC) and other frequently isolated nontuberculous mycobacteria (NTM) directly from smear-positive sputum samples. A nested PCR-restriction fragment length polymorphism analysis (nested-PRA) assay that focuses on the analysis of the hsp65 gene was developed and evaluated for its efficiency compared to that of traditional culture methods and 16S rRNA gene sequencing identification. A total of 204 smear-positive and culture-positive sputum specimens were prospectively collected for analysis between November 2005 and May 2006. The samples were classified according to an acid-fast bacillus (AFB) staining scale as rare/1+, 2+, or 3+. The results of the nested-PRA showed that the identification rate for AFB 3+, AFB 2+, and AFB rare/1+ samples was 100, 95, and 53%, respectively, and that the overall identification rate was 89%. All positive results by the nested-PRA method agreed with the results by culture and 16S rRNA gene sequence analysis. The nested-PRA appears to have clinical applicability when used for the direct identification of mycobacterial organisms (both MTBC and NTM) that are present in smear-positive sputum samples, especially for countries in which MTBC is endemic.     

PCR-based restriction fragment length polymorphism typing of Helicobacter pylori.

We applied a molecular typing approach for Helicobacter pylori that uses restriction fragment length polymorphism (RFLP) analyses of an 820-bp PCR-amplified portion of the ureC gene in H. pylori. The PCR products were digested with restriction enzyme HhaI, MboI, or MseI, and the fragments generated were analyzed by agarose electrophoresis. Among 25 independent clinical isolates, each showed a different pattern when a combination of the three RFLP patterns was used. Using this method, we studied isolates from the antrum or the body of the stomach of 14 patients before and after antibiotic therapy. Before treatment, successful isolation of H. pylori from the two sites of the stomach was possible for 12 of the 14 patients. For 10 of these 12 patients, each pair of isolates had identical RFLP profiles. For the other two patients (16.7%), however, isolates from the antrum and the body of the stomach had different RFLP profiles. Treatment was successful for 6 of the 14 patients; of the 8 patients with treatment failures, 5 had identical isolate pairs. In each case, the isolates found posttreatment were the same as the pretreatment isolates. For one of the patients who was colonized with two different isolates pretreatment, one of the isolates was identified at both sites after unsuccessful treatment. We also studied six long-term follow-up patients who had sequential biopsies at intervals of up to 5 months. Each follow-up isolate from each patient had the same RFLP profile as the initial isolate. This typing method provides a reliable and reproducible typing scheme for the study of H. pylori infections and indicates that infection with more than one H. pylori isolate is not rare.     

Rapid fingerprinting of Helicobacter pylori by polymerase chain reaction and restriction fragment length polymorphism analysis.

A simple and reliable technique was developed for differentiating Helicobacter pylori strains by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified DNAs. Oligonucleotide primer pairs developed to the urease, 48-kDa stress protein (htrA), and 26-kDa antigen-encoding genes were used to amplify fragments of the appropriate size from crude boiled cell preparations. The PCR-amplified products were digested with Sau3A, HaeIII, MspI, AluI, MluI, HinfI, and XbaI restriction endonucleases. Restriction fragment length polymorphisms were particularly evident within the urease and htrA genes and were easily detected by Sau3A, HaeIII, MspI, and AluI restriction endonuclease analysis. Double digestion of these separately amplified products or restriction analysis of multiple PCR-amplified fragments was found to discriminate 17 of 17 (100%) H. pylori strains which had unique genomic DNA fingerprints. Results of an investigation of multiple isolate sets obtained from patients before and after therapy was consistent with the hypothesis that treatment failures were due to the persistence of the same strain but did not discount the possibility that the patients were reinfected with a strain shared by family members or close contacts. The results indicate that the PCR-restriction endonuclease analysis method can be applied directly to biopsy samples, has the potential to fingerprint H. pylori isolates rapidly, and may permit detailed epidemiological investigations on the transmission of this important pathogen.     

Rapid screening of fluoroquinolone resistance determinants in Streptococcus pneumoniae by PCR-restriction fragment length polymorphism and single-strand conformational polymorphism

A rapid method, using PCR-restriction fragment length and single-strand conformation polymorphism (SSCP), was applied to screen for mutations of the fluoroquinolone resistance determinants in Streptococcus pneumoniae. One hundred nonduplicate Streptococcus pneumoniae isolates with ciprofloxacin MICs of > or = 4.0 microg/ml from the Prince of Wales Hospital, Hong Kong, years 2000 to 2003, were examined. For each isolate, PCR amplicons of quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE genes were digested with AluI, HinfI, Sau3AI, and MspI, respectively, and analyzed by SSCP. Each SSCP pattern was given a number, and each isolate obtained a four-digit code, e.g., 1111, that represented the SSCP profile. The SSCP patterns were correlated to mutations characterized from sequence analyses of PCR amplicons. The most common SSCP profile obtained was no. 5232 (40%), which included strains with two amino acid substitutions in the ParC (Lys-137-Asn) and ParE (Ile-460-Val) genes, followed by the SSCP profile 5223 (17%), which included strains with amino acid substitutions in the ParE (Ile-460-Val) gene only. Ten isolates (10%) with amino acid substitutions at GyrA and ParE (+/-ParC) genes were resistant to levofloxacin with a MIC of > or = 16 microg/ml. Other SSCP profiles were unique in distinguishing the common amino acid substitutions in GyrA (Ser-81-Phe) and ParC (Lys-137-Asn, Ser-79-Phe plus Lys-137-Asn, Asp-83-Asn plus Lys-137-Asn, Ser-79-Phe, and Glu-96-Asp). SSCP analysis of restricted fragments generated patterns that were highly discriminative for mutations present in the QRDRs of gyrA, gyrB, parC, and parE. This method provides a database of high resolution profiles on these mutations and allows rapid screening for new mutations of the fluoroquinolone resistance genes.     

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.     

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.     

Identification of cagA tyrosine phosphorylation DNA motifs in Helicobacter pylori isolates from peptic ulcer patients by novel PCR-restriction fragment length polymorphism and real-time fluorescence PCR assays

Cag pathogenicity island-containing Helicobacter pylori (type I) induces signal transduction pathways resulting in tyrosine phosphorylation of proteins adjacent to the site of bacterial adhesion on host gastric epithelial cells. Conventional block PCR-restriction fragment length polymorphism (RFLP) and real-time LightCycler (LC) PCR hybridization assays, validated by direct sequencing, were designed to test for the presence of three nucleotide sequences corresponding to tyrosine phosphorylation motifs (TPMs) A, B, and C in 84 isolates of H. pylori type I from patients in England. Overall, the PCR assays demonstrated that one or more TPMs were present in 62 strains (75%). Motif A was common (71% of strains), whereas motifs B and C were rarer (8% of strains). Strains lacking a TPM were typically vacuolating cytotoxin genotype vacA m2. Motif A was widely distributed in relation to disease severity and was more commonly (but not significantly [P = 0.071]) associated with gastric ulcer than with duodenal ulcer (86 versus 56%). The LC hybridization assay provided a rapid means of detecting all three motifs, but RFLP analysis was more specific for TPM-A. TPMs provide novel additional strain markers for defining cagA variation, including identification of RFLP types within TPM-A. The presence of a particular TPM was not of direct diagnostic value, either singly or in combination, but the higher proportion of TPM-A strains in gastric ulcer patients merits further investigation.     

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.     

Genetic identification of the main opportunistic Mucorales by PCR-restriction fragment length polymorphism

Mucormycosis is a rare and opportunistic infection caused by fungi belonging to the order Mucorales. Recent reports have demonstrated an increasing incidence of mucormycosis, which is frequently lethal, especially in patients suffering from severe underlying conditions such as immunodeficiency. In addition, even though conventional mycology and histopathology assays allow for the identification of Mucorales, they often fail in offering a species-specific diagnosis. Due to the lack of other laboratory tests, a precise identification of these molds is thus notoriously difficult. In this study we aimed to develop a molecular biology tool to identify the main Mucorales involved in human pathology. A PCR strategy selectively amplifies genomic DNA from molds belonging to the genera Absidia, Mucor, Rhizopus, and Rhizomucor, excluding human DNA and DNA from other filamentous fungi and yeasts. A subsequent digestion step identified the Mucorales at genus and species level. This technique was validated using both fungal cultures and retrospective analyses of clinical samples. By enabling a rapid and precise identification of Mucorales strains in infected patients, this PCR-restriction fragment length polymorphism-based method should help clinicians to decide on the appropriate treatment, consequently decreasing the mortality of mucormycosis.     

iceA genotypes of Helicobacter pylori strains isolated from Brazilian children and adults

Data concerning the geographic distribution of iceA alleles are scarce, and information on the association of the gene with the disease is rare and still controversial. Furthermore, no such study has been developed in Brazil, where duodenal ulcer and gastric adenocarcinoma are very common. We investigated, by PCR, the frequency of iceA alleles and cagA status in Helicobacter pylori strains isolated from 142 patients (62 children and 80 adults; 66 female; mean age, 30.0 years; age range, 3 to 78 years) with gastritis, duodenal ulcer, or gastric adenocarcinoma. iceA was identified in bacterium samples obtained from all patients. Eleven (7.7%) of them were infected with multiple strains. Among the patients with nonmixed infection, iceA2 allele was detected in 118 (90.1%). iceA2 allele was associated with ulcer (P = 0.02) and with carcinoma (P = 0.001). iceA2 amplicons of 229, 334, or 549 bp were detected, but none of them was associated with the patient's disorder. iceA2 strains were more frequent in patients older than 7 years (P = 0.001). The gene was also more frequent in strains obtained from males (P = 0.02). cagA was more common in strains obtained from carcinoma (P = 0.0008) and ulcer patients (P < 0.006). cagA-positive strains were more frequent in children older than 7 years (P < 0.003). No association between cagA status and sex was found (P = 0.28). In conclusion, we think iceA should not be used as a reliable marker for predicting the clinical outcome of H. pylori infection.     

Recognition of a Nocardia transvalensis complex by resistance to aminoglycosides, including amikacin, and PCR-restriction fragment length polymorphism analysis.

Amikacin resistance, rare among nocardiae, was observed in 58 clinical isolates of nocardiae. All of these isolates hydrolyzed hypoxanthine, and 75 to 100% utilized citrate, D-galactose, and D-trehalose as sole carbon sources. Based on utilization of I-erythritol, D-glucitol, i-myo-inositol, D-mannitol, and ribitol and susceptibility to amoxicillin-clavulanic acid, the 58 isolates were separable into four groups. One group was negative for I-erythritol and ribitol and included all the isolates belonging to Nocardia asteroides complex antibiogram type IV. The remaining three groups were positive for I-erythritol and ribitol and were grouped within Nocardia transvalensis. The group that included the type strain was designated N. transvalensis sensu stricto, and the other two groups were designated new taxons 1 and 2. PCR-restriction fragment length polymorphism (RFLP) analysis of a 439-bp segment of the 65-kDa heat shock protein gene with XhoI and HinfI produced identical patterns for 53 (91%) and 58 (100%) isolates, respectively, and differentiated them from all other Nocardia taxa. NarI- and HaeIII-derived RFLP patterns clearly differentiated each of the four biochemically defined taxa. These four groups were also distinguishable by using the chromogenic substrates in Dade MicroScan test panels. By high-performance liquid chromatography, these isolates exhibited the same unique mycolic acid-ester elution patterns that differed from those of all other clinically significant nocardiae. Gas-liquid chromatographic analysis of fatty acids also produced similar patterns for all isolates that distinguished them from all other Nocardia taxa, but did not differentiate the four taxa within the complex. We propose the designation N. transvalensis complex for these four groups of nocardiae, pending further genetic evaluation.     

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 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.     

Differentiation between reinfection and recrudescence of helicobacter pylori strains using PCR-based restriction fragment length polymorphism analysis

The aim of this study was to evaluate whether PCR-based restriction fragment length polymorphism (RFLP) analysis was effective in differentiating between reinfection and recrudescence of H. pylori strains. Following a 1-2 week regimen of omeprazole 20 mg, amoxicillin 1.0 g, and clarithromycin 500 mg twice daily, twenty patients with duodenal ulcer were enrolled in the study. Ten patients (group 1, control) were not successfully treated, and another 10 patients (group 2) exhibited recurrence of infection 6-24 months following the therapy. Follow-up diagnosis was performed by Giemsa stain and CLO test. RFLP profiles of antral and midbody biopsy specimens were compared before and after therapy. PCR products using the ureC gene were digested with restriction enzymes Hha I, Mbo I, and Hind III, and the fragments generated were analyzed by agarose gel electrophoresis. Hha I, Mbo I, and Hind III digestion produced 13, 7, and 2 distinguishable digestion patterns, respectively. There was no difference in RFLP profiles seen before and after the therapy in 17 duodenal ulcer patients, while different RFLP profiles were discovered in 3 patients. Following treatment, one (group 2) patient differed in Mbo I, and two (one each from both groups) patients differed in Hha I and Mbo I RFLP patterns. Eight of group 2 patients showed recrudescence of previous infection and two patients had reinfection by another strain. This study supports the hypothesis that PCR-based RFLP analysis can be effective for differentiating reinfection and recrudescence of H. pylori strains following triple therapy.     

Distribution study of Chlamydia trachomatis serovars among high-risk women in China performed using PCR-restriction fragment length polymorphism genotyping

This was one of the first epidemiological studies in China focused on genital Chlamydia trachomatis serotype distribution in high-risk female populations using omp1 gene-based restriction fragment length polymorphism analysis. One thousand seven hundred seventy cervical swab samples from women attending sexually transmitted disease clinics and female sex workers in six cities in China (Shenzhen and Guangzhou in southern China, Nanjing and Shanghai in eastern China, and Nanning and Chengdu in southwestern China) were subjected to serovar genotyping. The proportion of omp1 genes successfully amplified in 240 C. trachomatis plasmid-positive samples was 94.2% (226/240). Serotypes E (n = 63; 27.9%), F (n = 53; 23.5%), G (n = 28; 12.4%), and D (n = 25; 11.1%) were most prevalent. Though there was no significant difference in the geographic distribution of C. trachomatis, serotype E was predominant in the South (32.1%) and East (27.1%), while serotype F was predominant in the Southwest (28.3%). Serotype F infection was associated with young age and single status. Serovar G was associated with lower abdominal pain; 47.5% of asymptomatic patients were infected with serovar E. These results provide information on distribution of genital C. trachomatis serotypes among high-risk women in China and indicate that high-risk women, including those who are asymptomatic, can be infected with multiple serovars of C. trachomatis, revealing exposure to multiple sources of infection. Although the scope for generalizations is limited by our small sample size, our results showing clinical correlations with genotypes are informative.     

Antibiotic resistance of Helicobacter pylori strains in Japanese children

The resistance of Helicobacter pylori to the recently available antibiotic treatment regimens has been a growing problem. We investigated the prevalence of H. pylori resistance to clarithromycin, metronidazole, and amoxicillin among 51 H. pylori isolates from Japanese children. In addition, the mutations of the corresponding gene were studied by PCR and restriction fragment length polymorphism analysis. Primary resistance to clarithromycin, metronidazole, and amoxicillin was detected in 29, 24, and 0% of strains, respectively. The eradication rates in clarithromycin-susceptible and -resistant strains were 89 and 56%, respectively (P < 0.05). The prevalence of strains with acquired resistance to clarithromycin (78%) was higher than that of strains with primary resistance (P < 0.01). Among the clarithromycin-resistant strains studied, 92% showed cross-resistance to azithromycin. No acquired resistance to amoxicillin was demonstrated. The A2144G mutation in the 23S rRNA gene was detected in 11 of 12 (92%) clarithromycin-resistant strains tested, whereas the mutation was not detected in any of the 15 susceptible strains. The deletion of the rdxA gene was not demonstrated in any of the strains. The results indicate that a high prevalence of clarithromycin-resistant strains is associated with eradication failure. Testing of susceptibility to clarithromycin is recommended.