Development of a genus-specific PCR assay for the molecular detection, confirmation and identification of Fusobacterium spp

A genus-specific polymerase chain reaction (PCR)-based assay is developed for the detection and identification of clinically relevant Fusobacterium species, including F. nucleatum and F. necrophorum. Two 16S ribosomal DNA (rDNA) primers, FUSO1 (forward primer: 5'-GAG AGA GCT TTG CGT CC-3' [17-mer]) and FUSO 2 (reverse primer: 5'-TGG GCG CTG AGG TTC GAC -3' [18-mer]) are designed to target conserved regions of the 16S rDNA gene for Fusobacterium spp. Subsequent proof-of-principle studies employing this assay detected Fusobacterium spp. in the faeces of eight (10%) out of 80 patients with suspected gastrointestinal infection. This assay may be used for the genus-specific detection of Fusobacterium spp. from clinical specimens and for subsequent species identification.     

Identification of helicobacter species in human liver samples from patients with primary hepatocellular carcinoma

AIMS: Several studies have shown the presence of helicobacter species in the human biliary tract and in the intestinal tract of animals. Experimental infection by Helicobacter hepaticus in mice causes chronic hepatitis and hepatocellular carcinoma (HCC). This study investigated whether helicobacter species could be detected in the liver of patients with HCC. METHODS: Liver samples from 20 patients with primary liver carcinoma diagnosed by histopathology and 16 controls without primary liver carcinoma were studied. Histology with standard and immunohistochemical stains, culture, and polymerase chain reaction (PCR) amplification using helicobacter genus specific 16S rRNA primers were used to detect the presence of bacteria. Amplified products were identified by Southern hybridisation and sequencing. A search for other genes specific for Helicobacter pylori was also performed. RESULTS: Helicobacter species 16S rDNA was found in eight of 20 samples of primary liver carcinoma, whereas none of the controls harboured this rDNA. Six helicobacter specific PCR amplicons were sequenced and were found to have 98.5-99.0% similarity to the 16S rDNA of H pylori. Of the eight positive samples, seven were positive in PCR using 26 kDa protein primers and six showed morphological and immunohistochemical evidence of H pylori. The cagA and glmM genes were detected in only two samples. The vacA and rps4 genes were not detected. CONCLUSIONS: Helicobacter can be present in the liver of patients with primary liver carcinoma and is probably linked to the carcinogenic process in the liver.     

Routine molecular identification of enterococci by gene-specific PCR and 16S ribosomal DNA sequencing

For 279 clinically isolated specimens identified by commercial kits as enterococci, genotypic identification was performed by two multiplex PCRs, one with ddl(E. faecalis) and ddl(E. faecium) primers and another with vanC-1 and vanC-2/3 primers, and by 16S ribosomal DNA (rDNA) sequencing. For 253 strains, phenotypic and genotypic results were the same. Multiplex PCR allowed for the identification of 13 discordant results. Six strains were not enterococci and were identified by 16S rDNA sequencing. For 5 discordant and 10 concordant enterococcal strains, 16S rDNA sequencing was needed. Because many supplementary tests are frequently necessary for phenotypic identification, the molecular approach is a good alternative.     

Polymerase chain reaction, with sequencing, as a diagnostic tool in culture-negative bacterial meningitis

Objective: To evaluate the feasibility of using 16S rDNA universal primer PCR (followed by sequencing) and 65-kDa heat shock Mycobacterium tuberculosis protein gene PCR as a method to determine a bacterial etiology in culture-negative cerebrospinal fluid (CSF) samples.
Methods: One hundred and forty-nine CSF samples from 128 patients were processed. DNA was extracted from the CSF samples and amplified with the eubacterial 16S rDNA primers P11E and P13B, and with the 65-kDa heat shock protein gene mycobacterial primers. The amplicons were identified by sequencing and specific oligoprobe hybridization.
Results: Overall, a microbiological diagnosis was made in 11 of 125 ultimately culture-negative cases. The use of 65-kDa heat shock protein gene PCR was needed to improve the diagnosis of tuberculous meningitis; in four patients, prospectively studied, the outcome of antituberculous therapy could also be followed.
Conclusions: In culture-negative bacterial meningitis it is possible to improve the microbiological diagnosis by use of 16S rDNA amplification and sequencing, together with amplification of a more specific gene in mycobacteria.     

Multiplex PCR identification of <Emphasis Type="Italic">Taenia</Emphasis> spp. in rodents and carnivores

The genus Taenia includes several species of veterinary and public health importance, but diagnosis of the etiological agent in definitive and intermediate hosts often relies on labor intensive and few specific morphometric criteria, especially in immature worms and underdeveloped metacestodes. In the present study, a multiplex PCR, based on five primers targeting the 18S rDNA and ITS2 sequences, produced a species-specific banding patterns for a range of Taenia spp. Species typing by the multiplex PCR was compared to morphological identification and sequencing of cox1 and/or 12S rDNA genes. As compared to sequencing, the multiplex PCR identified 31 of 32 Taenia metacestodes from rodents, whereas only 14 cysts were specifically identified by morphology. Likewise, the multiplex PCR identified 108 of 130 adult worms, while only 57 were identified to species by morphology. The tested multiplex PCR system may potentially be used for studies of Taenia spp. transmitted between rodents and carnivores.     

Identification of Mycobacterium spp. by using a commercial 16S ribosomal DNA sequencing kit and additional sequencing libraries

Current methods for identification of Mycobacterium spp. rely upon time-consuming phenotypic tests, mycolic acid analysis, and narrow-spectrum nucleic acid probes. Newer approaches include PCR and sequencing technologies. We evaluated the MicroSeq 500 16S ribosomal DNA (rDNA) bacterial sequencing kit (Applied Biosystems, Foster City, Calif.) for its ability to identify Mycobacterium isolates. The kit is based on PCR and sequencing of the first 500 bp of the bacterial rRNA gene. One hundred nineteen mycobacterial isolates (94 clinical isolates and 25 reference strains) were identified using traditional phenotypic methods and the MicroSeq system in conjunction with separate databases. The sequencing system gave 87% (104 of 119) concordant results when compared with traditional phenotypic methods. An independent laboratory using a separate database analyzed the sequences of the 15 discordant samples and confirmed the results. The use of 16S rDNA sequencing technology for identification of Mycobacterium spp. provides more rapid and more accurate characterization than do phenotypic methods. The MicroSeq 500 system simplifies the sequencing process but, in its present form, requires use of additional databases such as the Ribosomal Differentiation of Medical Microorganisms (RIDOM) to precisely identify subtypes of type strains and species not currently in the MicroSeq library.     

rpoB gene sequencing for identification of Corynebacterium species

The genus Corynebacterium is a heterogeneous group of species comprising human and animal pathogens and environmental bacteria. It is defined on the basis of several phenotypic characters and the results of DNA-DNA relatedness and, more recently, 16S rRNA gene sequencing. However, the 16S rRNA gene is not polymorphic enough to ensure reliable phylogenetic studies and needs to be completely sequenced for accurate identification. The almost complete rpoB sequences of 56 Corynebacterium species were determined by both PCR and genome walking methods. In all cases the percent similarities between different species were lower than those observed by 16S rRNA gene sequencing, even for those species with degrees of high similarity. Several clusters supported by high bootstrap values were identified. In order to propose a method for strain identification which does not require sequencing of the complete rpoB sequence (approximately 3,500 bp), we identified an area with a high degree of polymorphism, bordered by conserved sequences that can be used as universal primers for PCR amplification and sequencing. The sequence of this fragment (434 to 452 bp) allows accurate species identification and may be used in the future for routine sequence-based identification of Corynebacterium species.     

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.     

Molecular probes for diagnosis of clinically relevant bacterial infections in blood cultures

Broad-range real-time PCR and sequencing of the 16S rRNA gene region is a widely known method for the detection and identification of bacteria in clinical samples. However, because of the need for sequencing, such identification of bacteria is time-consuming. The aim of our study was to develop a more rapid 16S real-time PCR-based identification assay using species- or genus-specific probes. The Gram-negative bacteria were divided into Pseudomonas species, Pseudomonas aeruginosa, Escherichia coli, and other Gram-negative species. Within the Gram-positive species, probes were designed for Staphylococcus species, Staphylococcus aureus, Enterococcus species, Streptococcus species, and Streptococcus pneumoniae. The assay also included a universal probe within the 16S rRNA gene region for the detection of all bacterial DNA. The assay was evaluated with a collection of 248 blood cultures. In this study, the universal probe and the probes targeting Pseudomonas spp., P. aeruginosa, E. coli, Streptococcus spp., S. pneumoniae, Enterococcus spp., and Staphylococcus spp. all had a sensitivity and specificity of 100%. The probe specific for S. aureus showed eight discrepancies, resulting in a sensitivity of 100% and a specificity of 93%. These data showed high agreement between conventional testing and our novel real-time PCR assay. Furthermore, this assay significantly reduced the time needed for identification. In conclusion, using pathogen-specific probes offers a faster alternative for pathogen detection and could improve the diagnosis of bloodstream infections.     

Differentiation of Ehrlichia platys and E. equi Infections in Dogs by Using 16S Ribosomal DNA-Based PCR

We have encountered a previously unrecognized specificity problem when using the small-subunit ribosomal DNA (16S rDNA)-based PCR primers recommended for use in the identification of Ehrlichia equi in clinical samples. These PCR primers annealed to E. platys 16S rDNA in blood samples containing high levels of E. platys organisms. Therefore, we designed and tested new PCR primers for the identification of E. equi.     

Identification ofMycobacterium shimoidei in a tuberculosis-like cavity by 16S ribosomal DNA direct sequencing

A new, well-documented case ofMycobacterium shimoidei infection is reported. This bacterium was isolated from a patient with preexisting pulmonary lesions who developed a tuberculosis-like cavity in the lung. Isolation of fewer than ten strains, not epidemiologically related, has been reported worldwide; all originated from human pulmonary samples. Identification of the strain described here was first accomplished by 16S rDNA direct sequencing after in vitro amplification. This method allows rapid identification of unusual bacteria. The identification was confirmed by specific phenotypical tests and by chromatographic methods.     

Novel diagnostic algorithm for identification of mycobacteria using genus-specific amplification of the 16S-23S rRNA gene spacer and restriction endonucleases

A novel genus-specific PCR for mycobacteria with simple identification to the species level by restriction fragment length polymorphism (RFLP) was established using the 16S-23S ribosomal RNA gene (rDNA) spacer as a target. Panspecificity of primers was demonstrated on the genus level by testing 811 bacterial strains (122 species in 37 genera from 286 reference strains and 525 clinical isolates). All mycobacterial isolates (678 strains among 48 defined species and 5 indeterminate taxons) were amplified by the new primers. Among nonmycobacterial isolates, only Gordonia terrae was amplified. The RFLP scheme devised involves estimation of variable PCR product sizes together with HaeIII and CfoI restriction analysis. It yielded 58 HaeIII patterns, of which 49 (84%) were unique on the species level. Hence, HaeIII digestion together with CfoI results was sufficient for correct identification of 39 of 54 mycobacterial taxons and one of three or four of seven RFLP genotypes found in Mycobacterium intracellulare and Mycobacterium kansasii, respectively. Following a clearly laid out diagnostic algorithm, the remaining unidentified organisms fell into five clusters of closely related species (i.e., the Mycobacterium avium complex or Mycobacterium chelonae-Mycobacterium abscessus) that were successfully separated using additional enzymes (TaqI, MspI, DdeI, or AvaII). Thus, next to slowly growing mycobacteria, all rapidly growing species studied, including M. abscessus, M. chelonae, Mycobacterium farcinogenes, Mycobacterium fortuitum, Mycobacterium peregrinum, and Mycobacterium senegalense (with a very high 16S rDNA sequence similarity) were correctly identified. A high intraspecies sequence stability and the good discriminative power of patterns indicate that this method is very suitable for rapid and cost-effective identification of a wide variety of mycobacterial species without the need for sequencing. Phylogenetically, spacer sequence data stand in good agreement with 16S rDNA sequencing results, as was shown by including strains with unsettled taxonomy. Since this approach recognized significant subspecific genotypes while identification of a broad spectrum of mycobacteria rested on identification of one specific RFLP pattern within a species, this method can be used by both reference (or research) and routine laboratories.     

Helicobacter spp. and liver diseases

To test whether Helicobacter species play a role in the enhancement of liver necro-inflammation and fibrosis and in the development of hepatocellular carcinoma (HCC), we sought DNA sequences of Helicobacter species in liver specimens from patients with viral-related chronic hepatitis, HCC or metastatic liver carcinoma. We enrolled 28 consecutive patients with ultrasound evidence of hepatic nodule(s) on their first liver biopsy: 21 had histological evidence of HCC (Group I) and 7 of metastatic liver carcinoma (Group II). In the same period we observed 27 consecutive patients with chronic hepatitis on their first liver biopsy (Group III). Helicobacter sequences were sought by PCR using primers for the 16S rDNA of Helicobacter spp, designed to amplify a 400 base-pair fragment, and detected by 2% agarose gel and hybridization with a specific biotinylated probe. We used, as positive controls for the DNA extraction from liver tissue, hepatic biopsy sections in which HBV infection was confirmed by HBcAg positivity and in which we amplified HBV-DNA by specific primers; positive controls for the amplification of Helicobacter spp were obtained from gastric biopsy sections in which Helicobacter pylori infection was confirmed by biochemical and histochemical tests. HBV-DNA was found in all five HBcAg positive liver biopsies. Helicobacter spp 16S rDNA was detected in all five biopsy specimens of gastric mucosa and in none of liver specimens from patients in any group. Our data suggest that Helicobacter species were not involved in the pathogenesis of virus-related HCC, chronic hepatitis or liver carcinoma metastasis.     

Rapid identification of bacteria by PCR-single-strand conformation polymorphism.

A new molecular biological approach for the identification of bacteria is described. This approach employs PCR of bacterial cell lysates with conserved primers located in the 16S rRNA sequence flanking a variable region, and analysis of the amplified product was based on the principle of single-strand conformation polymorphism (SSCP). The PCR product was denatured and separated on a nondenaturing polyacrylamide gel. SSCP patterns were detected by silver staining the nucleic acids. The mobility of the single-stranded DNA is sequence dependent and could be used to identify the unknown bacteria. Feasibility of the technique was demonstrated for a broad panel of gram-negative and gram-positive bacteria. We tested over 100 strains of bacteria representing 15 genera and 40 species. With the use of only two primer sets, P11P-P13P and ER10-ER11, we were capable to discriminate the tested species at the genus and species levels. Species-specific patterns were obtained for, e.g., Clostridium spp., Listeria spp., Pseudomonas spp., and Enterobacter spp. PCR-SSCP is a sensitive technique; e.g., the sensitivity obtained for Escherichia coli cells was 30 CFU. This technique is a simple and rapid method for the detection and identification of a wide spectrum of bacteria by whole-cell-based PCR amplification with the use of conserved primers and identification by nondenaturing gel electrophoresis.     

Evaluation of three oligonucleotide primer sets in PCR for the identification of Burkholderia cepacia and their differentiation from Burkholderia gladioli.

AIMS: To evaluate three oligonucleotide primer pairs--two specific for 16S and 23S rRNA sequences of Burkholderia cepacia, and the third specific for internal transcribed spacer region of 16S-23S sequences of B gladioli--for the identification and differentiation of reference and clinical strains of these and other species. METHODS: The three primers sets were applied in polymerase chain reaction (PCR) to a collection of 177 clinical isolates submitted for identification from diagnostic laboratories as presumed B cepacia. RESULTS: At an annealing temperature of 63 degrees C, all eight B cepacia and four B gladioli reference strains reacted with their specific primers. B vandii was the only other species that was positive with both B cepacia primers but five Burkholderia or Ralstonia species reacted with one of these primers. Seventy eight isolates were typical of B cepacia in biochemical tests and 75 of these reacted with specific primers; three, however, were positive with the B gladioli primers. Fifteen asaccharolytic isolates were confirmed as B cepacia by PCR but other non-fermenting Gram negative species were negative with each of the primers. CONCLUSIONS: PCR using 16S rRNA sequences is recommended for identification of B cepacia that give atypical results in biochemical tests.     

Molecular identification of zygomycetes from culture and experimentally infected tissues

Mucormycosis is an emerging infection associated with a high mortality rate. Identification of the causative agents remains difficult and time-consuming by standard mycological procedures. In this study, internal transcribed spacer (ITS) sequencing was validated as a reliable technique for identification of Zygomycetes to the species level. Furthermore, species identification directly from infected tissues was evaluated in experimentally infected mice. Fifty-four Zygomycetes strains belonging to 16 species, including the most common pathogenic species of Rhizopus spp., Absidia spp., Mucor spp., and Rhizomucor spp., were used to assess intra- and interspecies variability. Ribosomal DNA including the complete ITS1-5.8S-ITS2 region was amplified with fungal universal primers, sequenced, and compared. Overall, for a given species, sequence similarities between isolates were >98%. In contrast, ITS sequences were very different between species, allowing an accurate identification of Zygomycetes to the species level in most cases. Six species (Rhizopus oryzae, Rhizopus microsporus, Rhizomucor pusillus, Mucor circinelloides, and Mucor indicus) were also used to induce disseminated mucormycosis in mice and to demonstrate that DNA extraction, amplification of fungal DNA, sequencing, and molecular identification were possible directly from frozen tissues.     

Genotypic identification of erythromycin-resistant campylobacter isolates as helicobacter species and analysis of resistance mechanism

The correct identification of Campylobacter species remains cumbersome, especially when conventional biochemical tests and antimicrobial susceptibility patterns are used for a phenotypical identification. Correct identification is important for epidemiological purposes and for studying changes in antimicrobial resistance patterns. Six erythromycin-resistant campylobacter strains were investigated by 16S ribosomal DNA (rDNA) sequencing, 23S rDNA sequencing, and restriction fragment length polymorphism analysis of a putative heme-copper oxidase domain described as being specific for thermophilic Campylobacter species. Three erythromycin-resistant isolates from feces of human immunodeficiency virus (HIV)-seropositive patients with diarrhea and one blood isolate of from HIV-seropositive patient with cellulitis were identified by 16S rDNA analysis as Helicobacter cinaedi, whereas 23S rDNA sequencing suggested Wolinella succinogenes. The 16S rDNA sequence data of fecal isolates of two patients with travelers diarrhea revealed Helicobacter pullorum and were also in contrast with 23S rDNA sequencing. Of 4 H. cinaedi isolates, 1 contained the putative heme-copper oxidase gene thought to be specific for thermophilic species. The six erythromycin-resistant Helicobacter species had a similar point mutation A2143G in 23S rDNA resembling the macrolides resistance in Helicobacter pylori. We conclude that 16S rDNA sequencing should be preferred to 23S rDNA analysis and that macrolide-resistant campylobacter strains should be investigated by this approach for a correct identification.     

Rapid identification of Campylobacter spp. by melting peak analysis of biprobes in real-time PCR

We describe rapid PCR-biprobe identification of Campylobacter spp. This is based on real-time PCR with product analysis in the same system. The assay identifies enteropathogenic campylobacters to the species level on the basis of their degree of hybridization to three 16S ribosomal DNA (rDNA) biprobes. First-round symmetric PCR is performed with genus-specific primers which selectively target and amplify a portion of the 16S rRNA gene common to all Campylobacter species. Second-round asymmetric PCR is performed in a LightCycler in the presence of one of three biprobes; the identity of an amplified DNA-biprobe duplex is established after determination of the species-specific melting peak temperature. The biprobe specificities were determined by testing 37 reference strains of Campylobacter, Helicobacter, and Arcobacter spp. and 59 Penner serotype reference strains of Campylobacter jejuni and C. coli. From the combination of melting peak profiles for each probe, an identification scheme was devised which accurately detected the five taxa pathogenic for humans (C. jejuni/C. coli, C. lari, C. upsaliensis, C. hyointestinalis, and C. fetus), as well as C. helveticus and C. lanienae. The assay was evaluated with 110 blind-tested field isolates; when the code was broken their previous phenotypic species identification was confirmed in every case. The PCR-biprobe assay also identified campylobacters directly from fecal DNA. PCR-biprobe testing of stools from 38 diarrheic subjects was 100% concordant with PCR-enzyme-linked immunosorbent assay identification (13, 20) and thus more sensitive than phenotypic identification following microaerobic culture.     

Discrimination of Burkholderia multivorans and Burkholderia vietnamiensis from Burkholderia cepacia genomovars I, III, and IV by PCR

We present a PCR procedure for identification of Burkholderia cepacia, Burkholderia multivorans, and Burkholderia vietnamiensis. 16S and 23S ribosomal DNAs (rDNAs) of B. multivorans and B. vietnamiensis were sequenced and aligned with published sequences for definition of species-specific 18-mer oligonucleotide primers. Specific antisense 16S rDNA primers (for B. cepacia, 5'-AGC ACT CCC RCC TCT CAG-3'; for B. multivorans, 5'-AGC ACT CCC GAA TCT CTT-3') and 23S rDNA primers (for B. vietnamiensis, 5'-TCC TAC CAT GCG TGC AA-3') were paired with a general sense primer of 16S rDNAs (5'-AGR GTT YGA TYM TGG CTC AG-3') or with a sense primer of 23S rDNA (5'-CCT TTG GGT CAT CCT GGA-3'). PCR with these primers under optimized conditions is appropriate to specifically and rapidly identify B. multivorans, B. vietnamiensis, and B. cepacia (genomovars I, III, and IV are not discriminated). In comparison with the polyphasic taxonomic analyses presently necessary for species and genomovar identification within the B. cepacia complex, our procedure is more rapid and easier to perform and may contribute to clarifying the clinical significance of individual members of the complex in cystic fibrosis.