16S rRNA terminal restriction fragment length polymorphism for the characterization of the nasopharyngeal microbiota

A novel non-culture based 16S rRNA Terminal Restriction Fragment Length Polymorphism (T-RFLP) method using the restriction enzymes Tsp509I and Hpy166II was developed for the characterization of the nasopharyngeal microbiota and validated using recently published 454 pyrosequencing data. 16S rRNA gene T-RFLP for 153 clinical nasopharyngeal samples from infants with acute otitis media (AOM) revealed 5 Tsp509I and 6 Hpy166II terminal fragments (TFs) with a prevalence of >10%. Cloning and sequencing identified all TFs with a prevalence >6% allowing a sufficient description of bacterial community changes for the most important bacterial taxa. The conjugated 7-valent pneumococcal polysaccharide vaccine (PCV-7) and prior antibiotic exposure had significant effects on the bacterial composition in an additive main effects and multiplicative interaction model (AMMI) in concordance with the 16S rRNA 454 pyrosequencing data. In addition, the presented T-RFLP method is able to discriminate S. pneumoniae from other members of the Mitis group of streptococci, which therefore allows the identification of one of the most important human respiratory tract pathogens. This is usually not achieved by current high throughput sequencing protocols. In conclusion, the presented 16S rRNA gene T-RFLP method is a highly robust, easy to handle and a cheap alternative to the computationally demanding next-generation sequencing analysis. In case a lot of nasopharyngeal samples have to be characterized, it is suggested to first perform 16S rRNA T-RFLP and only use next generation sequencing if the T-RFLP nasopharyngeal patterns differ or show unknown TFs.

Clonal spread of methicillin-resistant Staphylococcus pseudintermedius in Europe and North America: an international multicentre study

OBJECTIVES: The aim of this study was to determine the phenotypic and genotypic resistance profiles of methicillin-resistant Staphylococcus pseudintermedius (MRSP) and to examine the clonal distribution in Europe and North America. METHODS: A total of 103 MRSP isolates from dogs isolated from several countries in Europe, the USA and Canada were characterized. Isolates were identified by PCR-restriction fragment length polymorphism (RFLP), antimicrobial susceptibility was determined by broth dilution or gradient diffusion, and antimicrobial resistance genes were detected using a microarray. Genetic diversity was assessed by multilocus sequence typing (MLST), PFGE and spa typing. Staphylococcal cassette chromosome mec (SCCmec) elements were characterized by multiplex PCR. RESULTS: Thirteen different sequence types (STs), 18 PFGE types and 8 spa types were detected. The hybrid SCCmec element II-III described in a MRSP isolate was present in 75 (72.8%) isolates. The remaining isolates either had SCCmec type III (n=2), IV (n=6), V (n=14) or VII-241 (n=4) or were non-typeable (n=2). The most common genotypes were ST71(MLST)-J(PFGE)-t02(spa)-II-III(SCCmec) (56.3%) and ST68-C-t06-V (12.6%). In addition to mecA-mediated beta-lactam resistance, isolates showed resistance to trimethoprim [dfr(G)] (90.3%), gentamicin/kanamycin [aac(6')-Ie-aph(2')-Ia] (88.3%), kanamycin [aph(3')-III] (90.3%), streptomycin [ant(6')-Ia] (90.3%), streptothricin (sat4) (90.3%), macrolides and/or lincosamides [erm(B), lnu(A)] (89.3%), fluoroquinolones (87.4%), tetracycline [tet(M) and/or tet(K)] (69.9%), chloramphenicol (cat(pC221)) (57.3%) and rifampicin (1.9%). CONCLUSIONS: Two major clonal MRSP lineages have disseminated in Europe (ST71-J-t02-II-III) and North America (ST68-C-t06-V). Regardless of their geographical or clonal origin, the isolates displayed resistance to the major classes of antibiotics used in veterinary medicine and thus infections caused by MRSP isolates represent a serious therapeutic challenge.

Genotyping of Francisella tularensis strains by pulsed-field gel electrophoresis, amplified fragment length polymorphism fingerprinting, and 16S rRNA gene sequencing

We evaluated three molecular methods for identification of Francisella strains: pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphism (AFLP) analysis, and 16S rRNA gene sequencing. The analysis was performed with 54 Francisella tularensis subsp. holarctica, 5 F. tularensis subsp. tularensis, 2 F. tularensis subsp. novicida, and 1 F. philomiragia strains. On the basis of the combination of results obtained by PFGE with the restriction enzymes XhoI and BamHI, PFGE revealed seven pulsotypes, which allowed us to discriminate the strains to the subspecies level and which even allowed us to discriminate among some isolates of F. tularensis subsp. holarctica. The AFLP analysis technique produced some degree of discrimination among F. tularensis subsp. holarctica strains (one primary cluster with three major subclusters and minor variations within subclusters) when EcoRI-C and MseI-A, EcoRI-T and MseI-T, EcoRI-A and MseI-C, and EcoRI-0 and MseI-CA were used as primers. The degree of similarity among the strains was about 94%. The percent similarities of the AFLP profiles of this subspecies compared to those of F. tularensis subsp. tularensis, F. tularensis subsp. novicida, and F. philomiragia were less than 90%, about 72%, and less than 24%, respectively, thus permitting easy differentiation of this subspecies. 16S rRNA gene sequencing revealed 100% similarity for all F. tularensis subsp. holarctica isolates compared in this study. These results suggest that although limited genetic heterogeneity among F. tularensis subsp. holarctica isolates was observed, PFGE and AFLP analysis appear to be promising tools for the diagnosis of infections caused by different subspecies of F. tularensis and suitable techniques for the differentiation of individual strains.

Prevalence and genotypes of Toxoplasma gondii in feline faeces (oocysts) and meat from sheep, cattle and pigs in Switzerland

The protozoan parasite Toxoplasma gondii infects almost all warm blooded animal species including humans, and is one of the most prevalent zoonotic parasites worldwide. Post-natal infection in humans is acquired through oral uptake of sporulated T. gondii oocysts or by ingestion of parasite tissue cysts upon consumption of raw or undercooked meat. This study was undertaken to determine the prevalence of oocyst-shedding by cats and to assess the level of infection with T. gondii in meat-producing animals in Switzerland via detection of genomic DNA (gDNA) in muscle samples. In total, 252 cats (44 stray cats, 171 pet cats, 37 cats with gastrointestinal disorders) were analysed coproscopically, and subsequently species-specific identification of T. gondii oocysts was achieved by Polymerase Chain Reaction (PCR). Furthermore, diaphragm samples of 270 domestic pigs (120 adults, 50 finishing, and 100 free-range animals), 150 wild boar, 250 sheep (150 adults and 100 lambs) and 406 cattle (47 calves, 129 heifers, 100 bulls, and 130 adult cows) were investigated by T. gondii-specific real-time PCR. For the first time in Switzerland, PCR-positive samples were subsequently genotyped using nine PCR-restriction fragment length polymorphism (PCR-RFLP) loci (SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) for analysis. Only one of the cats shed T. gondii oocysts, corresponding to a T. gondii prevalence of 0.4% (95% CI: 0.0-2.2%). In meat-producing animals, gDNA prevalence was lowest in wild boar (0.7%; 95% CI: 0.0-3.7%), followed by sheep (2.0%; 95% CI: 0.1-4.6%) and pigs (2.2%; 95% CI: 0.8-4.8%). The highest prevalence was found in cattle (4.7%; 95% CI: 2.8-7.2%), mainly due to the high prevalence of 29.8% in young calves. With regard to housing conditions, conventional fattening pigs and free-range pigs surprisingly exhibited the same prevalence (2.0%; 95% CI: 0.2-7.0%). Genotyping of oocysts shed by the cat showed T. gondii with clonal Type II alleles and the Apico I allele. T. gondii with clonal Type II alleles were also predominantly observed in sheep, while T. gondii with mixed or atypical allele combinations were very rare in sheep. In pigs and cattle however, genotyping of T. gondii was often incomplete. These findings suggested that cattle in Switzerland might be infected with Toxoplasma of the clonal Types I or III, atypical T. gondii or more than one clonal Type.

Genotyping of human and porcine Yersinia enterocolitica, Yersinia intermedia, and Yersinia bercovieri strains from Switzerland by amplified fragment length polymorphism analysis

In this study, 231 strains of Yersinia enterocolitica, 25 strains of Y. intermedia, and 10 strains of Y. bercovieri from human and porcine sources (including reference strains) were analyzed using amplified fragment length polymorphism (AFLP), a whole-genome fingerprinting method for subtyping bacterial isolates. AFLP typing distinguished the different Yersinia species examined. Representatives of Y. enterocolitica biotypes 1A, 1B, 2, 3, and 4 belonged to biotype-related AFLP clusters and were clearly distinguished from each other. Y. enterocolitica biotypes 2, 3, and 4 appeared to be more closely related to each other (83% similarity) than to biotypes 1A (11%) and 1B (47%). Biotype 1A strains exhibited the greatest genetic heterogeneity of the biotypes studied. The biotype 1A genotypes were distributed among four major clusters, each containing strains from both human and porcine sources, confirming the zoonotic potential of this organism. The AFLP technique is a valuable genotypic method for identification and typing of Y. enterocolitica and other Yersinia spp.

Use of the Agilent 2100 bioanalyzer for rapid and reproducible molecular typing of Streptococcus pneumoniae

Restriction fragment length polymorphism (RFLP) analysis is an economic and fast technique for molecular typing but has the drawback of difficulties in accurately sizing DNA fragments and comparing banding patterns on agarose gels. We aimed to improve RFLP for typing of the important human pathogen Streptococcus pneumoniae and to compare the results with the commonly used typing techniques of pulsed-field gel electrophoresis and multilocus sequence typing. We designed primers to amplify a noncoding region adjacent to the pneumolysin gene. The PCR product was digested separately with six restriction endonucleases, and the DNA fragments were analyzed using an Agilent 2100 bioanalyzer for accurate sizing. The combined RFLP results for all enzymes allowed us to assign each of the 47 clinical isolates of S. pneumoniae tested to one of 33 RFLP types. RFLP analyzed using the bioanalyzer allowed discrimination between strains similar to that obtained by the more commonly used techniques of pulsed-field gel electrophoresis, which discriminated between 34 types, and multilocus sequence typing, which discriminated between 35 types, but more quickly and with less expense. RFLP of a noncoding region using the Agilent 2100 bioanalyzer could be a useful addition to the molecular typing techniques in current use for S. pneumoniae, especially as a first screen of a local population.

Detection of Streptococcus pneumoniae strain cocolonization in the nasopharynx

Colonization with more than one distinct strain of the same species, also termed cocolonization, is a prerequisite for horizontal gene transfer between pneumococcal strains that may lead to change of the capsular serotype. Capsule switch has become an important issue since the introduction of conjugated pneumococcal polysaccharide vaccines. There is, however, a lack of techniques to detect multiple colonization by S. pneumoniae strains directly in nasopharyngeal samples. Two hundred eighty-seven nasopharyngeal swabs collected during the prevaccine era within a nationwide surveillance program were analyzed by a novel technique for the detection of cocolonization, based on PCR amplification of a noncoding region adjacent to the pneumolysin gene (plyNCR) and restriction fragment length polymorphism (RFLP) analysis. The numbers of strains and their relative abundance in cocolonized samples were determined by terminal RFLP. The pneumococcal carriage rate found by PCR was 51.6%, compared to 40.0% found by culture. Cocolonization was present in 9.5% (10/105) of samples, most (9/10) of which contained two strains in a ratio of between 1:1 and 17:1. Five of the 10 cocolonized samples showed combinations of vaccine types only (n = 2) or combinations of nonvaccine types only (n = 3). Carriers of multiple pneumococcal strains had received recent antibiotic treatment more often than those colonized with a single strain (33% versus 9%, P = 0.025). This new technique allows for the rapid and economical study of pneumococcal cocolonization in nasopharyngeal swabs. It will be valuable for the surveillance of S. pneumoniae epidemiology under vaccine selection pressure.

Speciating Campylobacter jejuni and Campylobacter coli isolates from poultry and humans using six PCR-based assays

Six previously published polymerase chain reaction (PCR) assays each targeting different genes were used to speciate 116 isolates previously identified as Campylobacter jejuni using routine microbiological techniques. Of the 116 isolates, 84 were of poultry origin and 32 of human origin. The six PCR assays confirmed the species identities of 31 of 32 (97%) human isolates and 56 of 84 (67%) poultry isolates as C. jejuni. Twenty eight of 84 (33%) poultry isolates were identified as Campylobacter coli and the remaining human isolate was tentatively identified as Campylobacter upsaliensis based on the degree of similarity of 16S rRNA gene sequences. Four of six published PCR assays showed 100% concordance in their ability to speciate 113 of the 116 (97.4%) isolates; two assays failed to generate a PCR product with four to 10 isolates. A C. coli-specific PCR identified all 28 hippuricase gene (hipO)-negative poultry isolates as C. coli although three isolates confirmed to be C. jejuni by the remaining five assays were also positive in this assay. A PCR-restriction fragment length polymorphism assay based on the 16S rRNA gene was developed, which contrary to the results of the six PCR-based assays, identified 28 of 29 hipO-negative isolates as C. jejuni. DNA sequence analysis of 16S rRNA genes from four hipO-negative poultry isolates showed they were almost identical to the C. jejuni type strain 16S rRNA sequences ATCC43431 and ATCC33560 indicating that assays reliant on 16S rRNA sequence may not be suitable for the differentiation of these two species.

Isolation and genotyping of Toxoplasma gondii causing fatal systemic toxoplasmosis in an immunocompetent 10-year-old cat

A 10-year-old male, neutered domestic shorthair cat was presented with fever, anorexia, vomiting, and diarrhea. Serologic testing for Feline immunodeficiency virus and Feline leukemia virus were negative. Fine-needle aspirates of mesenteric lymph nodes revealed the presence of banana-shaped apicomplexan parasites. The cat died after 4 days of hospitalization. Postmortem polymerase chain reaction (PCR) analysis confirmed the presence of Toxoplasma gondii in all examined organs. Parasites were ex vivo isolated in outbred mice and subsequently transferred into cell culture. Genotyping, using genetic markers for SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico for PCR-restriction fragment length polymorphism, revealed infection with type II T. gondii displaying type II alleles at all loci except Apico, which exhibited a type I allele. This is the most frequently identified genotype among cats acting as definitive hosts in central Europe, but to the authors' knowledge, it has never been associated with systemic toxoplasmosis in an adult, immunocompetent cat.

Genetic diversity in fluoroquinolone and macrolide-resistant Campylobacter coli from pigs

The genetic diversity of 115 Campylobacter coli strains, isolated from pigs of 59 geographical distant farms in Switzerland, were characterized on the basis of their DNA fingerprints and resistance to macrolides and fluoroquinolones. Sequence analysis showed that the macrolide-resistant isolates had a point mutation in the 23S ribosomal RNA (rRNA) genes (A2075G) and that the fluoroquinolone-resistant isolates had a point mutation in the gyrase gene gyrA (C257T). One fluoroquinolone-resistant strain had an additional transition mutation in the gyrB gene (A1471C). The flaA restriction fragment length polymorphism (RFLP) genotyping revealed that 57% of the isolates were genetically different. Point mutations in the 23S rRNA and gyrA genes could be found in both genetically distant and genetically related isolates. Additionally, isolates with and without point mutations were found within individual farms and on different farms. This study showed that the ciprofloxacin and erythromycin-resistant C. coli population present on the pig farms is not issued from a common ancestral clone, but individual Campylobacter strains have most likely mutated independently to acquire resistances under the selective pressure of an antibiotic.

Japanese-US common-arm analysis of paclitaxel plus carboplatin in advanced non-small-cell lung cancer: a model for assessing population-related pharmacogenomics

PURPOSE To explore whether population-related pharmacogenomics contribute to differences in patient outcomes between clinical trials performed in Japan and the United States, given similar study designs, eligibility criteria, staging, and treatment regimens. METHODS We prospectively designed and conducted three phase III trials (Four-Arm Cooperative Study, LC00-03, and S0003) in advanced-stage, non-small-cell lung cancer, each with a common arm of paclitaxel plus carboplatin. Genomic DNA was collected from patients in LC00-03 and S0003 who received paclitaxel (225 mg/m(2)) and carboplatin (area under the concentration-time curve, 6). Genotypic variants of CYP3A4, CYP3A5, CYP2C8, NR1I2-206, ABCB1, ERCC1, and ERCC2 were analyzed by pyrosequencing or by PCR restriction fragment length polymorphism. Results were assessed by Cox model for survival and by logistic regression for response and toxicity. Results Clinical results were similar in the two Japanese trials, and were significantly different from the US trial, for survival, neutropenia, febrile neutropenia, and anemia. There was a significant difference between Japanese and US patients in genotypic distribution for CYP3A4*1B (P = .01), CYP3A5*3C (P = .03), ERCC1 118 (P < .0001), ERCC2 K751Q (P < .001), and CYP2C8 R139K (P = .01). Genotypic associations were observed between CYP3A4*1B for progression-free survival (hazard ratio [HR], 0.36; 95% CI, 0.14 to 0.94; P = .04) and ERCC2 K751Q for response (HR, 0.33; 95% CI, 0.13 to 0.83; P = .02). For grade 4 neutropenia, the HR for ABCB1 3425C-->T was 1.84 (95% CI, 0.77 to 4.48; P = .19). CONCLUSION Differences in allelic distribution for genes involved in paclitaxel disposition or DNA repair were observed between Japanese and US patients. In an exploratory analysis, genotype-related associations with patient outcomes were observed for CYP3A4*1B and ERCC2 K751Q. This common-arm approach facilitates the prospective study of population-related pharmacogenomics in which ethnic differences in antineoplastic drug disposition are anticipated.

Novel mitochondrial tRNA(Ile) m.4282A>G gene mutation leads to chronic progressive external ophthalmoplegia plus phenotype

BACKGROUND/AIM To investigate the underlying pathomechanism in a 33-year-old female Caucasian patient presenting with chronic progressive external ophthalmoplegia (CPEO) plus symptoms. METHODS Histochemical analysis of skeletal muscle and biochemical measurements of individual oxidative phosphorylation (OXPHOS) complexes. Genetic analysis of mitochondrial DNA in various tissues with subsequent investigation of single muscle fibres for correlation of mutational load. RESULTS The patient's skeletal muscle showed 20% of cytochrome c oxidase-negative fibres and 8% ragged-red fibres. Genetic analysis of the mitochondrial DNA revealed a novel point mutation in the mitochondrial tRNA(Ile) (MTTI) gene at position m.4282G>A. The heteroplasmy was determined in blood, buccal cells and muscle by restriction fragment length polymorphism (RFLP) combined with a last fluorescent cycle. The total mutational load was 38% in skeletal muscle, but was not detectable in blood or buccal cells of the patient. The phenotype segregated with the mutational load as determined by analysis of single cytochrome c oxidase-negative/positive fibres by laser capture microdissection and subsequent LFC-RFLP. CONCLUSIONS We describe a novel MTTI transition mutation at nucleotide position m.4282G>A associated with a CPEO plus phenotype. The novel variant at position m.4282G>A disrupts the middle bond of the D-stem of the tRNA(Ile) and is highly conserved. The conservation and phenotype-genotype segregation strongly suggest pathogenicity and is in good agreement with the MTTI gene being frequently associated with CPEO. This novel variant broadens the spectrum of MTTI mutations causing CPEO.

Cyclin D1 (CCND1) A870G gene polymorphism modulates smoking-induced lung cancer risk and response to platinum-based chemotherapy in non-small cell lung cancer (NSCLC) patients

PURPOSE: The cyclin D1 (CCND1) A870G gene polymorphism is linked to the outcome in patients with resectable non-small cell lung cancer (NSCLC). Here, we investigated the impact of this polymorphism on smoking-induced cancer risk and clinical outcome in patients with NSCLC stages I-IV. METHODS: CCND1 A870G genotype was determined by polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis (RFLP) of DNA extracted from blood. The study included 244 NSCLC patients and 187 healthy control subjects. RESULTS: Patient characteristics were: 70% male, 77% smokers, 43% adenocarcinoma, and 27% squamous cell carcinoma. Eighty-one percent of the patients had stages III-IV disease. Median age at diagnosis was 60 years and median survival was 13 months. Genotype frequencies of patients and controls both conformed to the Hardy Weinberg equilibrium. The GG genotype significantly correlated with a history of heavy smoking (>or=40 py, P=0.02), and patients with this genotype had a significantly higher cigarette consumption than patients with AA/AG genotypes (P=0.007). The GG genotype also significantly correlated with tumor response or stabilization after a platinum-based first-line chemotherapy (P=0.04). Survival analysis revealed no significant differences among the genotypes. CONCLUSION: Evidence was obtained that the CCND1 A870G gene polymorphism modulates smoking-induced lung cancer risk. Further studies are required to explore the underlying molecular mechanisms and to test the value of this gene polymorphism as a predictor for platinum-sensitivity in NSCLC patients.

Origin and prognostic value of circulating KRAS mutations in lung cancer patients

Because of the current controversy on the origin and clinical value of circulating KRAS codon 12 mutations in lung cancer, we screened 180 patients using a combined restriction fragment-length polymorphism and polymerase chain reaction (RFLP-PCR) assay. We detected KRAS mutations in 9% plasma samples and 0% matched lymphocytes. Plasma KRAS mutations correlated significantly with poor prognosis. We validated the positive results in a second laboratory by DNA sequencing and found matching codon 12 sequences in blood and tumor in 78% evaluable cases. These results support the notion that circulating KRAS mutations originate from tumors and are prognostically relevant in lung cancer.

Genetic polymorphisms associated with the inflammatory response in bacterial meningitis.

BACKGROUND Bacterial meningitis (BM) is an infectious disease that results in high mortality and morbidity. Despite efficacious antibiotic therapy, neurological sequelae are often observed in patients after disease. Currently, the main challenge in BM treatment is to develop adjuvant therapies that reduce the occurrence of sequelae. In recent papers published by our group, we described the associations between the single nucleotide polymorphisms (SNPs) AADAT +401C > T, APEX1 Asn148Glu, OGG1 Ser326Cys and PARP1 Val762Ala and BM. In this study, we analyzed the associations between the SNPs TNF -308G > A, TNF -857C > T, IL-8 -251A > T and BM and investigated gene-gene interactions, including the SNPs that we published previously. METHODS The study was conducted with 54 BM patients and 110 healthy volunteers (as the control group). The genotypes were investigated via primer-introduced restriction analysis-polymerase chain reaction (PIRA-PCR) or polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) analysis. Allelic and genotypic frequencies were also associated with cytokine and chemokine levels, as measured with the x-MAP method, and cell counts. We analyzed gene-gene interactions among SNPs using the generalized multifactor dimensionality reduction (GMDR) method. RESULTS We did not find significant association between the SNPs TNF -857C > T and IL-8 -251A > T and the disease. However, a higher frequency of the variant allele TNF -308A was observed in the control group, associated with changes in cytokine levels compared to individuals with wild type genotypes, suggesting a possible protective role. In addition, combined inter-gene interaction analysis indicated a significant association between certain genotypes and BM, mainly involving the alleles APEX1 148Glu, IL8 -251 T and AADAT +401 T. These genotypic combinations were shown to affect cyto/chemokine levels and cell counts in CSF samples from BM patients. CONCLUSIONS In conclusion, this study revealed a significant association between genetic variability and altered inflammatory responses, involving important pathways that are activated during BM. This knowledge may be useful for a better understanding of BM pathogenesis and the development of new therapeutic approaches.