Novel isolates of Cydia pomonella granulovirus (CpGV): deciphering the molecular mechanism for overcoming CpGV resistance in codling moth (Cydia pomonella)

During the last twenty years, Cydia pomonolla granulovirus (CpGV, Baculoviridae) has become the most important biological control agent for the codling moth (CM) in organic and integrated apple production. All registered products in Europe are based on the isolate CpGV-M, which was discovered 1964 in Mexico. A serious threat to future application of CpGV is the occurrence of CM field populations resistant to CpGV. Since 2003, populations with up to 10,000-fold reduced susceptibility were reported from orchards in Germany, France, Italy, Switzerland, Austria and the Netherlands. A putative alternative to CpGV-M are novel CpGV isolates which are able to overcome CM resistance. This thesis focuses on the identification and characterisation of resistance overcoming CpGV isolates and the analysis of their molecular difference to CpGV-M.rnSixteen CpGV isolates were tested against CM lab strains in bioassays. Hereby, five isolates were identified which were able to completely overcome resistance. The genomes of these isolates were compared to CpGV-M by restriction fragment length polymorphism (RFLP) analysis. To identify the molecular factor responsible for improved virulence of some CpGV isolates, major genomic differences were sequenced and analysed. A 0.7 kb insertion was found in CpGV-I01, -I12 and -E2, but not in other resistance overcoming isolates. Analysis of the insertions sequence revealed that it might be due to a transposition event, but not involved in overcoming resistance. rnFor unequivocal identification of CpGV isolates, a new method based on molecular analysis was established. Partial sequencing of the conserved polyhedrin/granulin (polh/gran), late expression factor-8 (lef-8) and late expression factor-9 (lef-9) genes revealed single nucleotide polymorphisms (SNPs). SNP analysis correlated with the grouping obtained by RFLP analysis. A phylogenetic classification due to different genome types A-E is proposed. Phylogenetic analysis suggested that CpGV-M was the phylogenetically youngest of the tested CpGV isolates.rnWhole genome sequencing of two resistance overcoming isolates CpGV-I12 (type D genome) and -S (type E genome) and CpGV-M (type A genome) was performed. Comparison of the three genomes revealed a high sequence identity. Several insertions and deletions ranging from 1-700 nucleotides (nt) were found. Comparison on open reading frame (ORF) level revealed that CpGV-I12 and -S shared only one protein alteration when compared to CpGV-M: a stretch of 24 nt present in ORF cp24 was not found in any of the resistance overcoming isolates. Cp24 codes for the early gene pe38. Combined with the results of phylogenetic analysis, it is proposed that these 24 nt are a recent insertion into the CpGV-M genome. The role of pe38 in overcoming resistance was investigated by knocking out pe38 of a CpGV-M based bacmid and swapping of CpGV-I12 pe38 of into the k.o. bacmid. When pe38 of CpGV-I12 was inserted into the k.o. bacmid, the infectivity could not be rescued, suggesting that the genomic portion of pe38 might play a role in its function.rnIt can be concluded that the recently observed CpGV resistance in CM is only related to type A genomes. RFLP and SNP analysis provide tools for identifying and characterising different CpGV isolates reliably, a pre-condition for a future registration of CpGV products based on novel CpGV isolates.rnrnrn

Genus Bifidobacterium: taxonomy studies and gene expression analysis on folate pathway

Folates (vitamin B9) are essential water soluble vitamins, whose deficiency in humans may contribute to the onset of several diseases, such as anaemia, cancer, cardiovascular diseases, neurological problems as well as defects in embryonic development. Human and other mammals are unable to synthesize ex novo folate obtaining it from exogenous sources, via intestinal absorption. Recently the gut microbiota has been identified as an important source of folates and the selection and use of folate producing microorganisms represents an innovative strategy to increase human folate levels. The aim of this thesis was to gain a fundamental understanding of folate metabolism in Bifidobacterium adolescentis. The work was subdivided in three main phases, also aimed to solve different problems encountered working with Bifidobacterium strains. First, a new identification method (based on PCR-RFLP of hsp60 gene) was specifically developed to identify Bifidobacterium strains. Secondly, Bifidobacterium adolescentis biodiversity was explored in order to recognize representing strains of this species to be screened for their folate production ability. Results showed that this species is characterized by a wide variability and support the idea that a possible new taxonomic re-organization would be required. Finally B. adolescentis folate metabolism was studied using a double approach. A quantitative analysis of folate content was complemented by the examination of expression levels of genes involved in folate related pathways. For the normalization process, required to increase the robustness of the qRT-PCR analysis, an appropriate set of reference genes was tested using two different algorithms. Results demonstrate that B.adolescentis strains may represent an endogenous source of natural folate and they could be used to fortify fermented dairy products. This bio-fortification strategy presents many advantages for the consumer, providing native folate forms more bio-available, and not implicated in the discussed controversy concerning the safety of high intake of synthetic folic acid.

Identification and genetic diversity in phytoplasmas associated with diseases of cassava and other agronomic relevant crops in south-east Asia and Latin America

Identification and genetic diversity of phytoplasmas infecting tropical plant species, selected among those most agronomically relevant in South-east Asia and Latin America were studied. Correlation between evolutionary divergence of relevant phytoplasma strains and their geographic distribution by comparison on homologous genes of phytoplasma strains detected in the same or related plant species in other geographical areas worldwide was achieved. Molecular diversity was studied on genes coding ribosomal proteins, groEL, tuf and amp besides phytoplasma 16S rRNA. Selected samples infected by phytoplasmas belonging to diverse ribosomal groups were also studied by in silico RFLP followed by phylogenetic analyses. Moreover a partial genome annotation of a ‘Ca. P. brasiliense’ strain was done towards future application for epidemiological studies. Phytoplasma presence in cassava showing frog skin (CFSD) and witches’ broom (CWB) diseases in Costa Rica - Paraguay and in Vietnam – Thailand, respectively, was evaluated. In both cases, the diseases were associated with phytoplasmas related to aster yellows, apple proliferation and “stolbur” groups, while only phytoplasma related to X-disease group in CFSD, and to hibiscus witches’ broom, elm yellows and clover proliferation groups in CWB. Variability was found among strains belonging to the same ribosomal group but having different geographic origin and associated with different disease. Additionally, a dodder transmission assay to elucidate the role of phytoplasmas in CWB disease was carried out, and resulted in typical phytoplasma symptoms in periwinkle plants associated with the presence of aster yellows-related strains. Lethal wilt disease, a severe disease of oil palm in Colombia that is spreading throughout South America was also studied. Phytoplasmas were detected in symptomatic oil palm and identified as ‘Ca. P. asteris’, ribosomal subgroup 16SrI-B, and were distinguished from other aster yellows phytoplasmas used as reference strains; in particular, from an aster yellows strain infecting corn in the same country.

Entwicklung und Evaluierung von Methoden zur zeitnahen Identifizierung weinrelevanter Mikroorganismen

Hefen der Gattung Saccharomyces und Milchsäurebakterien sind bei der Weinbereitung von besonderer Bedeutung. Neben der alkoholischen Gärung sind Hefen an der Ausbildung von Aromastoffen beteiligt. Milchsäurebakterien spielen eine Rolle beim biologischen Säureabbau (malolaktische Fermentation), können jedoch aufgrund ihrer Stoffwechseleigenschaft weitere Aromamodifikationen bewirken. Die Zusammensetzung der mikrobiellen Flora zu verschiedenen Zeitpunkten der Weinbereitung hat einen direkten Einfluss auf die Qualität der Weine, welche sich sowohl positiv als auch negativ verändern kann. Daher ist die zuverlässige Identifizierung und Differenzierung verschiedener Mikroorganismen auf Art- aber auch Stamm-Ebene während der Vinifikation von Bedeutung.rnDer erste Teil dieser Arbeit beschäftigte sich mit der Differenzierung von Hefearten der Gattung Saccharomyces, welche mit Hilfe konventioneller Methoden nicht eindeutig identifiziert werden können. Unter Verwendung des DNA-Fingerprintverfahrens Specifically Amplified Polymorphic DNA (SAPD)-PCR sowie der Matrix-Assisted-Laser-Desorption/Ionization-Time-Of-Flight-Mass-Spectrometry (MALDI-TOF-MS) war eine Differenzierung dieser taxonomisch sehr nah verwandten Arten möglich. Weiterhin konnten interspezifische Hybridstämme detektiert werden. In diesem Zusammenhang wurde der Hybridcharakter des Stammes NCYC 3739 (S. cerevisiae x kudriavzevii) entdeckt. Um die Elternspezies eines Hybridstamms zuverlässig zu bestimmen, sind weiterführende Genanalysen notwendig. Hierzu konnte eine Restriktionsfragmentlängenpolymorphismus (RFLP)-Analyse verschiedener genetischer Marker erfolgreich herangezogen werden.rnIm Rahmen dieser Arbeit wurde weiterhin ein Schnellidentifizierungssystem zum Nachweis weinrelevanter Milchsäurebakterien entwickelt. Mit Hilfe der Sequence Characterized Amplified Region (SCAR)-Technik konnten artspezifische Primer generiert werden, welche auf der Grundlage charakteristischer Fragmente der SAPD-PCR abgeleitet wurden. Durch die Anwendung dieser Primer in einer Multiplex-PCR-Reaktion war die Detektion verschiedener, einerseits häufig in Wein vorkommender und andererseits potentiell an der Ausbildung von Weinfehlern beteiligter Milchsäurebakterien-Arten möglich. Die ermittelte Nachweisgrenze dieser Methode lag mit 10^4 - 10^5 Zellen/ml im Bereich der Zelltiter, die in Most und Wein anzutreffen sind. Anhand der Untersuchung verschiedener Weinproben von Winzern in Rheinhessen wurde die Praxistauglichkeit dieser Methode demonstriert. rnUm die gesamten Milchsäurebakterien-Population im Verlauf der Weinbereitung zu kontrollieren, kann die Denaturierende Gradienten-Gelelektrophorese herangezogen werden. Hierzu wurden in dieser Arbeit Primer zur Amplifikation eines Teilbereichs des rpoB-Gens abgeleitet, da dieses Gen eine Alternative zur 16S rDNA darstellt. Die DNA-Region erwies sich als geeignet, um zahlreiche weinrelevante Milchsäurebakterien-Arten zu differenzieren. In einigen ersten Versuchen konnte gezeigt werden, dass diese Methode für eine praktische Anwendung in Frage kommt.rnOenococcus oeni ist das wichtigste Milchsäurebakterien während der malolaktischen Fermentation und wird häufig in Form kommerzieller Starterkulturen eingesetzt. Da verschiedene Stämme unterschiedliche Eigenschaften aufweisen können, ist es von Bedeutung, die Identität eines bestimmten Stammes zweifelsfrei feststellen zu können. Anhand der Analyse verschiedener O. oeni-Stämme aus unterschiedlichen Weinbaugebieten konnte gezeigt werden, dass sowohl die nested SAPD-PCR als auch die MALDI-TOF-MS genügend Sensitivität aufweisen, um eine Unterscheidung auf Stamm-Ebene zu ermöglichen, wobei die mittels nSAPD-PCR ermittelten Distanzen der Stämme zueinander mit deren geographischer Herkunft korrelierte.rnDie in der vorliegenden Arbeit entwickelten Methoden können dazu beitragen, den Prozess der Weinherstellung besser zu kontrollieren und so eine hohe Qualität des Endproduktes zu gewährleisten.rn

Temporal variation of genetic diversity and differentiation of albacore, Thunnus alalunga, in the Mediterranean Sea

This study is on albacore (Thunnus alalunga, Bonnaterre 1788), an epi- and mesopelagic oceanic tuna species cosmopolitan in the tropical and temperate waters of all oceans including the Mediterranean Sea, extending in a broad band between 40°N and 40°S. What it’s known about albacore population structure is based on different studies that used fisheries data, RFLP, mtDNA control region and nuDNA markers, blood lectins analysis, individual tags and microsatellite. At the moment, for T. alalunga six management units are recognized: the North Pacific, South Pacific, Indian, North Atlantic, South Atlantic and Mediterranean stocks. In this study I have done a temporal and spatial comparison of genetic variability between different Mediterranean populations of Thunnus alalunga matching an historical dataset ca. from 1920s composed of 43 individuals divided in 3 populations (NADR, SPAIN and CMED) with a modern dataset composed of 254 individuals and 7 populations (BAL, CYP, LIG, TYR, TUR, ADR, ALB). The investigation was possible using a panel of 94 nuclear SNPs, built specifically for the target species at the University of Basque Country UPV/EHU. First analysis done was the Hardy-Weinberg, then the number of clusters (K) was determined using STRUCTURE and to assess the genetic variability, allele frequencies, the average number of alleles per locus, expected (He) and observed (Ho) heterozygosis, and the index of polymorphism (P) was used the software Genetix. Historical and modern samples gives different results, showing a clear loss of genetic diversity over time leading to a single cluster in modern albacore instead of the two found in historical samples. What this study reveals is very important for conservation concerns, and additional research endeavours are needed.

Populationsgenetik der ersten Bauern Mitteleuropas

Die vorliegende Dissertation ist eine molekulargenetische Studie an humanem neolithischem Skelettmaterial. Im zentralen Blickpunkt stand die Bestimmung der Variabilität der mitochondrialen Haplogruppen einer frühneolithischen Stichprobe aus drei unterschiedlichen Kulturkreisen, welche die Linearbandkeramik (LBK und AVK), die Körös-Kultur und eine Sammelkategorie osteuropäischer spätmeso- und frühneolithischer Kulturen umfasste. Im Vergleich dieser Gruppen untereinander sowie mit Rezentdaten moderner Populationen aus vergleichbaren Gebieten Mittel- und Osteuropas sowie dem Nahen Osten sollten bestehende Modelle und Hypothesen zur Neolithisierung Mitteleuropas geprüft werden. Insgesamt konnte für 43 neolithische Individuen aus 16 Fundorten der reproduzierbare Nachweis endogener DNA erbracht werden. Eine eindeutige Haplogruppenbestimmung konnte durch die Sequenzierung vier überlappender Fragmente der mitochondrialen Hypervariablen Region I sowie durch RFLP-Analyse zusätzlicher charakteristischer Nukleotidpositionen für alle 43 Individuen durchgeführt werden. Die neolithischen Individuen der Linearbandkeramik sowie der Körös-Kultur zeigten eine hohe Diversität an bekannten europäischen Haplogruppen, wohingegen die kleinere Stichprobe aus dem Gebiet Osteuropas eine auffällige Homogenität aufwies. Neben Frequenzunterschieden zur modernen mitteleuropäischen Bevölkerung war innerhalb der LBK/AVK-Stichprobe eine hohe Frequenz der Haplogruppe N1a festzustellen, welche nicht in den beiden anderen neolithischen Stichproben zu finden war und auch in der heutigen Rezentbevölkerung Eurasiens und Nordafrikas nur mit einer durchschnittlichen Frequenz von 0,2% vertreten ist. Innerhalb der Individuen der Körös-Kultur fanden sich zwei Haplotypen, welche heute nicht in Europa bekannt sind, dagegen jedoch in Süd- bzw. Nordostasien gehäuft vorkommen. Die Ergebnisse der aDNA-Analysen bestätigten im Wesentlichen das komplexe Bild der Neolithischen Transition in Mitteleuropa und konnten die, für diesen Raum postulierte, Hypothese der leap frog colonization weitestgehend unterstützen. Auch für den geographischen Vergleichsraum des nördlichen Osteuropa konnten Parallelen zur etablierten Sichtweise der archäologischen Forschung zu diesem Gebiet und den vorliegenden Ergebnissen der aDNA-Analysen aufgezeigt werden. Die zeitlich verzögerte Annahme der neolithischen Lebensweise im waldreichen nördlichen Osteuropa spiegelt sich in der reduzierten Diversität an mtDNA-Haplogruppen wider. Die vorliegende Dissertation konnte nicht nur durch die Ergebnisse der Haplogruppen-Bestimmung, sondern vor allem durch die umfangreichen und elaborierten Reproduktions- und Authentifizierungprotokolle deutlich machen, dass der Nachweis von humaner alter DNA trotz der allgegenwärtigen, methodenimmanenten Kontaminationsgefahr unter streng kontrollierten Bedingungen möglich ist. Gleichermaßen konnte veranschaulicht werden, dass die aDNA-Analyse wertvolle Hinweise auf das genetische status quo einer Population liefern kann, welche nicht bzw. nur in sehr eingeschränkten Maße von rezenten DNA-Daten abgeleitet werden können. Als sekundäres Ergebnis erlaubte der bislang größte vorliegende Datensatz von ~2500 Klonsequenzen zudem einen detaillierten Einblick in Häufigkeiten und Verteilungsmuster von post mortem Sequenzveränderungen. Es konnten für den mitochondrialen Bereich der Nukleotidpositionen 15997-16409 so genannte hot bzw. cold spots definiert werden, welche für die Auswertung und Interpretation von zukünftigen Sequenzierungen der Hypervariablen Region I des mt-Genoms von entscheidender Bedeutung sein werden.

Interaction between serotonin 5-HT2A receptor gene and dopamine transporter (DAT1) gene polymorphisms influences personality trait of persistence in Austrian Caucasians

We examined 89 normal volunteers using Cloninger's Temperament and Character Inventory (TCI). Genotyping the 102T/C polymorphism of the serotonin 5HT2A receptor gene and the ser9gly polymorphism in exon 1 of the dopamine D3 receptor (DRD3) gene was performed using PCR-RFLP, whereas the dopamine transporter (DAT1) gene variable number of tandem repeats (VNTR) polymorphism was investigated using PCR amplification followed by electrophoresis in an 8% acrylamide gel with a set of size markers. We found a nominally significant association between gender and harm avoidance (P=0.017; women showing higher scores). There was no association of either DAT1, DRD3 or 5HT2A alleles or genotypes with any dimension of the TCI applying Kruskal-Wallis rank-sum tests. Comparing homozygote and heterozygote DAT1 genotypes, we found higher novelty seeking scores in homozygotes (P=0.054). We further found a nominally significant interaction between DAT1 and 5HT2A homo-/heterozygous gene variants (P=0.0071; DAT1 and 5HT2A genotypes P value of 0.05), performing multivariate analysis of variance (MANOVA). Examining the temperamental TCI subscales, this interaction was associated with persistence (genotypes: P=0.004; homo-/heterozygous gene variants: P=0.0004). We conclude that an interaction between DAT1 and 5HT2A genes might influence the temperamental personality trait persistence.

SNPs in DNA repair genes associated to meningitis and host immune response

In vitro and in animal models, APE1, OGG1, and PARP-1 have been proposed as being involved with inflammatory response. In this work, we have investigated if the SNPs APE1 Asn148Glu, OGG1 Ser326Cys, and PARP-1 Val762Ala are associated to meningitis. The patient genotypes were investigated by PIRA-PCR or PCR-RFLP. DNA damages were detected in genomic DNA by Fpg treatment. IgG and IgA were measured from plasma and the cytokines and chemokines were measured from cerebrospinal fluid samples using Bio-Plex assays. A higher frequency (P<0.05) of APE1 Glu allele in bacterial meningitis (BM) and aseptic meningitis (AM) patients was observed. The genotypes Asn/Asn in control group and Asn/Glu in BM group was also higher. For the SNP OGG1 Ser326Cys, the genotype Cys/Cys was more frequent (P<0.05) in BM group. The frequency of PARP-1 Val/Val genotype was higher in control group (P<0.05). The occurrence of combined SNPs is significantly higher in BM patients, indicating that these SNPs may be associated to the disease. Increasing in sensitive sites to Fpg was observed in carriers of APE1 Glu allele or OGG1 Cys allele, suggesting that SNPs affect DNA repair activity. Alterations in IgG production were observed in the presence of SNPs APE1 Asn148Glu, OGG1 Ser326Cys or PARP-1 Val762Ala. Moreover, reduction in the levels of IL-6, IL-1Ra, MCP-1/CCL2 and IL-8/CXCL8 was observed in the presence of APE1 Glu allele in BM patients. In conclusion, we obtained indications of an effect of SNPs in DNA repair genes on the regulation of immune response in meningitis.

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.

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.

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.

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.

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.

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.