Charakterisierung der mikrobiellen Population und Untersuchungen zum Quorum Sensing in der Rhizosphäre vom Weidelgras (Lolium perenne) auf einer Rückstandshalde der Kaliindustrie

Im Rahmen dieser Dissertation wurden die Dynamik und die Kommunikation innerhalb der mikrobiellen Population der Rhizosphäre von Deutschem Weidelgras (Lolium perenne) untersucht, welches auf einer teilweise rekultivierten Rückstandshalde der Kaliindustrie wuchs. Um die niederschlagsbedingte Auswaschung von Salzen zu reduzieren, wird die Rückstandshalde des Kaliwerks Sigmundshall (in Bokeloh bei Hannover) schrittweise mit dem technogenen Abdecksubstrat REKAL/SAV ummantelt. Dieses weist eine hohe Standfestigkeit und Wasserspeicherkapazität auf und kann zudem begrünt werden, wofür als Pionierpflanze Lolium perenne dient. Durch diese Rekultivierung wird Niederschlag besser gespeichert und über Evapotranspiration wieder in die Luft abgegeben, was letztendlich die Bildung von Salzwasser vermindert. Da das Abdecksubstrat neben alkalischem pH-Wert auch teilweise hohe Schwermetallkonzentrationen aufweist, sollte in der vorliegenden Arbeit erstmals die mikrobielle Rhizosphären-Gemeinschaft in diesem extremen Habitat mittels einer kulturunabhängigen Methode erforscht werden. Zudem wurden erste Untersuchungen angestellt, ob im Substrat die zelldichte-abhängige bakterielle Kommunikation (Quorum Sensing) nachgewiesen werden kann. Mittels extrahierter Gesamt-DNA wurde anhand der 16S rDNA die Analyse des „Terminalen Restriktonsfragmentlängenpolymorphismus“ (TRFLP) verwendet, um die komplexe bakterielle Rhizosphären-Gemeinschaft unter zeitlichen und lokalen Aspekten zu vergleichen. Auftretende Veränderungen bei den bakteriellen Populationen der jeweiligen Proben wurden durch eine Zu- oder Abnahme der auch als Ribotypen bezeichneten terminalen Restriktionsfragmente (TRF) erfasst. Hierbei zeigten sich am Südhang der Halde während der Sommermonate der Jahre 2008 und 2009 zwar Schwankungen in den bakteriellen Gemeinschaftsprofilen, es lagen jedoch keine eindeutigen Dynamiken vor. Im Vergleich zum Südhang der Halde wies der Nordhang eine höhere Ribotyp-Diversität auf, was mit der fortgeschritteneren Rekultivierung dieses Haldenabschnitts zusammenhängen könnte. Zusätzlich wurden Bakterien aus der Rhizosphäre von Lolium perenne isoliert und mithilfe der Biosensoren Agrobacterium tumefaciens A136 pCF218 pCF372 und Chromobacterium violaceum CV026 auf die Produktion von N-Acylhomoserinlactonen (AHLs) überprüft. Diese AHLs werden von Gram-negativen Mikroorganismen als Signalmoleküle verwendet, um ihre Genexpression zelldichteabhängig zu kontrollieren. Von den 47 getesteten Gram-negativen Rhizosphärenisolaten konnten nur bei einem reproduzierbar AHL-Moleküle mithilfe des Reporterstamms A. tumefaciens nachgewiesen werden. Der AHL-Produzent wurde als Pseudomonas fluorescens identifiziert. Mittels dünnschichtchromatographischer Analysen konnten die extrahierten bakteriellen AHL-Moleküle den N-Octanoyl-L-homoserinlactonen zugeordnet werden.

Anàlisi polifàsica de soques de "Pseudomonas fluorescens" potencials agents de biocontrol de malalties de fruiters

Molts bacteris del grup fluorescent del gènere Pseudomonas són capaços de controlar malalties de les plantes causades per fongs i bacteris fitopatògens (ACBs) o mostren activitat com a bacteris promotors del creixement de les plantes (BPCPs). S'han descrit diversos metabòlits que intervenen de manera important en la seva activitat com a ACBs i BPCPs entre els quals en destaquen el 2,4-diacetilfloroglucinol (Phl), àcid fenazin-1-carboxílic (PCA), Pirrolnitrina (Prn), àcid cianhídric (HCN), àcid 3-indolacètic (IAA), sideròfors i quitinases. L'objectiu principal del nostre treball ha estat la comparació de les característiques d'un grup de Pseudomonas del grup fluorescent utilitzant una aproximació polifàsica amb la finalitat d'establir possibles relacions entre algunes de les característiques i la capacitat d'actuar com a ACB o BPCP. Atesa la importància en el biocontrol de la producció de metabòlits com Phl, PCA i Prn, l'objectiu preliminar ha estat la recerca i obtenció de soques productores d'aquests metabòlits. Per assolir aquest objectiu s'ha emprat una aproximació molecular basada en la detecció dels gens biosintètics implicats en la seva producció en lloc de la detecció directa dels metabòlits per evitar els efectes que poden tenir les condicions de cultiu en la inducció o repressió de la seva síntesi. S'han realitzat diferents protocols basats (i) en la cerca assistida de productors mitjançant l'ús de marcadors fenotípics i posterior confirmació per PCR i, (ii) en l'ús de la PCR per a la detecció dels gens directament dels extractes bacterians, d'enriquiments d'aquests extractes i la realització de la hibridació en colònies per al posterior aïllament. La cerca assistida de productors de Phl mitjançant marcadors fenotípics i posteriorment la utilització de tècniques moleculars (amplificació per PCR del gen phlD), ha estat el millor mètode en el tipus de mostres processades en el nostre treball, on la proporció de productors és relativament baixa. En total s'han aïllat a partir de diversos ambients 4 soques portadores dels gens de la síntesi de PCA, 15 de Phl i 1 de Prn. S'ha constituït una col·lecció de 72 soques de Pseudomonas del grup fluorescent que inclou 18 aïllats propis portadors dels gens biosintètics necessaris per la producció de Phl PCA i Prn; 6 soques de referència procedents de col·leccions de cultius tipus, 14 soques productores dels diferents antibiòtics cedides per altres investigadors i una selecció de 34 soques procedents d'un treball previ realitzat en el nostre grup de recerca. A la col·lecció s'hi troben soques candidates a ACB i BPCP de diverses malalties i plantes. Les 72 soques s'han caracteritzat fenotípica i genotípicament. La caracterització fenotípica s'ha portat a terme mitjançant la identificació a nivell d'espècie amb galeries API 20NE i proves bioquímiques específiques; la producció de metabòlits com PCA, Phl, Prn, IAA, HCN, quitinases i sideròfors mitjançant l'ús de diferents tècniques; antagonisme in vitro en diversos medis enfront dos fongs (Stemphylium vesicarium i Penicillium expansum) i tres bacteris fitopatògens (Erwinia amylovora, Pseudomonas syringae pv. syringae i Xanthomonas arboricola pv. juglandis); l'eficàcia de la inhibició de la infecció en bioassaigs in vivo sobre material vegetal enfront els fongs P. expansum en poma i S. vesicarium en fulles de perera i enfront el bacteri E. amylovora en fruits immadurs de perera i, finalment, en assaigs de promoció de creixement en dos portaempelts comercials de Prunus. Cal destacar que P. expansum causa la podridura blava en pomes i peres en postcollita, S. vesicarium la taca bruna de la perera i E. amylovora el foc bacterià de les rosàcies. El nombre de soques de Pseudomonas, sobre el total de les 72 estudiades, productores d'IAA (4) i quitinases (6) és baix, mentre que és elevat en el cas del HCN (32), que a més està associat a la producció de Phl. Els resultats obtinguts en l'antagonisme in vitro han mostrat en el cas dels bacteris que és dependent del patogen indicador i del medi de cultiu. La presència o absència de ferro no sembla ser un factor que potencií l'antagonisme. En el cas dels fongs no s'ha observat però, influència del medi de cultiu emprat. En el total de 72 soques s'ha observat un percentatge baix de soques que manifesten antagonisme en tots els medis assajats vers 3 o 4 dels patògens (7). Solament 2 d'aquestes 7 soques han mostrat ser també efectives en bioassaigs d'inhibició de les infeccions causades per 2 dels 3 patògens assajats. Algunes de les soques efectives en els bioassaigs no són antagonistes in vitro en cap dels medis assajats enfront el mateix patogen. En el cas de la promoció del creixement, s'han observat més soques promotores del creixement del portaempelts de prunera Marianna 2624 que no en l'híbrid de presseguer-ametller GF677 i les eficàcies assolides són també majors en el cas de Marianna 2624, detectant una elevada especificitat soca/portaempelts La caracterització genotípica s'ha realitzat mitjançant l'anàlisi dels polimorfismes en la longitud dels fragments de restricció de DNA ribosomal (RFLP-rDNA) i l'anàlisi dels polimorfismes en la longitud dels fragments de macrorestricció genòmica de DNA cromosòmic separats per electroforesi en camp polsant (MRFLP-PFGE). Ambdues anàlisis van mostrar una gran heterogeneïtat genètica entre les soques caracteritzades i no s'ha pogut relacionar les agrupacions obtingudes amb les característiques fenotípiques o capacitat d'actuar com a ACB o BPCP. Els patrons de macrorestricció genòmica (MRFLP-PFGE) del bacteri model P. fluorescens EPS288 són estables en el temps i independents de les condicions de cultiu assajades al laboratori o en mostres naturals, mostrant ser una tècnica eficaç en la identificació de reaïllats de mostres naturals inoculades prèviament amb el bacteri. Una selecció de soques que comparteixen el fet de produir floroglucinol s'han caracteritzat mitjançant RFLP i seqüenciació del gen phlD. S'ha establert una relació entre les agrupacions obtingudes en les anàlisis RFLP-rDNA, RFLP-phlD i les seqüències del gen. En l'anàlisi filogenètica de les seqüències del gen phlD s'ha observat un elevat grau de polimorfisme obtenint-se 3 agrupacions principals. Les agrupacions semblen relacionar-se amb els patrons de producció de metabòlits (Phl, HCN i Prn en una primera agrupació; Phl i HCN en la segona i solament Phl en la tercera), però aquestes no s'han pogut relacionar amb l'origen geogràfic de les soques o la seva activitat com a ACBs i/o BPCP. Amb les dades obtingudes de la caracterització fenotípica i genotípica s'ha realitzat una anàlisi multivariant (correspondències, correlacions d'Spearman i de freqüències amb variables categòriques). S'ha demostrat la importància de disposar d'una tècnica que permeti depurar una col·lecció de soques descartant les soques genèticament idèntiques, ja que influeixen en els resultats de les anàlisis. Pels tres patògens assajats com a indicadors i els dos portaempelts emprats, no s'ha observat cap correlació entre la inhibició de la infecció o la promoció del creixement amb les característiques fenotípiques i genotípiques de les soques que fos significatiu i consistent en les tres tècniques emprades.

Characterization and mechanism of action of the biological control agent Pantoea agglomerans EPS125

La soca EPS125 ha mostrat ser un efectiu agent de control biològic de diferents patògens fúngics de postcollita en diferents fruits. Degut a la seva elevada eficàcia, es va plantejar desenvolupar aquesta soca comercialment i per aquest motiu en el present treball es plantejà complementar la informació necessària pel seu registre. D'acord amb els resultats obtinguts mitjançant proves fenotípiques i genotípiques, la soca EPS125 queda inclosa dins l'espècie Pantoea agglomerans (Enterobacter agglomerans-Erwinia herbicola). En relació a la utilització de fonts de carboni, en el perfil i contingut d'àcids grassos cel·lulars i en el polimorfisme en la longitud dels fragments de macrorestricció genòmica (MRFLP), la soca EPS125 mostrà trets característics que la diferencien d'altres soques. Els dos marcadors moleculars (125.2 i 125.3) específics per la soca EPS125 dissenyats en el present treball mostraren ser semiespecífics per la seva detecció mitjançant la tècnica PCR i Real Time PCR. Quedant pendent l'anàlisi d'especificitat de l'ús combinat dels dos marcadors moleculars en una reacció PCR multiplex. P. agglomerans EPS125 ha mostrat ser molt efectiva en el control de Penicillium expansum en poma amb una dosi efectiva mitjana de 2.7x105 a 7x105 ufc/ml, i una ratio de 25-101 cèl·lules de la soca EPS125 per inactivar una espora del patogen segons el model de saturació hiperbòlica. Segons les aproximacions fenotípiques i estudis genotípics realitzats, sembla que els mecanismes de biocontrol utilitzats per la soca EPS125 contra P. expansum en poma estan directament relacionats amb la capacitat de formació de biofilm per aquesta soca.

Genomic analysis of recombinant Sabin clinical isolates

Recombination in Poliovirus vaccine strains is a very frequent phenomenon. In this report 23 polio/Sabin strains isolated from healthy vaccinees or from VAPP patients after OPV administration, were investigated in order to identify recombination sites from 2C to 3D regions of the poliovirus genome. RT-PCR, followed by Restriction Fragment Length Polymorphism (RFLP) screening analysis were applied in four distant genomic regions (5' UTR, VP1, 2C and 3C-3D) in order to detect any putative recombinant. The detected recombinants were sequenced from 2C to the end of the genome (3' UTR) and the exact recombination sites were determined with computational analysis. Five of the 23 isolated strains were recombinant in one genomic region, two of them in 2C, isolates EP16:S3/S2, EP23:S3/S1, two in 3D isolates EP6:S2/S1, EP12:S2/S1 and one in 3A isolate EP9:S2/Sl. Point mutations were found in strains EP3, EP6, EP9 and EP12. Recombination specific types and sites re-occurrence along with point mutations are discussed concerning the polioviruses evolution.

Comparison of the euryarchaeal microbial community in guts and food-soil of the soil-feeding termite Cubitermes fungifaber across different soil types

Termites are an important component of tropical soil communities and have a significant affect on the structure and nutrient content of soil. Digestion in termites is related to gut structure, gut physico-chemical conditions and gut symbiotic microbiota. Here we describe the use of 16S rRNA gene sequencing and Terminal-restriction Fragment Length Polymorphism (T-RFLP) analysis to examine methanogenic Archaea (MA) in the guts and food-soil of the soil-feeder Cubitermes fungifaber Sjostedt across a range of soil types. If they are strictly vertically inherited, then MA in guts should be the same in all individuals even if the soils differ across sites. In contrast, gut MA should reflect what is present in soil if populations are merely a reflection of what is ingested as the insects forage. We show clear differences between the euryarchaeal communities in termite guts and in food-soils from five different sites. Analysis of 16S rRNA gene clones indicated little overlap between the gut and soil communities. Gut clones were related to a termite-derived Methanomicrobiales cluster, to Methanobrevibacter and, surprisingly, to the haloalkaliphile Natronococcus. Soil clones clustered with Methanosarcina, Methanomicrococcus or Rice Cluster I. T-RFLP analysis indicated that the archaeal communities in the soil samples differed from site to site, whereas those in termite guts were similar between sites. There was some overlap between the gut and soil communities but these may represent transient populations in either guts or soil. Our data does not support the hypothesis that termite gut MA are derived from their food soil but also does not support a purely vertical transmission of gut microflora.

Comparison of Euryarchaea strains in the guts and food-soil of the soil-feeding termite Cubitermes fungifaber across different soil types

Termites are an important component of tropical soil communities and have a significant effect on the structure and nutrient content of soil. Digestion in termites is related to gut structure, gut physicochemical conditions, and gut symbiotic microbiota. Here we describe the use of 16S rRNA gene sequencing and terminal-restriction fragment length polymorphism (T-RFLP) analysis to examine methanogenic archaea (MA) in the guts and food-soil of the soil-feeder Cubitermes fungifaber Sjostedt across a range of soil types. If these MA are strictly vertically inherited, then the MA in guts should be the same in all individuals even if the soils differ across sites. In contrast, gut MA should reflect what is present in soil if populations are merely a reflection of what is ingested as the insects forage. We show clear differences between the euryarchaeal communities in termite guts and in food-soils from five different sites. Analysis of 16S rRNA gene clones indicated little overlap between the gut and soil communities. Gut clones were related to a termite-derived Methanomicrobiales cluster, to Methanobrevibacter and, surprisingly, to the haloalkaliphile Natronococcus. Soil clones clustered with Methanosarcina, Methanomicrococcus, or rice cluster I. T-RFLP analysis indicated that the archaeal communities in the soil samples differed from site to site, whereas those in termite guts were similar between sites. There was some overlap between the gut and soil communities, but these may represent transient populations in either guts or soil. Our data do not support the hypothesis that termite gut MA are derived from their food-soil but also do not support a purely vertical transmission of gut microflora.

DNA variation in a 13-Mb region including the F9 gene: inferring the genealogical history and causal role of a hemophilia B mutation (IVS 5+13 A -> G)

About 5.5% of all UK hemophilia B patients have the base substitution IVS 5+13 A-->G as the only change in their factor (F)IX gene (F9). This generates a novel donor splice site which fits the consensus better than the normal intron 5 donor splice. Use of the novel splice site should result in a missense mutation followed by the abnormal addition of four amino acids to the patients' FIX. In order to explain the prevalence of this mutation, its genealogical history is examined. Analysis of restriction fragment length polymorphism in the 21 reference UK individuals (from different families) with the above mutation showed identical haplotypes in 19 while two differed from the rest and from each other. In order to investigate the history of the mutation and to verify that it had occurred independently more than once, the sequence variation in 1.5-kb segments scattered over a 13-Mb region including F9 was examined in 18 patients and 15 controls. This variation was then analyzed with a recently developed Bayesian approach that reconstructs the genealogy of the gene investigated while providing evidence of independent mutations that contribute disconnected branches to the genealogical tree. The method also provides minimum estimates of the age of the mutation inherited by the members of coherent trees. This revealed that 17 or 18 mutant genes descend from a founder who probably lived 450 years ago, while one patient carries an independent mutation. The independent recurrence of the IVS5+13 A-->G mutation strongly supports the conclusion that it is the cause of these patients' mild hemophilia.

Contrasting arbuscular mycorrhizal communities colonizing different host plants show a similar response to a soil phosphorus concentration gradient

High soil phosphorus (P) concentration is frequently shown to reduce root colonization by arbuscular mycorrhizal (AM) fungi, but the influence of P on the diversity of colonizing AM fungi is uncertain. We used terminal restriction fragment length polymorphism (T-RFLP) of 18S rDNA and cloning to assess diversity of AM fungi colonizing maize (Zea mays), soybean (Glycene max) and field violet (Viola arvensis) at three time points in one season along a P gradient of 10280mgl1 in the field. Percentage AM colonization changed between sampling time points but was not reduced by high soil P except in maize. There was no significant difference in AM diversity between sampling time points. Diversity was reduced at concentrations of P > 25mgl1, particularly in maize and soybean. Both cloning and T-RFLP indicated differences between AM communities in the different host species. Host species was more important than soil P in determining the AM community, except at the highest P concentration. Our results show that the impact of soil P on the diversity of AM fungi colonizing plants was broadly similar, despite the fact that different plants contained different communities. However, subtle differences in the response of the AM community in each host were evident.

Evidence for an association with type 2 diabetes mellitus at the PPARG locus in a South Indian population

Peroxisome proliferator-activated receptor-gamma2 (PPARG2) is a nuclear hormone receptor of ligand-dependent transcription factor involved in adipogenesis and a molecular target of the insulin sensitizers thiazolidinediones. We addressed the question of whether the 3 variants (-1279G/A, Pro12Ala, and His478His) in the PPARG2 gene are associated with type 2 diabetes mellitus and its related traits in a South Indian population. The study subjects (1000 type 2 diabetes mellitus and 1000 normal-glucose-tolerant subjects) were chosen randomly from the Chennai Urban Rural Epidemiology Study, an ongoing population-based study in southern India. The variants were screened by single-stranded conformational variant, direct sequencing, and restriction fragment length polymorphism. Linkage disequilibrium was estimated from the estimates of haplotypic frequencies. The -1279G/A, Pro12Ala, and His478His variants of the PPARG2 gene were not associated with type 2 diabetes mellitus. However, the 2-loci analyses showed that, in the presence of Pro/Pro genotype of the Pro12Ala variant, the -1279G/A promoter variant showed increased susceptibility to type 2 diabetes mellitus (odds ratio, 2.092; 95% confidence interval, 1.22-3.59; P = .008), whereas in the presence of 12Ala allele, the -1279G/A showed a protective effect against type 2 diabetes mellitus (odds ratio, 0.270; 95% confidence interval, 0.15-0.49; P < .0001). The 3-loci haplotype analysis showed that the A-Ala-T (-1279G/A-Pro12Ala-His478His) haplotype was associated with a reduced risk of type 2 diabetes mellitus (P < .0001). Although our data indicate that the PPARG2 gene variants, independently, have no association with type 2 diabetes mellitus, the 2-loci genotype analysis involving -1279G/A and Pro12Ala variants and the 3-loci haplotype analysis have shown a significant association with type 2 diabetes mellitus in this South Indian population.

Lack of association between serum adiponectin levels and the Pro12Ala polymorphism in Asian Indians

AIMS: The aim of the study was to investigate the association of serum adiponectin levels with the Pro12Ala polymorphism of the peroxisome proliferator activated receptor-gamma (PPARG) gene in Asian Indians. METHODS: We selected 400 diabetic subjects, 200 with the Pro12Pro genotype (100 male and 100 female) and 200 with the Pro12Ala genotype (100 male and 100 female) and 400 age- and sex-matched normal glucose tolerance subjects with similar genotype profiles from the Chennai Urban Rural Epidemiology Study. Fasting serum adiponection levels were measured using radioimmunoassay. The Pro12Ala polymorphism was genotyped by PCR-restriction fragment length polymorphism using BstUI. RESULTS: All clinical and biochemical parameters were similar in the subjects with the Pro12Pro and Pro12Ala genotypes. There was no significant difference in serum adiponectin values between subjects with the Pro12Pro and Pro12Ala genotypes (males 5.4 vs. 5.8 microg/ml, P = 0.546; females 6.9 vs. 7.2 microg/ml, P = 0.748). Adiponectin values did not differ among these two genotypes even when categorized based on their diabetes status (normal glucose tolerance Pro12Pro 7.9 vs. Pro12Ala 7.7 microg/ml, P = 0.994; diabetes Pro12Pro 4.7 vs. Pro12Ala 5.4 microg/ml, P = 0.622). CONCLUSION: The Pro12Ala polymorphism of the PPARG gene is not associated with serum adiponectin levels in Asian Indians.

Thr54 allele carriers of the Ala54Thr variant of FABP2 gene have associations with metabolic syndrome and hypertriglyceridemia in urban South Indians

The intestinal fatty acid-binding protein gene is proposed as a candidate gene for diabetes because the protein it codes is involved in fatty acid absorption and metabolism. This study investigates the association of the Ala54Thr variant of the intestinal fatty acid-binding protein gene on type 2 diabetes mellitus and other related metabolic traits in Asian Indians. Ala54Thr polymorphism was genotyped by using polymerase chain reaction-restriction fragment length polymorphism in unrelated 773 type 2 diabetic and 899 normal glucose-tolerant (NGT) subjects, randomly chosen from the Chennai Urban Rural Epidemiology Study, an ongoing population-based study in South India. The Ala54Thr polymorphism was not associated with type 2 diabetes mellitus or obesity. However, genotype-phenotype study revealed that the NGT subjects carrying the Thr54 allele had significantly higher 2-hour plasma glucose (P = .007), glycated hemoglobin (P = .004), 2-hour insulin (P = .027), and fasting low-density lipoprotein cholesterol (P = .032) levels compared with those with the Ala54 allele. Normal glucose-tolerant subjects with Ala54Thr and Thr54Thr genotypes had significantly higher fasting serum triglyceride levels (P = .003) compared with those with Ala54Ala. The subjects were stratified into those with hypertriglyceridemia (serum triglyceride levels >or=150 mg/dL) and those without. The odds ratio for hypertriglyceridemia for the individuals carrying the Ala54Thr genotype was 1.491 (95% confidence interval [CI], 1.22-1.83, P < .0001), and for those carrying the Thr54Thr genotype, it was 1.888 (95% CI, 1.34-2.67; P < .0001). Subjects were also stratified into those with metabolic syndrome (MS) and those without, according to modified Adult Treatment Panel III guidelines. The odds ratio (adjusted for age and sex) for MS for the individuals carrying the Ala54Thr genotype was 1.240 (95% CI, 1.02-1.51; P = .03), whereas for those carrying the Thr54Thr genotype, it was 1.812 (95% CI, 1.28-2.57; P = .001). Carriers of the Thr54 allele have associations with MS and hypertriglyceridemia in this urban South Indian population.

Role of genetic polymorphism peroxisome proliferator-activated receptor-gamma2 Pro12Ala on ethnic susceptibility to diabetes in South-Asian and Caucasian subjects: Evidence for heterogeneity

OBJECTIVE: To determine whether the peroxisome proliferator-activated receptor (PPAR)-gamma Pro12ala polymorphism modulates susceptibility to diabetes in South Asians. RESEARCH DESIGN AND METHODS: South Asians (n = 697) and Caucasians (n = 457) living in Dallas/Forth Worth, Texas, and South Asians living in Chennai, India (n = 1,619), were enrolled for this study. PPAR-gamma Pro12Ala was determined using restriction fragment-length polymorphism. Insulin responsiveness to an oral glucose tolerance test (OGTT) was measured in nondiabetic subjects. RESULTS: The Caucasian diabetic subjects had significantly lower prevalence of PPAR-gamma 12Ala when compared with the Caucasian nondiabetic subjects (20 vs. 9%, P = 0.006). However, there were no significant differences between diabetic and nondiabetic subjects with reference to the Pro12Ala polymorphism among the South Asians living in Dallas (20 vs. 23%) and in India (19 vs. 19.3%). Although Caucasians carrying PPAR-gamma Pro12Ala had lower plasma insulin levels at 2 h of OGTT than the wild-type (Pro/Pro) carriers (76 +/- 68 and 54 +/- 33 microU/ml, respectively, P = 0.01), no differences in either fasting or 2-h plasma insulin concentrations were found between South Asians carrying the PPAR-gamma Pro12Ala polymorphism and those with the wild-type genotype at either Chennai or Dallas. CONCLUSIONS: Although further replication studies are necessary to test the validity of the described genotype-phenotype relationship, our study supports the hypothesis that the PPAR-gamma Pro12Ala polymorphism is protective against diabetes in Caucasians but not in South Asians.

Effect of inclusion of lactulose and Lactobacillus plantarum on the intestinal environment and performance of piglets at weaning

The prebiotic lactulose, a probiotic strain of Lactobacillus plantarum (L. plantarum) and a synbiotic combination of these two agents were evaluated as growth promoters in 25–39-day old commercial weaning pigs. Ninety-six weaning pigs were allocated into 32 pens, taking initial weight into account, and distributed into four groups as follows: a control diet (CTR), the same diet supplemented daily with L. plantarum (109 CFU/mL sprayed on top; 20 mL/pig) (LPN); 10 g/kg lactulose (LAC) or a combination of both treatments (SYN). At day 14, eight piglets from each group were euthanized and proximal colon digesta was sampled for luminal pH, short-chain fatty acids (SCFA) and lactic acid concentrations. Deoxyribonucleic acid was extracted from colonic digesta and the microbial community was profiled by terminal restriction fragment length polymorphism analysis (T-RFLP) and qPCR. Blood urea nitrogen (BUN) and acute-phase proteins (Pig-MAP) were measured. Lactulose treatment (LAC) improved feed intake (P<0.05), average daily gain (P<0.01), feed:gain ratio (P<0.05) and reduced BUN (P<0.01). Both, LAC and LPN treatment, decreased the Enterobacteriaceae:Lactobacillus spp. ratio in the colonic luminal contents (P<0.05). Moreover LPN treatment promoted a decrease in the percentage of branched fatty acids (P<0.01) suggesting a reduction in proteolytic microbial activity. Microbial profiling of colonic luminal contents by T-RFLP revealed changes in some microbial species. Terminal restriction fragments (TRFs) compatible with Bifidobacterium thermoacidophilum were more frequently detected in experimental diets compared to CTR (P<0.05). Pigs receiving SYN diet demonstrated the combined positive effects of individual LAC and LPN treatment although we were not able to show a specific increase in the probiotic strain with the inclusion of lactulose. Collectively, these data suggest the combination of lactulose and L. plantarum acts as a complementary synbiotic, but not as a synergistic combination.

Analysis of the bacterial communities present in lungs of patients with cystic fibrosis from American and British centers

The aim of this study was to determine whether geographical differences impact the composition of bacterial communities present in the airways of cystic fibrosis (CF) patients attending CF centers in the United States or United Kingdom. Thirty-eight patients were matched on the basis of clinical parameters into 19 pairs comprised of one U.S. and one United Kingdom patient. Analysis was performed to determine what, if any, bacterial correlates could be identified. Two culture-independent strategies were used: terminal restriction fragment length polymorphism (T-RFLP) profiling and 16S rRNA clone sequencing. Overall, 73 different terminal restriction fragment lengths were detected, ranging from 2 to 10 for U.S. and 2 to 15 for United Kingdom patients. The statistical analysis of T-RFLP data indicated that patient pairing was successful and revealed substantial transatlantic similarities in the bacterial communities. A small number of bands was present in the vast majority of patients in both locations, indicating that these are species common to the CF lung. Clone sequence analysis also revealed that a number of species not traditionally associated with the CF lung were present in both sample groups. The species number per sample was similar, but differences in species presence were observed between sample groups. Cluster analysis revealed geographical differences in bacterial presence and relative species abundance. Overall, the U.S. samples showed tighter clustering with each other compared to that of United Kingdom samples, which may reflect the lower diversity detected in the U.S. sample group. The impact of cross-infection and biogeography is considered, and the implications for treating CF lung infections also are discussed.

The use of culture-independent tools to characterize bacteria in endo-tracheal aspirates from pre-term infants at risk of bronchopulmonary dysplasia

Although premature infants are increasingly surviving the neonatal period, up to one-third develop bronchopulmonary dysplasia (BPD). Despite evidence that bacterial colonization of the neonatal respiratory tract by certain bacteria may be a risk factor in BPD development, little is known about the role these bacteria play. The aim of this study was to investigate the use of culture-independent molecular profiling methodologies to identify potential etiological agents in neonatal airway secretions. This study used terminal restriction fragment length polymorphism (T-RFLP) and clone sequence analyses to characterize bacterial species in endo-tracheal (ET) aspirates from eight intubated pre-term infants. A wide range of different bacteria was identified in the samples. Forty-seven T-RF band lengths were resolved in the sample set, with a range of 0-15 separate species in each patient. Clone sequence analyses confirmed the identity of individual species detected by T-RFLP. We speculate that the identification of known opportunistic pathogens including S. aureus, Enterobacter sp., Moraxella catarrhalis, Pseudomonas aeruginosa and Streptococcus sp., within the airways of pre-term infants, might be causally related to the subsequent development of BPD. Further, we suggest that culture-independent techniques, such as T-RFLP, hold important potential for the characterization of neonatal conditions, such as BPD.