Usefulness of real time PCR for the differentiation and quantification of 652 and JP2 Actinobacillus actinomycetemcomitans genotypes in dental plaque and saliva

BACKGROUND: The aim of our study is to describe a fast molecular method, able to distinguish and quantize the two different genotypes (652 and JP2) of an important periodontal pathogen: Actinobacillus actinomycetemcomitans. The two genotypes show differences in the expression of an important pathogenic factor: the leukotoxin (ltx). In order to evidence this, we performed a real time PCR procedure on the ltx operon, able to recognize Aa clinical isolates with different leukotoxic potentials. METHODS: The specificity of the method was confirmed in subgingival plaque and saliva specimens collected from eighty-one Italian (Sardinian) subjects with a mean age of 43.9, fifty five (68 %) of whom had various clinical forms of periodontal disease. RESULTS: This procedure showed a good sensitivity and a high linear dynamic range of quantization (10(7)-10(2 )cells/ml) for all genotypes and a good correlation factor (R2 = 0.97–0.98). Compared with traditional cultural methods, this real time PCR procedure is more sensitive; in fact in two subgingival plaque and two positive saliva specimens Aa was only detected with the molecular method. CONCLUSION: A low number of Sardinian patients was found positive for Aa infections in the oral cavity, (just 10 positive periodontal cases out of 81 and two of these were also saliva positive). The highly leukotoxic JP2 strain was the most representative (60 % of the positive specimens); the samples from periodontal pockets and from saliva showed some ltx genotype for the same patient. Our experience suggests that this approach is suitable for a rapid and complete laboratory diagnosis for Aa infection

Leer en comunidad: creación de clubes de lectura de literatura escrita por mujeres en la universidad

Depto. de Lengua Española y Teoría de la LiteraturaFac. de FilologíaFALSEUniversidad Complutense de Madridsubmitte

Absence, loss-of-function, or inhibition of Escherichia coli AcrB does not increase expression of other efflux pump genes supporting the discovery of AcrB inhibitors as antibiotic adjuvants

OBJECTIVES: To determine whether expression of efflux pumps and antibiotic susceptibility are altered in Escherichia coli in response to efflux inhibition. METHODS: The promoter regions of nine efflux pump genes (acrAB, acrD, acrEF, emrAB, macAB, cusCFBA, mdtK, mdtABC, mdfA) were fused to gfp in pMW82 and fluorescence from each reporter construct was used as a measure of the transcriptional response to conditions in which AcrB was inhibited, absent or made non-functional. Expression was also determined by RT-qPCR. Drug susceptibility of efflux pump mutants with missense mutations known or predicted to cause loss of function of the encoded efflux pump was investigated. RESULTS: Data from the GFP reporter constructs revealed that no increased expression of the tested efflux pump genes was observed when AcrB was absent, made non-functional, or inhibited by an efflux pump inhibitor/competitive substrate, such as PAβN or chlorpromazine. This was confirmed by RT-qPCR for PAβN and chlorpromazine; however, a small but significant increase in macB gene expression was seen when acrB is deleted. Efflux inhibitors only synergized with antibiotics in the presence of a functional AcrB. When AcrB was absent or non-functional, there was no impact on MICs when other efflux pumps were also made non-functional. CONCLUSIONS: Absence, loss-of-function, or inhibition of E. coli AcrB did not significantly increase expression of other efflux pump genes, which suggests there is no compensatory mechanism to overcome efflux inhibition and supports the discovery of inhibitors of AcrB as antibiotic adjuvants

Molecular identification of bacteriocins produced by Lactococcus lactis dairy strains and their technological and genotypic characterization

In this study, the bacteriocins produced by ten Lactococcus lactis subsp. lactis strains, previously isolated from raw milk and traditional Sardinian cheeses, were identified by targeting and sequencing the bacteriocin encoding genes. The inhibitory activity against different food borne and spoilage bacteria, and the technological and genotypic characteristics of the bacteriocinogenic L.lactis strains were also analysed. The presence of the nisin structural gene was confirmed for all L.lactis strains. Three strains were shown to harbour the Lactococcins B structural gene. The sequences of PCR products for the nisin gene from the ten bacteriocinogenic strains were compared with that of L. lactis strain ATCC 11454 (nisin A producer strain). Differences were observed in strain 6LS5 indicating its ability to produce the nisin Z variant. Five strains presented a wider inhibitory spectrum resulting in activity towards Pseudomonas aeruginosa and all the Bacillus, Listeria, Staphylococcus and Clostridium strains tested. High β-galactosidase and moderate aminopeptidase activities, that promote the desirable flavours in cheese, were detected in the majority of the strains. Rep-PCR with primer (GTG)5 revealed high diversity among the strains and allowed discrimination at both interspecific and intraspecific level. The autochthonous bacteriocinogenic isolates from Sardinian dairy products described in this work may potentially find application as starter, co-starter or protective adjunct cultures in the manufacturing of cheeses

Azole resistance and ERG11 464 polymorphism in oral Candida albicans clinical strains isolated in Sardinia

The in vitro activity of three different azoles (fluconazole, FLC, voriconazole, VRC and ketoconazole, KTC) was determined and correlated with the single nucleotide polymorphisms (SNPs) in a “hot spot” region of the ERG11 gene in a collection of 52 arbitrarily selected C. albicans strains isolated from Sardinian subjects with oropharyngeal candidiasis. Among the strains evaluated, 23.1% were resistant, 9.6% Sensible Dose Dependent (SDD) and 67.3% susceptible to FLC. Among the FLC resistant strains, 83.3% were also cross-resistant to VRC (10/12) and 66.6% to KTC (8/12). The homozygous point mutation G464S was only detected in four out of five SDD to fluconazole strains. These data showed that the resistance of Candida albicans to azoles and the mutation at codon 464 of ERG11 are not associated. In addition the results also indicate a high prevalence of azole-resistant and cross resistant strains among these patients

Analisi dei possibili fattori correlati a ipovitaminosi D nella popolazione detenuta negli istituti penitenziari di Parma

INTRODUZIONE I carcerati sono ritenuti a rischio di ipovitaminosi D. La presente analisi ha rilevato la calcemia e/o la concentrazione ematica di vitamina D (25OHD) nei detenuti degli Istituti Penitenziari di Parma ed indagato possibili fattori in relazione ad esse. METODI Dei 133 soggetti (maschi, maggiorenni) detenuti da almeno 3 anni, sono stati esclusi quelli con patologie o terapie tali da influenzare il metabolismo di calcio e/o 25OHD. Sono stati quindi valutate calcemia e/o 25OHD di 110 detenuti, per metterle in relazione con età, indice di massa corporea (IMC), fototipo, regime detentivo e positività alla Mantoux. E’stata effettuata un’indagine descrittiva e sono state studiate possibili relazioni con analisi multivariata. RISULTATI Il 30% dei soggetti presenta ipovitaminosi D conclamata e il 62% è a rischio di ipovitaminosi; l’età media risulta di 53 anni (29-89); il 40% dei detenuti è normopeso, il 57,3% è sovrappeso o obeso (mediana IMC=26), il 2,7 % è affetto da anoressia; la carnagione è prevalentemente chiara (fototipo 1-2 nell’80%); il 27,2% è in regime di alta sicurezza, il 72,8% è ubicato nelle sezioni comuni o in semilibertà; la Mantoux è risultata positiva nel 16% dei casi, negativa nel 36%, non rilevabile nel 48%. La regressione logistica evidenzia che età, IMC e fototipo non sembrano essere in relazione con calcemia e/o 25OHD; la detenzione ad alta sicurezza (OR=3,7; IC 95%=1,5-9,1; p=0,004) e la Mantoux positiva (OR=4,9; IC 95%=1,2-19,9; p=0,025) sono invece in relazione con ipovitaminosi D. CONCLUSIONI La detenzione, ed in particolare il regime di alta sicurezza, risulta influenzare il rischio di ipovitaminosi D. Anche la positività alla Mantoux è in relazione a deficit di 25OHD. Tali dati confermano l’utilità di interventi di integrazione vitaminica nei detenuti in regime 41Bis e/o Mantoux positivi. Questa indagine preliminare suggerisce approfondimenti, anche relativamente a dieta e anni di detenzione

Mutations upstream of fabI in triclosan resistant Staphylococcus aureus strains are associated with elevated fabI gene expression

Background The enoyl-acyl carrier protein (ACP) reductase enzyme (FabI) is the target for a series of antimicrobial agents including novel compounds in clinical trial and the biocide triclosan. Mutations in fabI and heterodiploidy for fabI have been shown to confer resistance in S. aureus strains in a previous study. Here we further determined the fabI upstream sequence of a selection of these strains and the gene expression levels in strains with promoter region mutations. Results Mutations in the fabI promoter were found in 18% of triclosan resistant clinical isolates, regardless the previously identified molecular mechanism conferring resistance. Although not significant, a higher rate of promoter mutations were found in strains without previously described mechanisms of resistance. Some of the mutations identified in the clinical isolates were also detected in a series of laboratory mutants. Microarray analysis of selected laboratory mutants with fabI promoter region mutations, grown in the absence of triclosan, revealed increased fabI expression in three out of four tested strains. In two of these strains, only few genes other than fabI were upregulated. Consistently with these data, whole genome sequencing of in vitro selected mutants identified only few mutations except the upstream and coding regions of fabI, with the promoter mutation as the most probable cause of fabI overexpression. Importantly the gene expression profiling of clinical isolates containing similar mutations in the fabI promoter also showed, when compared to unrelated non-mutated isolates, a significant up-regulation of fabI. Conclusions In conclusion, we have demonstrated the presence of C34T, T109G, and A101C mutations in the fabI promoter region of strains with fabI up-regulation, both in clinical isolates and/or laboratory mutants. These data provide further observations linking mutations upstream fabI with up-regulated expression of the fabI gene