Maria Santagati

A variation in 3 ' UTR of hPTP1B increases specific gene expression and associates with insulin resistance

Protein tyrosine phosphatase 1B (PTP1B) inhibits insulin signaling and, when overexpressed, plays a role in insulin resistance (Ahmad et al. 1997). We identified, in the 3 untranslated region of the PTP1B gene, a 1484insG variation that, in two different populations, is associated with several features of insulin resistance: among male individuals, higher values of the insulin resistance HOMA(IR) index (P=.006), serum triglycerides (P=.0002), and total/HDL cholesterol ratio (P=.025) and, among female individuals, higher blood pressure (P=.01). Similar data were also obtained in a family-based association study by use of sib pairs discordant for genotype (Gu et al. 2000). Subjects carrying the 1484insG variant showed also PTP1B mRNA overexpression in skeletal muscle (6,166 plus minus 1,879 copies/40 ng RNA vs. 2,983 plus minus 1,620; P<.01). Finally, PTP1B mRNA stability was significantly higher (P<.01) in human embryo kidney 293 cells transfected with 1484insG PTP1B, as compared with those transfected with wild-type PTP1B. Our data indicate that the 1484insG allele causes PTP1B overexpression and plays a role in insulin resistance. Therefore, individuals carrying the 1484insG variant might particularly benefit from PTP1B inhibitors, a promising new tool for treatment of insulin resistance (Kennedy and Ramachandran 2000).

Characterization of a Genetic Element Carrying the Macrolide Efflux Gene mef(A) in Streptococcus pneumoniae

ABSTRACT The mef(A) gene from a clinical isolate ofStreptococcus pneumoniae exhibiting the M-type resistance to macrolides was found to be part of the 7,244-bp chromosomal element Tn1207.1, which contained 8 open reading frames.orf2 encodes a resolvase/invertase, and orf5 is a homolog of the macrolide-streptogramin B resistance genemsr(SA).

The Novel Conjugative Transposon Tn1207.3 Carries the Macrolide Efflux Gene mef(A) in Streptococcus pyogenes

The macrolide efflux gene mef(A) of the Streptococcus pyogenes clinical strain 2812A was found to be carried by a 52-kb chromosomal genetic element that could be transferred by conjugation to the chromosome of other streptococcal species. The characteristics of this genetic element are typical of conjugative transposons and was named Tn1207.3. The size of Tn1207.3 was established by pulsed-field gel electrophoresis (PFGE), and DNA sequencing analysis showed that the 7,244 bp at the left end of Tn1207.3 were identical to those of the pneumococcal Tn1207.1 element. Tn1207.3-like genetic elements were found to be inserted at a single specific chromosomal site in 12 different clinical isolates S. pyogenes exhibiting the M phenotype of resistance to macrolides and carrying the mef(A) gene. Tn1207.3 was transferred from S. pyogenes 2812A to Streptococcus pneumoniae, and sequence analysis carried out on six independent transconjugants showed that insertion of Tn1207.3 in the pneumococcal genome always occurred at a single specific site as in Tn1207.1. Using MF2, a representative S. pneumoniae transconjugant, as a donor, Tn1207.3 was transferred again by conjugation to S. pyogenes and Streptococcus gordonii. The previously described nonconjugative element Tn1207.1 of S. pneumoniae appears to be a defective element, part of a longer conjugative transposon that carries mef(A) and is found in clinical isolates of S. pyogenes.

Bacteriocin-producing oral streptococci and inhibition of respiratory pathogens

The use of bacteria as probiotics is in continuous development, thanks to their capacity to maintain or restore a hosts natural microbiome by interference with and/or inhibition of other microorganisms mediated by antimicrobial peptide production such as bacteriocins. In the oral cavity, Streptococcus salivarius, a non-pathogenic and predominant oral species, is one of the major bacteriocin producers that is able to coexist in this environment and reduce the frequency of colonization of the main pathogens involved in upper respiratory tract infections. The aim of this study was to screen oral bacteria colonizing healthy children for their use as potential oral probiotics. Eighty-one α-hemolytic streptococci isolated from nasal and/or pharyngeal swabs of 31 healthy children aged between two and twelve years were isolated. Among them, 13 α-hemolytic streptococci were selected for their bacteriocin-like inhibitory activity against potential pathogens. These strains were tested for bacteriocin production and assayed for their capacity to adhere to HEp-2 cell lines. Our data showed that 13 bacteriocin producer strains were able to inhibit different gram-positive pathogens. Among them one strain, S. salivarius 24SMB, deposited as DSM 23307, was selected as a potential oral probiotic, thanks to its safety assessment, ability to inhibit Streptococcus pneumoniae and the absence of virulence and antibiotic resistance genes.

DNA methylase modifications and other linezolid resistance mutations in coagulase-negative staphylococci in Italy

BACKGROUNDnDespite 10 years of clinical use, linezolid resistance in Staphylococcus aureus and coagulase-negative staphylococci (CoNS) is still a rare phenomenon. This study reports the mechanisms of resistance and strain types seen in clusters of linezolid-resistant CoNS from two different hospitals in Italy during the period 2008-09.nnnMETHODSnGenes associated with linezolid resistance were subjected to molecular analysis and isolates were characterized by PFGE macrorestriction analysis using SmaI.nnnRESULTSnThirty-three linezolid-resistant isolates of methicillin-resistant CoNS comprising Staphylococcus epidermidis (24), Staphylococcus hominis (5) and Staphylococcus simulans (4) were studied. The isolates showed varying levels of linezolid resistance. Almost all isolates for which linezolid MICs were 64 mg/L possessed point mutations in domain V of 23S rRNA, while isolates for which the MICs were 256 mg/L expressed methylase activity at position A2503 mediated by the cfr gene. Overall, the isolates showed reduced susceptibility to vancomycin (MICs 1-2 mg/L) and 11 of the 33 isolates showed no susceptibility to teicoplanin. These strains were also resistant to chloramphenicol (28 of 33), lincomycin (24 of 33), erythromycin (17 of 33) and quinupristin/dalfopristin (13 of 33). S. epidermidis isolates, showing mutations or methylase modifications, belonged to different PFGE profiles and to two different sequence types (ST2 and ST23), in which the cfr gene was carried on a plasmid of ∼50 kb.nnnCONCLUSIONSnClinical CoNS strains with resistance to linezolid and other second-line antibiotics, as well as reduced susceptibility to glycopeptides, have emerged in Italy.

High affinity and selectivity of [[(arylpiperazinyl)alkyl]thio]thieno[2,3-d]pyrimidinone derivatives for the 5-HT1A receptor. Synthesis and structure-affinity relationships

In this work we report the affinity of new thienopyrimidinones for 5-HT(1A)Rs and the selectivity versus alpha(1)ARs. The 3-amino-2-[[3-[4-(2-methoxyphenyl)-1-piperazinyl]propyl]thio]-6-ethyl -thieno[2,3-d]pyrimidin-4(3H)-one 27 is the most potent and selective (Ki 0.19 nM, selectivity 115). Compound 31 with the N4 piperazine orthonitrophenyl nucleus instead of the orthomethoxyphenyl also shows a good affinity and selectivity (Ki 1. 46 nM, selectivity 84). The results of derivatives 28, 29 and 30 (Ki 3.28, 12.59 and 4.38 nM; selectivity 24, 4 and 5, respectively), which have, respectively, an ethyl, an allyl and an acetylamino group instead of an N3 amino group, indicate the importance of this last group for the interaction with 5-HT(1A)R. Comparison of the results for the superior homologue 53 (Ki 3.72 nM, selectivity 51) and the inferior homologue 52 (5-HT(1A) Ki 1499 nM, alpha(1)A Ki NA) of 2-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-6,7-dimethyl-8H-[1, 3,4]thiadiazolo[3,2-a]thieno[2,3-d]pyrimidin-8-one 57 (Ki 23 nM, selectivity 5) shows how important the length of the chain binding the two heterocyclic systems is in the interaction with 5-HT(1A)Rs and alpha(1)ARs.

Design, synthesis and binding properties of novel and selective 5-HT3 and 5-HT4 receptor ligands.

This work reports the synthesis and the binding tests on the 5-HT3 and 5-HT4 receptors of new thienopyrimidopiperazine and piperazinylacylaminodimethylthiophene derivatives, in order to identify potent and selective ligands for each receptor. The 3-amino-2-(4-benzyl-1-piperazinyl)-5,6-dimethyl-thieno[2,3-d]pyrimidin-4(3H)-one derivative 28 showed the highest affinity and selectivity for the 5-HT3 over the 5-HT4 receptor (5-HT3 Ki=3.92 nM, 5-HT4 not active), whereas the 2-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butanoylamino]-4,5-dimethyl-3-thiophenecarboxylic acid ethyl ester (41) showed the highest affinity and selectivity for the 5-HT4 over the 5-HT3 receptor (5-HT4 Ki=81.3 nM, 5-HT3 not active). Conformational analyses were carried out on the compounds of the piperazinylacylaminodimethylthiophene series (39-42) taking compound 41 as the template.

Genetic elements carrying macrolide efflux genes in streptococci.

Type M resistance to macrolides in Streptococcus pyogenes and Streptococcus pneumoniae is associated to the presence of macrolide efflux genes (mef). These genes are carried by 3 very conserved chromosomal genetic elements: (i) mega, 5.5 kb, typical of S. pneumoniae, carries mef(E), can insert at different sites in the bacterial chromosome and in other genetic elements, does not have genes encoding putative recombinases or transposases, and it is not conjugative; (ii) Tn1207.3, 54, 52 kb, typical of S. pyogenes, carries mef(A), it integrates at a single specific site in the bacterial chromosome, carries 3 ORFs encoding for putative recombinases, it is transferred by conjugation among different streptococcal species, and its genome resembles that of a bacteriophage; (iii) Tn1207.1, 7.2 kb, is a defective form of Tn1207.3 found in a clonal population of S. pneumoniae, it carries mef(A), it integrates at a single specific site in the bacterial chromosome (the same of Tn1207.3), and it is not conjugative.

Insights and clinical perspectives of daptomycin resistance in Staphylococcus aureus: A review of the available evidence

The emergence of glycopeptide reduced susceptibility and resistance in Staphylococcus aureus strains is a growing clinical problem that poses significant clinical challenges in treatment. Its development is a complex and novel process involving many subtle physiological changes in the micro-organism. Daptomycin is the first cyclic lipopeptide approved for clinical use. Unlike most other antimicrobials, a trend towards increased daptomycin resistance has not been reported, although several cases of daptomycin non-susceptibility have been reported. The present review will present the available evidence on daptomycin resistance of S. aureus, with particular attention to its development. In addition to a literature overview, we have compiled the reported cases of daptomycin non-susceptibility to shed light on possible clinical mechanisms of resistance. In the 36 reports describing 62 clinical cases, infections caused by meticillin-resistant S. aureus (MRSA) strains with a vancomycin minimum inhibitory concentration (MIC) between 1mg/L and 2mg/L often led to vancomycin treatment failure, which may be associated with the development of non-susceptibility to daptomycin. Additional evidence suggests that underdosage of daptomycin is an important clinical aspect that merits further study. The current analysis highlights the importance of determining the MIC when using vancomycin to treat patients with severe S. aureus infections and that when failure is suspected, testing for heterogeneous vancomycin-intermediate S. aureus (hVISA) may also be necessary. Whilst further investigation is needed, it can be hypothesised that MRSA strains become hVISA during prolonged bacteraemia, which may predispose to the development of daptomycin resistance.

Methicillin resistance and vancomycin heteroresistance in Staphylococcus aureus in cystic fibrosis patients.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are increasingly being reported among cystic fibrosis (CF) populations worldwide. In this paper, we sought to examine at the epidemiology, the molecular characterisation and the antibiotic resistance of MRSA isolates in our cohort of CF patients. All MRSA strains were collected prospectively at the University Hospital of Catania, Italy, during a two-year study between mid 2005 to mid 2007 and underwent molecular, pathotype and susceptibility characterisations. Our study demonstrates persisting infections with both hospital-associated (HA-) and community-associated (CA-)MRSA, including Panton–Valentine leukocidin (PVL)-positive strains, in our CF population with an overall prevalence of 7.8%. We demonstrated that, in these patients, persistence was sustained by either identical clones that underwent subtle changes in their toxin content or by different clones over time. The isolation of MRSA in our CF population aged 7–24 years was associated with an increased severity of the disease even if, due to the small sample of patients included and the paucity of data on the clinical outcome, these results cannot be conclusive. Furthermore, three strains were heteroresistant vancomycin-intermediate S. aureus (hVISA), questioning the use of glycopeptides in the treatment of MRSA infections in these patients.