Colonization, safety, and tolerability study of the Streptococcus salivarius 24SMBc nasal spray for its application in upper respiratory tract infections.

Streptococcus salivarius, a non-pathogenic species and the predominant colonizer of the oral microbiota, finds a wide application in the prevention of upper respiratory tract infections, also reducing the frequency of their main pathogens. In this pilot study, the primary objective was to evaluate the safety and tolerability of a nasal spray, S. salivarius 24SMBc, as a medical device in a clinical study involving 20 healthy adult subjects. The secondary aim was to determine the ability of colonization assessed by molecular fingerprinting. Twenty healthy adult subjects, aged between 30 and 54 years, without a medical history of recurrent otitis media, were enrolled. All patient characteristics fulfilled the inclusion criteria. All subjects were treated daily for 3 days with the nasal spray containing S. salivarius 24SMBc at a concentration of 5 × 10(9) colony-forming units (CFU)/ml. The persistence of S. salivarius in the nasopharynx was investigated by the antagonism test and random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR). The tolerability and safety were clinically assessed by clinical examinations during treatment. Our results demonstrate the capability of S. salivarius 24SMBc to colonize the rhinopharynx tissues in 95% of subjects and persist in 55% of them after 6 days from the last dose of the formulation, maintaining a concentration of 10(5) CFU/ml. The treatment was well tolerated by all healthy patients and no adverse effects were found. The topical application of streptococcal probiotics is a relatively undeveloped field but is becoming an attractive approach for both prevention and therapy, especially for pediatric age patients. S. salivarius 24SMBc possess characteristics making this strain suitable for use in bacteriotherapy.

Streptococcus salivarius 24SMB administered by nasal spray for the prevention of acute otitis media in otitis-prone children.

This paper reports the results of the first study in which Streptococcus salivarius 24SMB, a safe α-haemolytic strain capable of producing bacteriocin-like substances with significant activity against acute otitis media (AOM) pathogens, was intranasally administered in an attempt to reduce the risk of new episodes of AOM in otitis-prone children. In this prospective, randomized, double-blind, placebo-controlled study, 100 children aged 1-5 years with histories of recurrent AOM were randomized 1:1 to receive an intranasal S. salivarius 24SMB or placebo twice daily for 5 days each month for 3 consecutive months. Fifty treated children and 47 who received placebo who were compliant with study protocol were followed monthly for 6 months. The number of children who did not experience any AOM was higher among the children treated with the S. salivarius 24SMB preparation than among those in the placebo group (30.0 vs 14.9%; p = 0.076). Moreover, the number of children who received antibiotics during the study period was lower among the children treated with S. salivarius 24 SMB than among those who received placebo (70 vs 83.0%; p = 0.13). Compared with the children who were not colonized by S. salivarius 24SMB after treatment, the number of colonized children who experienced any AOM was significantly lower (42.8 vs 13.6%; p = 0.03). Similar results were observed when the children treated with antibiotics for AOM were analysed (67.8 vs 95.5%; p = 0.029). This study revealed the ability of intranasally administered S. salivarius 24SMB to reduce the risk of AOM in otitis-prone children.

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.

A cluster of three single nucleotide polymorphisms in the 3'-untranslated region of human glycoprotein PC-1 gene stabilizes PC-1 mRNA and is associated with increased PC-1 protein content and insulin resistance-related abnormalities.

Glycoprotein PC-1 inhibits insulin signaling and, when overexpressed, plays a role in human insulin resistance. Mechanisms of PC-1 overexpression are unknown. We have identified a haplotype in the 3'-untranslated region of the PC-1 gene that may modulate PC-1 expression and confer an increased risk for insulin resistance. Individuals from Sicily, Italy, carrying the "P" haplotype (i.e., a cluster of three single nucleotide polymorphisms: G2897A, G2906C, and C2948T) were at higher risk (P < 0.01) for insulin resistance and had higher (P < 0.05) levels of plasma glucose and insulin during an oral glucose tolerance test and higher levels of cholesterol, HDL cholesterol, and systolic blood pressure. They also had higher (P < 0.05-0.01) PC-1 protein content in both skeletal muscle and cultured skin fibroblasts. In CHO cells transfected with either P or wild-type cDNA, specific PC-1 mRNA half-life was increased for those transfected with P (t/2 = 3.73 +/- 1.0 vs. 1.57 +/- 0.2 h; P < 0.01). In a population of different ethnicity (Gargano, East Coast Italy), patients with type 2 diabetes (the most likely clinical outcome of insulin resistance) had a higher P haplotype frequency than healthy control subjects (7.8 vs. 1.5%, P < 0.01), thus replicating the association between the P allele and the insulin resistance-related abnormalities observed among Sicilians. In conclusion, we have identified a possible molecular mechanism for PC-1 overexpression that confers an increased risk for insulin resistance-related abnormalities.

Activity of telithromycin against multi-drug resistant Streptococcus pneumoniae and molecular characterization of macrolide and tetracycline resistance determinants.

The antistreptococcal activity of telithromycin and 11 different comparators was evaluated in 26 multi-drug resistant (MDR) Streptococcus pneumoniae strains collected during 2002-2003 as part of the ongoing PROTEKT (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin) Italian Surveillance Program. The strains were characterized for their susceptibility to antibiotics both at the phenotypic and genotypic levels; furthermore, the association of erm(B), mef(A) class and tet(M) genes, as well as the mobile elements carrying them were determined. The strains in this study were resistant to penicillin (MIC > or = 2 mg/l) in 23.1% of cases, resistant to tetracycline in 88.4%, to cotrimoxazole in 34.6% and cefuroxime in 26.9% while only telithromycin and levofloxacin retained 100% activity against all microorganisms. Co-existence of different resistance determinants was found in 19.2% of all isolates collected in our laboratory, coming from southern Italy. Twenty-three isolates showing the MLSB phenotype of resistance possessing the erm(B) gene (88.5%), associated with tet(M), were carried on the same Tn1545-like element, while two isolates showing the M phenotype possessing the mef(A) gene alone, were carried on Tn1207.1. In only one strain were mef(E) and tet(M) together carried on Tn2009.

[[(Arylpiperazinyl)alkyl]thio]thieno[2,3-d]pyrimidinone derivatives as high-affinity, selective 5-HT1A receptor ligands.

A series of 2-[[(4-aryl-1-piperazinyl)alkyl]thio]thieno[2,3-d]pyrimidin-4 (1H)-one and 3-substituted 2-[[(4-aryl-1-piperazinyl)alky]thio]thieno[2,3-d]pyrimidin-4 (3H)-one derivatives was prepared and evaluated for in vitro 5-HT1A receptor affinity by radioligand binding assays; the selectivity for 5-HT1A receptors rather than alpha 1-adrenoceptors was also examined (ratio of the IC50 alpha 1 to IC50 5-HT1A). The binding tests gave indications about the best features of the [(arylpiperazinyl)alkyl]thio moiety and of the substituents on the thiophene and pyrimidinone rings for efficacious and selective 5-HT1A ligands. The most effective derivative for displacing [3H]-8-OH-DPAT from rat hippocampal membranes was the 3-amino-2-[[3-[4-(2-methoxyphenyl)-1-piperazinyl] propyl]thio]-5,6-dimethylthieno[2,3-d]pyrimidin-4(3H)-one (70) (IC50 = 0.3 nM) with selectivity of 24 for the 5-HT1A over the alpha 1-adrenoceptor. Compound 73, where the 2-methoxyphenyl on the N4 piperazine ring was replaced with a pyrimidine group, showed the best selectivity, with a ratio of 74, while its affinity IC50 for 5-HT1A was 6.8 nM. These results, compared to those for compounds 46 (IC50 24 nM; selectivity 2) and 49 (IC50 226 nM; selectivity 5), N3 unsubstituted analogues of derivatives 70 and 73, show the importance of an amino group in position 3 of the thienopyrimidine system for the interaction with 5-HT1A receptor binding sites, although this fragment can affect the affinity and selectivity only if linked to the (arylpiperazinyl)alkyl moiety. The better selectivity of piperidine 74 (IC50 0.8; selectivity 45) compared to the analogous piperazine 70 is also noteworthy. Twenty of the 30 molecules used for determining the binding affinity to 5-HT1A and alpha 1-adrenergic receptors were selected for QSAR analysis using a series of molecular descriptors and calculated with the TSAR software.

rrn operons in Haemophilus parainfluenzae and mosaicism of conserved and species-specific sequences in the 16S-23S rDNA long spacer.

The mosaic organisation of short-sequence boxes was analysed in the cloned and sequenced long ribosomal spacer (547 bp) of Haemophilus parainfluenzae GR. Comparison and alignment of both the long and the short spacer were performed in H. parainfluenzae and H. influenzae Rd. The long spacer contained two tRNA genes (tRNA(Ala) and tRNA(Ile)) which are highly homologous to the corresponding genes found in the spacers of other species, such as Haemophilus spp., Actinobacillus spp., and Plesiomonas shigelloides. At the 3' end of tRNA(Ala) a putative ribosomal spacer loop was found, showing a strong secondary structure. Pulsed field gel electrophoresis (PFGE) analysis after restriction of the genome of H. parainfluenzae GR with I-Ceu I and subsequent polymerase chain reaction (PCR) analysis of PFGE-separated DNA fragments demonstrated that the H. parainfluenzae genome contained six operons and that the long spacer was present in three copies of them. Two short DNA segments were identified as being species-specific, allowing us to design PCR primers which were useful in the molecular identification of H. parainfluenzae isolates.

Molecular epidemiology of enterococci with high-level resistance to aminoglycosides.

DNA-based methodologies are considerably more powerful than other phenotype-based typing systems, providing a finer level of epidemiological discrimination, differentiating both closely and distantly related independent isolates that otherwise may appear as identical. In this study, plasmid analysis and pulsed-field gel electrophoresis were used to compare 28 isolates of Enterococci (respectively 13 strains of Enterococcus faecalis and 15 strains of Enterococcus faecium) with high-level resistance to aminoglycosides, isolated in Catania (Italy). Plasmid profile analysis resolved 20 different patterns among 24 plasmid harboring strains; many isolates showed one or two plasmids of the same size, but different plasmid content. Analysis of the PFGE-based RFLP patterns after SmaI digestion of genomic DNA resolved 26 different clones from 28 isolates: particularly, it resolved two different clones from three isolates showing identical plasmid profiles, and it identified as a single clone two isolates exhibiting different plasmid profiles. Thus, on the basis of our PFGE-based RFLP analysis data, we concluded that all the strains included in the study were genetically unrelated with two exceptions.

Clonal types and multidrug resistance patterns of methicillin-resistant Staphylococcus aureus (MRSA) recovered in Italy during the 1990s.

A large number (272) of methicillin-resistant Staphylococcus aureus (MRSA) isolates recovered from Italian hospitals during the early and late 1990s were characterized for multidrug resistance pattern and clonal type using a combination of genotyping methods, including pulsed-field gel electrophoresis (PFGE), spaA typing, multilocus sequence typing (MLST), determination of SCC mec type, and hybridization pattern with Tn 554. The majority of MRSA belonged to four genetic lineages: the pandemic Iberian and Brazilian clones, and two unique clonal types-the "Italian" and "Rome" clones of MRSA. The Italian clone carried the SCC mec type I in the genetic background of ST228, which is a double-locus variant of the sequence type of the multidrug-resistant New York/Japanese clone (ST5). The properties of the Rome clone showed several striking similarities to those of the Archaic clone of MRSA that was dominant among MRSA isolates in the mid-1960s to 1970s, but has not been detected since then in recent global surveillance studies.

Presence of the ica operon in clinical isolates of Staphylococcus epidermidis and its role in biofilm production.

Staphylococcus epidermidis is an important cause of catheter-associated infections, which are attributed to its ability to form a multilayered biofilm on polymeric surfaces. This ability depends, in part, on the activity of the icaADBC locus and the icaR gene, which are involved in the production of the polysaccharide intercellular adhesin (PIA) that is functionally necessary for cell-to-cell adhesion and biofilm accumulation. The present study determined: (1) the prevalence of the icaADBC operon in S. epidermidis isolates from catheter-related and other nosocomial infections; (2) the correlation between the presence of this operon, biofilm production and resistance to antibiotics; (3) the expression of ica genes and biofilm production; and (4) the genetic relatedness of the isolates. The results showed that icaRADBC was present in 45% of the isolates included in the study, and that such isolates were significantly more resistant to the main antibiotics tested than were ica-negative isolates. The presence of the entire cluster did not always correlate with biofilm production, determined under different culture conditions, but there was evidence to suggest a correlation when at least two genes (icaAD) were co-transcribed. Eight of 18 ica-positive isolates had the entire operon in the same restriction fragment after pulsed-field gel electrophoresis, but the isolates were not clonal. Estimation of genetic relatedness indicated that ica-positive S. epidermidis isolates belonged to different lineages, distributed in only one of two major clusters, with a genetic distance of c. 0.12.

In vitro selection of glycopeptide-resistant variants of Enterococci.

In order to study the possible phenotypic and genotypic changes related to glycopeptide pressure on enterococci, a study was undertaken using stepwise in vitro exposure to achieve the following objectives: (i) to evaluate the development of resistance and cross-resistance between vancomycin and teicoplanin; (ii) to determine the stability of the acquired level of resistance; (iii) to determine the phenotypic and genotypic changes related to glycopeptide pressure; and (iv) to assess the spectrum of antibiotic-susceptibility of all strains. Our results showed that no variants resistant to glycopeptides could be selected after in vitro glycopeptide exposure experiments. However some strains showed increased MIC values: 8 mg/l to vancomycin in eight strains selected by vancomycin itself, while teicoplanin produced intermediate values to vancomycin in only three strains. The phenotypes were stable in vitro after numerous passages in antibiotic-free medium and three out of nine strains with a changed MIC level, showed 40, 42 and 43 kDa proteins in cell membrane preparations. The profile of antibiotic resistance was comparable in all isogenic strains tested with the exception of three selected strains that became susceptible to penicillin G. The pressure produced by glycopeptides, particularly vancomycin has contributed to an increased level of MIC that can influence the acquisition and/or full expression of this resistance.

High affinity and selectivity of [[(arylpiperazinyl)alkyl]thio]thieno[2,3-d]pyrimidinone derivatives for the 5-HT(1A) 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-HT(3) and 5-HT(4) receptor ligands.

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

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.

In vitro activity of levofloxacin against coagulase-positive and -negative staphylococci.

The in vitro activity of levofloxacin compared with that of ciprofloxacin, ofloxacin and norfloxacin were examined by conventional in vitro tests against 150 clinical isolates of staphylococci, subdivided according to species and susceptibility to methicillin. Although the minimum inhibitory concentrations (MICs) of all quinolones were highest in methicillin-resistant Staphylococcus aureus strains, the activity of levofloxacin was almost complete in methicillin-resistant S. epidermidis and methicillin-resistant S. haemolyticus when compared with ciprofloxacin and ofloxacin, which showed more than 30% resistance. Methicillin-susceptible S. aureus and S. epidermidis strains were susceptible to all quinolones with few differences between the antibiotics tested. The minimal bactericidal activity of levofloxacin was within the double dilution range of MIC values for all strains tested, demonstrating its potent role against staphylococci. In time-kill studies, levofloxacin exerted bactericidal activity within 3 h against all staphylococci. These in vitro results suggest that levofloxacin is a potent fluoroquinolone against coagulase-negative staphylococci and that it is both methicillin-susceptible and resistant. Further studies are necessary to determine the role of this drug in the treatment of infections sustained by these microorganisms.

Serum levels of myoglobin in aged thyreopathic patients.

Serum concentrations of triiodothyronine (T-3), thyroxine (T-4) and myoglobin were determined by radioimmunoassay in 10 hypothyroid, 15 hyperthyroid and 14 euthyroid aged patients. The average ages were between 69 and 71 yr for these groups. The serum levels of T-3 and T-4 were typical for the clinical diagnosis and were accompanied by characteristic changes in the serum myoglobin concentrations. The hypothyroid, the euthyroid and the hyperthyroid groups displayed 107.0, 33.1 and 17.0 ng myoglobin per ml of serum, respectively. These differences are statistically highly significant. The authors are of the opinion that the serum myoglobin level depends on the myoglobin content of the muscle tissue, being higher in hypothyroid and lower in hyperthyroid patients as compared to the euthyroid persons.

Rapid method for detection of gyrA and grlA mutations in unrelated strains of Staphylococci susceptible and resistant to levofloxacin.

A panel of 150 clinical isolates of methicillin resistant and susceptible Staphylococci were investigated using a rapid and simple PCR-RFLPs technique to detect DNA nucleotide changes at the site of the most frequently reported mutations in grlA (codons 79, 80) and gyrA (codons 83, 84) genes which confer fluorquinolone resistance in Staphylococci. Convergent dual mutations in and gyrA and grlA were found in all strains exhibiting resistance to ciprofloxacin (MIC, 8 to > or =128 mg/l) and levofloxacin (MIC, 8 to > or =64 mg/l). Mutations in grlA and gyrA were also found in strains susceptible to levofloxacin and resistant to ciprofloxacin. In our sample no strains with only grlA mutations were found. Our data indicate that methicillin-resistant fluorquinolone-resistant strains are likely to have mutations in both grlA and gyrA. In contrast, methicillin-susceptible strains do not show any mutation. The genetic relatedness of a sample of representative epidemiologically unrelated MRSA strains, tested by PFGE and rep-PCR, are in agreement with the hypothesis of a clonal selection of these resistant strains.

Synthesis and pharmacological properties of thieno[2',3':4,5]pyrimido [2,1-b][1,3,4]thiadiazine derivatives.

A new series of 2-substituted-7,8-dimethyl-3H,9H-thieno[2',3':4,5]pyrimido[2,1- b][1,3,4]thiadiazin-9-ones 5a-d and 6a,b was synthesized through the hydrazinium(1+) salt of 3-amino-2,3-dihydro-5,6-dimethyl-2-thioxo- thieno[2,3-d]pyrimidin-4(1H)-one, 2. These derivatives and two analogs 10a,b were tested for their analgesic and antiinflammatory properties. The pharmacological results are discussed in comparison with those of related compounds previously tested.

Synthesis of aminothienopyrimidine and thienotriazolopyrimidine derivatives as potential anticonvulsant agents.

New aminothienopyrimidines and derivatives of the new thienotriazolopyrimidine ring system were synthesized to investigate their anticonvulsant activity. The tricyclic compounds 6a, c, e, f, m, o and p and two bicyclic derivatives 4f and 4o were tested. The pharmacological screening results are reported and discussed.

Synthesis of new [1,3,4]thiadiazolo[3,2-a]thieno[2,3-d]pyrimidinone derivatives with antiinflammatory activity.

New thiadiazolothienopyrimidinones were synthesized in continuation of efforts to prepare thienopyrimidine derivatives with analgesic and antiinflammatory activities. In this study, the effect of various substituents in the thiophene ring on the pharmacological activity of the compounds was investigated.