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.

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.

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.

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.

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.

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.

Macrolide efflux genes mef(A) and mef(E) are carried by different genetic elements in Streptococcus pneumoniae.

Susceptibilities to macrolides were evaluated in 267 Streptococcus pneumoniae isolates, of which 182 were from patients with invasive diseases and 85 were from healthy carriers. Of the 98 resistant isolates, 20 strains showed an M phenotype and carried mef. Strains that carried both mef(A) and mef(E) were found: 17 strains carried mef(A) and 3 carried mef(E). The characteristics of the strains carrying the mef genes and the properties of the mef-containing elements were studied. Strains carrying mef(A) belonged to serotype 14, were susceptible to all the antibiotics tested except erythromycin, and appeared to be clonally related by pulsed-field gel electrophoresis (PFGE). The three mef(E) strains belonged to different serotypes, showed different susceptibility profiles, and did not appear to be related by PFGE. The sequences of a fragment of the mef-containing element, which encompassed mef and the msr(A) homolog, were identical among the three mef(E)-positive strains and among the three mef(A)-positive strains, although there were differences between the sequences for the two variants at 168 positions. In all mef(A)-positive strains, the mef element was inserted in celB, which led to impairment of the competence of the strains. In line with insertion of the mef(E) element at a different site, the competence of the mef(E)-positive strains was maintained. Transfer of erythromycin resistance by conjugation was obtained from two of three mef(A) strains but from none of three mef(E) strains. Due to the important different characteristics of the strains carrying mef(A) or mef(E), we suggest that the distinction between the two genes be maintained.

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.

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.

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.

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.

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.

[Correlation between methicillin-resistance and resistance to fluoroquinolones in Staphylococcus aureus and Staphylococcus epidermidis]. / Correlazione della resistenza alla meticillina e ai fluorochinoloni in Staphylococcus aureus e Staphylococcus epidermidis.

Fluoroquinolones resistance in Staphylococci is associated to point mutations in grlA (80,84 and 116) grlB, gyrA (84,88) and gyrB genes. Almost all MRSA strains are ciprofloxacin and levofloxacin resistant while, in a lesser degree, MRCoN staphylococci show to be resistant to levofloxacin. This observation made possible to predict a different correlation between methicillin-resistance and the resistance to FQs in this two different species. In this study, we compare genomic analysis of S. aureus and S. epidermidis with the resistance to FQs. Our results show that strains of MRSA are distributed in 4 different PFGE-types while 12 MRSE strains are distributed in 9. MRSA resistant to FQs showed a unique PFGE pattern; on the contrary of FQs susceptible MRSA and MSSA. Furthermore mecA and gyrA genes are located in the same SmaI fragment in MRSA and in different in MRSE. MSSE and MRSE show more ClaI/mecA polymorphisms than MRSA. All this data confirm the clonal origin of MRSA and show that FQs resistance is linked to the presence of mec locus and both clonally spread. On the contrary in MRSE FQs-resistance is independent from MR and arise with the normal frequence of antibiotic induction.

Synthesis and pharmacological screening of 1,3,4-thiadiazino[2,3-b]quinazoline derivatives.

Derivatives of 1,3,4-thiadiazino[2,3-b]quinazoline 7, 9, 9a, 12, 12a, and 13 were prepared from the 3-amino-6-bromo-2,3-dihydro-2-thioxo-4-(1H)-quinazolinone (2) and its analogue without bromine. A series of the title derivatives with or without bromine was tested and the results of pharmacological screening are 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.

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.

Characterization of a genetic element carrying the macrolide efflux gene mef(A) in Streptococcus pneumoniae.

The mef(A) gene from a clinical isolate of Streptococcus 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 gene msr(SA).

High potent and selective arylpiperazine derivatives as ligands for the 5-HT1A receptor.

This paper reports the synthesis and affinities on the 5-HT1A versus the alpha1A receptors of new arylpiperazinylalkylthiothienopyrimidine and thiadiazole derivatives 16-24. Arylpiperazines 16-23 show affinities values in the nanomolar range for the 5-HT1A receptor. The compound 16 is highly potent (Ki 0.26 nM, selectivity 28), the derivatives 20 and 21 are less potent, but highly selective (Ki 9.40 and 5.06 nM, selectivity 207 and 73, respectively).

Replicase complex genes of Semliki Forest virus confer lethal neurovirulence.

Semliki Forest virus (SFV) is a mosquito-transmitted pathogen of small rodents, and infection of adult mice with SFV4, a neurovirulent strain of SFV, leads to lethal encephalitis in a few days, whereas mice infected with the avirulent A7(74) strain remain asymptomatic. In adult neurons, A7(74) is unable to form virions and hence does not reach a critical threshold of neuronal damage. To elucidate the molecular mechanisms of neurovirulence, we have cloned and sequenced the entire 11,758-nucleotide genome of A7(74) and compared it to the highly neurovirulent SFV4 virus. We found several sequence differences and sought to localize determinants conferring the neuropathogenicity by using a panel of chimeras between SFV4 and a cloned recombinant, rA774. We first localized virulence determinants in the nonstructural region by showing that rA774 structural genes combined with the SFV4 nonstructural genome produced a highly virulent virus, while a reciprocal recombinant was asymptomatic. In addition to several amino acid mutations in the nonstructural region, the nsp3 gene of rA774 displayed an opal termination codon and an in-frame 21-nucleotide deletion close to the nsp4 junction. Replacement in rA774 of the entire nsp3 gene with that of SFV4 reconstituted the virulent phenotype, whereas an arginine at the opal position significantly increased virulence, leading to clinical symptoms in mice. Completion of the nsp3 deletion in rA774 did not increase virulence. We conclude that the opal codon and amino acid mutations other than the deleted residues are mainly responsible for the attenuation of A7(74) and that the attenuating determinants reside entirely in the nonstructural region.

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.