Application of capsular sequence typing (CST) to serotype non-viable Streptococcus pneumoniae isolates from an old collection.

Serotyping of Streptococcus pneumoniae is essential for monitoring changes in the pneumococcal population and the impact of vaccines. Recently, various DNA-based methods have become available and are increasingly used because they are cheaper and easier to perform than the Quellung reaction. Our aim was to apply a DNA-based method, capsular sequence typing (CST), to a collection of non-viable lyophilized pneumococcal isolates dating from the 1980s to elucidate the serotypes circulating in Italy 30 years ago. As a preliminary evaluation of the method, CST was applied to 68 recent pneumococcal isolates representative of the most common serotypes circulating in Italy in invasive pneumococcal disease (IPD) previously serotyped by the Quellung reaction. CST was then applied to 132 lyophilized non-viable isolates. A serotype-specific polymerase chain reaction (PCR), using primers suggested by the Centers for Disease Control and Prevention (CDC), was performed when CST did not yield a univocal serotype. Considering the control isolates, CST concordance with the Quellung reaction was 95.6 %. For the non-viable lyophilized isolates, CST identified a univocal serotype for 59.4 % of the isolates. This percentage increased to 78.1 % if CST was combined with serotype-specific PCR. The most frequent serotypes in the collection of non-viable strains were: 3 (15.6 %), 14 (11.7 %), 35B (5.5 %), 19A (5.5 %), and 8 (4.7 %). CST proved to be a valid method for serotyping pneumococcal strains and provided information about pneumococcal serotypes present in Italy 30 years ago. The combination of CST with serotype-specific PCR was an effective strategy to identify pneumococcal serotypes that can be suggested also for routine laboratories.

Present status of accelerator-based BNCT: Focus on developments in Argentina.

In this work we provide some information on the present status of accelerator-based BNCT (AB-BNCT) worldwide and subsequently concentrate on the recent accelerator technology developments in Argentina.

Typing of Panton-Valentine leukocidin-encoding phages carried by methicillin-susceptible and methicillin-resistant Staphylococcus aureus from Italy.

Panton-Valentine leukocidin (PVL) is the hallmark of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) but can also be found in methicillin-susceptible S. aureus (MSSA) sharing pathogenic and epidemiological characteristics of CA-MRSA. PVL is encoded by two co-transcribed genes that are carried by different staphylococcal bacteriophages. We applied an extended PCR-based typing scheme for the identification of two morphological groups (elongated-head group and icosahedral-head group I phages) and specific PVL phage types in S. aureus isolates recovered in Italy. We examined 48 PVL-positive isolates (25 MSSA and 23 MRSA) collected from different hospital laboratories from April 2005 to May 2011. spa typing, multilocus sequence typing and staphylococcal cassette chromosome mec typing were applied to categorize the isolates. Phage typeability was 48.0% in MSSA and 91.3% in MRSA, highlighting the limitation of the PCR typing scheme when applied to PVL-positive MSSA. Five different PVL phages and two variants of a known phage were detected, the most prevalent being ΦSa2usa, recovered in 15 out of 48 (31.2%) isolates, and carried by both MSSA and MRSA belonging to CC8 and CC5. The recently described ΦTCH60 was recovered in four isolates. A PVL phage (ΦSa119) from an ST772 MRSA, that was not detected using the previous typing scheme, was sequenced, and new primers were designed for the identification of the icosahedral-head group II PVL phages present in ST772 and ST59 MRSA. A comprehensive PVL-phage typing can contribute to the understanding of the epidemiology and evolution of PVL-positive MSSA and MRSA.

Accelerator-based BNCT.

The activity in accelerator development for accelerator-based BNCT (AB-BNCT) both worldwide and in Argentina is described. Projects in Russia, UK, Italy, Japan, Israel, and Argentina to develop AB-BNCT around different types of accelerators are briefly presented. In particular, the present status and recent progress of the Argentine project will be reviewed. The topics will cover: intense ion sources, accelerator tubes, transport of intense beams, beam diagnostics, the (9)Be(d,n) reaction as a possible neutron source, Beam Shaping Assemblies (BSA), a treatment room, and treatment planning in realistic cases.

Characterization of macrolide efflux pump mef subclasses detected in clinical isolates of Streptococcus pyogenes isolated between 1999 and 2005.

The macrolide efflux mechanism of resistance, mef, was characterized in community-acquired respiratory tract infections with Streptococcus pyogenes. Fifty-four (4.6%) M phenotype isolates were screen tested as negative for mef(A). Of these 54 isolates, 5 (0.4%), 27 (2.3%), and 1 (0.1%) were considered to be mef(I) positive, a novel mosaic variant of mef, or a novel subclass of mef, respectively. This study shows (i) the definitive presence of mef(E) in S. pyogenes and its global distribution, (ii) the presence of a mosaic variant of mef composed of mef(A) and mef(E), (iii) the previously undescribed presence of mef(I) in S. pyogenes, and (iv) the presence of a novel subclass of mef in S. pyogenes.

[Antibiotic-resistance in Italy: activity of the first year of the surveillance project AR-ISS]. / Antibiotico-resistenza in Italia: un anno di attività del progetto di sorveglianza AR-ISS.

The antibiotic resistance surveillance project AR-ISS, started in 2001, is based on a network of 62 sentinel microbiological laboratories throughout the country. The laboratories collect and transmit data to the Istituto Superiore di Sanità on the antibiotic susceptibility of bloodstream isolates of 7 species: Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis/faecium, Klebsiella pneumoniae/oxytoca ed Escherichia coli. They also send selected bacterial strains for further characterization. Results of the first year of surveillance are presented and are compared with data from the previous study EARSS-Italia and from other European countries. Oxacillin resistance in S. aureus appears to be stable, however, it remains one of the highest in Europe (41,5%). No strain with intermediate susceptibility or resistance to vancomycin has been isolated. In S. pneumoniae, the level of penicillin resistance is moderate (10,8%), but macrolide resistance has increased greatly (37,6% versus 28,6% of the previous study), following a tendency common to several European countries. Unexpectedly, vancomycin resistance in E. faecium was found to be 18%, the highest in Europe. Presumptive ESBL production in Gram-negative organisms can be estimated at 20% in Klebsiella and 1% in E. coli. Ampicillin and ciprofloxacin resistance in E. coli (respectively 50% and 18%) are among the highest in Europe. In conclusion, the rate of antibiotic resistance in the species studied is worrisome and requires continuing monitoring. Although some activities of AR-ISS need improvements, the surveillance has the potentiality to produce relevant and representative data about antibiotic resistance in Italy that can be used for comparison at the European level.

Prevalence, determinants, and molecular epidemiology of Streptococcus pneumoniae isolates colonizing the nasopharynx of healthy children in Rome.

The aim of this study was to determine the factors favouring Streptococcus pneumoniae nasopharyngeal colonization of healthy children attending daycare centres and to describe the circulation of penicillin-nonsusceptible strains using molecular techniques. A single nasopharyngeal swab was obtained from 610 children attending daycare centres in the southeast area of Rome. Streptococcus pneumoniae isolates were serotyped, and antibiotic susceptibility was assayed by the E test. The genetic determinants of erythromycin resistance were detected by a duplex polymerase chain reaction, and the penicillin-nonsusceptible isolates were typed by pulsed-field gel electrophoresis. The overall carriage rate of Streptococcus pneumoniae was 14.9%. Living with more than three persons in the same household was the only risk factor statistically associated with carriage. Sixteen of 85 (18.8%) strains were nonsusceptible to penicillin, and 44 (52%) were resistant to erythromycin. Of the erythromycin-resistant strains, the vast majority showed a high level of resistance and carried the erm(B) gene. The penicillin-nonsusceptible strains belonged to six different serotypes; molecular typing showed that in only one case (2 strains) was there a circulation of the same clone in the same daycare centre. In view of the high rate of resistant Streptococcus pneumoniae strains, risk factors for carriage of resistant strains were evaluated. Children who received macrolides in the previous month had a higher risk of being colonized by macrolide-resistant strains as well as by strains resistant to both penicillin and erythromycin. Limiting the use of antibiotics in children seems the most appropriate measure to control the spread of antibiotic-resistant strains.

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.

Activity of quinupristin-dalfopristin in invasive isolates of Streptococcus pneumoniae from Italy.

Eighty-five recent isolates of Streptococcus pneumoniae from patients with invasive disease were examined for their susceptibility to erythromycin, clindamycin, penicillin and quinupristin-dalfopristin by E test. A novel duplex PCR assay was used to detect the presence of the erm(B) or mef(A) genes in all of the erythromycin-resistant isolates. All of the strains tested were susceptible to the combination quinupristin-dalfopristin, regardless of their susceptibility to penicillin or to erythromycin. By duplex PCR, two-thirds of the erythromycin-resistant strains harbored erm, and one-third harbored mef. The activity of quinupristin-dalfopristin was not influenced by the genetic determinant of erythromycin resistance. The in vitro susceptibility of S. pneumoniae to quinupristin-dalfopristin is promising for future use; however, it is important to monitor the possible emergence of resistance.

Prosthetic biologic valve endocarditis caused by a vancomycin-resistant (vanA) Enterococcus faecalis: case report.

We recently observed (February 1999) a 68-year old patient with endocarditis on a prosthetic biologic valve caused by a vancomycin-resistant Enterococcus faecalis. Broth dilution tests showed susceptibility to ampicillin (MIC=0.5 microg/ml), no high resistance to aminoglycosides (MIC for gentamicin <500 microg/ml) and resistance to vancomycin (MIC >256 microg/ml) and teicoplanin (MIC >16 microg/ml). A PCR assay detected vanA gene in this strain. A transthoracic echocardiogram did not show valvular vegetations. A possible endocarditis was diagnosed and the patient received ampicillin for 8 weeks and gentamicin for 6 weeks. The patient remained afebrile after a 4-month follow-up when he underwent surgical replacement of the dysfunctional bioprosthetic valve. Mitral valve was sterile on culture, but histology confirmed the diagnosis of previous endocarditis. This is the third case of endocarditis caused by vancomycin-resistant E. faecalis reported to date.

The alleles of the bft gene are distributed differently among enterotoxigenic Bacteroides fragilis strains from human sources and can be present in double copies.

Enterotoxigenic Bacteroides fragilis (ETBF) strains are associated with diarrheal disease in children. These strains produce a zinc metalloprotease enterotoxin, or fragilysin, that can be detected by a cytotoxicity assay with HT-29 cells. Recently, three different isoforms or variants of the enterotoxin gene, designated bft-1, bft-2, and bft-3, have been identified and sequenced. We used restriction fragment length polymorphism analysis of the PCR-amplified enterotoxin gene to detect the isoforms bft-1 and bft-2 or bft-3 borne by ETBF. By sequencing the portion of the bft gene corresponding to the mature toxin in some strains and applying allele-specific PCR for strains categorized as bft-2 or bft-3, we found in our collection two strains harboring bft-3, a variant that had been described for isolates from East Asia. Analysis of 66 ETBF strains from different sources showed that bft-1 is the most frequent allele, being present in 65% of isolates; it is largely predominant in isolates from feces of adults, while bft-2 is present in isolates from feces of children. This association is statistically significant (P, 0.0064). Sixteen strains were examined by Southern hybridization using, as probes, the bft and second metalloprotease genes, both included in a pathogenicity islet. Five strains were found to harbor double copies of both genes, suggesting that the whole islet was duplicated. Four of these strains, harboring bft-1 (three strains) or bft-2 (one strain), were found to produce a large amount of biologically active toxin, as determined by a cytotoxicity assay with HT-29 cells. The strains harboring bft-3, either in a single copy or in double copies, produced the smallest amount of toxin in our collection.

Detection and characterization of vancomycin-resistant enterococci in farm animals and raw meat products in Italy.

The emergence of vancomycin-resistant enterococci (VRE) in Europe has been ascribed to the long-time use of the glycopeptide antibiotic avoparcin as feed additive in food animals, until its ban in 1997 in EU. The pres- ence of VRE in food of animal origin is believed to represent a potential risk for the consumer. We studied the fecal carriage of VRE in broiler chickens and slaughter pigs in Italy before the avoparcin ban and eval- uated the impact of avoparcin withdrawal on the presence of VRE in raw meat products. Broilers and pigs were both found to be frequently colonized by VRE, as 36% and 24.6% of the flocks or the herds, respec- tively, were positive. Molecular typing of VRE strains by PFGE showed that animals housed in different pens within the same farm were colonized by clonally related strains. After the avoparcin ban, a decrease in the rate of VRE contamination in meat products was observed. Such a decrease was statistically significant in poultry (from 18.8% to 9.6%) but not in pork products (from 9.7% to 6.9%). The majority of VRE from all sources carried the vanA resistance gene and included Enterococcus faecium, E. faecalis, E. hirae, E. durans, and E. gallinarum. None of the strains carried the vanB gene, whereas constitutively resistant vanC-positive strains were frequently found. Our results show that avoparcin withdrawal has been successful in reducing VRE contamination in meat products. However, this measure needs to be complemented by a prudent use of glycopeptide antibiotics in human medicine.

Decrease of vancomycin-resistant enterococci in poultry meat after avoparcin ban.

In Italy, 18 months after the ban of avoparcin, the percentage of poultry meat samples containing vanA gene-positive vancomycin-resistant enterococci fell from 14.6% to 8%.

Genetic tests to reveal TAT homodimer formation and select TAT homodimer inhibitor.

The HIV Tat protein is essential for productive infection and is a potent activator of viral gene expression. By constructing a genetic fusion between the amino-terminal DNA-binding domain of the lambda repressor (as a reporter for dimerization) and Tat, we show that Tat forms dimers in vivo. By deletion analysis and site-directed mutagenesis, we show that (i) the peptide encoded by exon-1 of Tat is sufficient to promote dimerization and (ii) cys37 is essential for homo-dimerization of Tat protein. Furthermore, by using a new E. coli strain in which the expression of beta-galactosidase is under the negative control of the cl::Tat repressor, we select a protein (CD10/Nep) expressed by human Jurkat T-cells which inhibits Tat dimerization.

pR plasmid replication provides evidence that single-stranded DNA induces the SOS system in vivo.

Evidence is presented that the pR bat gene is essential for plasmid replication and for spontaneous induction of the SOS response in Escherichia coli. Mutations preventing single-stranded DNA production, needed for pR plasmid replication, also prevent the induction of the SOS system. The following experimental design was used. Firstly, we identified the minima rep region, defined as the minimal DNA sequence necessary for pR plasmid replication and, secondly, analyzed the nucleotide sequence of this region. This identified structures and functions (ori-plus, ori-minus and Rep protein) homologous to those found in phages and plasmids replicating by the rolling-circle mechanism. Finally, mutations were introduced either in the replication protein catalytic site or in the nick site consensus sequence, which caused the pR plasmid to lose its ability to induce the SOS system. We conclude that, in this system, the in vivo SOS-inducing signal appears to be the single-stranded DNA produced during pR replication.