Molecular epidemiology of Enterococcus faecium isolates from an Italian hospital.

BACKGROUND: Outbreaks of vancomycin-resistant Enterococcus faecium (VRE) strains is an emerging problem worldwide. Even if still relatively uncommon in European hospitals, infections caused by VRE have also been increasing recently in this continent. METHODS: In this study, we characterized 50 consecutive VRE and 23 vancomycin-sensitive E. faecium (VSE) isolates collected in an Italian hospital. The presence of the esp gene and that of genes encoding resistance to glycopeptides was investigated by polymerase chain reaction (PCR). All of the isolates were typed by multi-locus sequence typing (MLST), and a selection of them also by pulsed-field gel electrophoresis (PFGE). RESULTS: We found that all of the VRE and 18 (78%) of the VSE strains belonged to the single clonal complex-17 (CC17). The most represented sequence type (ST) was ST78 (34% of the isolates). When further analyzed by PFGE, ST78 isolates were subdivided into five pulsotypes, four of them closely related. The strong association between the esp gene and CC17 was confirmed. Interestingly, such an association was higher among vancomycin-resistant isolates. Most of the esp-positive isolates (34/46, 74%) encoded Esp4, a rare variant of this protein characterized by the absence of A repeats. CONCLUSIONS: Our findings underscore the role of the CC17 lineage in the nosocomial spread of VRE and VSE, and its rapid local evolution, underscoring the need for programs designed to provide early detection in order to prevent its spreading among the nosocomial population.

The Mycobacterium tuberculosis sigma factor sigmaB is required for full response to cell envelope stress and hypoxia in vitro, but it is dispensable for in vivo growth.

The numerous sigma (sigma) factors present in Mycobacterium tuberculosis are indicative of the adaptability of this pathogen to different environmental conditions. In this report, we describe the M. tuberculosis sigma(B) regulon and the phenotypes of an M. tuberculosis sigB mutant strain exposed to cell envelope stress, oxidative stress, and hypoxia. The sigB mutant was especially defective in survival under hypoxic conditions in vitro, but it was not attenuated for growth in THP-1 cells or during mouse and guinea pig infection.

Assessment of the lightcycler PCR assay for diagnosis of invasive aspergillosis in paediatric patients with onco-haematological diseases.

A reliable diagnosis of invasive aspergillosis (IA) is hampered by the difficulty in obtaining suitable tissue samples. To evaluate the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the LightCycler PCR for the diagnosis of IA, 536 blood samples were collected over a 22-month period from 62 paediatric patients (median age 10 years, range 1-18) considered at risk of IA. The galactomannan antigen (GM) and fungal DNA were assessed on serial blood samples. IA was diagnosed in eight of 62 patients (13%): proven, five, probable, three. Sensitivity, specificity, PPV and NPV of LightCycler PCR varied according to the number of positive samples used to define positivity: 88%; 37%; 17% and 95% for single sample positivity; and 63%, 81%, 33% and 94% for serial sample positivity respectively. The concordance between positivity of LightCycler PCR assay and the diagnosis of IA was 79%. The single positivity of LightCycler PCR assay showed a good sensitivity for the diagnosis of IA in paediatric patients. The high NPV makes LightCycler PCR a promising tool in addition to GM testing to design a strategy of pre-emptive antifungal therapy, although further validation studies are needed.

[Diagnostic pathway in a case with severe degree of hypertension]. / Iter diagnostico di un caso di ipertensione arteriosa di grado severo.

A 29- year-old male was admitted because of exertion dyspnea and intense headache. These symptoms were associated with severe hypertension, small multiple areas of cerebral ischemia, thrombocytopenia, prolonged aPTT and renal failure. The diagnostic tests performed during hospitalization resulted in a diagnosis of Primary Antiphospholipids Syndrome. The renal biopsy sample suggested histopathological features of uncommon simultaneous occurrence of antiphospholipids nephropathy and a "collapsing variant" of segmental focal glomerulosclerosis. It is fundamental to be aware that this syndrome is very likely to occur, and therefore to perform antiphospholipids antibodies assessment, since only an anticoagulant therapy proves effective; nevertheless, in view of the pathological renal findings, other therapies such as steroids might be added.

Antiviral activity in vitro of Urtica dioica L., Parietaria diffusa M. et K. and Sambucus nigra L.

Parietaria diffusa M. et K., Urtica dioica L. (Urticaceae) and Sambucus nigra L. (Caprifoliaceae) are plants usually used in popular medicine of central Italy for treating numerous diseases, first of all Herpes zoster. Several plant products have been described as potential antiviral agents, with special attention being devoted to those having retroviruses as etiological agents, including acquired immunodeficiency syndrome (AIDS), in which a retrovirus, the designated human immunodeficiency virus HIV, has been clearly identified as the primary cause of this disease. The present study proposes a preliminary screening of the antiviral activity of Parietaria diffusa, Sambucus nigra and Urtica dioica preparation against the feline immunodeficiency virus (FIV) infection. The feline immunodeficiency virus is a widespread lentivirus of domestic cats sharing numerous biological and pathogenic features with the human immunodeficiency virus (HIV). FIV infection in cats has therefore been proposed as an animal model for AIDS studies with respect to pathogenesis, chemotherapy, and vaccine development [Pedersen, N.C., 1993. Feline immunodeficiency virus infection. In: Levy, J.A. (Ed.), The Retroviridae. Plenum Press, New York; Bendinelli, M., Pistello, M., Lombardi, S., Poli, A., Garzelli, C., Matteucci, D., Ceccherini-Nelli, L., Malvaldi, G., Tozzini, F., 1995. Feline immunodeficiency virus: an interesting model for AIDS studies and an important cat pathogen. Clinical Microbiology Revue 8, 87-112; North, T.W., LaCasse, R.A., 1995. Testing anti-HIV drugs in the FIV model. Nature Medicine 1, 410-411; Matteucci, D., Pistello, M., Mazzetti, P., Giannechini, S., Isola, P., Merico, A., Zaccaro, L., Rizzati, A., Bendinelli, M., 2000. AIDS vaccination studies using feline immunodeficiency virus as a model: immunisation with inactivated whole virus suppresses viraemia levels following intravaginal challenge with infected cells but non-following intravenous challenge with cell-free virus. Vaccine 18, 119-130]. Early studies showed that some of them presented antiviral activity against infection of FIV as assayed by syncytia formation using feline kidney Crandell cells (CrFK).

Expression of M6 protein gene of Streptococcus pyogenes in Streptococcus gordonii after chromosomal integration and transcriptional fusion.

The M6 protein of Streptococcus pyogenes was expressed on the cell surface and secreted in Streptococcus gordonii Challis (formerly Streptococcus sanguis) after chromosomal integration of a promoterless M6 protein gene (emm-6.1). The ermC gene, conferring resistance to erythromycin, was cloned downstream of emm-6.1, within the same ClaI fragment. The initiation codon of emm-6.1 was 19 bp downstream of a ClaI site, so that ClaI cleavage would leave the gene promoterless. The ClaI fragment containing the promoterless emm-6.1 and ermC was ligated in vitro with a ClaI digest of S. gordonii chromosomal DNA. Random chromosomal integration of the heterologous DNA was obtained by using the ligation mixture to transform the naturally competent S. gordonii Challis. Twenty-eight percent of transformants selected for erythromycin resistance also expressed M6. Among the best M6 producers, 10 clones were selected for the stability of their phenotype. Nine of the 10 clones were shown to harbour one intact copy of the emm-6.1/ermC ClaI fragment integrated into the chromosome. These strains both expressed M6 protein on the surface and secreted different amounts of the molecule, since in each case the protein was produced after a transcriptional fusion of emm-6.1 with a different chromosomal promoter. A S. gordonii strain expressing large amounts of surface M6 protein, as judged by immunofluorescence and Western blot, was compared to the M- parental strain in a standard opsonophagocytosis assay. Of the isogenic pair, M6+ S. gordonii survived better in human blood and was phagocytosed at a slower rate.

Method and parameters for genetic transformation of Streptococcus sanguis Challis.

A simple procedure for genetic transformation of Streptococcus sanguis Challis was developed and standardized. During the exponential phase of growth, cells became competent while growing as diplococci in broth containing 10% foetal calf serum. High levels of competence were maintained by the cultures for 60 min. Competent cells could be stored frozen without loss of competence for at least three years. Using total chromosomal DNA as donor, the dose-response curve for transformation of a point mutation (streptomycin resistance) showed one-hit kinetics, as the DNA concentration varied from 0.000001 to 10 micrograms/ml. At 10 micrograms/ml, more than 2.2% of the colony-forming units were transformed to streptomycin resistance, while transforming activity remained detectable with 1 pg of DNA/ml. Optimal time of exposure of competent cells to transforming DNA was 30 min. The transformation reaction was inhibited at 0 and 4 degrees C, whereas it occurred efficiently both at 25 and 37 degrees C.

Characterization of conjugative transposon Tn5251 of Streptococcus pneumoniae.

Tn5251 belongs to the Tn916-Tn1545 family of conjugative transposons (CT) and was found integrated into CT Tn5252, to form the composite element Tn5253 of Streptococcus pneumoniae. We show that Tn5251 is identical in structure and size to Tn916. DNA sequence analysis of a 4,419-bp segment containing the tet(M) gene showed that only 73 nucleotides out of 4,419 were different in the two CT. Essentially all differences (66/73) were clustered in a 688-bp segment of tet(M), which was 90% identical to Tn916 and 100% identical to the tet(M) genes of Tn1545 from S. pneumoniae and pOZ101 from Neisseria gonorrhoeae. DNA sequence analysis of the Tn5251/Tn5252 junction fragments allowed us (i) to determine Tn5251 termini, (ii) to define the 6-bp coupling sequences flanking the CT, and (iii) to infer the structure of the integration site (attB) of Tn5251 into Tn5252. Conjugal transfer of Tn5251 independent from Tn5253 could not be detected, even if we could show excision and formation of Tn5251 circular intermediates at a level of 5.4 copies per 10(6) chromosomes.

Insertion vectors for construction of recombinant conjugative transposons in Bacillus subtilis and Enterococcus faecalis.

The broad-host range of conjugal transfer and the chromosomal location make conjugative transposons (CT) attractive candidates as tools for genetic manipulation of a large variety of bacteria. In this paper we describe insertion vectors capable of integrating into Tn916, the prototype of CT in Gram-positive bacteria. The integration of vectors into a single chromosomal copy of Tn916 was studied both after natural transformation of Bacillus subtilis, and after electroporation in Enterococcus faecalis. Integration occurred either by double or by single crossover, and the integrated DNA segment was shown to be highly stable. All recombinant CT (rCT) were still able to excise from the chromosome to form circular intermediates, the first step of both transposition and conjugal transfer. All classes of rCT generated by insertion vector pSMB47 were capable of conjugal transfer, while using pVMB11 it was possible to generate non-conjugative rCT.

Electrotransformation of Streptococcus agalactiae with plasmid DNA.

A protocol for efficient electrotransformation of Streptococcus agalactiae (group B streptococcus) Lancefield's strain O90R (NTCT 9993) (an unencapsulated derivative of type Ia strain O90) was developed. The Escherichia coli-Streptococcus shuttle vector pDP28 (7.8 kb) carrying the ermB gene for resistance to erythromycin was used as donor DNA. Frozen 'electrocompetent' cells were prepared by repeated washes in 10% glycerol. A 50-microliters aliquot containing about 5 x 10(9) colony forming units of bacteria was subjected to the electric pulse. Optimal conditions for electrotransformation were determined using different media, harvesting cells at different points of the growth curve, and using different field strengths. The dose-response curve for transformation of S. agalactiae with pDP28 showed one-hit kinetics as donor DNA varied between 0.01 and 3 micrograms. The efficiency of electrotransformation for this range of amounts of donor DNA was 1.2 x 10(4) cfu micrograms-1. The transformation frequencies obtained with this electroporation protocol are high enough to allow both subcloning and shotgun cloning of streptococcal DNA in S. agalactiae.

Real Time PCR Using Molecular Beacons : A New Tool to Identify Point Mutations and to Analyze Gene Expression in Mycobacterium tuberculosis.

Molecular beacons are a novel family of hybridization probes, which emit fluorescence upon interaction with their target. They are hairpin-shaped oligonucleotides with a central part complementary to the target, flanked by two 5 6 base pair (bp) inverted repeats, which can form a stable stem. A fluorescent moiety is covalently linked to the 5' end of the molecule, whereas the quenching moiety, 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL), is covalently linked to the 3' end. The stem keeps the two moieties in close proximity to each other, causing the fluorescence of the fluorophore to be quenched by energy transfer. When molecular beacons bind to their target, they undergo a conformational change that results in the restoration of fluorescence of the internally quenched fluorophore (1) (Fig. 1). Molecular beacons are extremely specific, and can clearly discriminate between targets differing only by a single nucleotide (2,3). When present in a PCR reaction where their target is the amplification product, molecular beacons can form a stable hybrid with the amplicon during the annealing step. The intensity of fluorescence at the annealing step in each amplification cycle is a direct measure of amplicon concentration (2,4) (Fig. 2). Another interesting feature of molecular beacons is that they can be coupled to a variety of differently colored fluorophores. This allows multiplex PCR reactions where different DNA fragments can be amplified and detected simultaneously in the same tube (2,3). Fig. 1. Operation of molecular beacons. On their own, these molecules are nonfluorescent, because the stem hybrid keeps the fluorophore (◯) close to the quencher (•). When the probe sequence in the loop hybridizes to its target, forming a rigid double helix, a conformational reorganization occurs that separates the quencher from the fluorophore, restoring fluorescence (1). Fig. 2. Real time measurement of amplicon synthesis during PCR using molecular beacons. (A) Four PCR reactions were initiated with a different number of template molecules (indicated). The concentration of amplicons present after each cycle of amplification was determined by measuring fluorescence during the last few seconds of the annealing step. (B) Inverted relationship between the threshold cycle (the cycle at which the fluorescent signal becomes detectable above the background) and the logarithm of the initial number of template molecules. In this example, the target is M. tuberculosis H37Rv chromosomal DNA. The primers-molecular beacon set used in the reaction was specific for sigA (reprinted from ref. 4).

Ethnopharmacobotanical studies of the Tuscan Archipelago.

From an ethno-pharmacobotanical point of view, Tuscany is a region with very rich and interesting traditions. The Tuscan Archipelago, particularly due to its geographical position and its history, presents a large variety of plant species used in popular medicine in numerous pathologies, including several viral infections. Over 100 species of plants are used in popular medicine in this region.

Curing animals with plants: traditional usage in Tuscany (Italy).

Tuscany is an area rich in traditions, many of an ethnobotanical nature, and those of veterinary practice are of special interest. Almost a 100 different plant species are used to treat animals; sometimes old remedies are used to cure similar human ailments, other times the cure is used exclusively for veterinary treatment.

Ethnopharmacobotany in Tuscany: plants used as antihypertensives.

Among the more than 400 plants used in popular medicine in Tuscany, over 30 are used in the therapy of hypertension. For some of them their use is already known while for others there is no documentation. In this work we present the first results obtained from research carried out on antihypertensive plants belonging to Gentianaceae and in particular Gentiana kokiana.

An ethno-pharmacobotanical survey in the Sarrabus district (south-east Sardinia).

The therapeutic uses and methods of administration of 70 plants in the traditional medicine of Sarrabus (south-east Sardinia, Italy) are documented. Among these species, some were not reported previously for Sardinia, while others turn out to have an original therapeutic use.

[Renal biopsy: outpatient procedure?]. / Biopsia renale: possibile procedura ambulatoriale?

BACKGROUND: The renal biopsy is usually performed as an in-patient procedure, with patients admitted to hospital for at least 24 hours. We have carried out renal biopsies on two groups of patients. In the first group, patients rest in the hospital for 8 hours following the procedure. They are discharged after undergoing ultrasonography and a TC scan. These patients return to the hospital after 24 hours to verify possible post-biopsy complications. In the comparison group, patients remain in hospital for 24 hours. RESULTS: In both groups, the only observed complication was asymptomatic postbiopsy hematoma. No major complications were present in either group. CONCLUSIONS: In selected cases, renal biopsy performed by an expert practitioner as an outpatient procedure is safe and does not require 24-hour observation.

[Aspects of the on-line hemodiafiltration with regeneration and reinfusion of the ultrafiltrate (HFR): multicenter study]. / Aspetti dell'emodiafiltrazione on-line con rigenerazione e reinfusione dell'ultrafiltrato (HFR): uno studio multicentrico.

Despite technological advances in dialysis treatment, survival, morbidity and the quality of life in hemodialysis (HD) patients are affected by long-term complications, often related to the treatment itself. Among these complications, moderate protein and caloric malnutrition are present in approximately 30% of dialysis patients and are viewed as major contributors to increased mortality. In malnutrition pathogenesis, great importance is given to protein catabolism and to the loss of somatic protein and amino acids during dialysis. On the contrary, toxin clearance is believed to influence, positively, both protein anabolism and dietary protein intake. In hemodiafiltration (HDF), the clearance process is potentiated by three mechanisms (diffusion, convection and adsorption) and this could have a favorable effect on malnutrition. In addition, the reinfusion of regenerated ultrafiltrate (UF) would avoid the loss of large amounts of useful solutes as occurs with standard HD. In fact, all amino acids are present in the UF, which is not important in standard HD, but could be a problem in hemodiafiltration reinfusion (HFR). We treated 16 patients with HFR during the previous 3 months (the study will last for 12 months). Patients had been previously treated with bicarbonate dialysis for at least 6 months. The clinical tolerance of HFR was excellent and the technique appeared to be quite simple. The preliminary biochemical results demonstrated the stabilization of some parameters (such as urea and uric acid) with an adequate clearance of small molecules, while variables related to nutritional status (body weight, serum albumin and serum transferrin) did not change substantially. Surprisingly, the loss of both branched chain amino acids (BCAA) and essential amino acids (EAA) seemed slightly lower in HFR compared with standard HD. However, the reduced loss of amino acids (AA) observed with HFR should take into account other factors, such as absorption on adsorbent material and the basal plasma AA concentrations. Therefore, although each patient is in control of himself, it is difficult to draw any definite conclusions after only 3 months. However, it is evident that the loss of AA in HFR is quite modest and is not increased by the fact that it is a hemofiltration technique with all the consequent positive effects.

Critical research concepts in tuberculosis vaccine development.

A new and improved vaccine against tuberculosis (TB) would provide a powerful tool to conquer one of the most insidious infectious diseases of mankind. Protection afforded by bacillus Calmette-Guérin (BCG) has been shown to be limited and inconsistent, especially in adults that are known to transmit TB disease. In the last two decades, several new vaccines have been developed and tested with the aim to elicit robust and long-lived T-cell responses against Mycobacterium tuberculosis antigens. Although much progress has been made in the TB vaccine field, there is an urgent need to address critical research questions about TB immunity with a special focus on designing vaccines aimed at preventing infection and transmission of TB. Here, we discuss the rationale behind the current immunization strategies being implemented for TB vaccines and provide some suggestions for hypothesis driven research to encourage the development of novel TB vaccines.

Engineered E. coli delivers therapeutic genes to the colonic mucosa.

Taking advantage of the proximity of bowel mucosa to luminal bacteria, we have attempted to deliver a therapeutic gene to the colonic mucosa by oral administration of an invasive and non-pathogenic Escherichia coli. E. coli diamenopimelate (dap) auxotroph, harboring plasmid pGB2Omegainv-hly, express the inv gene from Yersinia pseudotubercolosis that confers the ability to invade nonprofessional phagocytic cells and the hly gene from Listeria monocytogenes that allows expression of lystreriolysin O, a perforin cytolysin able to perfore phagosomal membranes. This bacterial vector invades and transfers functional DNA to epithelial cells in vitro. We have shown that this strain carrying a therapeutic gene (pC1OmegaTGF-beta1) can significantly reduce the severity of experimental colitis in mice. However, as a consequence of mucosal barrier disruption during colitis, vector-specific mRNA transcripts could be recovered from the colon and also from extra-colonic tissues. We therefore replaced the constitutive CMV promoter in pC1OmegaTGF-beta1 by the inflammation-inducible interleukin-8 promoter generating plasmid pC1OmegaTGF-beta1IND. Plasmid-specific TGF-beta1 mRNA transcripts were detectable in mouse CMT-93 epithelial cells incubated with E. coli BM2710/pGB2Omegainv-hly carrying pC1OmegaTGF-beta1IND following exposure to inflammatory cytokines. Furthermore, the transcripts were detectable only within inflamed tissues and the therapeutic effects were comparable to those in animals treated with E. coli BM2710/pGB2Omegainv-hly+pC1OmegaTGF-beta1. In summary, engineered enteric bacteria can efficiently deliver in vivo therapeutic genes to the intact intestinal mucosa and regulation expression of the therapeutic gene by an inflammation-inducible promoter prevents its dissemination during colitis.

The joint of Tn916 circular intermediates is a homoduplex in Enterococcus faecalis.

Tn916 is a 18-kb conjugative transposon originally identified in Enterococcus faecalis. The first step for Tn916 movement is its excision from the donor replicon with the formation of a nonreplicating covalently closed circular intermediate. Studies on formation of circular intermediates in Escherichia coli have shown that the joint between the Tn916 termini is a 6-bp heteroduplex formed by the two regions flanking the transposon before its excision (coupling sequences). In this work we studied the joint of Tn916 termini in circular intermediates formed in both E. coli and E. faecalis. Our strategy was to use direct sequencing of amplification products obtained from the joint region of single target molecules. In E. coli, 50% of circular intermediates contained a heteroduplex joint, while the remaining 50% displayed a homoduplex joint formed by one of the two coupling sequences. In E. faecalis, we could not demonstrate the presence of any heteroduplex joint. In this case 77.7% of the analyzed joints were formed by the left coupling sequence.