Enterococcus hirae ATCC 9790 produces a penicillin-binding protein (PBP5) of low penicillin affinity which under certain conditions can take over the functions of all the other PBPs. The 7.1-kb EcoRI fragment containing the pbp5 gene of this strain and of two mutants, of which one (E. hirae R40) overproduces PBP5 and the other (E. hirae Revl4) does not produce PBP5, was cloned in pUC18 and sequenced. In (4,7,12,23,30). These PBPs share several properties: they can be produced by the cells in large amounts, have similar molecular masses (about 75,000 Da), have an unusually low affinity for beta-lactam antibiotics, and play an important role in the mechanism of resistance to these antibiotics in enterococci and staphylococci.In enterococci, the low-affinity PBPs either are components of the normal PBP pattern or are generated by mutations in genes of the normal PBPs (1,12,21,32,33), whereas in staphylococci their synthesis is due to the acquisition of an additional gene (mec) which is practically identical in Staphylococcus aureus and several coagulase-negative species (22,27,29).In contrast to the evident and well-documented role of the low-affinity PBPs in the mechanism of resistance of grampositive cocci to beta-lactam antibiotics, very little is known about the role these proteins play in cell physiology. The most accepted hypothesis is that these proteins are multifunctional PBPs which are not essential for cell growth, at least under laboratory conditions, and can synthesize peptidoglycans under conditions which impair the activity of the other PBPs (4-6, 12, 13). Such an alternative system of peptidoglycan synthesis would represent an interesting and novel aspect of bacterial physiology.Previous studies of the low-affinity PBP of Enterococcus hirae ATCC 9790 (PBP5) based on mutants with a cell division defect and mutants with altered synthesis (either overproduction or lack) of PBP5 have elucidated certain aspects of the role of low-affinity PBPs in cell physiology (5, * Corresponding author. 6, 12, 13). Recently, the E. hirae PBP5-encoding gene (pbp5) has been cloned and sequenced (9), thus making possible investigations into the function of this PBP by a genetic approach.In this study, we sequenced the 7.1-kb EcoRI fragment, containing thepbp5 gene, cloned from chromosomal DNA of the wild-type E. hirae ATCC 9790, the PBP5 overproducer mutant E. hirae R40, and the PBP5 nonproducer mutant E. hirae Revl4. We found that the overproducer phenotype was associated with an 87-bp deletion in a genetic element, which we calledpsr (PBP5 synthesis repressor), located 1 kb upstream of the pbp5 gene, whereas the lack of PBP5 synthesis in E. hirae Revl4 was found to be caused by a nucleotide substitution in the coding frame of the protein which converted the 42nd codon, TCA, to the stop codon, TAA. MATERIALS AND METHODSBacterial strains and plasmids. The relevant properties of E. hirae strains and the recombinant plasmids constructed in this study are listed in Table 1. E. hirae strains were grown in brain heart infu...

Penicillin resistance development in enterococci has been associated with overproduction of a low-affinity penicillin-binding protein (PBP) that is a normal component of the PBP pattern of these bacteria and is apparently able to substitute the functions of the other PBPs. In resistant mutants of Enterococcus hirae ATCC 9790 the low-affinity PBP (PBP5) overproduction was associated with a deletion in a genetic element, located 1 kb upstream of the pbp5 gene, which negatively controlled PBP5 synthesis. Hypersusceptibility to penicillin was associated with a point mutation in the pbp5 gene, which causes premature termination of translation. Structural homologies between low-affinity PBPs of the different enterococcal species have been suggested by cross-reactivity of antibodies raised against E. hirae PBP5 with PBP5 of Enterococcus faecium and Enterococcus faecalis. Acquisition of a high-level ampicillin resistance in E. faecium was associated with overproduction of PBP5, which, compared with PBP5 of moderately resistant strains, appeared to be modified in its penicillin-binding capability. The modified phenotype of PBP5 was found to be associated to some amino acid substitutions in the region between the SDN and KTG motifs. In particular, the substitution converting a polar residue (T) in a nonpolar one (A or I) could play an important role in remodeling the penicillin-binding domain and determining the decrease in penicillin affinity.

Patients previously infected with hepatitis B virus (HBV) undergoing an allograft and recipients from HBV carrier donors are at risk of posttransplant viral reactivation. The role of prophylaxis with lamivudine remains unclear. One hundred seventeen patients, with a median age of 52 years (20-67 years), with various hematologic malignancies transplanted between 1999 and 2007 entered the study. Eighty-seven recipients negative for HBV surface antigen (HBsAg), antihepatitis B core antigen antibodies (anti-HBc), and HBV-DNA with HBsAg and HBV-DNA negative donors were defined as at low risk of HBV reactivation, whereas all the remaining 30 patients were defined as at high risk. Patients at high risk transplanted in 2005 or after received lamivudine to prevent HBV reactivation as per the Italian guidelines by the Associazione Italiana per lo Studio del Fegato (AISF). Patients at low risk did not experience HBV reactivation/hepatitis. Among the recipients at high risk, 11 of 25 anti-HBc positive, those HBsAg positive (2 of 2) or negative but transplanted from HBsAg positive donors (3 of 3) were treated with lamivudine. None of these developed HBV reactivation/hepatitis after a median follow-up of 40 months (17-55 months). Hepatitis developed in 3 anti-HBc positive untreated patients conditioned with a reduced-intensity regimen. Hepatitis B was not observed in recipients at low risk, transplanted from HBsAg negative/anti-HBc positive or negative donors. Lamivudine was effective in controlling reactivation in: HBsAg positive recipients, in patients transplanted from HBsAg positive donors and in HBsAg negative/antiHBc positive recipients, who showed a significant risk of reactivation if not given prophylaxis (NCT 00876148).

Hepatitis B virus (HBV) infection is a major cause of chronic liver disease. It is estimated that nearly 2 billion people are infected worldwide by HBV and that more than 350 million have persistent and chronic infection (38). HBV carriers have a high risk of developing long-term sequelae of hepatitis B, including cirrhosis and hepatocellular carcinoma that, in countries such as Italy with a moderate prevalence of HBV infection, account for nearly 25% of the indications for liver transplantation in reference centers (29). Recent advances in antiviral therapy, based on the development of new and more powerful nucleos(t)ide analogues, have dramatically improved chronic hepatitis B management, including the prevention of allograft reinfection in those patients undergoing liver transplantation for HBV-related disease.The success of antiviral therapy has been supported by the introduction of highly sensitive tests for monitoring HBV DNA. Molecular tests help to determine the activity of HBV infection, the selection of patients for treatment, and the efficacy of antiviral therapy, identifying the development of HBV drug-resistant strains (16,36,38).Several assays based on PCR are currently commercially available for HBV DNA. The recently introduced real-time PCR technique represents the method of choice compared to previous, conventional endpoint PCR due to a very sensitive quantification of the viral load over a wide dynamic range (7,12,15,18,20,21,24,31,32,35,41). At present, sample preparation is a major weakness in molecular tests, and improvements are constantly introduced to decrease the variability of the techniques and the risk of contamination, such as ready-to-use reagents and automation of the extraction procedure.A fully automated system, the COBAS AmpliPrep-COBAS TaqMan HBV test (CAP-CTM; Roche Molecular Systems, Inc., Branchburg, NJ) consisting of two integrated platformsthe COBAS AmpliPrep for automated nucleic acid extraction from plasma specimens and the COBAS TaqMan 48, a realtime PCR assay based on TaqMan technology-has recently been developed (13,35). Important improvements compared to other real-time PCR assays for HBV DNA are the incorporation of an internal quantitation standard to monitor the efficiency of the entire process and the introduction of a system to prevent carryover contamination. CAP-CTM is suitable for large routine series and has been demonstrated to equally quantitate HBV genotypes A through G (13, 35), but at present there are only few clinical data (13,28).In the present study, the performance of the CAP-CTM was evaluated and compared to the endpoint PCR assay COBAS AMPLICOR HBV monitor (CAHBM; Roche Molecular Systems). Analytical sensitivity and precision were assessed with an HBV proficiency panel, while correlation and differences in * Corresponding author. Mailing address: Laboratory of Microbiology, Molinette Hospital, Corso Bramante 88/90,

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