Molecular typing of vancomycin-resistant Enterococcus faecium with an automated repetitive sequence-based PCR microbial typing system compared with pulsed-field gel electrophoresis and multilocus sequence typing.

BACKGROUND: Pulsed-field gel electrophoresis (PFGE) is the main typing method used for the molecular typing of vancomycin-resistant Enterococcus faecium (VREfm). However, more rapid and unambiguous typing methods are needed. DiversiLab, a repetitive sequence-based PCR (rep-PCR), offers an alternative method for strain typing. METHODS: Thirty-nine VREfm isolates with known epidemiological relationships were characterized by semi-automated rep-PCR (DiversiLab), PFGE, and multilocus sequence typing (MLST). RESULTS: The DiversiLab results were analysed in 2 ways: first relying solely on the DiversiLab software, and second by DiversiLab analysis combined with manual interpretation. The analysis with interpretation yielded more DiversiLab profiles, correlated better with PFGE and MLST, and grouped the isolates better according to their relatedness in time and space. However, most of the DiversiLab groups also included isolates with different PFGE and MLST types. CONCLUSIONS: DiversiLab provides rapid information when investigating a potential hospital outbreak. However, the interpretation of E. faecium DiversiLab results cannot be fully automated and is not always straightforward. Other typing methods may be necessary to confirm the analysis.

Human beta1-adrenergic receptor is subject to constitutive and regulated N-terminal cleavage.

The beta(1)-adrenergic receptor (beta(1)AR) is the predominant betaAR in the heart, mediating the catecholamine-stimulated increase in cardiac rate and force of contraction. Regulation of this important G protein-coupled receptor is nevertheless poorly understood. We describe here the biosynthetic profile of the human beta(1)AR and reveal novel features relevant to its regulation using an inducible heterologous expression system in HEK293(i) cells. Metabolic pulse-chase labeling and cell surface biotinylation assays showed that the synthesized receptors are efficiently and rapidly transported to the cell surface. The N terminus of the mature receptor is extensively modified by sialylated mucin-type O-glycosylation in addition to one N-glycan attached to Asn(15). Furthermore, the N terminus was found to be subject to limited proteolysis, resulting in two membrane-bound C-terminal fragments. N-terminal sequencing of the fragments identified two cleavage sites between Arg(31) and Leu(32) and Pro(52) and Leu(53), which were confirmed by cleavage site and truncation mutants. Metalloproteinase inhibitors were able to inhibit the cleavage, suggesting that it is mediated by a matrix metalloproteinase or a disintegrin and metalloproteinase (ADAM) family member. Most importantly, the N-terminal cleavage was found to occur not only in vitro but also in vivo. Receptor activation mediated by the betaAR agonist isoproterenol enhanced the cleavage in a concentration- and time-dependent manner, and it was also enhanced by direct stimulation of protein kinase C and adenylyl cyclase. Mutation of the Arg(31)-Leu(32) cleavage site stabilized the mature receptor. We hypothesize that the N-terminal cleavage represents a novel regulatory mechanism of cell surface beta(1)ARs.

Cell-based beta2-adrenergic receptor-ligand binding assay using synthesized europium-labeled ligands and time-resolved fluorescence.

High-sensitivity, high-throughput, and user-friendly lanthanide-based assays for receptor-ligand interactions provide an attractive alternative to the traditional radioligand displacement assays. In this study, three small-molecule pindolol ligand derivatives were synthesized and their binding properties were tested in a radioligand displacement assay. The ligand derivatives were further labeled with fluorescent europium(III) chelate for beta(2)-adrenergic receptor-ligand binding assay. The europium-labeled pindolol ligands having no spacer (C0) or a 12-carbon spacer (C12) arm bound to the human beta(2)-adrenergic receptors overexpressed in human embryonic kidney HEK293(i) cells. Europium ligand with a 6-carbon spacer arm (C6) showed no binding. Competitive binding assays were developed with the functional labeled ligands. The IC(50) values for beta(2)-adrenergic antagonist propranolol were 60 and 37 nM, the Z' values were 0.51 and 0.77, and the signal-to-background ratios were 5.5 and 16.0 for C0 and C12, respectively. This study shows that functional time-resolved fluorescent assays can be constructed using fluorescent lanthanide chelates conjugated to small-molecule ligands.

Methicillin-sensitive Staphylococcus aureus bacteraemia and endocarditis among injection drug users and nonaddicts: host factors, microbiological and serological characteristics.

BACKGROUND: Endocarditis has been associated with lower mortality and fewer complications among injection drug users (IDUs) than nonaddicts in Staphylococcus aureus bacteraemia (SAB). The better prognosis of IDUs has not been clarified but it has generally been explained by younger age and other host factors. In this study, bacterial strains, their virulence factors, and host immune responses were compared among IDUs and nonaddicts with SAB, including those with and without endocarditis. METHODS: A total of 430 consecutive adult patients with methicillin-sensitive SAB were followed prospectively for 3 months. All 44 IDUs were included, and 44 nonaddicts as controls for them. According to the modified Duke criteria, 20 patients in both groups had endocarditis. For each addict without endocarditis, an age and sex matched nonaddict was selected as a control. S. aureus isolates were assigned a genotype by PFGE, Panton-Valentine leukocidin (PVL), staphylokinase (SAK), protease, and haemolysin production. Acute and convalescent sera were tested for antibodies to alpha-haemolysin (ASTA) and teichoic acid (TAA). RESULTS: There were no differences between IDUs and nonaddicts with SAB in the proportion of patients with a deep infection (98% vs 86%, P=0.06) or a thromboembolic complication (30% vs 14%, P=0.12). Endocarditis among IDUs was not associated with any specific strains, and only the FIN-4 strain was observed more often in IDUs than in nonaddicts (21% vs 5%, P=0.03). The majority of isolates (98%) were PVL negative, and there were no differences in the numbers of SAK, protease and haemolysin production among strains between IDUs and nonaddicts. However, haemolytic properties were found more frequently in strains from IDUs without endocarditis than those with endocarditis (88% vs 47%, P=0.007). IDUs displayed more often elevated TAA titers than nonaddicts, especially in endocarditis at acute phase (33% vs 5%, P=0.04) and at convalescent phase (50% vs 10%, P=0.01). The ASTA titer was more frequently initially positive among IDUs without endocarditis than with endocarditis (44% vs 6%, P=0.01). CONCLUSIONS: Characterization of the bacterial strains and their virulence factors, and host immune responses did not reveal significant differences between IDUs and nonaddicts with similar clinical picture of SAB. Serological tests were not helpful in identifying patients with endocarditis.

Nationwide trends in molecular epidemiology of methicillin-resistant Staphylococcus aureus, Finland, 1997-2004.

BACKGROUND: In Finland, the annual number of MRSA notifications to the National Infectious Disease Register (NIDR) has constantly increased since 1995, and molecular typing has revealed numerous outbreak isolates of MRSA. We analyzed the data on MRSA notifications of the NIDR, and MRSA isolates were identified mainly by pulsed-field gel electrophoresis (PFGE) at the National Reference Laboratory (NRL) in Finland during 1997-2004. One isolate representative of each major PFGE type was further characterized by multilocus sequence (MLST)-, staphylococcal cassette chromosome mec (SCCmec)-, and Panton-Valentine leukocidin (PVL)-typing. RESULTS: The annual number of MRSA notifications to the NIDR rose over ten-fold, from 120 in 1997 to 1458 in 2004, and the proportion of MRSA among S. aureus blood isolates tripled, from <1% during 1997-2003 to 2.8% in 2004. During the same period of time, 253 different strains among 4091 MRSA isolates were identified by PFGE: 215 were sporadic and 38 outbreak/epidemic strains, including 24 new strains. Two epidemic strains resembling internationally recognized MRSA clones accounted for most of the increase: FIN-16 (ST125:IA) from <1% in 1997 to 25% in 2004, and FIN-21 (ST228:I) from 6% in 2002 to 28% in 2004. Half of the ten most common strains carried SCCmec IV or V. CONCLUSION: The predominant MRSA strains seem to change over time, which encourages us to continue implementing active control measures with each new MRSA case.