Coxsackievirus B3-induced myocarditis in different immunocompetent mouse strains was used as a model to investigate interrelationships between virus replication and development of chronic enteroviral heart disease. Using in situ hybridization to detect enteroviral RNA, we show that heart muscle infection is not only detected in acute myocarditis but is also detected during the chronic phase of the disease. Coxsackievirus B3 could evade immunological surveillance in a host-dependent fashion, thus inducing a persistent infection of the myocardium in association with ongoing inflammation. Patterns of acute and persistent myocardial infection were quantitatively assessed in one representative mouse strain (A.CA/SnJ, H-2f) by applying computer-assisted digital image processing; these patterns were then related to the extent of myocardial tissue damage as well as to inflammation. We observed a strong correlation, both spatial and temporal, between viral replication and development of myocardial lesions, indicating that acute and chronic myocardial injuries are a consequence of multifocal organ infection. Analysis of strandspecific in situ hybridization revealed that viral replication in persistent infection is restricted at the level of RNA synthesis. The described procedure for quantitating organ infection provides a powerful tool for evaluating virus-host interactions and will be of particular interest to those studying human enterovirus-induced cardiomyopathies.

Mechanisms of resistance were studied in 22 macrolide-resistant mutants selected in vitro from 5 parental strains of macrolide-susceptible Streptococcus pneumoniae by serial passage in various macrolides (T. A. Davies, B. E. Dewasse, M. R. Jacobs, and P. C. Appelbaum, Antimicrob. Agents Chemother., 44:414-417, 2000). Portions of genes encoding ribosomal proteins L22 and L4 and 23S rRNA (domains II and V) were amplified by PCR and analyzed by single-strand conformational polymorphism analysis to screen for mutations. The DNA sequences of amplicons from mutants that differed from those of parental strains by their electrophoretic migration profiles were determined. In six mutants, point mutations were detected in the L22 gene (G95D, P99Q, A93E, P91S, and G83E). The only mutant selected by telithromycin (for which the MIC increased from 0.008 to 0.25 g/ml) contained a combination of three mutations in the L22 gene (A93E, P91S, and G83E). L22 mutations were combined with an L4 mutation (G71R) in one strain and with a 23S rRNA mutation (C2611A) in another strain. Nine other strains selected by various macrolides had A2058G (n ‫؍‬ 1), A2058U (n ‫؍‬ 2), A2059G (n ‫؍‬ 1), C2610U (n ‫؍‬ 1), and C2611U (n ‫؍‬ 4) mutations (Escherichia coli numbering) in domain V of 23S rRNA. One mutant selected by clarithromycin and resistant to all macrolides tested (MIC, >32 g/ml) and telithromycin (MIC, 4 g/ml) had a single base deletion (A752) in domain II. In six remaining mutants, no mutations in L22, L4, or 23S rRNA could be detected.Resistance to macrolides is increasingly reported in clinical isolates of Streptococcus pneumoniae worldwide (10, 14). Resistance to macrolides was primarily related to modification of the ribosomal targets of these antibiotics. This mechanism relies on N-6 dimethylation of a specific adenine residue in 23S rRNA which confers cross-resistance to macrolides, lincosamides, and streptogramins B, the so-called MLS B phenotype, and is encoded in pneumococci by genes belonging to the erm(B) or erm(A) class (11,18,26). Subsequently, target modification was reported in the majority if not all macrolideresistant pneumococci (11,26). More recently, a mechanism of resistance by active efflux of erythromycin due to the mefE gene, renamed mef(A) (18), was reported in S. pneumoniae and appeared to be predominant in the United States and Canada, with prevalences ranging from 41 to 85% (9,19,20). The efflux phenotype, which is also called the M phenotype, is characterized by resistance to 14-membered-ring (erythromycin, clarithromycin, and roxithromycin) and 15-membered-ring (azithromycin) macrolides only. Ribosomal mutation has been reported only recently in a few clinical isolates of S. pneumoniae (5,21,22). The changes were clustered in a highly conserved sequence of L4 and in nucleotide residues of domain V of 23S rRNA which have a key role in macrolide binding. A recent study by Davies et al. (4) showed that mutants can readily be selected in pneumococci in the presence of any of several MLS B antibiotics in vit...

We have developed an in situ hybridization assay capable of detecting enteroviral RNA in myocardial cells, using molecularly cloned coxsackievirus B3 cDNA as a diagnostic probe. Because of the high degree of nucleic acid sequence homology among the numerous enteroviral serotypes, including the group A and B coxsackieviruses and the echoviruses, detection of these various agents commonly implicated in human viral heart disease is possible in a single hybridization assay. We demonstrate the considerable potential of this method for an unequivocal diagnosis of enteroviral heart disease as well as for pathogenicity studies. Using athymic mice persistently infected with coxsackievirus B3 as a model system, we show that the myocardium is affected in a disseminated, multifocal manner.In North America and Europe, acute myocarditis is most commonly associated with infections by coxsackie B viruses (types 1 to 5). Other members of the human enteroviruses comprising at present over 70 serotypes (e.g., various coxsackie A viruses and echoviruses) are also considered to be relatively frequent causes of human viral heart disease (1-4). These agents appear to be capable of producing dilated cardiomyopathy of acute onset or lead to a variety of cardiac arrhythmias. Some of the acute cases may also evolve into a chronic form of dilated cardiomyopathy.The difficulty of establishing a specific diagnosis of viral heart disease is a major problem in clinical cardiology. Confirmation ofthe clinical suspicion of viral heart disease demands demonstration of replicating virus inside myocardial cells, which is exceedingly difficult by conventional methods (5). In addition, the increasing availability of potential antiviral agents has accentuated the need for methods by which endomyocardial biopsy specimens of patients with suspected viral heart disease can be diagnosed conclusively (6, 7).In order to introduce in situ nucleic acid hybridization (8-10) as a diagnostic tool in suspected enteroviral heart disease, we have recently cloned the single-stranded RNA genome of coxsackievirus B3 (CVB3) (11) that had been propagated in cultured human heart cells (12). Full-length reverse-transcribed cloned viral cDNA generated infectious CVB3 upon transfection into mammalian cells, demonstrating the molecular cloning of a faithful transcript ofthe original viral RNA. We now report the use of radioactively labeled cloned CVB3 cDNA as a diagnostic probe for the in situ detection of viral RNA in infected cultured cells and in myocardial tissue sections of CVB3-infected T-cell-deficient mice. We show that the molecular hybridization approach permits the detection of infected myocardial cells at the single-cell level, thus providing a unique possibility for an unequivocal diagnosis.A further advantage of the nucleic acid hybridization approach is provided by the high degree of genetic homology shared among the different serotypes of the human enterovirus group (11, 13-17), making possible their detection in a single hybridization assay. Detectio...

The plasmid-borne mph(A) gene that confers resistance to azithromycin and has recently emerged in Shigella sonnei is present in multidrug- and non–multidrug-resistant Escherichia coli isolates from 4 continents. Further spread of mph(A) to Shigella and Salmonella spp. may be expected.

The mechanism of resistance to the streptogramin antibiotics quinupristin and dalfopristin was studied in a Staphylococcus aureus clinical isolate selected under quinupristin-dalfopristin therapy, in four derivatives of S. aureus RN4220 selected in vitro, and in a mutant selected in a model of rabbit aortic endocarditis. For all strains the MICs of erythromycin, quinupristin, and quinupristin-dalfopristin were higher than those for the parental strains but the MICs of dalfopristin and lincomycin were similar. Portions of genes for domains II and V of 23S rRNA and the genes for ribosomal proteins L4 and L22 were amplified and sequenced. All mutants contained insertions or deletions in a protruding ␤ hairpin that is part of the conserved C terminus of the L22 protein and that interacts with 23S rRNA. Susceptible S. aureus RN4220 was transformed with plasmid DNA encoding the L22 alteration, resulting in transformants that were erythromycin and quinupristin resistant. Synergistic ribosomal binding of streptogramins A and B, studied by analyzing the fluorescence kinetics of pristinamycin I A -ribosome complexes, was abolished in the mutant strain, providing an explanation for quinupristin-dalfopristin resistance.Streptogramin antibiotics are a mixture of two classes of chemically distinct components, designated streptogramins A and B. Quinupristin-dalfopristin is a semisynthetic injectable streptogramin, a mixture of quinupristin and dalfopristin, which are semisynthetic derivatives of pristinamycin I A (PI A ; streptogramin B) and pristinamycin II A (PII A ; streptogramin A), respectively. The binding of these factors to the 50S ribosomal subunit causes inhibition of protein synthesis. Alone, each factor has a moderate bacteriostatic activity, but the combination can display a bactericidal synergistic effect, which has been attributed to the synergistic binding of the factors to their ribosomal target site (11).Quinupristin-dalfopristin is active against a wide range of gram-positive organisms including methicillin-resistant staphylococci and vancomycin-resistant Enterococcus faecium (33). Since streptogramins A and B are chemically unrelated and have different binding sites, the mechanisms of resistance to these two streptogramins are different. Resistance to each component of the streptogramins in both staphylococci and enterococci has been reported (13, 32). The most common type of resistance to streptogramin B antibiotics is related to the production of ribosomal methylases encoded by erm genes. Resistance results from decreased component B binding to the ribosome. Cross-resistance between streptogramins B and all macrolides and lincosamides occurs because these antimicrobials have overlapping binding sites, yielding the so-called macrolide-lincosamide-streptogramin B (MLS B ) phenotype. The synergistic inhibitory activity of the two streptogramin components is conserved even when the 23S rRNA is modified by an Erm methylase although the bactericidal activity of the streptogramin combination is altered. By contr...