Amelia Tait-Kamradt

Streptococcus pneumoniae and Streptococcus pyogenes resistant to macrolides but sensitive to clindamycin: a common resistance pattern mediated by an efflux system.

Macrolide-resistant Streptococcus pyogenes isolates from Finland, Australia, and the United Kingdom and, more recently, Streptococcus pneumoniae and S. pyogenes strains from the United States were shown to have an unusual resistance pattern to macrolides, lincosamides, and streptogramin B antibiotics. This pattern, referred to as M resistance, consists of susceptibility to clindamycin and streptogramin B antibiotics but resistance to 14- and 15-membered macrolides. An evaluation of the macrolide-lincosamide-streptogramin B resistance phenotypes among our streptococcal strains collected from 1993 to 1995 suggested that this unusual resistance pattern is not rare. Eighty-five percent (n = 66) of the S. pneumoniae and 75% (n = 28) of the S. pyogenes strains in our collection had an M phenotype. The mechanism of M resistance was not mediated by target modification, as isolated ribosomes from a pneumococcal strain bearing the M phenotype were fully sensitive to erythromycin. Further, the presence of an erm methylase was excluded with primers specific for an erm consensus sequence. However, results of studies that determined the uptake and incorporation of radiolabeled erythromycin into cells were consistent with the presence of a macrolide efflux determinant. The putative efflux determinant in streptococci seems to be distinct from the multicomponent macrolide efflux system in coagulase-negative staphylococci. The recognition of the prevalence of the M phenotype in streptococci has implications for sensitivity testing and may have an impact on the choice of antibiotic therapy in clinical practice.

Mutations in 23S rRNA and Ribosomal Protein L4 Account for Resistance in Pneumococcal Strains Selected In Vitro by Macrolide Passage

ABSTRACT The mechanisms responsible for macrolide resistance inStreptococcus pneumoniae mutants, selected from susceptible strains by serial passage in azithromycin, were investigated. These mutants were resistant to 14- and 15-membered macrolides, but resistance could not be explained by any clinically relevant resistance determinant [mef(A),erm(A), erm(B), erm(C),erm(TR), msr(A), mph(A),mph(B), mph(C), ere(A),ere(B)]. An investigation into the sequences of 23S rRNAs in the mutant and parental strains revealed individual changes of C2611A, C2611G, A2058G, and A2059G (Escherichia colinumbering) in four mutants. Mutations at these residues in domain V of 23S rRNA have been noted to confer erythromycin resistance in other species. Not all four 23S rRNA alleles have to contain the mutation to confer resistance. Some of the mutations also confer coresistance to streptogramin B (C2611A, C2611G, and A2058G), 16-membered macrolides (all changes), and clindamycin (A2058G and A2059G). Interestingly, none of these mutations confer high-level resistance to telithromycin (HMR-3647). Further, two of the mutants which had no changes in their 23S rRNA sequences had changes in a highly conserved stretch of amino acids (63KPWRQKGTGRAR74) in ribosomal protein L4. One mutant contained a single amino acid change (G69C), while the other mutant had a 6-base insert, resulting in two amino acids (S and Q) being inserted between amino acids Q67 and K68. To our knowledge, this is the first description of mutations in 23S rRNA genes or ribosomal proteins in macrolide-resistant S. pneumoniae strains.

Two New Mechanisms of Macrolide Resistance in Clinical Strains of Streptococcus pneumoniae from Eastern Europe and North America

ABSTRACT Resistance to macrolides in pneumococci is generally mediated by methylation of 23S rRNA via erm(B) methylase which can confer a macrolide (M)-, lincosamide (L)-, and streptogramin B (SB)-resistant (MLSB) phenotype or by drug efflux via mef(A) which confers resistance to 14- and 15-membered macrolides only. We studied 20 strains with unusual ML or MSB phenotypes which did not harbor erm(B) ormef(A). The strains had been isolated from patients in Eastern Europe and North America from 1992 to 1998. These isolates were found to contain mutations in genes for either 23S rRNA or ribosomal proteins. Three strains from the United States with an ML phenotype, each representing a different clone, were characterized as having an A2059G (Escherichia coli numbering) change in three of the four 23S rRNA alleles. Susceptibility to macrolides and lincosamides decreased as the number of alleles in isogenic strains containing A2059G increased. Sixteen MSB strains from Eastern Europe were found to contain a 3-amino-acid substitution (69GTG71 to TPS) in a highly conserved region of the ribosomal protein L4 (63KPWRQKGTGRAR74). These strains formed several distinct clonal types. The single MSB strain from Canada contained a 6-amino-acid L4 insertion (69GTGREKGTGRAR), which impacted growth rate and also conferred a 500-fold increase in MIC on the ketolide telithromycin. These macrolide resistance mechanisms from clinical isolates are similar to those recently described for laboratory-derived mutants.

Multicity Outbreak of Linezolid-Resistant Staphylococcus epidermidis Associated with Clonal Spread of a cfr-Containing Strain

We report a multicity outbreak of cfr-containing linezolid-resistant Staphylococcus epidermidis in Ohio. Thirty-nine isolates were obtained from 2 hospitals. Two clones with different mechanisms of linezolid resistance were circulating in hospital A. One of these contained the cfr gene, and the other a ribosomal mutation. The clone containing cfr was identical in both hospitals.

Identification of an erm(A) Erythromycin Resistance Methylase Gene in Streptococcus pneumoniae Isolated in Greece

ABSTRACT In a serotype 11A clone of erythromycin-resistant pneumococci isolated from young Greek carriers, we identified the nucleotide sequence of erm(A), a methylase gene previously described as erm(TR) in Streptococcus pyogenes. Theerm(A) pneumococci were resistant to 14- and 15-member macrolides, inducibly resistant to clindamycin, and susceptible to streptogramin B. To our knowledge, this is the first identification of resistance to erythromycin in S. pneumoniae attributed solely to the carriage of the erm(A) gene.

Clonal relatedness of erythromycin-resistant Streptococcus pyogenes isolates in Germany.

ABSTRACT In a nationwide study in Germany, a total of 381 Streptococcus pyogenes were collected. Erythromycin A-resistant strains were characterized for the underlying resistance genotype, showing 55.6% had the efflux type mef(A), 31.5% had erm(A), and 13.0% had erm(B). A total of 23 different multilocus sequence types were observed.

Discovery of azetidinyl ketolides for the treatment of susceptible and multidrug resistant community-acquired respiratory tract infections.

Respiratory tract bacterial strains are becoming increasingly resistant to currently marketed macrolide antibiotics. The current alternative telithromycin (1) from the newer ketolide class of macrolides addresses resistance but is hampered by serious safety concerns, hepatotoxicity in particular. We have discovered a novel series of azetidinyl ketolides that focus on mitigation of hepatotoxicity by minimizing hepatic turnover and time-dependent inactivation of CYP3A isoforms in the liver without compromising the potency and efficacy of 1.

Macrolide-Resistant Pneumococcal Endocarditis and Epidural Abscess that Develop during Erythromycin Therapy

Suppurative complications of Streptococcus pneumoniae infections have become uncommon in the antibiotic era. We report a case of pneumococcal bacteremia and pneumonia complicated with epidural abscess and endocarditis in which macrolide resistance (the MLS(B) phenotype) emerged during erythromycin therapy. Genetic determinants known to mediate the most common mechanisms of macrolide resistance (methylation of the 23S rRNA and antibiotic efflux) were not detected by polymerase chain reaction or DNA hybridization. Sequence analysis of the DNA encoding the 23S rRNA of the macrolide-resistant isolate from the patient demonstrated the replacement of adenine by thymine at position 2058 (A2058T) in 2 of 4 alleles. Clinicians should be alert to the possibility of the emergence of resistance during macrolide therapy for community-acquired pneumonia, particularly if suppurative complications of pneumococcal infection are suspected.

Comparative genome analysis of high-level penicillin resistance in Streptococcus pneumoniae.

Streptococcus pneumoniae strains with very high levels of penicillin resistance (minimum inhibitory concentration [MIC] >or=8 microg/ml) emerged in the 1990 s. Previous studies have traced the changes in penicillin binding proteins (PBP) that result in decreased penicillin susceptibility, and the role of several PBP genes in high-level resistance. In the present study, we investigated the changes that occurred at the two highest levels of penicillin resistance using NimbleGens Comparative Genome Sequencing (CGS) technology. DNA from a highly resistant (Pen MIC 16 microg/ml) pneumococcus was used to serially transform the R6 strain to high-level resistance. Four distinct levels of penicillin resistance above the susceptible R6 strain (MIC 0.016 microg/ml) were identified. Using CGS technology, the entire genome sequences of the two highest levels of resistant transformants were examined for changes associated with the resistance phenotypes. At the third level of resistance, changes in PBPs 1a, 2b, and 2x were found, very similar to previous reports. At the fourth resistance level, two additional changes were observed in the R6 transformants. More changes were observed in PBP2x, as well as in peptidoglycan GlcNAc deacetylase (pdgA), which had a missense mutation in the coding region. Genetic transformation with polymerase chain reaction (PCR) products generated from the high-level resistant parent containing either the additional PBP2x or mutant pdgA gene did not increase the MIC of the third-level transformant. Only when both PCR products were simultaneously transformed into the third-level transformant did colonies emerge that were at the highest level of resistance (16-32 microg/ml), equivalent to the highly resistant parent strain. This is the first instance of the involvement of a variant pdgA gene in penicillin resistance. It is also clear from these experiments and the literature that there are multiple paths to the pneumococcus achieving high-level penicillin resistance.