Macrolide resistance mechanisms among Streptococcus pneumoniae isolated over 6 years of Canadian Respiratory Organism Susceptibility Study (CROSS) (1998 2004)

BACKGROUND: Resistance to macrolides in Streptococcus pneumoniae arises primarily due to Erm(B) or Mef(A). Erm(B) typically confers high-level resistance to macrolides, lincosamides and streptogramin B (MLS(B) phenotype), whereas Mef(A) confers low-level resistance to macrolides only (M phenotype). The purpose of this study was to investigate the incidence of macrolide resistance mechanisms in Canadian isolates of S. pneumoniae obtained between 1998 and 2004. Furthermore, the genetic relatedness, serotype distribution and antibiotic susceptibility profile among S. pneumoniae isolates with dual erythromycin ribosomal methylase [Erm(B)] and efflux pump [Mef(A)] were analysed. METHODS: A total of 865 macrolide-resistant (erythromycin MIC > or = 1 mg/L) S. pneumoniae isolates were collected from the Canadian Respiratory Organism Susceptibility Study (CROSS) from 1998 to 2004. The presence of erm(B) and mef(A) was determined for each isolate by PCR; mutations in the genes coding for L4 and L22 ribosomal proteins and for 23S rRNA were identified by DNA sequencing. Each isolate containing both erm(B)- and mef(A)-mediated macrolide resistance was genotyped by PFGE and serotyped using the Quellung reaction with antisera. RESULTS: Of the 865 isolates studied, 404 (46.7%) were mef(A)-positive, 371 (42.9%) were erm(B)-positive, 50 (5.8%) were positive for both mef(A) and erm(B) and 40 (4.6%) were negative for both mef(A) and erm(B). Of the macrolide-resistant isolates negative for both mef(A) and erm(B), 22 (2.5%) contained 23S rRNA A2058G, A2059G or A2059C mutations, 7 (0.8%) contained 23S rRNA A2058G or A2059G mutations along with an S20N mutation in L4 ribosomal protein, and 1 isolate contained an E30K ribosomal protein mutation alone. Of the macrolide-resistant strains positive for both mef(A) and erm(B), 36 (72%) were multidrug-resistant (macrolide-, penicillin- and trimethoprim/sulfamethoxazole-resistant), 39 (78%) isolates belonged to serotype 19A or 19F and 36 (72%) belonged to one clonal complex (> or =80% genetic relatedness) genetically related to the Taiwan 19F-14 clone. CONCLUSIONS: The prevalence of efflux-based macrolide resistance in S. pneumoniae in Canada remained steady between 1998 and 2004. Macrolide resistance due to erm(B) decreased over the same time period, with a rapid increase in isolates with both erm(B) and mef(A) macrolide resistance.     

Ribosomal mutations in Streptococcus pneumoniae clinical isolates

Eleven clinical isolates of Streptococcus pneumoniae, isolated in Finland during 1996 to 2000, had an unusual macrolide resistance phenotype. They were resistant to macrolides and streptogramin B but susceptible, intermediate, or low-level resistant to lincosamides. No acquired macrolide resistance genes were detected from the strains. The isolates were found to have mutations in domain V of the 23S rRNA or ribosomal protein L4. Seven isolates had an A2059C mutation in two to four out of the four alleles encoding the 23S rRNA, two isolates had an A2059G mutation in two alleles, one isolate had a C2611G mutation in all four alleles, and one isolate had a 69GTG71-to-69TPS71 substitution in ribosomal protein L4.     

High-level telithromycin resistance in laboratory-generated mutants of Streptococcus pneumoniae

Resistance to macrolides in Streptococcus pneumoniae is usually mediated by methylation of 23S ribosomal RNA, encoded by the erm(B) methylation gene, or by efflux mediated by the mef(A) gene. Changes in the L4 and L22 ribosomal proteins have also been associated with macrolide resistance and reduced telithromycin activity. This study generated in vitro mutants from three parent strains of S. pneumoniae: 02J1175 [mef(A) +], 02J1095 [erm(B) +] and NCTC 13593 (macrolide susceptible). The erm(B) and the erm(B) upstream region, the mef(A) genes and the mef(A) upstream and downstream regions, the 23S rRNA genes encoding domains II and V and the L4 and L22 genes of the telithromycin-resistant strains were all amplified by PCR and all, except the mef(A) upstream and downstream regions, were sequenced. No changes were present in any of the genes of the mef(A) + mutants. No changes were found in the erm(B) genes, the 23S rRNA genes or the L4 protein genes of the erm(B) + mutants. However, a Lys-94 to Gln-94 amino acid mutation did occur in a mutant derived from erm(B) + with a telithromycin MIC of >32 mg/L. A 210 base pair deletion in the erm(B) upstream region was also present in this strain. We believe this is the first incidence of a Lys-94 to Gln-94 change in L22 associated with telithromycin resistance and also the first time that such a large deletion in the erm(B) upstream region has been identified in S. pneumoniae.     

Resistance to macrolides in clinical isolates of Streptococcus pyogenes due to ribosomal mutations

OBJECTIVE: Two clinical strains of Streptococcus pyogenes, 237 and 544, one isolated in Slovakia and the other in Croatia, that were resistant to azithromycin (MIC 8 and 2 mg/L, respectively) but susceptible to erythromycin (MIC 0.5 and 0.12 mg/L, respectively) did not contain any gene known to confer macrolide resistance by ribosomal modification (erm gene) or efflux [mef(A) and msr(A) genes]. The aim of the study was to determine the mechanisms of macrolide resistance in both strains. METHODS: Portions of genes encoding ribosomal proteins L22 and L4, and 23S rRNA (domains II and V) in the two macrolide-resistant strains and in control strains susceptible to macrolides, were analysed by PCR and single-strand conformational polymorphism, to screen for mutations. The DNA sequences of amplicons from resistant strains that differed from those of susceptible strains, in terms of their electrophoretic migration profiles, were determined. RESULTS: S. pyogenes 237 displayed a KG insertion after position 69 in ribosomal protein L4. S. pyogenes 544 contained a C2611U mutation in domain V of 23S rRNA. CONCLUSION: Mutations at a similar position in ribosomal protein L4 and 23S rRNA have been reported previously in macrolide-resistant pneumococci. This report shows that similar mutations can be found in macrolide-resistant S. pyogenes.     

Emergence of a Streptococcus pneumoniae isolate resistant to streptogramins by mutation in ribosomal protein L22 during pristinamycin therapy of pneumococcal pneumonia

OBJECTIVES: The aim of this study was to characterize the mechanism of resistance to macrolides and streptogramins of a Streptococcus pneumoniae strain isolated from blood cultures in an 80-year-old patient suffering from severe pneumonia unsuccessfully treated with pristinamycin. METHODS: Resistance genes erm(B) and mef(A) were searched for by PCR. Portions of genes for domains V and II of the 23S rRNA (rrl) and genes for ribosomal proteins L4 (rplD) and L22 (rplV) were amplified by PCR from total genomic DNA and sequenced. RESULTS: Resistance genes erm(B) and mef(A) were not detected. Only mutation in the rplV gene encoding ribosomal protein L22 was detected. The strain contained a six amino acid insertion ((107)KRTAHI(108)) in the C-terminus of the ribosomal protein L22. CONCLUSIONS: This is the first report of emergence of a pneumococcus resistant to streptogramins by mutation in ribosomal protein L22 during treatment with pristinamycin.     

Antimicrobial susceptibility of Streptococcus pneumoniae in eight European countries from 2001 to 2003

Susceptibility testing results for Streptococcus pneumoniae isolates (n = 2,279) from eight European countries, examined in the PneumoWorld Study from 2001 to 2003, are presented. Overall, 24.6% of S. pneumoniae isolates were nonsusceptible to penicillin G and 28.0% were resistant to macrolides. The prevalence of resistance varied widely between European countries, with the highest rates of penicillin G and macrolide resistance reported from Spain and France. Serotype 14 was the leading serotype among penicillin G- and macrolide-resistant S. pneumoniae isolates. One strain (PW 158) showed a combination of an efflux type of resistance with a 23S rRNA mutation (A2061G, pneumococcal numbering; A2059G, Escherichia coli numbering). Six strains which showed negative results for mef(A) and erm(B) in repeated PCR assays had mutations in 23S rRNA or alterations in the L4 ribosomal protein (two strains). Fluoroquinolone resistance rates (levofloxacin MIC > or = 4 microg/ml) were low (Austria, 0%; Belgium, 0.7%; France, 0.9%; Germany, 0.4%; Italy, 1.3%; Portugal, 1.2%; Spain, 1.0%; and Switzerland, 0%). Analysis of quinolone resistance-determining regions showed eight strains with a Ser81 alteration in gyrA; 13 of 18 strains showed a Ser79 alteration in parC. The clonal profile, as analyzed by multilocus sequence typing (MLST), showed that the 18 fluoroquinolone-resistant strains were genetically heterogeneous. Seven of the 18 strains belonged to new sequence types not hitherto described in the MLST database. Europe-wide surveillance for monitoring of the further spread of these antibiotic-resistant S. pneumoniae clones is warranted.     

Ribosomal mutations as the main cause of macrolide resistance in Campylobacter jejuni and Campylobacter coli

The aim of this study was to examine macrolide resistance mutations in Campylobacter species. In 76 strains studied, point mutation A to G at position 2059 of the 23S rRNA gene was detected in 30 of the 33 erythromycin-resistant strains. An amino acid insertion in the ribosomal protein L22 was found in one resistant strain without a 23S rRNA mutation. The A2059G mutation is the main cause of macrolide resistance in Campylobacter species.     

Emergence of group A streptococcus strains with different mechanisms of macrolide resistance

The mechanisms of resistance to macrolides in seven group A streptococcal (Streptococcus pyogenes) isolates that were the cause of pharyngitis in children who were unsuccessfully treated with azithromycin (10 mg/kg of body weight/day for 3 days) were evaluated. All posttreatment strains were found to be genetically related to the pretreatment isolates by random amplified polymorphism DNA analysis and pulsed-field gel electrophoresis. Two isolates had acquired either a mef(A) or an erm(B) gene, responsible for macrolide efflux and ribosomal modification, respectively. Three isolates displayed mutations in the gene encoding the L4 ribosomal protein that is part of the exit tunnel within the 50S subunit of the bacterial ribosome. In the two remaining posttreatment strains, the mechanisms of macrolide resistance could not be elucidated.     

Antistreptococcal activity of telithromycin compared with seven other drugs in relation to macrolide resistance mechanisms in Russia

The susceptibilities of 468 recent Russian clinical Streptococcus pneumoniae isolates and 600 Streptococcus pyogenes isolates, from 14 centers in Russia, to telithromycin, erythromycin, azithromycin, clarithromycin, clindamycin, levofloxacin, quinupristin-dalfopristin, and penicillin G were tested. Penicillin-nonsusceptible S. pneumoniae strains were rare except in Siberia, where their prevalence rate was 13.5%: most were penicillin intermediate, but for three strains (two from Smolensk and one from Novosibirsk) the MICs of penicillin G were 4 or 8 micro g/ml. Overall, 2.5% of S. pneumoniae isolates were resistant to erythromycin. Efflux was the prevalent resistance mechanism (five strains; 41.7%), followed by ribosomal methylation encoded by constitutive erm(B), which was found in four isolates. Ribosomal mutation was the mechanism of macrolide resistance in three isolates; one erythromycin-resistant S. pneumoniae isolate had an A2059G mutation in 23S rRNA, and two isolates had substitution of GTG by TPS at positions 69 to 71 in ribosomal protein L4. All S. pyogenes isolates were susceptible to penicillin, and 11% were erythromycin resistant. Ribosomal methylation was the most common resistance mechanism for S. pyogenes (89.4%). These methylases were encoded by erm(A) [subclass erm(TR)] genes, and their expression was inducible in 96.6% of isolates. The rest of the erythromycin-resistant Russian S. pyogenes isolates (7.6%) had an efflux resistance mechanism. Telithromycin was active against 100% of pneumococci and 99.2% of S. pyogenes, and levofloxacin and quinupristin-dalfopristin were active against all isolates of both species.     

Streptococcus pyogenes isolates with characterized macrolide resistance mechanisms in Spain: in vitro activities of telithromycin and cethromycin

The in vitro activities of telithromycin and cethromycin (ABT-773) against 412 Streptococcus pyogenes isolates, consecutively collected in 17 Spanish hospitals from different geographical areas, were evaluated and compared with those of erythromycin A, penicillin G, clindamycin and quinupristin-dalfopristin. With a susceptibility testing breakpoint of < or = 1 mg/L for both compounds, 96.1% of isolates were susceptible to telithromycin and 99.8% to cethromycin. Erythromycin non-susceptible isolates (intermediate plus resistant, MIC > or = 0.5 mg/L) comprised 23% of those tested, and were analysed for the genetic basis of their resistance by PCR. Among these isolates (n = 95), 72.6% harboured mef(A), 8.4%, erm(B), and 3.2%, erm(A), as sole macrolide resistance gene, whereas the presence of mef(A) plus erm(A) (11.6%) or mef(A) plus erm(B) (4.2%) was also observed. Both ketolides displayed a significant in vitro activity against S. pyogenes regardless of the macrolide resistance mechanisms. Nevertheless, in the case of telithromycin, 11 out of 19 of the erm(B)-positive isolates (2.7% of total population) exhibited an MIC range of 4-32 mg/L. According to the present results, telithromycin and cethromycin offer a wide coverage against S. pyogenes isolates in a geographic area with a high incidence of resistance to currently used macrolides.     

Macrolide resistance by ribosomal mutation in clinical isolates of Streptococcus pneumoniae from the PROTEKT 1999-2000 study

Sixteen (1.5%) of the 1,043 clinical macrolide-resistant Streptococcus pneumoniae isolates collected and analyzed in the 1999-2000 PROTEKT (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin) study have resistance mechanisms other than rRNA methylation or efflux. We have determined the macrolide resistance mechanisms in all 16 isolates by sequencing the L4 and L22 riboprotein genes, plus relevant segments of the four genes for 23S rRNA, and the expression of mutant rRNAs was analyzed by primer extension. Isolates from Canada (n = 4), Japan (n = 3), and Australia (n = 1) were found to have an A2059G mutation in all four 23S rRNA alleles. The Japanese isolates additionally had a G95D mutation in riboprotein L22; all of these originated from the same collection center and were clonal. Three of the Canadian isolates were also clonal; the rest were not genetically related. Four German isolates had A2059G in one, two, and three 23S rRNA alleles and A2058G in two 23S rRNA alleles, respectively. An isolate from the United States had C2611G in three 23S rRNA alleles, one isolate from Poland had A2058G in three 23S rRNA alleles, one isolate from Turkey had A2058G in four 23S rRNA alleles, and one isolate from Canada had A2059G in two 23S rRNA alleles. Erythromycin and clindamycin resistance gradually increased with the number of A2059G alleles, whereas going from one to two mutant alleles caused sharp rises in the azithromycin, roxithromycin, and rokitamycin MICs. Comparisons of mutation dosage with rRNA expression indicates that not all alleles are equally expressed. Despite their high levels of macrolide resistance, all 16 isolates remained susceptible to the ketolide telithromycin (MICs, 0.015 to 0.25 microg/ml).     

Detection of macrolide resistance mechanisms in Streptococcus pneumoniae and Streptococcus pyogenes using a multiplex rapid cycle PCR with microwell-format probe hybridization

In this study, a multiplex rapid cycle PCR with microwell-format probe hybridization method was developed to perform high-volume screening for macrolide resistance determinants in isolates of Streptococcus pneumoniae and Streptococcus pyogenes. The method was then utilized to determine the distribution of macrolide resistance mechanisms in recent isolates of S. pneumoniae and S. pyogenes from Great Britain and Ireland. For 83 strains of macrolide resistant S. pneumoniae tested, 51 (61.4%) were positive for mef(A), 29 (34.9%) erm(B), two (2.4%) double mechanisms mef(A) + erm(B), and one (1.2%) negative for all mechanisms tested. For 56 strains of macrolide-resistant S. pyogenes tested, 33 (58.9%) were positive for erm(A) subclass erm(TR), 18 (32.1%) mef(A) and five (8.9%) erm(B).     

Antipneumococcal activities of two novel macrolides, GW 773546 and GW 708408, compared with those of erythromycin, azithromycin, clarithromycin, clindamycin, and telithromycin

The MICs of GW 773546, GW 708408, and telithromycin for 164 macrolide-susceptible and 161 macrolide-resistant pneumococci were low. The MICs of GW 773546, GW 708408, and telithromycin for macrolide-resistant strains were similar, irrespective of the resistance genotypes of the strains. Clindamycin was active against all macrolide-resistant strains except those with erm(B) and one strain with a 23S rRNA mutation. GW 773546, GW 708408, and telithromycin at two times their MICs were bactericidal after 24 h for 7 to 8 of 12 strains. Serial passages of 12 strains in the presence of sub-MICs yielded 54 mutants, 29 of which had changes in the L4 or L22 protein or the 23S rRNA sequence. Among the macrolide-susceptible strains, resistant mutants developed most rapidly after passage in the presence of clindamycin, GW 773546, erythromycin, azithromycin, and clarithromycin and slowest after passage in the presence of GW 708408 and telithromycin. Selection of strains for which MICs were >/=0.5 microg/ml from susceptible parents occurred only with erythromycin, azithromycin, clarithromycin, and clindamycin; 36 resistant clones from susceptible parent strains had changes in the sequences of the L4 or L22 protein or 23S rRNA. No mef(E) strains yielded resistant clones after passage in the presence of erythromycin and azithromycin. Selection with GW 773546, GW 708408, telithromycin, and clindamycin in two mef(E) strains did not raise the erythromycin, azithromycin, and clarithromycin MICs more than twofold. There were no change in the ribosomal protein (L4 or L22) or 23S rRNA sequences for 15 of 18 mutants selected for macrolide resistance; 3 mutants had changes in the L22-protein sequence. GW 773546, GW 708408, and telithromycin selected clones for which MICs were 0.03 to >2.0 microg/ml. Single-step studies showed mutation frequencies <5.0 x 10(-10) to 3.5 x 10(-7) for GW 773546, GW 708408, and telithromycin for macrolide-susceptible strains and 1.1 x 10(-7) to >4.3 x 10(-3) for resistant strains. The postantibiotic effects of GW 773546, GW 708408, and telithromycin were 2.4 to 9.8 h.     

Intrinsic macrolide resistance in Mycobacterium smegmatis is conferred by a novel erm gene, erm(38)

High-level, acquired macrolide resistance in mycobacteria is conferred by mutation within the 23S rRNA gene. However, several mycobacteria are naturally resistant to macrolides, including the Mycobacterium smegmatis group and Mycobacterium tuberculosis complex. Thus, the aim of this study was to characterize this resistance. Intrinsic macrolide resistance in M. smegmatis was inducible and showed cross-resistance to lincosamides but not to streptogramin B (i.e., ML resistance). A similar phenotype was found with Mycobacterium microti and macrolide-resistant Mycobacterium fortuitum. A search of the DNA sequence data for M. smegmatis strain mc(2)155 identified a novel erm gene, erm(38), and expression analysis showed that erm(38) RNA levels increased >10-fold after a 2-h incubation with macrolide. Inducible ML resistance was not expressed by an erm(38) knockout mutant, and complementation of this mutant with intact erm(38) in trans resulted in high-level ML resistance (e.g., clarithromycin MIC of >512 micro g/ml). Thus, the results indicate that erm(38) confers the intrinsic ML resistance of M. smegmatis. Southern blot analysis with an erm(38)-specific probe indicated that a similar gene may be present in macrolide-resistant M. fortuitum. This finding, with the presence of the erm(37) gene (Rv1988) in the M. tuberculosis complex, suggests that such genes are widespread in mycobacteria with intrinsic macrolide resistance.     

Genotypes of macrolide-resistant pneumococci from children in southwestern Japan: raised incidence of strains that have both erm(B) and mef(A) with serotype 6B clones

MICs of penicillin G, erythromycin, clarithromycin, clindamycin, azithromycin, and telithromycin were tested for 189 clinical isolates collected during 2002 to 2005 from children in southwestern Japan. Serotyping and polymerase chain reaction for presence of erm(B) and mef(A) were performed. All strains with erm(B) + mef(A) were analyzed by pulsed-field gel electrophoresis (PFGE) and compared to 3 global clones: Spain(23F)-1; Spain(9V)-3 and its variant -14; a South Korean strain same as Taiwan (19F)-14 clone and 5 strains with erm(B) + mef(A) from other countries. Of the 173 macrolide-resistant (erythromycin MIC >/=0.5 mug/mL) strains, 104 (60.1%) had erm(B), 47 (27.2%) had mef(A), and 22 (12.7%) had erm(B) + mef(A). Strains expressing erm(B) or both erm(B) and mef(A) had high macrolide MIC(90)s (>64 mug/mL), except telithromycin (MIC(90), 0.25 mug/mL). Of the 22 erm(B) + mef(A) strains, 10 had 4 distinct PFGE patterns and were mainly serotype 6B clones, which differed from those described in previous reports; 5 other strains had unique profiles.     

Diversity of Ribosomal Mutations Conferring Resistance to Macrolides, Clindamycin, Streptogramin, and Telithromycin in Streptococcus pneumoniae

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 microg/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 microg/ml) and telithromycin (MIC, 4 microg/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.     

Antimicrobial susceptibility and macrolide resistance inducibility of Streptococcus pneumoniae carrying erm(A), erm(B), or mef(A)

Erythromycin-resistant Streptococcus pneumoniae isolates from young carriers were tested for their antimicrobial susceptibility; additionally, inducibility of macrolide and clindamycin resistance was investigated in pneumococci carrying erm(A), erm(B), or mef(A). Of 125 strains tested, 101 (81%) were multidrug resistant. Different levels of induction were observed with erythromycin, miocamycin, and clindamycin in erm(B) strains; however, in erm(A) strains only erythromycin was an inducer. Induction did not affect macrolide MICs in mef(A) strains.     

Increased activity of 16-membered lactone ring macrolides against erythromycin-resistant Streptococcus pyogenes and Streptococcus pneumoniae: characterization of South African isolates

The susceptibility of 40 erythromycin-resistant isolates of Streptococcus pyogenes and 40 multiply-resistant isolates of Streptococcus pneumoniae to six macrolide antibiotics, representing 14-, 15- and 16-membered lactone ring structures, was tested. The genetic basis for macrolide resistance in the strains was also determined. Both erm and mef determinants were encountered in the 36 S. pneumoniae isolates tested, but only mef in the five S. pyogenes isolates tested. All isolates showed cross-resistance among the 14-membered macrolides erythromycin, clarithromycin and roxithromycin and the 15-membered macrolide, azithromycin. However, the erythromycin-resistant S. pyogenes isolates retained full susceptibility to spiramycin and josamycin (16-membered agents). These latter two antibiotics were also more active than the other macrolides against erythromycin-resistant S. pneumoniae isolates, especially josamycin which was 8-64 times more active than erythromycin; spiramycin was only two to eight times more active than erythromycin.