mef(A), mef(E) and a new mef allele in macrolide-resistant Streptococcus spp. isolates from Norway

OBJECTIVES: To type mef genes in a nationwide collection of clinical isolates of Streptococcus pneumoniae and Streptococcus pyogenes as well as pharyngeal carrier strains of viridans streptococci in Norway. METHODS: Erythromycin-resistant mef-positive multilocus sequence-typed (MLST) clinical isolates of S. pneumoniae (n = 36) and S. pyogenes (n = 12) from the National Surveillance Program for Antimicrobial Resistance (NORM) as well as viridans streptococci (n = 20) from healthy adults were included. PCR-amplified mef genes were initially discriminated by BamHI digestion. Selected mef genes from representatives of different sequence types (STs) of S. pneumoniae (n = 11) and S. pyogenes (n = 4), and viridans group streptococcal species (n = 8) were typed by sequencing and their strains examined for co-resistances. Hydropathy plots of different mef-encoded proteins were performed. RESULTS: A predominance of mef(A) was detected in S. pneumoniae (23/36) and S. pyogenes (9/12) due to the clonal spread of ST9 and ST39, respectively. mef(E) was the most widely distributed mef determinant occurring in nine different STs of S. pneumoniae and in four different viridans species. A new mef allele was identified in two STs of S. pyogenes. CONCLUSIONS: mef(E) is the most widely distributed mef determinant in Norwegian clinical strains of S. pneumoniae and pharyngeal carrier strains of various viridans streptococci. However, mef(A) is more prevalent in S. pneumoniae and S. pyogenes due to clonal spread. A new mef allele was found in two different STs of S. pyogenes.     

Mechanisms of macrolide resistance among Streptococcus pneumoniae isolates from Russia

Among 76 macrolide-nonsusceptible Streptococcus pneumoniae isolates collected between 2003 and 2005 from Central Russia, the resistance mechanisms detected in the isolates included erm(B) alone (50%), mef alone [mef(E), mef(I), or a different mef subclass; 19.7%], or both erm(B) and mef(E) (30.3%). Isolates with dual resistance genes [erm(B) and mef(E)] belonged to clonal complex CC81 or CC271.     

Rise of Streptococcus pneumoniae isolates containing both erm(B) and mef(E) genes from an adult tertiary care community hospital system

The emergence of macrolide- and lincosamide-resistant Streptococcus pneumoniae is a worldwide concern. Of particular interest is the increasing prevalence of erythromycin and clindamycin-resistant isolates containing both erm(B) and mef genes. This study determined the prevalence of erythromycin and clindamycin resistance in 596 clinical S. pneumoniae isolates from 2 adult tertiary care hospitals over a 4-year period (2001-2004). Erythromycin resistance increased from 24% to 34%, but S. pneumoniae isolates resistant to clindamycin as well as to erythromycin increased from 3% in 2001 to 15.5% in 2004 (5-fold increase). Among erythromycin-resistant isolates, those also resistant to clindamycin (MLS(B) phenotype) increased 3-fold (12.8-45%). Of forty-one erythromycin/clindamycin-resistant S. pneumoniae isolates tested, 29 (71%) contained both erm(B) and mef(E) genes. Pulsed-field gel electrophoresis performed on 28 erm(B) + mef(E) positive isolates identified 2 predominant and possibly related clones, which made up 64% of the isolates.     

Macrolide resistance mechanisms and in vitro susceptibility patterns of viridans group streptococci isolated from blood cultures

OBJECTIVES: Our aim was to study the macrolide resistance mechanisms and antimicrobial susceptibilities of viridans group streptococci (VGS) isolated from blood cultures. METHODS: In vitro susceptibilities to nine antimicrobials were studied for 85 VGS isolated from blood cultures by agar dilution. Pheno- and genotyping of erythromycin-resistant isolates were studied by the double disc test and PCR. RESULTS: Resistance to erythromycin was found in 27% (n = 23) of the isolates. Erythromycin-resistant Streptococcus oralis (n = 13) predominated among the other erythromycin-resistant species isolated. The phenotypes among 23 erythromycin-resistant isolates were as follows: 12 constitutive macrolide-lincosamide-streptogramin (cMLS(B)) resistance phenotype and 11 macrolide (M) resistance phenotype. Of the cMLS(B) isolates 11 had erm(B) genes and 11 of the M phenotype isolates had mef(A) genes. Four of the cMLS(B) isolates had both erm(B) and mef(A) genes. None of the isolates had erm(TR) genes. Combined resistance to erythromycin with penicillin, clindamycin, chloramphenicol, tetracycline and quinupristin/dalfopristin was found in 100, 61, 74, 100 and 100% of the isolates, respectively. No resistance was found for vancomycin, linezolid and levofloxacin. CONCLUSIONS: The macrolide resistance mechanisms of our VGS isolates revealed that the cMLS(B) phenotype associated with erm(B) and the M phenotype associated with mef(A) genes are found with similar frequencies.     

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.     

Molecular basis of resistance to macrolides and other antibiotics in commensal viridans group streptococci and Gemella spp. and transfer of resistance genes to Streptococcus pneumoniae

We assessed the mechanisms of resistance to macrolide-lincosamide-streptogramin B (MLS(B)) antibiotics and related antibiotics in erythromycin-resistant viridans group streptococci (n = 164) and Gemella spp. (n = 28). The macrolide resistance phenotype was predominant (59.38%); all isolates with this phenotype carried the mef(A) or mef(E) gene, with mef(E) being predominant (95.36%). The erm(B) gene was always detected in strains with constitutive and inducible MLS(B) resistance and was combined with the mef(A/E) gene in 47.44% of isolates. None of the isolates carried the erm(A) subclass erm(TR), erm(A), or erm(C) genes. The mel gene was detected in all but four strains carrying the mef(A/E) gene. The tet(M) gene was found in 86.90% of tetracycline-resistant isolates and was strongly associated with the presence of the erm(B) gene. The cat(pC194) gene was detected in seven chloramphenicol-resistant Streptococcus mitis isolates, and the aph(3')-III gene was detected in four viridans group streptococcal isolates with high-level kanamycin resistance. The intTn gene was found in all isolates with the erm(B), tet(M), aph(3')-III, and cat(pC194) gene. The mef(E) and mel genes were successfully transferred from both groups of bacteria to Streptococcus pneumoniae R6 by transformation. Viridans group streptococci and Gemella spp. seem to be important reservoirs of resistance genes.     

Simultaneous detection of nine antibiotic resistance-related genes in Streptococcus agalactiae using multiplex PCR and reverse line blot hybridization assay

Streptococcus agalactiae (group B streptococcus [GBS]) is the leading cause of neonatal and maternal sepsis. Penicillin is recommended for intrapartum prophylaxis, but erythromycin or clindamycin is used for penicillin-allergic carriers. Antibiotic resistance (AR) has increased recently and needs to be monitored. We have developed a multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay to detect, simultaneously, seven genes encoding AR--erm(A/TR), erm(B), mef(A/E), tet(M), tet(O), aphA-3, and aad-6--and two AR-related genes, int-Tn and mreA. We tested 512 GBS isolates from Asia and Australasia and compared mPCR/RLB with antibiotic susceptibility phenotype or single-gene PCR. Phenotypic resistance to tetracycline was identified in 450 (88%) isolates, of which 442 had tet(M) (93%) and/or tet(O) (6%). Of 67 (13%) erythromycin-resistant isolates, 18 were susceptible to clindamycin, i.e., had the M phenotype, encoded by mef(A/E); 39 had constitutive (cMLS(B)) and 10 inducible clindamycin resistance, and of these, 34 contained erm(B) and 12 erm(A/TR). Of four additional isolates with mef(A/E), three contained erm(B) with cMLS(B) and one was erythromycin susceptible. Of 61 (12%) clindamycin-resistant isolates, 20 were susceptible to erythromycin and two had intermediate resistance. Based on sequencing, 21 of 22 isolates with mef had mef(E), and 8 of 353 with int-Tn had an atypical sequence. Several AR genes, erm(B), tet(O), aphA-3, aad-6, and mef(A/E), were significantly more common among Asian than Australasian isolates, and there were significant differences in distribution of AR genes between GBS serotypes. Our mPCR/RLB assay is simple, rapid, and suitable for surveillance of antibiotic resistance in GBS.     

In vitro activity of telithromycin against Spanish Streptococcus pneumoniae isolates with characterized macrolide resistance mechanisms

The susceptibilities to telithromycin of 203 Streptococcus pneumoniae isolates prospectively collected during 1999 and 2000 from 14 different geographical areas in Spain were tested and compared with those to erythromycin A, clindamycin, quinupristin-dalfopristin, penicillin G, cefotaxime, and levofloxacin. Telithromycin was active against 98.9% of isolates (MICs, < or =0.5 microg/ml), with MICs at which 90% of isolates are inhibited being 0.06 microg/ml, irrespective of the resistance genotype. The corresponding values for erythromycin were 61.0% (MICs, < or =0.25 microg/ml) and >64 microg/ml. The erm(B) gene (macrolide-lincosamide-streptogramin B resistance phenotype) was detected in 36.4% (n = 74) of the isolates, which corresponded to 93.6% of erythromycin-intermediate and -resistant isolates, whereas the mef(A) gene (M phenotype [resistance to erythromycin and susceptibility to clindamycin and spiramycin without blunting]) was present in only 2.4% (n = 5) of the isolates. One of the latter isolates also carried erm(B). Interestingly, in one isolate for which the erythromycin MIC was 2 microg/ml, none of these resistance genes could be detected. Erythromycin MICs for S. pneumoniae erm(B)-positive isolates were higher (range, 0.5 to >64 microg/ml) than those for erm(B)- and mef(A)-negative isolates (range, 0.008 to 2 microg/ml). The corresponding values for telithromycin were lower for both groups, with ranges of 0.004 to 1 and 0.002 to 0.06 microg/ml, respectively. The erythromycin MIC was high for a large number of erm(B)-positive isolates, but the telithromycin MIC was low for these isolates. These results indicate the potential usefulness of telithromycin for the treatment of infections caused by erythromycin-susceptible and -resistant S. pneumoniae isolates when macrolides are indicated.     

Phenotypic and molecular characterization of tetracycline- and erythromycin-resistant strains of Streptococcus pneumoniae

Sixty-five clinical isolates of Streptococcus pneumoniae, all collected in Italy between 1999 and 2002 and resistant to both tetracycline (MIC, >or=8 microg/ml) and erythromycin (MIC, >or=1 microg/ml), were investigated. Of these strains, 11% were penicillin resistant and 23% were penicillin intermediate. With the use of the erythromycin-clindamycin-rokitamycin triple-disk test, 14 strains were assigned to the constitutive (cMLS) phenotype of macrolide resistance, 44 were assigned to the partially inducible (iMcLS) phenotype, 1 was assigned to the inducible (iMLS) phenotype, and 6 were assigned to the efflux-mediated (M) phenotype. In PCR assays, 64 of the 65 strains were positive for the tetracycline resistance gene tet(M), the exception being the one M isolate susceptible to kanamycin, whereas tet(K), tet(L), and tet(O) were never found. All cMLS, iMcLS, and iMLS isolates had the erythromycin resistance gene erm(B), and all M phenotype isolates had the mef(A) or mef(E) gene. No isolate had the erm(A) gene. The int-Tn gene, encoding the integrase of the Tn916-Tn1545 family of conjugative transposons, was detected in 62 of the 65 test strains. Typing assays showed the strains to be to a great extent unrelated. Of 16 different serotypes detected, the most numerous were 23F (n = 13), 19A (n = 10), 19F (n = 9), 6B (n = 8), and 14 (n = 6). Of 49 different pulsed-field gel electrophoresis types identified, the majority (n = 39) were represented by a single isolate, while the most numerous type included five isolates. By high-resolution restriction analysis of PCR amplicons with four endonucleases, the tet(M) loci from the 64 tet(M)-positive pneumococci were classified into seven distinct restriction types. Overall, a Tn1545-like transposon could reasonably account for tetracycline and erythromycin resistance in the vast majority of the pneumococci of cMLS, iMcLS, and iMLS phenotypes, whereas a Tn916-like transposon could account for tetracycline resistance in most M phenotype strains.     

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.     

The macrolide resistance genes erm(B) and mef(E) are carried by Tn2010 in dual-gene Streptococcus pneumoniae isolates belonging to clonal complex CC271

The genetic elements carrying macrolide resistance genes in Streptococcus pneumoniae isolates belonging to CC271 were investigated. The international clone Taiwan(19F)-14 was found to carry Tn2009, a Tn916-like transposon containing tet(M) and mef(E). The dual erm(B) mef(E) isolates carried Tn2010, which is similar to Tn2009 with the addition of a putative new transposon, the erm(B) genetic element.     

Serotypes, Clones, and Mechanisms of Resistance of Erythromycin-Resistant Streptococcus pneumoniae Isolates Collected in Spain

The aim of this study was to analyze the distributions of antibiotic susceptibility patterns, serotypes, phenotypes, genotypes, and macrolide resistance genes among 125 nonduplicated erythromycin-resistant Streptococcus pneumoniae clinical isolates collected in a Spanish point prevalence study. The prevalence of resistance to macrolides in this study was 34.7%. Multiresistance (to three or more antimicrobials) was observed in 81.6% of these strains. Among 15 antimicrobials studied, cefotaxime, moxifloxacin, telithromycin, and quinupristin-dalfopristin were the most active drugs. The most frequent serotypes of erythromycin-resistant isolates were 19F (25%), 19A (17%), 6B (12%), 14 (10%), and 23F (10%). Of the 125 strains, 109 (87.2%) showed the MLS(B) phenotype [103 had the erm(B) gene and 6 had both erm(B) and mef(E) genes]. Sixteen (12.8%) strains showed the M phenotype [14 with mef(E) and 2 with mef(A)]. All isolates were tested by PCR for the presence of the int, xis, tnpR, and tnpA genes associated with conjugative transposons (Tn916 family and Tn917). Positive detection of erm(B), tet(M), int, and xis genes related to the Tn916 family was found in 77.1% of MLS(B) phenotype strains. In 16 strains, only the tndX, erm(B), and tet(M) genes were detected, suggesting the presence of Tn1116, a transposon recently described for Streptococcus pyogenes. Five clones, namely, Sweden(15A)-25, clone(19F) ST87, Spain(23F)-1, Spain(6B)-2, and clone(19A) ST276, accounted for half of the MLS(B) strains. In conclusion, the majority of erythromycin-resistant pneumococci isolated in Spain had the MLS(B) phenotype, belonged to multiresistant international clones, and carried the erm(B), tet(M), xis, and int genes, suggesting the spread of transposons of the Tn916 family.     

Serotype 19f multiresistant pneumococcal clone harboring two erythromycin resistance determinants (erm(B) and mef(A)) in South Africa

One hundred eighteen erythromycin-resistant Streptococcus pneumoniae (ERSP) strains (MICs of > or = 0.5 microg/ml) from five laboratories serving the private sector in South Africa were analyzed for the genes encoding resistance to macrolides. Sixty-seven ERSP strains (56.8%) contained the erm(B) gene, and 15 isolates (12.7%) contained the mef(A) gene. Thirty-six isolates (30.5%) harbored both the erm(B) and mef(A) genes and were highly resistant to erythromycin and clindamycin. DNA fingerprinting by BOX-PCR and pulsed-field gel electrophoresis identified 83% of these strains as belonging to a single multiresistant serotype 19F clone.     

Distribution of resistance genes tet(M), aph3'-III, catpC194 and the integrase gene of Tn1545 in clinical Streptococcus pneumoniae harbouring erm(B) and mef(A) genes in Spain

The most prevalent macrolide resistance phenotype and genotype among pneumococcal isolates was the cMLSB phenotype [erm(B) or erm(B)/mef(A)] (91.3%). We studied the distribution of other resistance genes, tet(M), catpC194, aph3'-III, in these strains, seeing evolution at work in that some strains carried different combinations of resistance determinants. The most prevalent patterns associated with resistance to erythromycin [erm(B)] were resistance to tetracycline [tet(M)] and chloramphenicol (catpC194) (48.2%) or resistance to tetracycline [tet(M)] alone (42.2%). In our isolates of Streptococcus pneumoniae there was a strong association of the erm(B) and tet(M) genes with Tn1545-related elements.     

Activities of 16-membered ring macrolides and telithromycin against different genotypes of erythromycin-susceptible and erythromycin-resistant Streptococcus pyogenes and Streptococcus pneumoniae

OBJECTIVES: To test four 16-membered macrolides (josamycin, spiramycin, midecamycin and rokitamycin) along with other compounds in the same class (erythromycin, clarithromycin, roxithromycin and azithromycin) plus clindamycin and telithromycin, against Streptococcus pyogenes and Streptococcus pneumoniae isolates with well-characterized resistance genotypes. METHODS: Four hundred and eighty-six isolates of S. pyogenes and 375 isolates of S. pneumoniae were assayed for their macrolide susceptibilities and investigated by PCR to detect their different erythromycin resistance genes. All strains had been isolated over the period 2002-2003 from specimens of different human origin obtained in 14 different Italian centres. RESULTS: All 16-membered macrolides showed very low MICs (MIC(50)s and MIC(90)s, < or =0.06-0.5 mg/L) for the erythromycin-susceptible isolates and for those with the M phenotype, but the telithromycin MICs for the M-type isolates were at least four times higher (MIC(90)s, 0.5 mg/L). In S. pyogenes, the MIC(50)s of 16-membered macrolides for the cMLS(B) isolates were > or = 256 mg/L, whereas that for telithromycin was 4 mg/L; the MIC(50)s of 16-membered macrolides and telithromycin ranged from < or = 0.06 to 0.5 mg/L for the iMLS(B) isolates with erm(A) and from 0.12 to > or = 256 mg/L for those with erm(B). In S. pneumoniae, the MIC(50)s of the 16-membered macrolides for the cMLS(B) isolates ranged from 0.5 to 128 mg/L, whereas for the iMLS(B) isolates their values ranged from < or = 0.06 to 4 mg/L; the MIC(50)s and MIC(90)s of telithromycin for both the cMLS(B) and the iMLS(B) isolates ranged from < or = 0.06 to 0.12 mg/L. CONCLUSIONS: MICs ranged for all the drugs, except telithromycin, from < or = 0.06 to > or = 256 mg/L, with 15% to 30% resistant S. pyogenes for all drugs tested except clindamycin (8%) and telithromycin (5.4%) and 10% to 40% resistant S. pneumoniae for all drugs tested except telithromycin (0.3%). In both S. pyogenes and S. pneumoniae, erythromycin resistance related to a mef gene meant that telithromycin MICs were definitely higher than in erythromycin-susceptible isolates, although telithromycin susceptibility was preserved in all cases. In S. pyogenes, the activity of both 16-membered macrolides and telithromycin against the iMLS(B) strains proved to be dependent on the erm gene involved, being greater against isolates with erm(A).     

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.     

Evolution of erythromycin resistance in Streptococcus pneumoniae in Italy

OBJECTIVES: To evaluate erythromycin resistance in recent invasive isolates of Streptococcus pneumoniae in Italy, to study the phenotypic and genotypic characteristics of the isolates, and to compare data with those obtained in a previous survey. METHODS: Invasive pneumococcal isolates were obtained from 56 laboratories throughout the country, in 2001-2003. Isolates were serotyped and antimicrobial susceptibilities determined by Sensititre panels and Etest. A new PCR was performed to detect erythromycin resistance genes. Typing methods for selected erythromycin-resistant isolates included PFGE and multilocus sequence typing (MLST). RESULTS: One hundred and fifty-five isolates out of 444 (34.9%) were resistant to erythromycin: 95 isolates (21.4%) carried erm(B), 56 (12.6%) carried mef(A) and three carried both genes. One isolate, carrying neither erm(B) nor mef(A), showed a point mutation in domain V of the 23S rRNA genes. The mef(A)-positive isolates carried subtype mef(A) (47 isolates), subtype mef(E) (nine isolates), and both subtype mef(E) and erm(B) (three isolates). All subtype mef(A) strains, except two, belonged to serotype 14, appeared to be clonally related by PFGE and related to the England14-9 clone by MLST. The two isolates belonging to other serotypes showed different genetic backgrounds. CONCLUSIONS: Erythromycin resistance in S. pneumoniae has increased in the last few years in Italy. erm(B) is still the predominant resistance determinant; however, the increase in erythromycin resistance (34.9% versus 28.8% of the previous years) is mainly due to an increase in the proportion of isolates carrying the efflux pump mef(A), whereas the proportion of isolates carrying erm(B) has not changed.     

Molecular epidemiology of macrolide resistance in beta-haemolytic streptococci of Lancefield groups A, B, C and G and evidence for a new mef element in group G streptococci that carries allelic variants of mef and msr(D)

OBJECTIVES: To study the molecular mechanisms of erythromycin resistance in beta-haemolytic streptococci of Lancefield groups A, B, C and G. METHODS: Erythromycin-resistant clinical isolates from North East Scotland were collected over 2 years. Resistance phenotypes were determined by disc diffusion and MICs by Etest. Resistance genes mef, msr(D), erm(B) and erm(TR) were identified by PCR and mef and msr(D) were sequenced. RESULTS: Erythromycin resistance prevalence was 1.9% in group A streptococci (31 of 1625), 4.3% in group B (53 of 1233), 3.8% in group C (18 of 479) and 6.2% in group G (64 of 1034). The numbers of resistant isolates available were 26, 42, 9 and 52 in each group respectively. The majority of resistant isolates in groups A (57.7%, 15 of 26), B (88.1%, 37 of 42) and G (90.4%, 47 of 52) were MLS(B). The contribution of M phenotype was significant in groups C (77.8%, 7 of 9) and A (42.3%, 11 of 26). Group A isolates carried mef(A) and group B carried mef(E) exclusively. A mef sequence distinct from mef(A) and mef(E) was identified in group G and was associated with a new msr(D) sequence. These sequence variants appear to be part of a new genetic element that is inserted in the comEC gene. A bimodal distribution of erythromycin MICs was noted in erm(TR) isolates. CONCLUSIONS: The results demonstrate significant differences in the mechanisms of macrolide resistance amongst different Lancefield groups in the same geographical area. New sequences show that resistance mechanisms are still evolving.     

Distribution of mef(A) in gram-positive bacteria from healthy Portuguese children

We screened 615 gram-positive isolates from 150 healthy children for the presence of the erm(A), erm(B), erm(C), erm(F), and mef(A) genes. The mef(A) genes were found in 20 (9%) of the macrolide-resistant isolates, including Enterococcus spp., Staphylococcus spp., and Streptococcus spp. Sixteen of the 19 gram-positive isolates tested carried the other seven open reading frames (ORFs) described in Tn1207.1, a genetic element carrying mef(A) recently described in Streptococcus pneumoniae. The three Staphylococcus spp. did not carry orf1 to orf3. A gram-negative Acinetobacter junii isolate also carried the other seven ORFs described in Tn1207.1. A Staphylococcus aureus isolate, a Streptococcus intermedius isolate, a Streptococcus sp. isolate, and an Enterococcus sp. isolate had their mef(A) genes completely sequenced and showed 100% identity at the DNA and amino acid levels with the mef(A) gene from S. pneumoniae.     

Pharmacodynamic modeling of clarithromycin against macrolide-resistant [PCR-positive mef(A) or erm(B)] Streptococcus pneumoniae simulating clinically achievable serum and epithelial lining fluid free-drug concentrations

The association between macrolide resistance mechanisms and clinical outcomes remains understudied. The present study, using an in vitro pharmacodynamic model, assessed clarithromycin (CLR) activity against mef(A)-positive and erm(B)-negative Streptococcus pneumoniae isolates by simulating free-drug concentrations in serum and both total (protein-bound and free) and free drug in epithelial lining fluid (ELF). Five mef(A)-positive and erm(B)-negative strains, one mef(A)-negative and erm(B)-positive strain, and a control [mef(A)-negative and erm(B)-negative] strain of S. pneumoniae were tested. CLR was modeled using a one-compartment model, simulating a dosage of 500 mg, per os, twice a day (in serum, free-drug C(p) maximum of 2 micro g/ml, t(1/2) of 6 h; in ELF, C(ELF(total)) maximum of 35 micro g/ml, t(1/2) of 6 h; C(ELF(free)) maximum of 14 micro g/ml, t(1/2) of 6 h). Starting inocula were 10(6) CFU/ml in Mueller-Hinton broth with 2% lysed horse blood. With sampling at 0, 4, 8, 12, 20, and 24 h, the extent of bacterial killing was assessed. Achieving CLR T/MIC values of > or =90% (AUC(0-24)/MIC ratio, > or =61) resulted in bacterial eradication, while T>MIC values of 40 to 56% (AUC(0-24)/MIC ratios of > or =30.5 to 38) resulted in a 1.2 to 2.0 log(10) CFU/ml decrease at 24 h compared to that for the initial inoculum. CLR T/MIC values of < or =8% (AUC(0-24)/MIC ratio, < or =17.3) resulted in a static effect or bacterial regrowth. The high drug concentrations in ELF that were obtained clinically with CLR may explain the lack of clinical failures with mef(A)-producing S. pneumoniae strains, with MICs up to 8 micro g/ml. However, mef(A) isolates for which MICs are > or =16 micro g/ml along with erm(B) may result in bacteriological failures.