JAC Advance Access originally published online on November 16, 2005
Journal of Antimicrobial Chemotherapy 2006 57(1):139-141; doi:10.1093/jac/dki404
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Macrolide resistance mechanisms and in vitro susceptibility patterns of viridans group streptococci isolated from blood cultures
en Hasçelik21 School of Health Services, Hacettepe University, 06100, Ankara, Turkey; 2 Department of Microbiology and Clinical Microbiology, School of Medicine, Hacettepe University, 06100, Ankara, Turkey
* Corresponding author. Tel: +90-312-3051587; Fax: +90-312-3102730; E-mail: aergin{at}hacettepe.edu.tr
Received 26 May 2005; returned 26 July 2005; revised 6 October 2005; accepted 7 October 2005
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Abstract |
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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 (cMLSB) resistance phenotype and 11 macrolide (M) resistance phenotype. Of the cMLSB isolates 11 had erm(B) genes and 11 of the M phenotype isolates had mef(A) genes. Four of the cMLSB 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 cMLSB phenotype associated with erm(B) and the M phenotype associated with mef(A) genes are found with similar frequencies.
Keywords: erythromycin resistance , MLSB , M phenotype , erm(B) , mef(A) , S. oralis
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Introduction |
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Although they are part of the commensal flora of the human upper respiratory tract, viridans group streptococci (VGS) have been recognized in the aetiology of various diseases such as endocarditis and bacteraemia, and have been associated with shock and respiratory distress syndrome in febrile neutropenic patients.1
It has been shown that VGS can play a significant role as a reservoir of antimicrobial resistance genes transferring different resistance traits to more pathogenic organisms like Streptococcus pneumoniae and Streptococcus pyogenes.2 Penicillin has been the first choice treatment in VGS infections but resistance to ß-lactams and other antimicrobial agents is increasing.1 There are three different resistance mechanisms causing macrolide resistance in streptococcal isolates; first is the target site modification mediated by the erythromycin resistance methylases encoded by the erm(A) or erm(B) genes conferring resistance to macrolide, lincosamide and streptogramin B antibiotics (MLSB phenotype). Expression of MLSB resistance can be constitutive (cMLSB) or inducible (iMLSB).2 The second is the active drug efflux mediated by the membrane-bound efflux protein encoded by the mef(A) gene conferring resistance to 14- and 15-membered macrolides only (M phenotype). There are two subclasses of the mef(A) gene [the mef(A) gene in S. pyogenes and the mef(E) gene in S. pneumoniae strains]. They have been considered a single class of mef(A) gene and MefA protein due to their high homology but a recent study found that mef(A) and mef(E) elements had genetic differences and proposed to refer them as mef(A) subclass mef(A) or subclass mef(E). The element that contains the mef(E) gene is a macrolide efflux genetic assembly (MEGA) and the mef(A)-carrying element is a defective transposon (Tn1207.1). The elements both contain are an open reading frame adjacent to mef and mel in MEGA, which shares 35% identity with the msr(A) gene of Staphylococcus aureus.3 The third mechanism is mutation in the streptococcal 23S rRNA or ribosomal protein genes leading to resistance to macrolide and streptogramin B antibiotics (MS phenotype).2
The objectives of the present study were to determine the macrolide resistance mechanisms and antimicrobial susceptibilities of viridans streptococci isolated from blood cultures.
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Materials and methods |
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Bacterial strains and identification
Eighty-five VGS isolates recovered from blood cultures at Hacettepe University Hospital, Ankara during January 1996 to December 2004 were included in the study. The isolates were identified by colony morphology, Gram stain, catalase and optochin tests. Species identification was determined by standard methods and BD Phoenix Streptococci, SMIC/ID Panel [Becton and Dickinson Diagnostic Systems (BD), Pont de Claix, France].4
Antimicrobial susceptibility and macrolide pheno- and genotyping
Penicillin G, erythromycin, clindamycin, chloramphenicol, tetracycline, levofloxacin, linezolid, quinupristin/dalfopristin and vancomycin susceptibility were determined by the agar dilution method recommended by the CLSI (formerly the NCCLS), using Mueller–Hinton agar supplemented with 5% (v/v) sheep blood.5 Plates were incubated in a 5% CO2 atmosphere for 20–24 h at 35°C. S. pneumoniae ATCC 49619 and Staphylococcus aureus ATCC 29213 were used as quality controls for all antimicrobials tested. The phenotypes of erythromycin-resistant isolates were determined by the double disc test with erythromycin (15 µg) and clindamycin (2 µg) discs as described previously.2 Genomic DNA was extracted as described previously.6 The DNA from erythromycin-resistant isolates was amplified with primers specific for the erm(B), erm(TR) and mef(A) genes.6,7
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Results |
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The clinical isolates included Streptococcus oralis (n = 22), Streptococcus mitis (n = 21), Streptococcus sanguinis (n = 13), Streptococcus anginosus (n = 7), Streptococcus parasanguinis (n = 6), Streptococcus gordonii (n = 5), Streptococcus cristatus and Streptococcus sobrinus (n = 3, each), Streptococcus intermedius (n = 2), Streptococcus constellatus, Streptococcus salivarius and Streptococcus vestibularis (n = 1, each). To determine the association between erythromycin resistance and the resistance to the other antibiotics, the isolates were divided into two groups according to their erythromycin susceptibility (susceptible
0.25 mg/L; intermediate resistant = 0.5 mg/L; resistant 
1 mg/L) (Table 1). Twenty-three strains (27%) were resistant to erythromycin. Erythromycin-resistant strains were also resistant to penicillin, tetracycline and quinupristin/dalfopristin. Sixty-one and 74% of the clindamycin- and chloramphenicol-resistant strains were also resistant to erythromycin, respectively. No resistance to vancomycin, linezolid or levofloxacin was detected.
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The phenotypes among the 23 erythromycin-resistant isolates were as follows: 52% cMLSB (n = 12) and 48% M phenotype (n = 11). The strains with the cMLSB phenotype had higher MICs (all >256 mg/L) of erythromycin than the M phenotype (range = 2–8 mg/L) strains. Of the cMLSB isolates, 11 of the 12 carried erm(B) either alone or together with mef(A) but one isolate did not carry any of the macrolide-resistance genes studied here. All M phenotype isolates were explained by the presence of the mef(A) gene and none had erm(B) in combination. None of the isolates amplified the erm(TR) gene. The distribution of the erythromycin-resistant isolates in accordance to species type is shown in Table 2.
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Discussion |
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Of the 85 viridans streptococcal blood culture isolates of this study 27% were resistant to erythromycin. The erythromycin-resistance level was comparable to previous reports from different countries in which Rodriguez-Avial et al.8 from Spain reported 45.6% and Uh et al.9 from Korea reported 33.9% erythromycin resistance among their blood culture isolates.
The phenotype and genotype frequencies from different countries have usually shown that cMLSB and erm(B) gene (
60%) are the most prevalent resistance mechanism among the VGS isolates from blood cultures.8–10 We have detected methylase type resistance responsible for the cMLSB phenotype in 52% of the erythromycin-resistant isolates and all of the isolates have harboured the erm(B) gene, except one. Strains carrying erm(B) genes displayed higher MICs of erythromycin (>256 mg/L) than those with a mef(A) gene (MICs from 2–8 mg/L) but in 4 of 12 isolates with the cMLSB phenotype, the erm(B) gene was combined with a mef(A) gene. Combination of both genes has already been detected in erythromycin-resistant S. pneumoniae and Streptococcus agalactiae and this accumulation of genes in strains of VGS has been suspected of being a possible reservoir and source of genetic exchange of the resistance genes to pathogenic streptococci.10 Efflux type resistance (M phenotype) was found in 48% of our erythromycin-resistant isolates and in all these isolates mef(A) was detected while none carried erm(B). The molecular characterization of our VGS isolates revealed that the erm(B) and mef(A) genes are found with similar frequencies.
A common point among studies of VGS blood isolates is that the erythromycin-resistant isolates either did not show or very rarely harboured the iMLSB type of resistance.8–10 We also did not find this phenotype. In order not to misidentify the iMLSB phenotype, we also performed the double disc test with 4 mm rather than the 15–20 mm distance between the erythromycin and clindamycin discs as suggested by Seppala et al.,2 and could not find any difference from the conventional method. The PCR results have also revealed that our isolates do have erm(B) and mef(A) genes alone.
The most commonly isolated erythromycin-resistant species in our study in rank order were S. oralis, S. mitis, S. cristatus, S. intermedius, S. parasanguinis and S. salivarius. In accordance with a previous report from the UK (51%), we also noted a high rate (57%) of S. oralis (n = 13) isolates resistant to erythromycin.1 The M phenotype and mef(A) gene predominated among S. oralis isolates which was different from other reports.3,10
Erythromycin-resistant VGS isolates were also resistant to tetracycline, penicillin and quinupristin/dalfopristin (Table 1). The resistance to tetracycline might be due to a transposon encoding the genes of tet(M) and erm(B).3 Thus the erythromycin-resistant isolates were also resistant to tetracycline but the presence of intermediate penicillin- and quinupristin/dalfopristin-resistant isolates was noteworthy.
As the VGS have an ability to spread antibiotic resistance genes to other bacteria, especially to S. pneumoniae, and can exhibit cross-resistance to many antimicrobial agents, continuous surveillance of the isolates should be performed in routine laboratories.
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No declarations were made by the authors of this paper.
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References |
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1. Smith A, Jackson MS, Kennedy H. Antimicrobial susceptibility of viridans group streptococcal blood isolates to eight antimicrobial agents. Scand J Infect Dis 2004; 36: 259–63.[CrossRef][Medline]
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Seppala H, Haanpera M, Al-Juhaish M et al. Antimicrobial susceptibility patterns and macrolide resistance genes of viridans group streptococci from normal flora. J Antimicrob Chemother 2003; 52: 636–44.
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Zolezzi PC, Laplana LM, Calvo CR et al. 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. Antimicrob Agents Chemother 2004; 48: 3462–7.
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Facklam R. What happened to the streptococci: overview of the taxonomic and nomenclature changes. Clin Microbiol Rev 2002; 15: 613–30.
5. Clinical and Laboratory Standards Institute (formerly NCCLS). Performance Standards for Antimicrobial Susceptibility Testing; Fifteenth Informational Supplement: M100-S15. CLSI, Wayne, PA, USA, 2005.
6. Sutcliffe J, Grebe T, Tait-Kamradt A et al. Detection of erythromycin-resistant determinants by PCR. Antimicrob Agents Chemother 1996; 40: 2562–6.[Abstract]
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Seppala H, Skurnik M, Soini H et al. A novel erythromycin resistance methylase gene (ermTR) in Streptococcus pyogenes. Antimicrob Agents Chemother 1998; 42: 257–62.
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Rodriguez-Avial I, Rodriguez-Avial C, Culebras E et al. In vitro activity of telithromycin against viridans group streptococci and Streptococcus bovis isolated from blood: antimicrobial susceptibility patterns in different groups of species. Antimicrob Agents Chemother 2005; 49: 820–3.
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Uh Y, Shin DH, Jang IH et al. Antimicrobial susceptibility patterns and macrolide resistance genes of viridans streptococci from blood cultures in Korea. J Antimicrob Chemother 2004; 53: 1095–7.
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Achour W, Guenni O, Malbruny B et al. Phenotypic and molecular characterization of macrolide and streptogramin resistance in Streptococcus mitis from neutropenic patients. J Antimicrob Chemother 2004; 54: 117–21.
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