Oropharyngeal carriage of macrolide-resistant viridans group streptococci: a prevalence study among healthy adults in Belgium

Surbhi Malhotra-Kumar1,*, Christine Lammens1, An Martel2, Cecile Mallentjer1, Sabine Chapelle1, Jeanine Verhoeven1, Monique Wijdooghe1, Freddy Haesebrouck2 and Herman Goossens1

1 Department of Medical Microbiology, University of Antwerp, S3, Universiteitsplein 1, B-2610 Wilrijk, Antwerp; 2 Department of Pathology, Bacteriology and Poultry Diseases, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium

Received 31 July 2003; returned 26 August 2003; revised 8 October 2003; accepted 16 October 2003


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives: Viridans group streptococci (VGS) are gaining significance as reservoirs of resistance determinants for respiratory tract pathogens. Our aim was to investigate healthy adults for oropharyngeal carriage of VGS that are resistant to macrolides, as well as to other common antibiotics.

Methods: Macrolide-resistant VGS were isolated from throat samples of 154 healthy Belgian adults, and phenotyped and genotyped for erm(A), erm(B) and mef(A). In vitro susceptibilities to 10 antimicrobials and the presence of tetracycline resistance genes were also determined.

Results: Carriage was detected in 71% of the population screened, from whom 157 unique, macrolide-resistant VGS were isolated. A constitutive (cMLS) phenotype was present in 105 isolates, of which 102 isolates carried either erm(B) or erm(B) + mef(A). The remaining three isolates did not present with any of the genes studied. All 45 isolates showing the M phenotype carried mef(A). The least abundant inducible (iMLS) isolates (n = 7) carried either erm(B) or erm(B) + mef(A). The most abundant macrolide-resistant VGS species was Streptococcus mitis (51%). Co-resistance to tetracycline was identified in 114 isolates, of which tet(M) was present in 105, tet(O) in two and both tet(M) and tet(O) in one, while the remaining six isolates did not present with either gene. tet(M) was also present in four tetracycline susceptible and two intermediately resistant isolates. Fluoroquinolone resistance (ciprofloxacin MIC >= 4 mg/L) was detected in 16 isolates. Resistance to telithromycin, penicillin and chloramphenicol was appreciably low.

Conclusions: This study highlights a high oropharyngeal carriage of macrolide-resistant VGS and its co-resistance to tetracycline and fluoroquinolones among healthy Belgian adults.

Keywords: oral flora, commensals, tetracycline


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The aerobic, oropharyngeal commensal flora consists predominantly of viridans group streptococci (VGS), which play an important role in inhibiting colonization by pathogens. However, recent studies have indicated that VGS are increasingly becoming resistant to many antibiotics, including macrolides.1,2

Macrolides are an important group of antibiotics used frequently for Gram-positive bacterial infections, and act by blocking protein synthesis on assembled and functioning 50S rRNA subunits, and by inhibiting the assembly of new 50S ribosomal subunits. Streptococci acquire macrolide resistance by three mechanisms: (i) by post-transcriptional modification of the 23S rRNA subunit by adenine-N6 methyltransferases (encoded by erm) that add one to two methyl groups to a single adenine (A2058 in Escherichia coli) in the 23S rRNA moiety; (ii) by an efflux pump mechanism mediated by the mef(A) gene product that helps to create a low intracellular drug concentration; and (iii) by ribosomal mutations in the key antibiotic binding site. Although the last mechanism can be acquired de novo, the most common way that closely related bacterial species exchange resistance determinants is through in vivo genetic transfer, especially under the selective pressure of an antibiotic. Accordingly, although no causal relationship has yet been established, salivary concentrations of administered antibiotics show a strong correlation with an increased colonization of resistant strains.3 Macrolides can achieve moderate to high levels of salivary concentrations 3 and upon administration are known to cause an emergence of resistant viridans streptococci in the oropharynx.4 As many other pathogenic streptococcal species, i.e. Streptococcus pneumoniae or Streptococcus pyogenes, are increasingly becoming resistant to macrolides, the potential role of VGS as a reservoir of macrolide resistance cannot be ignored. Furthermore, resistance among oropharyngeal VGS poses a considerable threat on its own to neutropenic patients, e.g. cancer patients undergoing chemotherapy. Lastly, induction of macrolide resistance might also inadvertently select for tetracycline resistance as their major resistant determinants, erm(B) and tet(M), are often present on the same mobile element.

The purpose of this prevalence study among healthy Belgian adults was to assess the aforementioned threat by studying the oropharyngeal carriage of macrolide-resistant VGS, and their concomitant resistance to other major orally administered antibiotic groups. To our knowledge, this is one of the first comprehensive studies evaluating resistance to all major oral antibiotic groups in a well-characterized set of commensal strains from adult healthy carriers.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Strain collection and identification

Pharyngeal swabs were collected from 154 healthy (at the time of sampling), non-pregnant volunteers in 2001. All participants were 17–25 years old. The swabs obtained were directly plated onto Mueller–Hinton agar plates (BBL, Cockeysville, MD, USA) with 5% defibrinated horse blood (E&O Laboratories, Bonnybridge, UK) containing 1 mg/L erythromycin (Sigma Chemical Co., St Louis, MO, USA). Plates were incubated at 37°C for 18–24 h in 5% CO2.

Strain characterization and susceptibility testing

A total of five putative streptococcal colonies per volunteer were chosen from each selective plate based on their morphology ({alpha}-haemolysis) and were further confirmed by compatible Gram stain and catalase negative tests. These isolates were further speciated by tRNA intergenic spacer length polymorphism analysis (tDNA-PCR).5 Isolates that could not be differentiated on the basis of the tDNA-PCR, i.e. Streptococcus oralis/mitis/pneumoniae, Streptococcus thermophilus, Streptococcus parasanguinis and Streptococcus vestibularis, were further speciated by Rapid ID 32 STREP (bioMérieux, Marcy l’Étoile, France). Test strips were interpreted using the API LAB Plus Electronic Code book (version 3.3.3; bioMérieux). Assignment of isolates to groups/species was carried out according to the criteria of Facklam.6 At this stage, if two or more isolates of the same species were recovered from one volunteer, PFGE was performed (see below). Differentiation of erythromycin-resistant streptococcal isolates into the constitutive (cMLS), inducible (iMLS) and M phenotypes was done by conventional double disc diffusion test with erythromycin (78 µg) and clindamycin (25 µg) Neo-Sensitab discs (Rosco, Taastrup, Denmark). MICs of clarithromycin (Abbott, Ottignies, Belgium), azithromycin (Pfizer, Groton, CT, USA), telithromycin (Aventis, Romainville, France), erythromycin, clindamycin, penicillin, ciprofloxacin, chloramphenicol, tetracycline and minocycline (all from Sigma Chemical Co.) were determined by agar dilution using Mueller–Hinton agar with 5% defibrinated horse blood. A 104 cfu/spot inoculum was incubated under aerobic conditions at 37°C for 18–24 h. MIC resistance breakpoints, defined by the NCCLS,7 were as follows (mg/L): erythromycin, clindamycin and clarithromycin, >=1; azithromycin, >=2; tetracycline, >=8; chloramphenicol, >=16; penicillin, >=4. For minocycline, NCCLS breakpoints for tetracycline were applied; for ciprofloxacin, breakpoint of resistance was taken as >=4 mg/L; for telithromycin, breakpoints of susceptibility and resistance were taken as <=1 and >=4 mg/L, respectively. Staphylococcus aureus ATCC 29213 and S. pneumoniae ATCC 49619 were used as controls.

Detection of resistance determinants

Genomic DNA was extracted by the alkaline lysis method (0.25% SDS, 0.05 N NaOH). PCR was performed with a DNA thermal cycler (9600 GeneAmp PCR system; Perkin-Elmer, Zaventem, Belgium). The primers described previously for erm(A), erm(B) and mef(A) give PCR products of 590, 639 and 348 bp, respectively.8,9 PCR for erm(A), erm(B) and mef(A) was carried out as described previously.8,10 For the tetracycline resistance genes, tet(M) and tet(O), a duplex PCR was performed using primers described previously, giving PCR products of 406 and 515 bp, respectively.11 While the cycling conditions were the same as described previously,11 the PCR mixture (50 µL) had a few modifications and consisted of 50 mM KCl, 10 mM Tris–HCl (pH 9.0), 3 mM MgCl2, 0.1% Triton X-100, 0.01% gelatin, 300 µM dNTPs, primers [0.5 µM for tet(M) and 1.25 µM for tet(O)], 0.9 U of SuperTaq DNA polymerase, and 2 µL of template DNA. After amplification, the detection and visualization of PCR products was carried out as described previously.10 Positive and negative controls were included in each run.

PFGE

To prevent reporting of clonal isolates, PFGE was performed. For this, the original protocol outlined by Kennedy et al.12 was followed, with the following modifications: cultures were centrifuged at 16 000g for 5 min, and the clot washed and resuspended in 1 mL of cold ST buffer (1 M NaCl, 10 mM Tris; Gibco-BRL, Paisley, UK). Cells were lysed using lysozyme (0.00288 g) and mutanolysin (250 U) in EC lysis buffer (1 M Tris pH 7.6, 5 M NaCl, 0.5 M EDTA pH 8, 4% deoxycholate, 10% N-lauryl sarcosine and 10% polyoxyethylene cetyl ether). All chemicals were purchased from Sigma Chemical Co. unless otherwise indicated. Electrophoresis, gel visualization, and further analysis were carried out as described previously.10

Statistical analyses

Pearson’s {chi}2-test with Yates’ continuity correction was used to attribute statistical significance to the co-occurrence of erm(B) and tet(M). Significance between the genotypes or MICs with the phenotype treated as a continuous variable (with M phenotype, iMLS and cMLS treated as 1, 2 and 3, respectively) was performed by a two-way ANOVA (Splus; Insightful Co., Seattle, WA, USA). P < 0.05 was considered significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Of the 154 volunteers studied, 110 (71%) presented with oropharyngeal carriage of macrolide-resistant VGS. In 75 carriers, only one unique macrolide-resistant isolate was present, while in 35 volunteers (32% of the carriers) more than one such isolate was recovered from the same individual. These were two (24 subjects), three (10 subjects) or four (one subject) unique isolates, yielding a total of 157 unique macrolide-resistant VGS isolates. Uniqueness was determined by PFGE profiles, as depicted in Figure 1. In many instances, the multiple unique VGS isolated from a subject even belonged to the same species, for instance, S. mitis. Such same-species, unique isolates were isolated twice from 16 individuals and three times in two individuals. A further analysis of the unique isolates identified a cMLS phenotype in 105 isolates, while an iMLS and M phenotype were present in seven and 45 isolates, respectively (Table 1, first two columns). On speciation, 80 macrolide-resistant isolates belonged to S. mitis (51%) and the other viridans species identified are enumerated in the first two columns of Table 2, while the tDNA-PCR fingerprint patterns of some of the species isolated are shown in Figure 2. Results of the in vitro activities of 10 antimicrobials against the three macrolide-resistant phenotypes are presented in Table 3.



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Figure 1. PFGE pattern of unique iso-species macrolide-resistant VGS from three subjects. Lanes 1 and 2, S. salivarius strains isolated from subject 1; lanes 3 and 4, S. mitis recovered from subject 2; and lanes 5 and 6, S. mitis from subject 3. Chromosomal DNA was digested with SmaI. The PFGE profiles were obtained by analysis with the computer software Gelcompar version 4.0 (Applied Maths, Kortrijk, Belgium).

 

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Table 1. Prevalence of macrolide and tetracycline resistance genes according to macrolide resistance phenotype
 

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Table 2. Prevalence of macrolide and tetracycline resistance genes according to species
 


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Figure 2. tDNA-PCR fingerprint patterns of streptococcal strains. (a) S. vestibularis LMG 13516T; (b) Streptococcus constellatus LMG 14504; (c) S. oralis LMG 14533; (d) S. salivarius LMG 11489T; (e) S. sanguinis LMG 14702T. The x-axis represents the fragment length in base pairs ; the y-axis represents the peak intensity.

 

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Table 3. In vitro susceptibilities of 157 oropharyngeal isolates of VGS
 
Phenotypes of the macrolide-resistant isolates obtained on disc diffusion (i.e. cMLS, iMLS or M phenotype) correlated well with MICs of erythromycin and clindamycin (P < 0.001). These MICs, as expected, were higher for isolates with a cMLS phenotype than for those with an M phenotype. Only eight cMLS isolates had an erythromycin MIC <= 16 mg/L, while none exceeded 16 mg/L for the isolates with the M phenotype.

The phenotypic profiles also correlated well with the genotypes (P < 0.001) (Table 1). Of the cMLS isolates, 102 of the 105 carried erm(B) either alone or together with mef(A), but three cMLS isolates did not carry any of the macrolide resistance genes studied here. This suggests the existence in these isolates of other resistance genes or mechanisms, i.e. ribosomal mutations. All iMLS and M phenotype isolates were explained by the presence of either erm(B) or mef(A), or both. None of the isolates carried erm(A). Cumulatively, erm(B) + mef(A) was present in 28 isolates, of which 26 isolates belonged to the cMLS phenotype and two to the iMLS phenotype (Table 1); however, no significant difference existed in the macrolide MIC levels between strains carrying only erm(B) and those carrying erm(B) + mef(A). The resistance mechanisms also seemed to show some specificity for a given viridans species. Among the three major species isolated, S. mitis, Streptococcus salivarius and Streptococcus sanguinis (80, 31 and 21 isolates, respectively), S. mitis showed the highest percentage prevalence of mef(A) and of erm(B) + mef(A), while erm(B) alone occurred with maximum frequency among S. sanguinis (Table 2).

The 157 macrolide-resistant isolates were also analysed for resistance to other antibiotics. Tetracycline resistance (MIC >= 8 mg/L) was noted in 114 isolates, of which 105 isolates carried tet(M) alone, one carried tet(M) and tet(O), two isolates carried only tet(O) and six resistant isolates did not present with either gene (Table 1, last three columns). In addition, tet(M) was also detected in four completely susceptible isolates (MIC <= 2 mg/L) and two isolates that were intermediately resistant to tetracycline (MIC 4 mg/L). Co-occurrence of tet(M) and erm(B) showed a significant correlation (P < 0.001). The species showing the highest percentage prevalence of tet(M) was S. salivarius (Table 2). Fluoroquinolone resistance (defined as ciprofloxacin MIC >= 4 mg/L) was found in 16 isolates, and 12 of these were S. mitis. Penicillin and telithromycin resistance (both with MIC 4 mg/L) was observed in one and four isolates, respectively, and two isolates were intermediately resistant to chloramphenicol (MIC 8 mg/L).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Acquisition of resistance genes by commensals does not transform them into pathogens, since the necessary virulence factors required for invading an immunocompetent host are lacking. Nevertheless, antibiotic-resistant VGS poses two notable threats: (i) in patients with underlying neutropenia, where antibiotic-resistant VGS might itself cause life-threatening infections; and (ii) in healthy individuals, where antibiotic-resistant VGS might transfer the resistance determinants to pathogenic streptococci.

Here, we show macrolide-resistant VGS in >70% of the healthy Belgian population. Carriage rates in Belgium, although lower than those recently reported in Spain (94%),13 are still quite high. Measures were taken not to overestimate the prevalence of different macrolide-resistant VGS clones by performing PFGE if more than one iso-species isolate was recovered from any one individual. Half of our unique macrolide-resistant isolates were S. mitis, while the other half were distributed among eight different VGS species. Although speciating VGS is labour intensive, we believe that an accurate identification down to species level is important for studying macrolide-resistant VGS, as major differences among the species in relation to macrolide resistance are observed. Moreover, S. mitis is also the predominant and most resistant species isolated from blood cultures of patients with streptococcal infections,2 reiterating the importance of such resistant commensals becoming opportunistic pathogens.

We further demonstrated a predominance of the erm(B)-mediated cMLS phenotype among the macrolide-resistant VGS, which parallels an earlier study in Belgium on macrolide-resistant S. pneumoniae where cMLS was also the predominant phenotype.14 A predominance of the same resistance determinants in both commensals and pathogenic streptococci strongly suggests inter-species gene transfer. Furthermore, we identified one of the highest prevalences of erm(B) and mef(A) co-carriage reported so far among commensal or blood culture isolates. Although we did not show here any higher MIC levels for erm(B) + mef(A) isolates compared with erm(B) alone, the former might offer a subtle selective advantage not captured by the current MIC assays, or the samples were not enough to reach statistical significance.

Among the tet genes, tet(M) was most prevalent in our study, confirming recent data on VGS isolated from blood cultures.1 Furthermore, our observations of a much higher prevalence of tetracycline resistance in our macrolide-resistant isolates (72%) than reported in general isolates (12%),2 together with a strong statistical correlation between tet(M) and erm(B), supports earlier observations that both these genes are carried on the same transposon.

Penicillin resistance was observed in only one macrolide-resistant isolate. This contrasts with recent data on VGS where comparable levels of penicillin and macrolide resistance have been observed,2,15 and in S. pneumoniae, macrolides have been shown to co-select for both high-level macrolide and penicillin resistance.16

Lastly, we report a 10% prevalence of fluoroquinolone resistance in macrolide-resistant isolates, the majority of which were S. mitis, which is comparable to the prevalence noticed in VGS from healthy controls and blood culture isolates in other studies.15,17 A similar resistance rate in oral and blood culture isolates only serves to re-emphasize the strong possibility of endogenous VGS infections in neutropenic patients.

To conclude, we report here a high oropharyngeal carriage of macrolide-resistant VGS among the healthy Belgian population. Macrolide and tetracycline resistance co-occurred in the majority of the strains, with a strong statistical correlation between erm(B) and tet(M). Penicillin resistance was conspicuously absent from our isolates, while fluoroquinolone resistance was uniquely present. The latter finding in the macrolide-resistant VGS has prompted us to undertake a more extensive study to ascertain the actual carriage rate of fluoroquinolone-resistant VGS in Belgium.


    Acknowledgements
 
We gratefully acknowledge Dr Patrice Courvalin for providing control strains for tet(M) (S. pyogenes BM137) and tet(O) (Enterococcus faecalis BM4110).


    Footnotes
 
* Corresponding author. Tel: +32-3-820-25-51; Fax: +32-3-820-26-63; E-mail: surbhi.malhotra{at}ua.ac.be Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Rodriguez-Avial, I., Rodriguez-Avial, C., Culebras, E. et al. (2003). Distribution of tetracycline resistance genes tet(M), tet(O), tet(L) and tet(K) in blood isolates of viridans group streptococci harbouring erm(B) and mef(A) genes. Susceptibility to quinupristin/dalfopristin and linezolid. International Journal of Antimicrobial Agents 21, 536–41.[CrossRef][ISI][Medline]

2 . Doern, G. V., Ferraro, M. J., Brueggemann, A. B. et al. (1996). Emergence of high rates of antimicrobial resistance among viridans group streptococci in the United States. Antimicrobial Agents and Chemotherapy 40, 891–4.[Abstract]

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17 . Guerin, F., Varon, E., Hoi, A. B. et al. (2000). Fluoroquinolone resistance associated with target mutations and active efflux in oropharyngeal colonizing isolates of viridans group streptococci. Antimicrobial Agents and Chemotherapy 44, 2197–200.[Abstract/Free Full Text]