High proportion of pharyngeal carriers of commensal streptococci resistant to erythromycin in Spanish adults

Emilio Pérez-Tralleroa,b,*, Diego Vicentea, Milagrosa Montesa, José M. Marimona and Luis Piñeiroa

a Servicio de Microbiología, Hospital Donostia and b Departamento de Medicina Preventiva y Salud Pública, Facultad de Medicina, Universidad del País Vasco, San Sebastián, Spain


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The presence of erythromycin-resistant (ErR) commensal streptococci in the throat of 110 healthy subjects and 87 patients with pharyngitis was investigated. The resistance determinants were studied by PCR using the primers for mef and erm genes, followed by hybridization and sequencing analysis. Overall, 94.4% of the subjects carried one or more ErR strains in their pharynx. A total of 253 ErR strains was studied: 127 (50.2%) showed constitutive or inducible resistance to clindamycin (MLSB phenotype) and 126 (49.8%) were susceptible to clindamycin (M phenotype). In 50 subjects (25.4%) both phenotypes were detected. The ermB gene was predominant among the MLSB phenotype strains (97.6%). The mefA (mefA/mefE) gene was detected in 100% of the strains with the M phenotype. One Streptococcus oralis strain bearing the MLSB phenotype carried both mefA and ermB genes. The mefA gene from clinical isolates of Streptococcus mitis and S. oralis was transferred by conjugation to an erythromycin-susceptible Streptococcus pneumoniae strain.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
{alpha}-Haemolytic streptococci comprise 30–60% of the oral bacterial flora together with other bacteria such as anaerobes, Streptococcus pneumoniae and, in some cases, Streptococcus pyogenes.1 With the exception of the well-established association with infective endocarditis, {alpha}-haemolytic streptococci have a low infective potential. Some studies have found high percentages of resistance to erythromycin in clinical isolates of this group of bacteria.24 The possibility of transmission of genes encoding anti-biotic resistance from non-pathogenic bacteria such as {alpha}-haemolytic streptococci to pathogenic bacteria has been described.57 S. pneumoniae and S. pyogenes are frequently involved in clinical infections and erythromycin is considered to be an adequate alternative to penicillin in the treatment of both types of infection. Erythromycin-resistant S. pneumoniae and erythromycin-resistant S. pyogenes have been reported in various parts of the world with increased frequency during the past few years.814 This resistance is mediated mainly by two resistance genes: ermB and mefA. mefA is also the name presently used to refer to the genes originally described as mefE and mefA by Sutcliffe et al.13 and Clancy et al.,15 respectively. The aim of this study was to investigate the prevalence and type of macrolide resistance in non-haemolytic and {alpha}-haemolytic commensal streptococci from the pharynx. A high level of prevalence could be a possible source of macrolide resistance for S. pneumoniae and S. pyogenes.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pharyngeal swabs were collected from 197 subjects during two time periods: March–August 1998 and March 2000. In the first period, 87 subjects had pharyngitis and 56 were healthy. In the second period, all 54 were healthy. All healthy subjects were aged between 18 and 39 years. The samples were plated on CNA Columbia agar (Becton Dickinson, Cockeysville, MD, USA), which contains colistin (0.01 mg/L) and nalidixic acid (0.01 mg/L), supplemented with 5% horse blood (Oxoid Ltd, Basingstoke, UK) and erythromycin (Abbott Laboratories, Abbott Park, IL, USA). During the first period the erythromycin concentration in the selective plates was 2 mg/L; later we used 1 mg/L, in accordance with the most recent breakpoint for erythromycin non-susceptibility. Plates were incubated at 35°C for 18–24 h in 5% CO2.

Identification

Based on the different colony morphology, two or three colonies from each selective culture were studied. Catalase-negative, non-ß-haemolytic and compatible colonies by Gram's stain were identified by rapid ID 32 STREP (bioMérieux, Marcy l'Étoile, France). The test strips were read after 4 h, and identification was obtained using the API LAB Plus Electronic Code Book (Version 3.3.3; bioMérieux).

Susceptibility studies and detection of clindamycin-inducible strains

All streptococci isolated from the selective plates were confirmed as erythromycin resistant by disc diffusion16 after 18–24 h of incubation on Mueller–Hinton agar (Becton Dickinson) supplemented with 5% horse blood agar at 35°C in 5% CO2. At the same time clindamycin-inducible strains were detected. Two erythromycin discs were placed, one apart from and the other near to (at a distance of 15– 20 mm) a clindamycin disc. After incubation the presence or absence of blunting was recorded in the zone of inhibition of the clindamycin disc.

MICs of erythromycin and clarithromycin (Abbott Laboratories), roxithromycin (Hoechst-Marion-Roussel, Romainville, France), azithromycin (Pfizer Inc., New York, NY, USA), josamycin (ICN Biomedicals, Costa Mesa, CA, USA), midekamycin (Meiji Pharmaceutical Co., Ltd, Tokyo, Japan), clindamycin (Upjohn, Kalamazoo, MI, USA), streptogramin A and streptogramin B (Rhone-Poulenc, Vitry-Alforville, France) were determined by agar dilution using Mueller–Hinton agar supplemented with 5% horse blood. The inoculum was 104 cfu/spot and plates were incubated for 18–24 h in 5% CO2. S. pneumoniae ATCC 49619 and Staphylococcus aureus ATCC 29213 were included as controls.

Presence of the mef and erm genes

Genomic DNA was prepared with Qiagen columns (Chatsworth, CA, USA) after initial incubation with lysozyme (Sigma Chemical Co., St Louis, MO, USA) (4 g/L). A polymerase chain reaction (PCR) was performed using the specific primers (Amersham Pharmacia, Amersham, UK) identified by Clancy et al.15 and Tait-Kamradt et al.17 in order to detect the genes originally described as mefA and mefE, now both known as mefA.18 The primers described by Sutcliffe et al.19 were used for ermA, ermB and ermC genes, whereas the primers identified by Kataja et al.20 were used to detect ermTR. When the PCR products yielded the expected size, microwell hybridization analysis21 was performed using specific biotinylated capture probes. Negative and positive controls were included in each run.

Sequencing

Following PCR, the amplicons obtained for the erythromycin-resistant strains were sequenced automatically with the primers described for the original mefE and ermB genes by ABI 300 Genetic Analyzer (Perkin Elmer Applied Biosystem, Foster City, CA, USA). These sequences were compared with those described for the original S. pneumoniae mefE gene (GenBank accession no. U83667), the original S. pyogenes mefA gene (GenBank accession no. U70055) and the S. pneumoniae ermB gene (GenBank accession no. X52632), respectively, using the software available at the website of the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov).

Mating experiments

An erythromycin-susceptible (MIC < 0.5 mg/L) S. pneumoniae R6 strain22 made rifampicin resistant (MIC 64 mg/L) by stepwise selection was used as the recipient. Conjugation was performed as described previously.7 Transconjugants were selected on blood agar supplemented with erythromycin 2 mg/L and rifampicin (Sigma Chemical Co.) 8 mg/L. DNase (Sigma Chemical Co.) at a final concentration of 1 g/L was added to some matings to rule out transformation.

Detection of the mefA gene and its sequencing in the commensal and transconjugant strains were performed as described previously.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Overall, 186 of 197 subjects (94.4%) carried ErR commensal streptococci in their pharynx. There were no statistical differences in the number, the species or the resistance phenotypes of erythromycin-resistant {alpha}-haemolytic streptococci found in healthy subjects or subjects with pharyngitis, nor between the two time periods. Ninety-five of 110 healthy subjects had received no antibiotics during the 3 month period before throat sampling. The history of antibiotic therapy was not recorded for the subjects with pharyngitis. A total of 253 non-ß-haemolytic ErR strains was studied (one, two and three isolates of a different species or resistance phenotype from 123, 59 and four subjects, respectively). One hundred and twenty-six of them were clindamycin susceptible (M phenotype) and 127 were clindamycin resistant (120 showed a constitutive resistance and seven an inducible resistance) (MLSB phenotype). Both phenotypes were detected in 50 subjects.

Overall, 224 isolates belonged to the Streptococcus mitis group and 12 to the Streptococcus salivarius group. Seventeen isolates could not be attributed to any group (Table IGo). In vitro activities of nine antimicrobials in a randomly selected sample of 112 ErR strains were studied. MICs of macrolides, clindamycin and streptogramin B were higher for the strains with the MLSB phenotype than for the strains with the M phenotype (Table IIGo). Only five strains with the MLSB phenotype had an erythromycin MIC < 32 mg/L and those five were clindamycin inducible.


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Table I. Distribution of ErR commensal streptococci by phenotype and principal resistance genes
 

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Table II. MICs (mg/L) of macrolides, clindamycin and streptogramin antibiotics for 112 ErR commensal streptococci
 
In all 126 ErR clindamycin-susceptible strains the mefA gene was detected by PCR and the hybridization test was positive. Sequencing of the PCR product of the mefA gene of six ErR strains (three S. oralis, one S. salivarius and two S. mitis) exhibited >=90% identity when compared with the mefA gene. Four strains showed >=98% identity with the original mefE gene described for S. pneumoniae and two strains exhibited >=98% identity with the original mefA gene described for S. pyogenes.

The ermB gene was detected in 124 of the 127 ErR strains harbouring the MLSB resistance phenotype. In the other three strains no amplicons were detected using the primers described in Materials and methods. Sequencing the amplicon of the ermB gene of one S. oralis strain showed 96% identity when compared with the ermB gene described for S. pneumoniae.

One S. oralis strain bearing the MLSB phenotype carried both mefA and ermB genes. Sequencing the respective amplicons showed 98% identity with the original mefE gene described for S. pneumoniae and 98% identity with the ermB gene described for S. pneumoniae.

The mefA gene from clinical isolates of S. mitis and S. oralis was transferred by conjugation to an erythromycin-susceptible, rifampicin-resistant strain of S. pneumoniae. Both the donor and the transconjugant strains were resistant to erythromycin, clarithromycin and azithromycin but susceptible to clindamycin. The addition of DNase did not affect the gene transfer. The sequences obtained for the donor and transconjugant strains were >98% identical to the original mefA gene described for S. pyogenes.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
More than 90% of the subjects enrolled in this study carried ErR strains in their pharynx. This fact is disturbing, since in the past {alpha}-haemolytic streptococci were considered to be uniformly susceptible to macrolides. Detection of antibiotic-resistant strains after previous antibiotic treatment is fairly common.2325 However, 86.4% of the subjects interviewed indicated that they had not taken antibiotics during the previous 3 months. Some of them stated that they had never taken macrolides. Therefore, most of the strains isolated in the pharynx of these subjects were probably already erythromycin resistant when acquired. The transmission of resistant strains from person to person seems to be a frequent event.2427 The possibility that ErR strains transfer in vivo their resistance to pathogenic streptococci is an even greater cause for concern. The fact that gene transfer of macrolide-resistant determinants may occur has been known for more than a decade.28 S. pneumoniae and S. pyogenes inhabit the pharyngeal tract and they can interchange resistance genes with commensal streptococci.

With variations depending on which resistance phenotype is predominant in each country (M or MLSB), the macrolide resistance shown by S. pneumoniae or S. pyogenes is mediated mainly by two resistance genes: ermB and mefA. The ermB gene determines constitutive or inducible cross-resistance to macrolides, lincosamides and streptogramin B antibiotics. The mefA gene determines resistance only to 14- and 15-membered macrolides. In this study, 49.8% of the ErR strains harboured the mefA gene and 49% carried the ermB gene. With the exception of one strain that carried both resistance genes, each type of strain expressed the appropriate resistance phenotype: the M phenotype for those carrying the mefA gene and the MLSB phenotype for those harbouring the ermB gene. The strain that carried both the mefA and ermB genes showed the MLSB phenotype. This duplicity of genes and its expression as MLSB phenotype has already been identified in S. pneumoniae by other authors.7,29

The DNA of the original mefE13 and the original mefA15 genes is >90% identical. Nevertheless, this slight difference must be highlighted owing to the differences in prevalence depending on the Streptococcus species. Both genes have been identified in S. pneumoniae and S. pyogenes, although mefE is found more frequently in S. pneumoniae whereas mefA is predominant in S. pyogenes. Both variants of the new mefA gene have been detected in the ErR strains by sequencing. However, only the original mefA could be transferred by conjugation to the S. pneumoniae R6 strain. The experiment was not successful with the original mefE. Luna et al.7 transferred the mefA gene from a strain of {alpha}-haemolytic streptococci to Enterococcus faecalis and S. pneumoniae recipients but they did not specify which type of mefA gene was transferred. Whether or not the large number of subjects with ErR strains in their pharynx influences the high percentage of clinical isolates of erythromycin-resistant S. pneumoniae30 and S. pyogenes14,31 found in Spain is presently unknown.

Taking into account the growing public health problem of antimicrobial resistance, more studies are needed to clarify the exact role played by commensal streptococci of the pharynx as recipients and donors of resistance genes.


    Notes
 
* Correspondence address. Servicio de Microbiología, Hospital Donostia, Paseo Dr Beguiristain s/n, 20014 San Sebastián (Gipuzkoa), Spain. Tel: +34-943-00-70-46; Fax: +34-943-00-70-63; E-mail: labmikro{at}terra.es Back


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 Introduction
 Materials and methods
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 Discussion
 References
 
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Received 25 January 2001; returned 29 March 2001; revised 30 April 2001; accepted 4 May 2001