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Phenotype, genotype, and serotype distribution of macrolide resistant invasive and non-invasive Streptococcus pneumoniae strains, in Sousse, Tunisia Caractérisation phénotypique, génotypique et sérotypique des souches invasives et non invasives de Streptococcus pneumoniae résistantes aux macrolides, à Sousse, Tunisie M. Marzouk ∗,1 , A. Ferjani 2 , S. Amamou 3 , S. Alibi 4 , M. Haj Ali 5 , J. Boukadida 6 Laboratoire de microbiologie et immunologie, UR12SP34, CHU Farhat Hached, 4000, Sousse, Tunisia Received 24 February 2014; received in revised form 23 June 2014; accepted 28 July 2014
Abstract Objective. – We determined the macrolide resistance phenotypes and genotypes in Streptococcus pneumoniae isolates in Sousse and assessed the serotype distribution. Methods. – We included S. pneumoniae strains isolated at our laboratory (2010–2013). The antimicrobial susceptibility was tested according to CA-SFM specifications. Serotyping was performed by agglutination of latex particles, to identify a subset of serotypes included in pneumococcal conjugate vaccines. The presence of macrolide resistance genes (ermB, mefA, mel) was detected by PCR. Results. – A total of 52.8% of 140 S. pneumoniae isolates were macrolide-resistant: MLSB (89.2%) and M (10.8%). The MLSB phenotypes were genotypically confirmed by ermB gene presence. 62% had decreased susceptibility to penicillin. The serotypes were: 14, 1, 23F, and 19A. Serotype coverage by PCV7, PCV10 and PCV13 was 44.2%, 73.6%, and 75.6% respectively. Conclusion. – 50% of S. pneumoniae isolates were macrolide resistant. The MLSB phenotype encoded by the ermB gene was the most frequent. Serotype coverage seems inadequate. © 2014 Elsevier Masson SAS. All rights reserved. Keywords: Macrolides; Streptococcus pneumoniae
Résumé Objet. – Caractéristiques phénotypiques, génotypiques et sérotypiques des pneumocoques résistants aux macrolides à Sousse. Matériels et méthodes. – Étude des pneumocoques isolés dans notre laboratoire (2010–2013). Antibiogramme réalisé selon les recommandations du CA-SFM et sérotypage par méthode d’agglutination de particules de latex sensibilisées par des anticorps capsulaires. Les gènes de résistance aux macrolides (ermB, mefA, mel) ont été mis en évidence par PCR.
∗ 1 2 3 4 5 6
Corresponding author. E-mail addresses: [email protected], [email protected] (M. Marzouk). M. Marzouk wrote the study protocol and the article. A. Ferjani wrote the study protocol. S. Amamou managed the the technical part. S. Alibi contributed to the technical part. M. Haj Ali collected the data. J. Boukadida supervised the study.
http://dx.doi.org/10.1016/j.medmal.2014.07.016 0399-077X/© 2014 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Marzouk M, et al. Phenotype, genotype, and serotype distribution of macrolide resistant invasive and non-invasive Streptococcus pneumoniae strains, in Sousse, Tunisia. Med Mal Infect (2014), http://dx.doi.org/10.1016/j.medmal.2014.07.016
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Résultats. – Un total de 52,8 % des 140 souches étaient résistantes aux macrolides : MLSB (89,2 %) et M (10,8 %). Le gène ermB s’associait au phénotype MLSB. Soixante-deux pour cent étaient des PSDP. Les sérotypes étaient : 14, 1, 23 F et 19A. La couverture sérotypique par le PCV7, PCV10 et PCV13 était : 44,2 %, 73,6 % et 75,6 %. Conclusion. – Dans notre région, plus de 50 % des pneumocoques sont résistants aux macrolides. Le phénotype MLSB codé par le gène ermB demeure le plus fréquent. La couverture sérotypique de ces souches paraît insuffisante. © 2014 Elsevier Masson SAS. Tous droits réservés. Mots clés : Macrolides ; Streptococcus pneumoniae
1. Introduction Streptococcus pneumoniae is a major and frequent agent of severe infections occurring at any age. The antibiotic susceptibility of this bacterium has been continuously decreasing, thus complicating the treatment of pneumococcal infections. Pneumococcal ENT infection is the most frequent and often the initial condition for other more severe infections. Macrolides are the most frequently prescribed antibiotic therapy, especially in case of beta-lactam failure, for this type of infections, and especially since the emergence of strains with decreased susceptibility to penicillins (PDSP). Nevertheless, given the frequency of macrolide resistance, they can only be used as an empirical treatment, and only according to antibiogram data. It is thus strongly recommended to update data on pneumococcal susceptibility to the various macrolides. We focused our study on the phenotypes and genotypes of pneumococcal resistance to macrolides and related agents in strains isolated in Sousse (Tunisia) during the previous 4 years, and on the determination of capsular serotypes of these strains. 2. Materials and methods 2.1. Population and study location
telithromycin, tetracycline, chloramphenicol, cotrimoxazole, levofloxacin, rifampicin, vancomycin, and teicoplanin. 2.4. Phenotype of susceptibility to macrolides The results were analyzed according to CA-SFM recommendations. The constitutive or inducible phenotype MLSB (by modifying the target by methylation) means there is resistance to erythromycin, lincomycin, and telithromycin; pristinamycin remains active. The phenotype M (resistance by efflux pump) means there is resistance to erythromycin only. 2.5. Determination of serogroups and serotypes S. pneumoniae serogroups and serotypes were determined by the latex particle agglutination test sensitized by the anticapsular polysaccharide antibody (Pneumo-Test-Latex® kit, Statens Serum Institute, Copenhagen, Denmark) corresponding to the 13-valent pneumococcal vaccine, according to the manufacturer’s recommendations. The quality control was performed with S. pneumoniae serotype 1 (Statens Serum Institute). 2.6. Genotypic detection of genes of resistance to macrolides
The samples were seeded on fresh and cooked blood agar and incubated 18 to 24 h at 37 ◦ C with 5% CO2 . S. pneumoniae was identified according to the usual methods. The internal quality control was performed with the reference strain S. pneumoniae ATCC 49619.
The studied genes of resistance to macrolide (ermB, mefA, mel) were identified by gene amplification (PCR). The polymerase master mix contained Taq buffer (10X), dNTPs, Mgcl2. Taq polymerase, 1 L of every primer [1], and 2 L of DNA (extracted by thermal lysis). A negative control and a reference strain depending on the targeted gene (positive control) were included in every series. Pr. Leclercq provided the reference strains: Enterococcus faecalis JH2-2 (Tn 1545), Streptococcus pyogenes UCN64, and Staphylocococcus aureus RN 4220 for the genes ermB, mefA, and mel. A molecular weight marker (100pb) was used to determine the size of migrated bands. The amplified sequences were revealed with the Molecular Imager GelDoc XR (Biorad).
2.3. Phenotype of susceptibility to antibiotics
3. Results
The antibiogram was performed and analyzed according to the recommendations issued by the French microbiology antibiogram committee (French acronym CA-SFM). The tested antibiotics were: penicillin G, amoxicillin, cefotaxime, oxacillin, erythromycin, lincomycin, pristinamycin,
One hundred and forty non-redundant S. pneumoniae strains were collected during the study period in our laboratory. Seventy-four strains (52.8%) were resistant to macrolides. The invasive strains (from normally sterile sites) (50%) were identified in blood cultures (23%), CSF (16.2%), puncture fluids
We studied non-redundant S. pneumoniae strains isolated in the microbiology laboratory of the Sousse Farhat Hached Teaching Hospital (2010–2013), issued from various samples. 2.2. Culture and identification of S. pneumoniae strains
Please cite this article in press as: Marzouk M, et al. Phenotype, genotype, and serotype distribution of macrolide resistant invasive and non-invasive Streptococcus pneumoniae strains, in Sousse, Tunisia. Med Mal Infect (2014), http://dx.doi.org/10.1016/j.medmal.2014.07.016
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Table 1 Correlation between the phenotypes and the genotypes of MLS resistance of S. pneumoniae isolates. Phénotypes des résistances associés aux gènes ermB, mefA et mel seuls ou associés. Genotype
Phenotype MLSB M
ermB (n = 34)
mef A (n = 6)
mel (n = 0)
ermB + mefA (n = 7)
ermB +mel (n = 9)
mefA +mel (n = 2)
ermB+ mefA + mel (n = 1)
No gene
34 0
0 6
– –
7 0
9 0
0 2
1 0
15 0
A total of 75.6% of macrolide resistant pneumococci had an identifiable serotype: 14 (41.2%), 1 (29.4%), 23 F (2.5%), and 19A (2.5%). The serotype coverage by PCV7, PCV10, and PCV13 was respectively: 44.2%, 73.6%, and 75.6%. 4. Discussion Since the description of the first pneumococcal strain resistant to erythromycin, such strains were identified all over the world with increasingly higher prevalences related to the excessive prescription of these agents, especially by community physicians [2]. We studied the phenotypic and genotypic characteristics of macrolide resistant pneumococci isolated in Sousse during the 4 previous years, as well as their susceptibility to antibiotics, and the most frequently involved serotypes. Resistance to macrolides has been sharply increasing in Tunisia, currently superior to 50% [3]. This correlates with our results where more than half of isolated S. pneumoniae strains were resistant to macrolides. More than 80% of our strains carried the phenotype MLSB inducing a high level of resistance to erythromycin. These results correlate with published data [3–5]. The resistance by efflux pump is more rare (10%) inducing a lower level of resistance to erythromycin. Modification of target was the most frequent resistance mechanism in our study. The ermB gene related to the phenotype MLSB was the most frequent, followed by a much lower frequency of the gene mefA related to the phenotype M. The predominance of the ermB gene was reported in Tunisia and in Europe [3,6], whereas in the USA and in Australia the gene mefA is the most frequent [6]. The genotype (ermB + mefA), usually observed in South Africa [6], is
Fig. 1. MICs (mg/mL) of macrolide resistant S. pneumoniae isolates according to the genotype. CMIs (mg/mL) des souches de pneumocoques résistantes aux macrolides en fonction du gène.
(10.8%); non-invasive strains (50%) were identified in sputum (20.3%), superficial pus (17.6%), and other samples (12.1%). The phenotypes of resistance to macrolides were of the types MLSB (89.2%) and M (10.8%). No strain carried the phenotype MSB. The studied genes were alone or associated (Table 1). All the strains carrying the ermB gene presented with the phenotype MLSB. Phenotype M was identified in case of genotype (mefA ± mel) (Table 1). None of these 3 genes was detected in 15 cases (20.3%). The presence of the ermB gene alone or associated was correlated to high MIC (> 256 mg/mL) in most cases (Fig. 1). Forty-six macrolide resistant S. pneumoniae strains (62%) were also PDSP (Table 2); most were multi-resistant (Table 2).
Table 2 Antibiotic resistance patterns of erythromycin resistant S. pneumoniae isolates, distribution of phenotypes and genotypes. Profil de résistance des pneumocoques résistants aux macrolides aux autres familles d’antibiotiques en fonction de leur génotype. Antibiotics
ermB
mefA
ermB + mefA
ermB + mel
mel + mefA
ermB + mefA + mel
No gene
E EP E Te E Te P E Sxt Te E P Sxt E Te Ch E Te Ch P E Sxt P Ch
4 9 5 4 1 6 1 3 1
2 1 2 0 1 0 0 0 0
0 2 0 2 1 0 1 0 1
0 4 1 0 0 1 0 3 0
0 2 0 0 0 0 0 0 0
0 0 0 0 0 0 0 1 0
6 5 4 0 0 0 0 0 0
E: erythromycin; P: penicillin; Te: tetracycline; Sxt: cotrimoxazole; Ch: chloramphenicol.
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far less frequent (< 10% of our strains). Few authors have studied the gene mel in S. pneumoniae. This plasmid borne gene, better known in staphylococci as msrA, encodes for the efflux pump system in association with other chromosomal genes. This pump confers the phenotype MSB. When it is detected in strains harboring ermB, the phenotype MSB is masked by the phenotype MLSB [7]. Nevertheless, when it is associated to the gene mefA, only phenotype M is expressed; which could mean that this phenotype MSB is not present in pneumococci. Resistance to macrolides is more marked in PDSP (> 60% of our strains) [3,8,9]. Resistance to macrolides is also associated with resistance to tetracyclines, to chloramphenicol, and to cotrimoxazole, and varies according to regions [9]; 80% of pneumococci carrying the gene ermB were multi-resistant, especially to tetracyclines [10–12]. These combinations suggest that this resistance could be related to conjugative transposons Tn1545. All the multi-resistant strains in our study as well as in the literature [3], carried the gene ermB alone or associated. No studied gene was detected in 20% of the cases. These cases could correspond to ribosomal mutations. Some strains with a mutation of L4 and L22 were reported, 21% of which in strains resistant to erythromycin, in East Europe [13,14]. Furthermore, we did not study the other genes implicated in resistance to macrolides: ermC, ermA, ereA, and ereB. These genes of resistance have rarely been implicated in pneumococcal resistance to macrolides [15]. The gene ermA was detected in only one strain in Greece and could confer low-level resistance to erythromycin [16]. The serotypes of pneumococci resistant to macrolides in our region were predominantly serotypes 14 and 1. This is different from other countries where a predominance of 19F and 23 was reported [17–19]. The serotypic coverage of PCV7 in our region seems non-effective in more than half of the cases and that of PCV10 and PCV13 does not cover almost 25% of these strains. These rates are worrying given the severity of pneumococcal infections and the risk of spreading [20].
5. Conclusion The overconsumption of antibiotics not justified by laboratory tests has induced the increase of resistance, especially to macrolides. Most of this resistance’s mechanisms are known and well codified genetically. In our region, more than half of pneumococcal strains were resistant to macrolides; the phenotype MLSB encoded by the gene ermB was the most frequent. Nevertheless, some phenotypic resistances require more investigations. The serotypic coverage of strains resistant to macrolides in our region seems insufficient and is a true public health concern.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
Acknowledgements We warmly thank Pr. Leclercq (Caen University, France) for his invaluable help. This study was supported financially by the MESRSUR12SP34 (public financing). References [1] Angot P, Vergnaud M, Auzou M, Leclerq R. Macrolide resistance phenotypes and genotypes in French clinical isolates of Streptococcus pneumoniae. Observatoire de Normandie du pneumocoque. Eur J Clin Microbiol Infect Dis 2000;19:755–8. [2] Adriaenssens N, Coenen S, Versporten A, Muller A, Minalu G, Faes G, et al. European surveillance of antimicrobial consumption ESAC: outpatient antibiotic use in Europe (1997-2009). J Antimicrob Chemothe 2011;66(Suppl 6):vi3–12. [3] Rachdi M, Boutiba-Ben Boubaker I, Moalla S, Smaoui H, Hammami A, Kechrid A, et al. Phenotypic and genotypic characterization of macrolide resistant Streptococcus pneumoniae in Tunisia. Patho Biol 2008;56:125–9. [4] Okitsu N, Kaieda S, Yano H, Nakano R, Hosaka K, et al. Characterization of ermB gene transposition by Tn1545 and Tn917 in macrolide resistant Streptococcus pneumoniae isolates. J Clin Microbiol 2005;43:168–73. [5] Ambrose KD, Nisbet R, Stephens DS. Macrolide efflux in Streptococcus pneumoniae is mediated by a dual efflux pump (mel and mef) and is erythromycin inducible. Antimicrob Agents Chemother 2005;49:4203–9. [6] Farrell DJ, Couturier C, Hryniewicz W. Distribution and antibacterial susceptibility of macrolide resistance genotypes in Streptococcus pneumoniae: PROTEKT Year 5 (2003–2004). Intern J Antimic Agents 2008;31:245–9. [7] Eady EA, Ross JI, Tipper JL, Walters CE, Cove JH, Noble WC. Distribution of genes encoding erythromycin ribosomal methylases and an erythromycin efflux pump in epidemiologically distinct groups of staphylococci. J Antimicrob Chemother 1993;31:211–7. [8] Dias R, Louro D, Canica M. Antimicrobial susceptibility of invasive Streptococcus pneumoniae isolates in Portugal over an 11-year period. Antimicrob Agents Chemother 2006;50:2098–105. [9] Reinert R, Ringelstein A, Van Der Linden M, Yücel Cil M, Al-Lahham A, Schmitz FJ. Molecular epidemiology of macrolide-resistant Streptococcus pneumoniae isolates in Europe. J Clin Microbiol 2005;43:1294–300. [10] Doern GV, Heilmann KP, Huynh HK, Rhomberg PR, Coffman SL, Brueggemann AB. Antimicrobial resistance among clinical isolates of Streptococcus pneumoniae in the United States during 1999–2000, including a comparison of resistance rates since 1994–1995. Antimicrob Agents Chemother 2001;45:1721–9. [11] Marchese A, Tonoli E, Balistreri A, Debbia EA, Schito GC. Antibiotic susceptibility patterns and serotypes of antibiotic resistant and/or invasive Streptococcus pneumoniae strains circulating in Italy. Microb Drug Resist 2000;6:163–70. [12] Maria PR, Rocco M, Saralee B, Gianni V, Michael R. Antimicrobial susceptibility of Streptococcus pneumoniae from children attending day-care centers in a central italian city. Clin Microbiol Infect 1998;4:622–6. [13] Tait-Kamradt A, Davies T, Appelbaum PC, Depardieu F, Courvalin P, Petitpas J. Two new mechanisms of macrolide resistance in clinical strains of Streptococcus pneumonia from Eastern Europe and North America. Antimicrob Agents Chemother 2000;44:3395–401. [14] Depardieu F, Courvalin P. Mutation in 23s rRNA responsible for resistance to 16-membered macrolides and streptogramins in Streptococcus pneumoniae. Antimicrob Agents Chemother 2000;45:319–23. [15] Shortridge VD, Flamm RK, Ramer N, Beyer J, Tanaka SK. Novel Mechanism of Macrolide Resistance in Streptococcus pneumonia. Diagn Microbiol Infect Dis 1996;6:73–8. [16] Leclercq R, Courvalin P. Bacterial resistance to macrolide, lincosamide and streptogramin antibiotics by target modification. Antimicrob Agents Chemother 1991;35:1267–72. [17] Taha N, Araj GF, Wakim RH, Kanj SS, Kanafani ZA, Sabra A, et al. Genotypes and serotype distribution of macrolide resistant invasive and
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non- invasive Streptococcus pneumoniae isolates from Lebanon. Ann Clin Microbiol Antimicrobial 2012;11:2. [18] Gulay Z, Ozbek OA, Bicmen M, Gur D. Macrolide resistance determinants in erythromycin-resistant Streptococcus pneumoniae in Turkey. J Infect Dis 2008;61:490–3. [19] Isaacman DJ, McIntosh ED, Reinert RR. Burden of invasive pneumococcal disease and serotype distribution among Streptococcus pneumonia isolates
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in young children in Europe: impact of the 7-valent pneumococcal conjugate vaccine and considerations for future conjugate vaccines. Int J Infect Dis 2010;14:e197–209. [20] McIntosh ED, Reinert RR. Global prevailing and emerging pediatric pneumococcal serotypes. Expert Rev Vaccines 2011;10:109–29.
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Original article
Phenotype, genotype, and serotype distribution of macrolide resistant invasive and non-invasive Streptococcus pneumoniae strains, in Sousse, Tunisia Caractérisation phénotypique, génotypique et sérotypique des souches invasives et non invasives de Streptococcus pneumoniae résistantes aux macrolides, à Sousse, Tunisie M. Marzouk ∗,1 , A. Ferjani 2 , S. Amamou 3 , S. Alibi 4 , M. Haj Ali 5 , J. Boukadida 6 Laboratoire de microbiologie et immunologie, UR12SP34, CHU Farhat Hached, 4000, Sousse, Tunisia Received 24 February 2014; received in revised form 23 June 2014; accepted 28 July 2014
Abstract Objective. – We determined the macrolide resistance phenotypes and genotypes in Streptococcus pneumoniae isolates in Sousse and assessed the serotype distribution. Methods. – We included S. pneumoniae strains isolated at our laboratory (2010–2013). The antimicrobial susceptibility was tested according to CA-SFM specifications. Serotyping was performed by agglutination of latex particles, to identify a subset of serotypes included in pneumococcal conjugate vaccines. The presence of macrolide resistance genes (ermB, mefA, mel) was detected by PCR. Results. – A total of 52.8% of 140 S. pneumoniae isolates were macrolide-resistant: MLSB (89.2%) and M (10.8%). The MLSB phenotypes were genotypically confirmed by ermB gene presence. 62% had decreased susceptibility to penicillin. The serotypes were: 14, 1, 23F, and 19A. Serotype coverage by PCV7, PCV10 and PCV13 was 44.2%, 73.6%, and 75.6% respectively. Conclusion. – 50% of S. pneumoniae isolates were macrolide resistant. The MLSB phenotype encoded by the ermB gene was the most frequent. Serotype coverage seems inadequate. © 2014 Elsevier Masson SAS. All rights reserved. Keywords: Macrolides; Streptococcus pneumoniae
Résumé Objet. – Caractéristiques phénotypiques, génotypiques et sérotypiques des pneumocoques résistants aux macrolides à Sousse. Matériels et méthodes. – Étude des pneumocoques isolés dans notre laboratoire (2010–2013). Antibiogramme réalisé selon les recommandations du CA-SFM et sérotypage par méthode d’agglutination de particules de latex sensibilisées par des anticorps capsulaires. Les gènes de résistance aux macrolides (ermB, mefA, mel) ont été mis en évidence par PCR.
∗ 1 2 3 4 5 6
Corresponding author. E-mail addresses: [email protected], [email protected] (M. Marzouk). M. Marzouk wrote the study protocol and the article. A. Ferjani wrote the study protocol. S. Amamou managed the the technical part. S. Alibi contributed to the technical part. M. Haj Ali collected the data. J. Boukadida supervised the study.
http://dx.doi.org/10.1016/j.medmal.2014.07.016 0399-077X/© 2014 Elsevier Masson SAS. All rights reserved.
Please cite this article in press as: Marzouk M, et al. Phenotype, genotype, and serotype distribution of macrolide resistant invasive and non-invasive Streptococcus pneumoniae strains, in Sousse, Tunisia. Med Mal Infect (2014), http://dx.doi.org/10.1016/j.medmal.2014.07.016
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Résultats. – Un total de 52,8 % des 140 souches étaient résistantes aux macrolides : MLSB (89,2 %) et M (10,8 %). Le gène ermB s’associait au phénotype MLSB. Soixante-deux pour cent étaient des PSDP. Les sérotypes étaient : 14, 1, 23 F et 19A. La couverture sérotypique par le PCV7, PCV10 et PCV13 était : 44,2 %, 73,6 % et 75,6 %. Conclusion. – Dans notre région, plus de 50 % des pneumocoques sont résistants aux macrolides. Le phénotype MLSB codé par le gène ermB demeure le plus fréquent. La couverture sérotypique de ces souches paraît insuffisante. © 2014 Elsevier Masson SAS. Tous droits réservés. Mots clés : Macrolides ; Streptococcus pneumoniae
1. Introduction Streptococcus pneumoniae is a major and frequent agent of severe infections occurring at any age. The antibiotic susceptibility of this bacterium has been continuously decreasing, thus complicating the treatment of pneumococcal infections. Pneumococcal ENT infection is the most frequent and often the initial condition for other more severe infections. Macrolides are the most frequently prescribed antibiotic therapy, especially in case of beta-lactam failure, for this type of infections, and especially since the emergence of strains with decreased susceptibility to penicillins (PDSP). Nevertheless, given the frequency of macrolide resistance, they can only be used as an empirical treatment, and only according to antibiogram data. It is thus strongly recommended to update data on pneumococcal susceptibility to the various macrolides. We focused our study on the phenotypes and genotypes of pneumococcal resistance to macrolides and related agents in strains isolated in Sousse (Tunisia) during the previous 4 years, and on the determination of capsular serotypes of these strains. 2. Materials and methods 2.1. Population and study location
telithromycin, tetracycline, chloramphenicol, cotrimoxazole, levofloxacin, rifampicin, vancomycin, and teicoplanin. 2.4. Phenotype of susceptibility to macrolides The results were analyzed according to CA-SFM recommendations. The constitutive or inducible phenotype MLSB (by modifying the target by methylation) means there is resistance to erythromycin, lincomycin, and telithromycin; pristinamycin remains active. The phenotype M (resistance by efflux pump) means there is resistance to erythromycin only. 2.5. Determination of serogroups and serotypes S. pneumoniae serogroups and serotypes were determined by the latex particle agglutination test sensitized by the anticapsular polysaccharide antibody (Pneumo-Test-Latex® kit, Statens Serum Institute, Copenhagen, Denmark) corresponding to the 13-valent pneumococcal vaccine, according to the manufacturer’s recommendations. The quality control was performed with S. pneumoniae serotype 1 (Statens Serum Institute). 2.6. Genotypic detection of genes of resistance to macrolides
The samples were seeded on fresh and cooked blood agar and incubated 18 to 24 h at 37 ◦ C with 5% CO2 . S. pneumoniae was identified according to the usual methods. The internal quality control was performed with the reference strain S. pneumoniae ATCC 49619.
The studied genes of resistance to macrolide (ermB, mefA, mel) were identified by gene amplification (PCR). The polymerase master mix contained Taq buffer (10X), dNTPs, Mgcl2. Taq polymerase, 1 L of every primer [1], and 2 L of DNA (extracted by thermal lysis). A negative control and a reference strain depending on the targeted gene (positive control) were included in every series. Pr. Leclercq provided the reference strains: Enterococcus faecalis JH2-2 (Tn 1545), Streptococcus pyogenes UCN64, and Staphylocococcus aureus RN 4220 for the genes ermB, mefA, and mel. A molecular weight marker (100pb) was used to determine the size of migrated bands. The amplified sequences were revealed with the Molecular Imager GelDoc XR (Biorad).
2.3. Phenotype of susceptibility to antibiotics
3. Results
The antibiogram was performed and analyzed according to the recommendations issued by the French microbiology antibiogram committee (French acronym CA-SFM). The tested antibiotics were: penicillin G, amoxicillin, cefotaxime, oxacillin, erythromycin, lincomycin, pristinamycin,
One hundred and forty non-redundant S. pneumoniae strains were collected during the study period in our laboratory. Seventy-four strains (52.8%) were resistant to macrolides. The invasive strains (from normally sterile sites) (50%) were identified in blood cultures (23%), CSF (16.2%), puncture fluids
We studied non-redundant S. pneumoniae strains isolated in the microbiology laboratory of the Sousse Farhat Hached Teaching Hospital (2010–2013), issued from various samples. 2.2. Culture and identification of S. pneumoniae strains
Please cite this article in press as: Marzouk M, et al. Phenotype, genotype, and serotype distribution of macrolide resistant invasive and non-invasive Streptococcus pneumoniae strains, in Sousse, Tunisia. Med Mal Infect (2014), http://dx.doi.org/10.1016/j.medmal.2014.07.016
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Table 1 Correlation between the phenotypes and the genotypes of MLS resistance of S. pneumoniae isolates. Phénotypes des résistances associés aux gènes ermB, mefA et mel seuls ou associés. Genotype
Phenotype MLSB M
ermB (n = 34)
mef A (n = 6)
mel (n = 0)
ermB + mefA (n = 7)
ermB +mel (n = 9)
mefA +mel (n = 2)
ermB+ mefA + mel (n = 1)
No gene
34 0
0 6
– –
7 0
9 0
0 2
1 0
15 0
A total of 75.6% of macrolide resistant pneumococci had an identifiable serotype: 14 (41.2%), 1 (29.4%), 23 F (2.5%), and 19A (2.5%). The serotype coverage by PCV7, PCV10, and PCV13 was respectively: 44.2%, 73.6%, and 75.6%. 4. Discussion Since the description of the first pneumococcal strain resistant to erythromycin, such strains were identified all over the world with increasingly higher prevalences related to the excessive prescription of these agents, especially by community physicians [2]. We studied the phenotypic and genotypic characteristics of macrolide resistant pneumococci isolated in Sousse during the 4 previous years, as well as their susceptibility to antibiotics, and the most frequently involved serotypes. Resistance to macrolides has been sharply increasing in Tunisia, currently superior to 50% [3]. This correlates with our results where more than half of isolated S. pneumoniae strains were resistant to macrolides. More than 80% of our strains carried the phenotype MLSB inducing a high level of resistance to erythromycin. These results correlate with published data [3–5]. The resistance by efflux pump is more rare (10%) inducing a lower level of resistance to erythromycin. Modification of target was the most frequent resistance mechanism in our study. The ermB gene related to the phenotype MLSB was the most frequent, followed by a much lower frequency of the gene mefA related to the phenotype M. The predominance of the ermB gene was reported in Tunisia and in Europe [3,6], whereas in the USA and in Australia the gene mefA is the most frequent [6]. The genotype (ermB + mefA), usually observed in South Africa [6], is
Fig. 1. MICs (mg/mL) of macrolide resistant S. pneumoniae isolates according to the genotype. CMIs (mg/mL) des souches de pneumocoques résistantes aux macrolides en fonction du gène.
(10.8%); non-invasive strains (50%) were identified in sputum (20.3%), superficial pus (17.6%), and other samples (12.1%). The phenotypes of resistance to macrolides were of the types MLSB (89.2%) and M (10.8%). No strain carried the phenotype MSB. The studied genes were alone or associated (Table 1). All the strains carrying the ermB gene presented with the phenotype MLSB. Phenotype M was identified in case of genotype (mefA ± mel) (Table 1). None of these 3 genes was detected in 15 cases (20.3%). The presence of the ermB gene alone or associated was correlated to high MIC (> 256 mg/mL) in most cases (Fig. 1). Forty-six macrolide resistant S. pneumoniae strains (62%) were also PDSP (Table 2); most were multi-resistant (Table 2).
Table 2 Antibiotic resistance patterns of erythromycin resistant S. pneumoniae isolates, distribution of phenotypes and genotypes. Profil de résistance des pneumocoques résistants aux macrolides aux autres familles d’antibiotiques en fonction de leur génotype. Antibiotics
ermB
mefA
ermB + mefA
ermB + mel
mel + mefA
ermB + mefA + mel
No gene
E EP E Te E Te P E Sxt Te E P Sxt E Te Ch E Te Ch P E Sxt P Ch
4 9 5 4 1 6 1 3 1
2 1 2 0 1 0 0 0 0
0 2 0 2 1 0 1 0 1
0 4 1 0 0 1 0 3 0
0 2 0 0 0 0 0 0 0
0 0 0 0 0 0 0 1 0
6 5 4 0 0 0 0 0 0
E: erythromycin; P: penicillin; Te: tetracycline; Sxt: cotrimoxazole; Ch: chloramphenicol.
Please cite this article in press as: Marzouk M, et al. Phenotype, genotype, and serotype distribution of macrolide resistant invasive and non-invasive Streptococcus pneumoniae strains, in Sousse, Tunisia. Med Mal Infect (2014), http://dx.doi.org/10.1016/j.medmal.2014.07.016
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far less frequent (< 10% of our strains). Few authors have studied the gene mel in S. pneumoniae. This plasmid borne gene, better known in staphylococci as msrA, encodes for the efflux pump system in association with other chromosomal genes. This pump confers the phenotype MSB. When it is detected in strains harboring ermB, the phenotype MSB is masked by the phenotype MLSB [7]. Nevertheless, when it is associated to the gene mefA, only phenotype M is expressed; which could mean that this phenotype MSB is not present in pneumococci. Resistance to macrolides is more marked in PDSP (> 60% of our strains) [3,8,9]. Resistance to macrolides is also associated with resistance to tetracyclines, to chloramphenicol, and to cotrimoxazole, and varies according to regions [9]; 80% of pneumococci carrying the gene ermB were multi-resistant, especially to tetracyclines [10–12]. These combinations suggest that this resistance could be related to conjugative transposons Tn1545. All the multi-resistant strains in our study as well as in the literature [3], carried the gene ermB alone or associated. No studied gene was detected in 20% of the cases. These cases could correspond to ribosomal mutations. Some strains with a mutation of L4 and L22 were reported, 21% of which in strains resistant to erythromycin, in East Europe [13,14]. Furthermore, we did not study the other genes implicated in resistance to macrolides: ermC, ermA, ereA, and ereB. These genes of resistance have rarely been implicated in pneumococcal resistance to macrolides [15]. The gene ermA was detected in only one strain in Greece and could confer low-level resistance to erythromycin [16]. The serotypes of pneumococci resistant to macrolides in our region were predominantly serotypes 14 and 1. This is different from other countries where a predominance of 19F and 23 was reported [17–19]. The serotypic coverage of PCV7 in our region seems non-effective in more than half of the cases and that of PCV10 and PCV13 does not cover almost 25% of these strains. These rates are worrying given the severity of pneumococcal infections and the risk of spreading [20].
5. Conclusion The overconsumption of antibiotics not justified by laboratory tests has induced the increase of resistance, especially to macrolides. Most of this resistance’s mechanisms are known and well codified genetically. In our region, more than half of pneumococcal strains were resistant to macrolides; the phenotype MLSB encoded by the gene ermB was the most frequent. Nevertheless, some phenotypic resistances require more investigations. The serotypic coverage of strains resistant to macrolides in our region seems insufficient and is a true public health concern.
Disclosure of interest The authors declare that they have no conflicts of interest concerning this article.
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