Idrissa Diawara, Khalid Zerouali, Khalid Katfy, Abouddihaj Barguigua, Houria Belabbes, Mohammed Timinouni, Naima Elmdaghri
PII: S1567-1348(16)30074-0
DOI: doi:10.1016/j.meegid.2016.03.003 Reference: MEEGID 2662
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Received date: 16 December 2015 Revised date: 1 March 2016 Accepted date: 2 March 2016
Please cite this article as: Diawara, Idrissa, Zerouali, Khalid, Katfy, Khalid, Barguigua, Abouddihaj, Belabbes, Houria, Timinouni, Mohammed, Elmdaghri, Naima, Phenotypic and genotypic characterization of Streptococcus pneumoniae resistant to macrolide in Casablanca, Morocco, (2016), doi: 10.1016/j.meegid.2016.03.003
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Phenotypic and genotypic characterization of Streptococcus pneumoniae resistant to macrolide in Casablanca, Morocco
Idrissa Diawaraa,b*, Khalid Zeroualia,b, Khalid Katfyb, Abouddihaj Barguiguaa,c, Houria Belabbesa,b, Mohammed Timinounic and Naima Elmdaghria,b
a Laboratoire de Microbiologie, Faculté de Médecine et de Pharmacie, Hassan II University of Casablanca, B.P 5696, Casablanca, Maroc.
b Service de Microbiologie, CHU Ibn Rochd, B.P 2698, Casablanca, Maroc
c Molecular Bacteriology Laboratory, Institut Pasteur du Maroc, Casablanca, Morocco.
Email addresses:
Idrissa Diawara: diawaraidris@gmail.com
Khalid Zerouali: khalid.zerouali2000@gmail.com Khalid Katfy: khalidkatfy@hotmail.com
Abouddihaj Barguigua: dihaj82@yahoo.fr Houria Belabbes: belhor2001@yahoo.fr
Mohammed Timinouni:mohammed.timinouni@pasteur.ma Naima Elmdaghri: naimaelmdaghri@yahoo.fr
* Corresponding author
Department of Microbiology, Faculty of Medicine and Pharmacy, P.B. 5696, 19, Tarik Ibnou Ziad, Casablanca, Maroc
Tel: +212 648490217 / +212 681008541 Fax: +212 522271630, Casablanca, Morocco
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Abstract:
In Morocco, the 13-valent pneumococcal conjugate vaccine (PCV-13) was introduced in the national immunization program (NIP) in October 2010 and replaced by the PCV-10 in July 2012. The present study aimed to determine the prevalence of erythromycin-resistant Streptococcus pneumoniae (ERSP) and to analyze the phenotypic and genotypic
characteristics of these isolates in Casablanca, Morocco from January 2007 to December 2014.
Isolates were obtained from the Microbiology Laboratory of Ibn Rochd University Hospital Centre of Casablanca. Serogrouping was done using Pneumotest Kit and serotyping by the Quellung capsular swelling. Antibiotic susceptibility pattern was determined by disc diffusion and Etest methods.
A total of 655 S. pneumoniae isolates were collected from 2007 to 2014 from pediatric and adult patients. Fifty-five percent of these isolates were from invasive
pneumococcal diseases. Of the 655 isolates, 92 (14%) were ERSP. Globally, the proportion of ERSP from 2007 to 2010 (before vaccination) and from 2011 to 2014 (after vaccination) were 11.6% and 17.2 % (p= 0.04), respectively. Of the 92 ERSP, 89%, 4% and 7% displayed constitutive MLSB (resistance to macrolide, lincosamide and streptogramin B), inducible MLSB, and M phenotype (resistance to macrolide only), respectively. ERSP genotypic analysis showed that 90.2% carried the ermB gene, 6.5% the mefE gene, and 3.3% both the genes (ermB + mefE). The most prevalent ERSP serotypes were 6B, 19F and 23F before vaccination and 19F, 6B, 6A and 23F after vaccination. Erythromycin resistance among S.
pneumoniae is relatively high in Casablanca. The contribution of PCVs to the reduction in antibiotic use is encouraging but this should be accompanied by a rational use of antibiotic.
Keywords: Streptococcus pneumoniae; Macrolides resistance; ermB; mefE; Pneumococcal conjugate vaccine.
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1. Introduction
Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide, especially among children and the elderly (File et al., 2004). It is a major cause of
community-acquired respiratory tract infections such as community-acquired pneumonia and acute sinusitis, as well as life-threatening invasive pneumococcal diseases (IPD) such as bacteraemia, septicaemia and meningitis. Macrolides are extensively used for the treatment of respiratory infections due to their broad-spectrum activity against both typical and atypical respiratory pathogens as well as greater lung tissue penetration (Mandell et al., 2007). The clinical management of these infections has been complicated by the worldwide emergence and spread of resistance in S. pneumoniae to commonly used antibiotics, particularly to macrolides. Emergence of erythromycin resistance in S. pneumoniae is a growing concern because of the prevalence of this pathogen in infections of the respiratory tract (Jenkins et al., 2005).
The first erythromycin resistant strains of S. pneumoniae were isolated in the United States in 1967 (Dixon, 1967). Although the prevalence of resistant strains varies
geographically and temporally, erythromycin resistant strains are widespread (Farell et al., 2004). Macrolide resistance in S. pneumoniae is mediated by two major mechanisms:
methylation of the ribosomal macrolide target site, encoded by the ermB gene, and drug efflux, encoded by the mefA gene (Wierzbowski et al., 2007; Leclercq et al., 2002). The ermB gene methylates a specific adenine residue (A2058) in the peptidyl transferase center of 23S rRNA, thereby conferring high-level resistance to 14-, 15- and 16-membered ring macrolides, lincosamides and streptogramin B (MLSB phenotype) . The efflux mechanism confers
resistance to 14- and 15-member macrolides only (M phenotype) (Wierzbowski et al., 2007).
In S. pneumoniae, mefE, another variant of mefA, is found, presenting an identity of approximately 90% (Roberts et al., 1999). In addition to these major mechanisms, S.
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pneumoniae isolates positive for both ermB and mefA are becoming rapidly more common worldwide and have been identified in many countries (Rachdi et al., 2008; Monaco et al.
2005).
In Morocco, an increase in the rate of macrolide resistance among S. pneumoniae occurred after the introduction of newer macrolides in the 1990s and their extensive use thereafter. The prevalence rate in Casablanca reached 9.4% in 1999-2001, 12.2% in 2002- 2005 and 14.4% in 2006-2008 (Benbachir et al., 2012). The 13-valent pneumococcal conjugate vaccine (PCV-13) was introduced in the national immunization program (NIP) in October 2010 in a 2+1 schedule and replaced by the 10-valent pneumococcal conjugate vaccine (PCV-10) in July 2012 with the same schedule. There are no studies on the
characteristics of ERSP in Morocco. The present study aimed to determine the prevalence of erythromycin-resistant S. pneumoniae (ERSP) and to analyze the phenotypic and genotypic characteristics of these isolates in Casablanca, Morocco.
2. Materials and Methods
2.1. Bacterial isolates
Isolates were obtained from the Microbiology Laboratory of Ibn Rochd University Hospital Centre of Casablanca (IR-UHC), which is a tertiary care hospital comprising 1451 beds (this bed capacity represents 52% of capacity management of patients living in Grand Casablanca). The isolates were identified based on the typical colony morphology, Gram staining, optochin sensitivity test (Oxoid Company, Britain) on Mueller–Hinton agar plates supplemented with 5% sheep blood (BioMèrieux, Lyon, France) and bile solubility.
2.2. Pneumococcal serotyping
Serogrouping was done by the checkerboard method with Pneumotest-latex (Statens Serum Institute antisera, Copenhagen, Denmark), and serotyping was performed by Quellung capsule swelling using Statens Serum Institute antisera (Copenhagen, Denmark).
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Antibiotic susceptibility testing was done following Clinical Laboratory Standard Institute guidelines (CLSI, 2012). Erythromycin, tetracycline, chloramphenicol, trimethoprim- sulfamethoxazole (cotrimoxazole), pristinamycin, and telithromycin were tested by disk diffusion with antibiotic disks from Oxoid (Basingstoke, United Kingdom) on Mueller Hinton Agar supplemented with 5% sheep blood (BioMèrieux, Lyon, France). A Minimal Inhibitory Concentration (MIC) for erythromycin and penicillin G was determined on 5% sheep blood Mueller Hinton agar with E-tests from Oxoid (Oxoid, Basingstoke, UK). The breakpoints used for interpretation were those recommended by the CLSI in 2012. Quality control was conducted using S. pneumoniae ATCC 49619.
Macrolide resistance phenotyping was performed by double-disk diffusion using erythromycin disk (15 μg) and clindamycin disk (2 μg) as previously described (Seppala et al., 1993). A blunting of the clindamycin inhibition zone adjacent to the erythromycin disk indicated the presence of the inducible macrolide-resistant phenotype (inductive MLSB), whereas the absence of blunting indicated the presence of the constitutive macrolide-resistant phenotype (constitutive MLSB). The M phenotype was characterized by resistance to
erythromycin and susceptibility to clindamycin.
2.4. DNA extraction
Chromosomal DNA was extracted from the overnight cultures of the isolates that were grown on Columbia CNA Agar with 5% Sheep Blood by using the QIAamp®DNA mini kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions.
2.5. Detection and analysis of the ermB and mefA/E genes
The presence of macrolide resistance genes was detected by PCR as described previously (Del Grosso et al., 2002 Tait-Kamradt et al., 1997; Sutcliffe et al., 1996). In summary, we amplified the genes by PCR and analyzed the amplified DNA products by
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agarose gel electrophoresis. For ermB we used the primer pair 5’-
GAAAAGGTACTCAACCAAATA-3’ and 5’-AGTAACGGTACTTAAATTGTTTAC-3’
(Sutcliffe et al., 1996) and for mef A/E gene the primer pair 5’-
CTATGCGATTTTGGGACCTG-3’ and 5’-GAAAGCCCCATTATTGCACA-3’ (Tait-
Kamradt et al., 1997). In order to discriminate between mefA subclasses, mefA and mefE, PCR restriction fragment length polymorphism analysis was performed, as previously suggested (Del Grosso et al., 2002). The 1743-bp PCR product was digested with the BamHI or the DraI restriction enzyme. In mefA there is one BamHI site, so restriction generates two
fragments of 1,340 and 403 bp, while in mefE there are no BamHI restriction sites. Restriction of mefA with DraI yields two fragments of 1,493 and 250 bp, respectively, while restriction of mefE yields three fragments of 782, 711, and 250 bp.
2.6. Statistical analysis
Datawere analyzed with WHONET5.6, EpiInfo 7 (Centers for Disease Control, Atlanta, Georgia, USA) and Microsoft Excel. The chi square test or Fisher’s exact test was performed to compare proportion between collection periods. Differences were considered significant if the p-value was < 0.05.
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3. Results
3.1. Prevalence of macrolide resistance
A total of 655 S. pneumoniae isolates were collected from 2007 to 2014. Fifty-five percent of these isolates were from IPD cases. Of the 655 isolates, 56.5% were obtained before vaccination (2007 to 2010) and 43.5% after vaccination (2011 to 2014). Considering before and after vaccine introduction periods, isolates recovered from children (aged from 0 to 14 years) represented 40.5% and 30.2%, respectively.
Of the 655 isolates, 92 were ERSP (14%). Consecutive ERSP, one per patient, were collected from respiratory tract specimens (34.8%), cerebrospinal fluid (30.5%), blood cultures (18.4%) and other body fluids (16.3%). Globally, the proportion of ERSP between pre- and post-vaccination periods were 11.6% and 17.2 % (p= 0.04), respectively. The
proportion of ERSP isolated from pediatric and adult (age > 14 years) patients increased from 16.7% to 29.0% (p=0.02) and from 8.2% to 12.1% (p=0.18) before and after vaccine
implementation, respectively.
3.2. Mechanism of macrolide resistance
Of the 92 strains tested, 82 isolates (89%) displayed resistance to erythromycin and clindamycin (cMLSB) and 4 isolates (4%) were resistant to erythromycin with a blunting of the clindamycin inhibition zone (iMLSB). Resistance to erythromycin and susceptibility to clindamycin characteristic of the M phenotype was observed for 6 isolates (7%).
ERSP genotypic analysis by PCR and RFLP showed that 83 strains (90.2%) carried the ermB gene, 6 (6.5%) mefE genes, and 3 (3.3%) the ermB + mefE (Table 1).All mefE- positive S. pneumoniae isolates exhibited the M-phenotype. All dual ermB + mefE-positive isolates showed the cMLSB phenotype. MICs to erythromycin of mefE-positive isolates
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ranged from 2 to 8 μg/mL, ermB-positive (CMLSB) from 64 to >250 μg/mL, ermB-positive (iMLSB) from 64 to 256 μg/mL, and dual ermB + mefE-positive was >256 μg/mL (Table 1).
3.3. Co-resistance of ERSP
According to CLSI breakpoints, co-resistance to other antibiotic families was found in 87 (94.5%) of the 92 ERSP, constituting 8 different resistance patterns. Of these 87
nonsusceptible isolates, 88.5% were to tetracycline, 85% to penicillin G, 36.8% to
cotrimoxazole, and 12.6% to chloramphenicol. However, telithromycin and pristinamycin were shown to be highly active against all of the ERSP (100% susceptible).
3.4. Distribution of Serotyping among ERSP strains
Globally, vaccine serotypes and non-vaccine serotypes represented 80% and 20%
among the ERSP isolated in children before vaccination; after, they represented 76% and 24%, respectively. In the adult population, vaccine and non-vaccine serotypes accounted for 83% and 17% before vaccination, while they represented 62% and 38% of ERSP after vaccination, respectively.
Specific serotype distribution among ERSP isolated from children and adults showed that before vaccination, PCV-7 specific serotypes (i.e., 6B, 14, 19F and 23F) accounted for 76% and 61% of all ERSP, respectively. These rates decreased to 52% (p=0.07) in children and 58% (p=0.85) in adults in the post-vaccination period. The remaining vaccine serotypes were 6A and 19A (PCV-13 specific serotypes); their prevalence before and after PCV- 13/PCV-10 introduction was 4% and 24% (0.09) in children and 22% and 4% (p=0.41) in adults.
Among strains harboring the ermB gene, serotypes 6B, 23F, and 19F were most prevalent in children and adults before and after vaccine implementation (Figure 1 and Figure 2). In the post-vaccination periods, a non-significant decline of serotype 6B
prevalence (from 36% to 16 %) and 23F prevalence (from 20% to 12%) was observed among
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children. The same trend of non-significant decline was observed for adults with serotype 19F (from 33% to 17%) and 23F (from 11% to 4%). Inversely, serotype 19F in children and 6B in adult showed a non-significant increase after vaccination. Furthermore, serotypes 19A, 14 and 6A were found only after vaccination for children. In contrast for adults, 19A was found before vaccination only, and serotype 14 was not found in this population. As for non- vaccine serotypes, a non-significant increase was observed between the two periods among adults, while there was no-change in their prevalence among children.
As for the mefE-positive strains, 83.3% of them (5/6) belonged to serotype 6A, their prevalence was very low among the two age groups (Figure 1 and Figure 2). The ermB + mefE-positive isolates belonged to serogroup 19 (2 isolates among serotype 19F and 1 in 19A).
4. Discussion
Although the prevalence of resistance to macrolides in S. pneumoniae varies substantially among countries, the increase of macrolide-resistant pneumococci is a
worldwide problem (Calatayud et al., 2007; Schito and Felmingham, 2005). The recent use of PCVs has reduced the incidence of antibiotic-resistant serotypes in many countries (dos Santos et al., 2013; Tan, 2012; Tyrrell et al., 2009), but in Casablanca, Morocco we reported that in our context the evidence among ERSP isolates is unclear (Diawara et al., 2015). The present study, that aimed to determine the prevalence of ERSP and to analyze the phenotypic and genotypic characteristics of ERSP from 2007 to 20114, showed a global increase of ERSP prevalence after vaccine implementation in Casablanca. As reported elsewhere (Linares et al., 2010), the increase of antibiotic resistance rates is likely due to an increased antibiotic
consumption in Casablanca (Elmdaghri et al., 2012). Macrolides are the first antibiotics used empirically in association with β-lactams in the treatment of respiratory tract infection in
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Morocco. As previously described, antibiotic exposure is the most important risk for ERSP promotion and substantial diversity exists in the proportion of pneumococci resistant to macrolides (Dagan and Klugman, 2008). It has been demonstrated that resistance to
macrolides is low in countries where antibiotic prescribing is low, whereas the highest global resistance is in countries with easy access to antibiotics and where overcrowded institutions, such as day-care facilities, can amplify the selected resistant strains (Goossens et al., 2005). Selection for resistance has occurred predominantly in strains that colonize or infect children.
This could indicate increased opportunity for selection, in that children carry pneumococci more often and for longer than do adults, and their exposure to antibiotics is greater (Dagan and Klugman, 2008).
In this study, globally, 75% and 25% of ERSP belonged to vaccine and non-vaccine serotypes, respectively. The main vaccine serotypes responsible of macrolide resistance recovered before and after vaccination (6B, 19F, 23F and 6A) were known to be mainly associated with multidrug-resistant S. pneumoniae includingresistance to macrolide (Dagan and Klugman, 2008). Predominant serotypes can vary between countries (Wierzbowski et al., 2014, Daikos et al., 2008). Prevalence of antibiotics resistance is directly proportional to the frequency of antibiotic use in the community. In principle, if IPD and non-IPD infections are reduced by PCVs, this should result in a reduced antibiotic prescription rate in children and adults (Jacobs et al., 2003). Demonstration of this effect is difficult in regions where vaccines are introduced universally because antibiotic use can be influenced by secular trends that might mask the vaccine effect.
The use of the double-disc test with erythromycin-clindamycin may be a limitation of this study. Phenotypic and genotypic analysis of ERSP showed that enzymatic modification of MLS antimicrobial binding site was the predominant mechanism of macrolide resistance.
cMLSB and iMLSB phenotype isolates were highly resistant to erythromycin (MIC90: > 256
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mg/mL), clindamycin, penicillin G, cotrimoxazole and tetracycline. However, as it has been previously described (Chardon et al., 2002), strains with the M-phenotype showed lower macrolide-resistance levels with MICs not exceeding 8 mg/mL. Indeed, as previously reported, the use of double-disc test using erythromycin-clindamycin to differentiate cMLSB and iMLSB is less discriminating in ERSP because the constitutive or inducible character of MLS in the test must be inferred from the response to clindamycin, such that S. pneumoniae with MLSB resistance when tested by the double-disc test are invariably assigned to the cMLSB phenotype. This could explain the high proportion found of the cMLSB phenotype. A triple-disk test with addition of rokitamycin disc, not used in this study, could be
recommended instead of the double-disc test.
No discordance was found between the resistance phenotype, as detected by double disc diffusion, and the PCR detection of the ermB and mefA genes, respectively, in the cMLSB/iMLSB phenotype ERSP and M-phenotype ERSP.
Genotypic analysis of the strains showed that ermB-positive strains are predominant followed by other mechanisms mediated by mefE genes, and ermB + mefE. Notably, none of the strains was found to be mefA- and ermB-negative. Moreover, telithromycin and
pristinamycin were shown to be highly active against all of the ERSP isolates studied, demonstrating these molecules' potential for reducing the spread of ERSP. Several studies showed M phenotype with mefA gene to be the most represented in United-States, Canada and south Africa (up to 85% of erythromycin resistant pneumococcal isolates) (Wierzbowski et al., 2014; Wierzbowski et al., 2007; Sutcliffe et al., 1996), in contrast with European countries where MLSB phenotype is the most prevalent (Grivea et al., 2012; Reinert et al., 2005;
Marchandin et al., 2001; Descheemaeker et al., 2000). In North African countries, the dominant phenotype was MLSB in Tunisia (Rachdi et al., 2008). Nevertheless, this pattern of macrolide resistance determinants is not static and may be changing due to clonal spread of S.
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pneumoniae of certain serotypes and horizontal transfer of mefA elements among streptococci (Grivea et al., 2012). In this study, the predominant serotypes resistant to macrolide were 6B, 19F and 23F. As described (Wierzbowski et al., 2014), these three was also found to be the most prevalent serotypes of S. pneumoniae resistant to macrolide. Considering the periods before and after vaccination, there was no change in macrolide-resistant serotype distribution.
Furthermore, interestingly all but one ERSP carrying mefE gene belonged to serotype 6A. This finding differs from the Daikos et al study in Greece when serotype 19F was predominantly among mefE carrying ERSP (Daikos et al., 2008).
Nonetheless, we found that of all ERSP with dual combination of mefE and ermB belonged to serogroup 19. Worldwide, most isolates with the dual resistance mechanism belong to serotypes 19F or 19A (Wierzbowski et al., 2014; Grivea et al., 2012; Bowers et al., 2012).
The observed fluctuations of serotype distribution may be due to the intrinsic characteristics of S. pneumoniae, a highly transformable species. External factors, such as choice of PCV, antibiotic use, the socioeconomic status of populations and outbreaks of pneumococcal diseases have also been suggested as possible explanations for these serotype fluctuations (Elmdaghri et al., 2012).
This study described the prevalence and the distribution of macrolide resistance determinants in S. pneumoniae isolates in vaccine and non-vaccine area in Morocco. Further molecular studies are necessary to highlight genetic evolution of macrolide resistant S.
pneumoniae. These molecular studies using tools as genotyping by pulse field gel
electrophoresis (PFGE) and/or Multi Locus Sequence Typing (MLST) will be the future of this study.
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5. Conclusion
This is the first report describing the prevalence and the distribution of macrolide resistance determinants in S. pneumoniae isolates in Morocco. ERSP is relatively frequent in our country in spite of PCV implementation, and the cMLSB phenotype encoded by the ermB gene was the most prevalent. The contribution of PCVs to the reduction in antibiotic use is encouraging, but they should be accompanied by a rational use of antibiotic because antibiotic use is a powerful means of selecting and promoting multidrug-resistant S. pneumoniae,
including vaccine and non-vaccine serotype strains. Further surveillance studies are needed to understand the relationship between antibiotics use and pneumococcal vaccine effectiveness in Casablanca.
Competing interests
The authors declare that they have no competing interests.
Acknowledgments
This work was supported by an unrestricted, investigator-initiated grant from Pfizer (No. W1172148). The authors conceived the study, and the study design was developed and agreed to by the authors without any input from the funding body. The funding body was not involved in and, had no influence over, study design, data collection, data analyses,
interpretation of results, report writing or in the decision to submit the paper for publication.
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17 Figure captions
Figure 1: Distribution of serotype specific to macrolide resistance determinants from 2007 to 2010 and from 2011 to 2014 among children (aged from 0 to 14 years) in Casablanca, Morocco.
Figure 2: Distribution of serotype specific to macrolide resistance determinants from 2007 to 2010 and from 2011 to 2014 among adults (age > 14 years)in Casablanca, Morocco.
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19 Figure 2
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Table1: Distributions of phenotypes, genotypes, and antimicrobial resistance patterns of 92 erythromycin-resistant S. pneumoniae isolates
Phenotypea (no. of isolates)
Erythromycin MIC
Resistance pattern No. of isolates
No. of isolates with resistance determinant
MIC range (µg/mL) MIC50 (µg/mL) MIC90 (µg/mL) ermB mefE ermB +mefE
Constitutive MLSB (82) 64- > 250 >250 >250 Ery, Clin 5 5 0 0
Ery, Clin, PG, Chl, SXT, TE 6 5 0 1
Ery,Clin, PG, SXT, TE 21 21 0 0
Ery, Clin, PG, SXT 2 0 0 2
Ery, Clin, Chl, TE 5 5 0 0
Ery, Clin, PG, TE 36 36 0 0
Ery, Clin, SXT 2 2 0 0
Ery, Clin, TE 5 5 0 0
Inducible MLSB (4) 64- > 250 >250 >250 Ery, Clin, PG, SXT, TE 1 1 0 0
Ery, Clin, PG, TE 2 2 0 0
Ery, Clin, TE 1 1 0 0
M (6) 2-8 4 8 Ery, PG 6 0 6 0
aAccording to the results of the double-disk diffusion method
Ery: erythromycin; SXT: cotrimoxazole; TE: tetracycline; Chl: chloramphenicol; PG: penicillin G; Clin:
clindamycin.
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Highlights
The prevalence of erythromycin-resistant Streptococcus pneumoniae (ERSP) from 2007 to 2014 was 14%.
After vaccination (2010 to 2014), the rate of ERSP was increased significantly (11.6%
vs. 17.2%).
The main serotypes before and after vaccination were 6B, 19F, 23F and 6A.
More than 85% of ERSP were also resistant to tetracycline and penicillin G.
Enzymatic modification (ermB) was the main mechanism of macrolide resistance.
