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Molecular detection of genes responsible for macrolide resistance among Streptococcus pneumoniae isolated in North Lebanon
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ARTICLE IN PRESS Journal of Infection and Public Health xxx (2017) xxx–xxx
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Molecular detection of genes responsible for macrolide resistance among Streptococcus pneumoniae isolated in North Lebanon Salam El Ashkar a,1 , Marwan Osman a,1 , Rayane Rafei a , Hassan Mallat a , Marcel Achkar b , Fouad Dabboussi a , Monzer Hamze a,∗ a Laboratoire Microbiologie Santé et Environnement (LMSE), Ecole Doctorale des Sciences et de Technologie, Faculté de Santé Publique, Université Libanaise, Tripoli, Lebanon b Clinical Laboratory, Nini Hospital, Tripoli, Lebanon
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Article history: Received 2 July 2016 Received in revised form 10 October 2016 Accepted 18 November 2016 Keywords: Streptococcus pneumoniae Macrolide erm(B) mef(A/E) lin(A) Lebanon
a b s t r a c t In recent years, the increased use of macrolides was linked with the emergence of resistance Streptococcus pneumoniae worldwide. The main aim of this study was to determine the prevalence of S. pneumoniae resistant to macrolides and to identify the macrolide resistance genotypes among clinical isolates collected in North Lebanon. Disk diffusion susceptibility method was performed for 132 strains of S. pneumoniae isolated over a period of 5 years in North Lebanon. Polymerase Chain Reaction followed by pyrosequencing was carried out for confirmation of phenotypic diagnosis. The macrolide resistance genotypes were also identified by using PCR amplification of genes implicated in this resistance: erm(A), erm(B), erm(C), msr(A), lin(A) and mef(A/E). Macrolide resistance was found in 34.1% of S. pneumoniae isolates. We observed that the cMLSB phenotype (31/45, 68.9%) was the most common in these pneumococci and erm(B) was the most common resistance gene (32/45, 71.1%). This study shows that macrolide resistance in S. pneumoniae in North Lebanon is mainly related to target site modification with predominance of cMLSb phenotype but is also mediated by efflux pumps. lin(A) gene was reported for the first time in one S. pneumoniae strain in combination with erm(B) and mef(A/E) genes. © 2017 The Authors. Published by Elsevier Limited. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction Streptococcus pneumoniae (S. pneumoniae) is one of the major contributors to mortality and morbidity worldwide causing a variety of diseases ranging from simple respiratory infections such as otitis media and pneumococcal pneumonia to reach the threatening invasive infections such as meningitis and septicemia [1]. Even though proper antimicrobial treatments are available in the market, pneumococcal diseases kill 1.6 million people in developing countries each year, of which one million are children under the age of 5 years [2]. Beta-lactams, macrolides and fluoroquinolones are the three main classes of antibiotics used in the treatment of S. pneumoniae. The worldwide increase in Beta-lactams resistance coincided with an increase in macrolide resistant pneumococci [3]. There are two phenotypic resistance profiles among S. pneumoniae, designated M and MLSB , according to whether resistance to macrolides and/or lincosamides and/or streptogramin B agents is observed. Of
∗ Corresponding author. E-mail address: [email protected] (M. Hamze). 1 These authors contributed equally to this work.
these, MLSB and M phenotypes account for the majority of drug infection and macrolide resistance mainly caused by target site modification and active drug efflux [4]. MLSB phenotype is the end result of a methyltransferase, which is encoded by the erm(B) gene for erythromycin resistance methylase [5]. This gene confers high resistance to all macrolides (Minimum Inhibitory Concentration ≥ 64 g/mL) by reduction in the binding affinity to the 23S rRNA (domain V). This mechanism depends on methylation of specific position Adenine 2058 (A2058) in 23S rRNA [4]. The expression of the MLSB resistance may be constitutive (cMLSB ) or inducible (iMLSB ). Interestingly the constitutive resistance is characterized by erm(B) mRNA active methylase which is produced in the absence of an inducer [6,7]. However strains that carry an inducible erm gene are resistant to the inductor but remain susceptible to non-inductors macrolides. Macrolides C-14 and C-15 ring members are inducers as opposing to the 16-ring macrolides, lincosamides, and streptogramin, which are non-inducers [4,6]. The second mechanism of resistance in streptococci is the efflux mechanism that is encoded by mef (macrolide efflux) gene [8], which causes resistance to C-14 and C-15 membered macrolides compounds only, and the encoding phenotype is designed M [4].
http://dx.doi.org/10.1016/j.jiph.2016.11.014 1876-0341/© 2017 The Authors. Published by Elsevier Limited. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/).
Please cite this article in press as: El Ashkar S, et al. Molecular detection of genes responsible for macrolide resistance among Streptococcus pneumoniae isolated in North Lebanon. J Infect Public Health (2017), http://dx.doi.org/10.1016/j.jiph.2016.11.014
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2 Table 1 Primers used in the study. Gene
Primer sequence (5 -3 )
Size
Protocol cycle
Reference
erm(A)
F: 5 -AAGCGGTAAACCCCTCTGA-3 R: 5 -TTCGCAAATCCCTTCTCAAC-3
190
30 (30 at 94 ◦ C; 30 s at 52 ◦ C; 1 min at 72 ◦ C)
[15]
erm(B)
F: 5 -CTATCTGATTGTTGAAGAAGGATT-3 R: 5 -GTTTACTCTTGGTTTAGGATGAAA-3
142
Same as erm(A)
[15]
erm(C)
F: 5 -AATCGTCAATTCCTGCATGT-3 R: 5 -TAATCG TGGAATACGGGTTTG-3
299
Same as erm(A)
[15]
lin(A)
F: 5 -GGTGGCTGGGGGGTAGATGTATTAACTGG-3 R: 5 -GCTTCTTTTGAAATACATGGTATTTTTCGATC-3
323
30 (30 s at 94 ◦ C; 30 s at 57 ◦ C; 1 min at 72 ◦ C)
[16]
msr(A)
F: 5 -GGCACAATAAGAGTGTTTAAAGG-3 R: 5 -AAGTTATATCATGAATAGATTGTCCTGTT-3
940
25 (1 min at 94 ◦ C; 1 min at 50 ◦ C; 90sa at 72 ◦ C)
[16]
mef(E)
F: 5 -ATGGAAAAATACAACAATTGGAAACGA-3 R: 5 -TTATTTTAAATCTAATTTTCTAACCTC-3
1218
35 (30 s at 94 ◦ C; 30 s at 50 ◦ C; 90 s at 72 ◦ C)
[17]
mef(A)
F: 5 -AGTATCATTAATCACTAGTGC-3 R: 5 -TTCTTCTGGTACTAAAAGTTG-3
345
Same as mef(E)
[18]
In Streptococcus spp., mef genes include a number of subclasses, of which mef(A) and mef(E) are the most significant [9]. mef(A) gene, originally found in Strepococcus pyogenes [10] and mef(E) gene discovered in S. pneumoniae [11] are very common in S. pneumoniae. In addition, the cotranscription of mef(E) and msr(D), an msr-class gene with homology to msr(A) found in staphylococci, in S. pneumoniae suggested that the products of the two genes may act as a dual efflux system [12]. mef genes provides a low-level of macrolide resistance (Minimum Inhibitory Concentration = 1–32 g/mL) [13]. In order to better understand the epidemiology of macrolide resistance in S. pneumoniae in Lebanon, the main aim of this study was to determine the prevalence of resistance and to identify the genes responsible for this resistance among clinical S. pneumoniae strains isolated in North Lebanon.
geting V3 region was performed using pyrosequencer according to the instructions of the manufacturer [14]. The sequences obtained were aligned using the BioEdit v7.0.1 package (http://www.mbio. ncsu.edu/BioEdit/bioedit.html), then compared with sequences of S. pneumoniae published on the NCBI server (http://www.ncbi.nlm. nih.gov/BLAST/) using the basic local alignment search tool (BLAST) program. Detection of macrolide resistance genes In order to detect the genes known to be responsible for resistance to macrolide (erm(A), erm(B), erm(C), msr(A), mef(A/E) and lin(A) genes) in S. pneumoniae, PCR targeting these genes were performed using primers specific to each gene (Table 1).
Material and methods Results Sample collection This study was conducted in North Lebanon during the period 2010–2015. 132 non-duplicate S. pneumoniae isolates were collected from several clinical specimens including blood, sputum, bronchial wash, cerebrospinal fluid, deep tracheal aspirate and nasal and ear secretions. All isolates were identified by using gram staining and optochin susceptibility followed by latex agglutination testing (PastorexTM Meningitis, Biorad, France). Antimicrobial susceptibility test The susceptibility of different strains to antibiotics was performed by the disk diffusion according to CA-SFM 2015 recommendations. In order to highlight the macrolide resistance we tested the following antibiotics and its concentrations: Erythromycin (15 g), Clindamycin (2 g) and Pristinamycin (15 g) (Biorad, France). We also determined the sensitivity of isolates to penicillin using the oxacillin disk diffusion method (oxacillin disk charged of 1 g). DNA extraction and molecular identification All macrolide resistant S. pneumoniae isolates were used for molecular confirmation of the species identification. DNA was extracted using the QIAmp DNA Mini Kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer’s recommended procedures. The DNA was eluted in 100 L of elution buffer (Qiagen) and stored at −20 ◦ C until use. 16S rRNA sequencing analysis tar-
Molecular identification by pyrosequencing was accomplished and confirmed that all isolates were belonged to S. pneumoniae. Resistance to macrolide was found in 45 strains (34.1%) out of all 132 tested S. pneumoniae. Resistance phenotypes that were determined by disk diffusion susceptibility method with erythromycin, clindamycin and pristinamycin, showed that among 45 macrolideresistant isolates, 32 (71.1%) exhibited the MLSB phenotype: 31 (68.9%) belonged to the cMLSB , and 1 (2.2%) to the iMLSB phenotype. The remaining 13 isolates (28.9%) were confirmed as M phenotype (Table 2). PCR analysis of the 45 macrolide-resistant pneumococcal isolates showed that 37.8% (17/45) harbored the erm(B) gene and 28.9% (13/45) harbored the mef(A/E) gene. 14 isolates (31.1%) carried both erm(B) and mef(A/E) genes and only 1 strain (2.2%) possessed a combination of erm(B), lin(A) and mef(A/E). All the strains belonged to the MLSB phenotype harbored the erm(B) gene, while all the strains with M phenotype had the mef(A/E) gene (Table 3). In the other hand, S. pneumoniae with decreased susceptibility to penicillin (PNSP) is detected by oxacillin disk charged of 1 g (OXA1). Among the 45 strains of S. pneumoniae resistant to macrolide, 28 (62.2%) showed co-resistance to OXA-1 and 17 (37.8%) showed susceptibility to OXA-1. Discussion Nowadays, antibiotic resistance is one of the biggest threats to global health. It can affect anyone, of any age, in any country. This threat is influencing public health and patient safety by decreasing
Please cite this article in press as: El Ashkar S, et al. Molecular detection of genes responsible for macrolide resistance among Streptococcus pneumoniae isolated in North Lebanon. J Infect Public Health (2017), http://dx.doi.org/10.1016/j.jiph.2016.11.014
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Table 2 Distribution of macrolide resistant S. pneumoniae strains according to the nature of the samples. Nature of sample
Macrolide resistant S. pneumoniae strains
S. pneumoniae strains with cMLSb phenotype
S. pneumoniae strains with iMLSb phenotype
S. pneumoniae strains with M phenotype
Blood Sputum Bronchial wash Cerebrospinal fluid Nasal secretions Ear secretions Total
13 5 3 1 14 9 45
10 2 2 1 8 8 31
1 0 0 0 0 0 1
2 3 1 0 6 1 13
Table 3 Macrolide resistance phenotypes and genotypes of 45 erythromycin-resistant isolates. Phenotype
cMLSB iMLSB M
Genotype erm(B)
mef(A/E)
erm(B) and mef(A/E)
erm(B), mef(A/E) and lin(A)
17 – –
– – 13
13 1 –
1 – –
the availability of treatment options and increasing morbidity and mortality. Our study was conducted in North Lebanon, province suffering from a fall in the socio-economic status with a high unemployment rate, crowding and extreme poverty. Globally, resistance to macrolide was found in 45 strains (34.1%) out of all 132 tested S. pneumoniae. This prevalence was in the range to that reported in other neighboring countries, such as Turkey (14.5%) and Saudi Arabia (38.1%) but lower than that reported in East Asian countries, such as Japan (81.9%), Hong Kong (80.6%) and China (81.6%) [19]. There was a substantial geographic variability in the rate of erythromycin resistance with lower prevalence in Colombia (0%), Sweden (6%), Portugal (6.1%) and Brazil (6.2%) [19]. The majority of strains were isolated from several specimen types, including nasal secretions (14/45), blood (13/45), ear secretions (9/45), sputum (5/45), Bronchial wash (3/45), and cerebrospinal fluid (1/45). No statistically significant difference were detected between the prevalence of several resistance phenotypes and the nature of samples. Our data confirmed that S. pneumoniae can be spread from the upper respiratory tract to the sterile regions of the body [20]. In addition, in our study, a predominance of cMLSB phenotype was seen in 68.9% (31/45) of strains followed by the M phenotype that was detected in 28.9% of isolates (13/45). Finally, only one strain of S. pneumoniae (2.2%) exhibited the iMLSB phenotype. In parallel these findings were also supported by an earlier study in Serbia showing that cMLSB (79.8%) had the highest prevalence of resistance phenotypes in macrolide resistant S. pneumoniae, followed by M (16.7%) and iMLSB (3.5%) [21]. Another study conducted in Iran showed that cMLSB (84%) was reported as the predominant phenotype in macrolide resistant S. pneumoniae followed by M phenotype (16%) [22]. PCR analysis of the 45 macrolide-resistant S. pneumoniae isolates in North Lebanon showed that the erm(B) was the prevailing gene present in 37.8% of all strains. Furthermore, 31.1%, 28.9% and 2.2% of strains harbored both erm(B) and mef(A/E) genes, mef(A/E) gene and a combination of erm(B), lin(A) and mef(A/E) genes, respectively. The number of pneumococcal strains with a dual resistance mechanism related to the presence of both erm(B) and mef(A) genes has increased globally. The erm(B) gene was reported as predominant in several regions, such as Belgium (91.5%), France (90%), Spain (88.3%), Serbia (82.4%), Hungary (82.4%), Poland (80.8%), China (76.5%), Japan (58%) and Italy (55.8%) [23]. However, the mef(A) gene was found as more prevalent in UK (70.8%), Greece (66.2%), Australia (59.5%), Finland (55.4%), USA (55.2%), and Germany
(53.2%) [19]. The countries with a high prevalence of the dual resistance mechanism in pneumococci were South Korea and South Africa. The majority of these strains are multiresistant and clonally related [24,25]. On the other hand, lin(A) gene was identified in one strain in combination with erm(B) and mef(A/E) genes. To our knowledge, lin(A) gene conferring resistance only to lincosamide has been reported in many Staphylococcus spp. strains, but it is the first time reported in S. pneumoniae. Moreover, it has been observed that all the strains belonging to the MLSB phenotype harbored the erm(B) gene, while all the strains with M phenotype had the mef(A/E) gene. The presence of both resistance genes was confirmed in 14 strains with MLSB phenotype. In the same context, two recent studies conducted in Lebanon and Iran described the same distribution with predominance of macrolide resistant S. pneumoniae isolates harboring erm(B) gene followed by both erm(B) and mef(A/E) genes and mef(A/E) gene, respectively [22,26]. Furthermore all isolates were screened for Penicillin nonsusceptibility with a 1 g oxacillin disc. It was spotted in our study that 62.2% of macrolide resistant isolates are co-resistant to oxacillin. The prevalence of PNSP strains described in our study was similar to that reported in other neighboring countries [27,28]. These results confirm recent reports showing the dissemination of S. pneumoniae strains resistant to penicillin to several antibiotic classes [29–31]. These findings could be associated with the expansion of antibiotics use without any medical prescription, its misuse in postoperative periods and overuse in the food industry and prophylactic treatment in the Middle Eastern Region [32]. Moreover, our data accord with previous findings of association between inclusion of a macrolide in a -lactam-based empirical antibiotic treatment and mortality among patients with bacteremic S. pneumoniae pneumonia [33]. Conclusion In conclusion, a high rate of macrolide resistant S. pneumoniae (34.1%) was reported in North Lebanon with predominance of cMLSB phenotype and erm(B) gene. Furthermore, to our knowledge, lin(A) gene was reported for the first time in S. pneumoniae. For that, further studies are needed to molecular characterize a probable linkage to mobile genetic elements of this gene. On the other hand, it’s notable that the rate of resistance is rising in Lebanon as well as worldwide. The choice in the treatment against S. pneumoniae is complicated especially when we are
Please cite this article in press as: El Ashkar S, et al. Molecular detection of genes responsible for macrolide resistance among Streptococcus pneumoniae isolated in North Lebanon. J Infect Public Health (2017), http://dx.doi.org/10.1016/j.jiph.2016.11.014
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faced with a strain having co-resistance to both beta-lactams and macrolides (62.2%). Campaigns on behalf of better antibiotic use among prescribers should be organized with more standardized treatment guidelines. Funding This study was financed by the AZM center for research in biotechnology and its applications, Doctoral School of Science and Technology, Lebanese University, Tripoli, Lebanon. Competing interests None declared. Ethical approval Not required. Author contributions Conceived and designed the experiments: MO, RR, HM, FD, MH. Performed the experiments: SA. Analyzed the data: SA, MO, RR, FD, MH. Contributed reagents/materials/analysis tools: MA, MH. Wrote the paper: SA, MO, FD, MH. Acknowledgements We would like to thank Taha Abdou, Sara Amrieh and Mariam Yehya for their excellent technical assistance and Pr. Ghassan Matar from American University of Beirut for kindly providing us control strains. References [1] Klugman KP, Feldman C. Streptococcus pneumoniae respiratory tract infections. Curr Opin Infect Dis 2001;14(2):173–9. [2] Levine OS, O’Brien KL, Knoll M, Adegbola RA, Black S, Cherian T, et al. Pneumococcal vaccination in developing countries. Lancet 2006;367(9526):1880–2. [3] Cornick JE, Bentley SD. Streptococcus pneumoniae: the evolution of antimicrobial resistance to beta-lactams, fluoroquinolones and macrolides. Microbes Infect 2012;14(7–8):573–83. [4] Edelstein PH. Pneumococcal resistance to macrolides, lincosamides, ketolides, and streptogramin B agents: molecular mechanisms and resistance phenotypes. Clin Infect Dis 2004;38(Suppl. 4):S322–7. [5] Matsuoka M, Inoue M, Nakajima Y, Endo Y. New erm Gene in Staphylococcus aureus clinical isolates. Antimicrob Agents Chemother 2002;46(1):211–5. [6] Leclercq R, Courvalin P. Resistance to macrolides and related antibiotics in Streptococcus pneumoniae. Antimicrob Agents Chemother 2002;46(9):2727–34. [7] Charpentier E, Tuomanen E. Mechanisms of antibiotic resistance and tolerance in Streptococcus pneumoniae. Microbes Infect 2000;2(15):1855–64. [8] Zhong P, Shortridge VD. The role of efflux in macrolide resistance. Drug Resist Updat 2000;3(6):325–9. [9] Mingoia M, Morici E, Brenciani A, Giovanetti E, Varaldo PE. Genetic basis of the association of resistance genes mef(I) (macrolides) and catQ (chloramphenicol) in streptococci. Front Microbiol 2014;5:747. [10] Clancy J, Petitpas J, Dib-Hajj F, Yuan W, Cronan M, Kamath AV. Molecular cloning and functional analysis of a novel macrolide-resistance determinant, mefA, from Streptococcus pyogenes. Mol Microbiol 1996;22(5):867–79. [11] Tait-Kamradt A, Clancy J, Cronan M, Dib-Hajj F, Wondrack L, Yuan W. mefE is necessary for the erythromycin-resistant M phenotype in Streptococcus pneumoniae. Antimicrob Agents Chemother 1997;41(10):2251–5.
[12] Gay K, Stephens DS. Structure and dissemination of a chromosomal insertion element encoding macrolide efflux in Streptococcus pneumoniae. J Infect Dis 2001;184(1):56–65. [13] Doern GV. Macrolide and ketolide resistance with Streptococcus pneumoniae. Med Clin North Am 2006;90(6):1109–24. [14] Grahn N, Olofsson M, Ellnebo-Svedlund K, Monstein HJ, Jonasson J. Identification of mixed bacterial DNA contamination in broad-range PCR amplification of 16S rDNA V1 and V3 variable regions by pyrosequencing of cloned amplicons. FEMS Microbiol Lett 2003;219(1):87–91. [15] Strommenger B, Kettlitz C, Werner G, Witte W. Multiplex PCR assay for simultaneous detection of nine clinically relevant antibiotic resistance genes in Staphylococcus aureus. J Clin Microbiol 2003;41(9):4089–94. [16] Cetin ES, Gunes H, Kaya S, Aridogan BC, Demirci M. Distribution of genes encoding resistance to macrolides, lincosamides and streptogramins among clinical staphylococcal isolates in a Turkish university hospital. J Microbiol Immunol Infect 2010;43(6):524–9. [17] Nishijima T, Saito Y, Aoki A, Toriya M, Toyonaga Y, Fujii R. Distribution of mefE and ermB genes in macrolide-resistant strains of Streptococcus pneumoniae and their variable susceptibility to various antibiotics. J Antimicrob Chemother 1999;43:637–43. [18] Perez-Trallero E, Montes M, Orden B, Tamayo E, Garcia-Arenzana JM, Marimon JM. Phenotypic and genotypic characterization of Streptococcus pyogenes isolates displaying the MLSB phenotype of macrolide resistance in Spain, 1999 to 2005. Antimicrob Agents Chemother 2007;51(4):1228–33. [19] Farrell DJ, Couturier C, Hryniewicz W. Distribution and antibacterial susceptibility of macrolide resistance genotypes in Streptococcus pneumoniae: PROTEKT Year 5 (2003-2004). Int J Antimicrob Agents 2008;31(3):245–9. [20] Kadioglu A, Weiser JN, Paton JC, Andrew PW. The role of Streptococcus pneumoniae virulence factors in host respiratory colonization and disease. Nat Rev Microbiol 2008;6:288–301. [21] Hadnadev M, Gajic I, Mijac V, Kurucin T, Povazan A, Vulin A. Phenotypes and genotypes of macrolide-resistant Streptococcus pneumoniae in Serbia. Arch Biol Sci 2014;66(1):99–105. [22] Azadegan A, Ahmadi A, Lari AR, Talebi M. Detection of the efflux-mediated erythromycin resistance transposon in Streptococcus pneumoniae. Ann Lab Med 2015;35(1):57–61. [23] Felmingham D, Canton R, Jenkins SG. Regional trends in beta-lactam, macrolide, fluoroquinolone and telithromycin resistance among Streptococcus pneumoniae isolates 2001-2004. J Infect 2007;55(2):111–8. [24] Jenkins SG, Brown SD, Farrell DJ. Trends in antibacterial resistance among Streptococcus pneumoniae isolated in the USA: update from PROTEKT US Years 1-4. Ann Clin Microbiol Antimicrob 2008;7:1. [25] McGee L, McDougal L, Zhou J, Spratt BG, Tenover FC, George R. Nomenclature of major antimicrobial-resistant clones of Streptococcus pneumoniae defined by the pneumococcal molecular epidemiology network. J Clin Microbiol 2001;39(7):2565–71. [26] Taha N, Araj GF, Wakim RH, Kanj SS, Kanafani ZA, Sabra A. Genotypes and serotype distribution of macrolide resistant invasive and non-invasive Streptococcus pneumoniae isolates from Lebanon. Ann Clin Microbiol Antimicrob 2012;11:2. [27] Borg MA, Tiemersma E, Scicluna E, van de Sande-Bruinsma N, de Kraker M, Monen J. Prevalence of penicillin and erythromycin resistance among invasive Streptococcus pneumoniae isolates reported by laboratories in the southern and eastern Mediterranean region. Clin Microbiol Infect 2009;15(3):232–7. [28] Holstein A, Amirault P, Arnould JP, Bachelier MN, Benseddik Z, Bret L. 19972007, 10 years of monitoring the evolution of resistance of Streptococcus pneumoniae to antibiotics in the region Centre; review of the Pneumococcus network. Pathol Biol (Paris) 2010;58(1):62–6. [29] Maraki S, Papadakis IS. Antimicrobial resistance trends among communityacquired respiratory tract pathogens in Greece, 2009-2012. Sci World J 2014;2014, 941564. [30] Aspa J, Rajas O, de Castro FR. Pneumococcal antimicrobial resistance: therapeutic strategy and management in community-acquired pneumonia. Expert Opin Pharmacother 2008;9(2):229–41. [31] Pihlajamaki M, Kaijalainen T, Huovinen P, Jalava J. Rapid increase in macrolide resistance among penicillin non-susceptible pneumococci in Finland, 19962000. J Antimicrob Chemother 2002;49(5):785–92. [32] Ventola CL. The antibiotic resistance crisis: part 1: causes and threats. P T 2015;40:277–83. [33] Martinez JA, Horcajada JP, Almela M, Marco F, Soriano A, García E. Addition of a macrolide to a beta-lactam-based empirical antibiotic regimen is associated with lower in-hospital mortality for patients with bacteremic pneumococcal pneumonia. Clin Infect Dis 2003;36:389–95.
Please cite this article in press as: El Ashkar S, et al. Molecular detection of genes responsible for macrolide resistance among Streptococcus pneumoniae isolated in North Lebanon. J Infect Public Health (2017), http://dx.doi.org/10.1016/j.jiph.2016.11.014
ARTICLE IN PRESS Journal of Infection and Public Health xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Journal of Infection and Public Health journal homepage: http://www.elsevier.com/locate/jiph
Molecular detection of genes responsible for macrolide resistance among Streptococcus pneumoniae isolated in North Lebanon Salam El Ashkar a,1 , Marwan Osman a,1 , Rayane Rafei a , Hassan Mallat a , Marcel Achkar b , Fouad Dabboussi a , Monzer Hamze a,∗ a Laboratoire Microbiologie Santé et Environnement (LMSE), Ecole Doctorale des Sciences et de Technologie, Faculté de Santé Publique, Université Libanaise, Tripoli, Lebanon b Clinical Laboratory, Nini Hospital, Tripoli, Lebanon
a r t i c l e
i n f o
Article history: Received 2 July 2016 Received in revised form 10 October 2016 Accepted 18 November 2016 Keywords: Streptococcus pneumoniae Macrolide erm(B) mef(A/E) lin(A) Lebanon
a b s t r a c t In recent years, the increased use of macrolides was linked with the emergence of resistance Streptococcus pneumoniae worldwide. The main aim of this study was to determine the prevalence of S. pneumoniae resistant to macrolides and to identify the macrolide resistance genotypes among clinical isolates collected in North Lebanon. Disk diffusion susceptibility method was performed for 132 strains of S. pneumoniae isolated over a period of 5 years in North Lebanon. Polymerase Chain Reaction followed by pyrosequencing was carried out for confirmation of phenotypic diagnosis. The macrolide resistance genotypes were also identified by using PCR amplification of genes implicated in this resistance: erm(A), erm(B), erm(C), msr(A), lin(A) and mef(A/E). Macrolide resistance was found in 34.1% of S. pneumoniae isolates. We observed that the cMLSB phenotype (31/45, 68.9%) was the most common in these pneumococci and erm(B) was the most common resistance gene (32/45, 71.1%). This study shows that macrolide resistance in S. pneumoniae in North Lebanon is mainly related to target site modification with predominance of cMLSb phenotype but is also mediated by efflux pumps. lin(A) gene was reported for the first time in one S. pneumoniae strain in combination with erm(B) and mef(A/E) genes. © 2017 The Authors. Published by Elsevier Limited. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Introduction Streptococcus pneumoniae (S. pneumoniae) is one of the major contributors to mortality and morbidity worldwide causing a variety of diseases ranging from simple respiratory infections such as otitis media and pneumococcal pneumonia to reach the threatening invasive infections such as meningitis and septicemia [1]. Even though proper antimicrobial treatments are available in the market, pneumococcal diseases kill 1.6 million people in developing countries each year, of which one million are children under the age of 5 years [2]. Beta-lactams, macrolides and fluoroquinolones are the three main classes of antibiotics used in the treatment of S. pneumoniae. The worldwide increase in Beta-lactams resistance coincided with an increase in macrolide resistant pneumococci [3]. There are two phenotypic resistance profiles among S. pneumoniae, designated M and MLSB , according to whether resistance to macrolides and/or lincosamides and/or streptogramin B agents is observed. Of
∗ Corresponding author. E-mail address: [email protected] (M. Hamze). 1 These authors contributed equally to this work.
these, MLSB and M phenotypes account for the majority of drug infection and macrolide resistance mainly caused by target site modification and active drug efflux [4]. MLSB phenotype is the end result of a methyltransferase, which is encoded by the erm(B) gene for erythromycin resistance methylase [5]. This gene confers high resistance to all macrolides (Minimum Inhibitory Concentration ≥ 64 g/mL) by reduction in the binding affinity to the 23S rRNA (domain V). This mechanism depends on methylation of specific position Adenine 2058 (A2058) in 23S rRNA [4]. The expression of the MLSB resistance may be constitutive (cMLSB ) or inducible (iMLSB ). Interestingly the constitutive resistance is characterized by erm(B) mRNA active methylase which is produced in the absence of an inducer [6,7]. However strains that carry an inducible erm gene are resistant to the inductor but remain susceptible to non-inductors macrolides. Macrolides C-14 and C-15 ring members are inducers as opposing to the 16-ring macrolides, lincosamides, and streptogramin, which are non-inducers [4,6]. The second mechanism of resistance in streptococci is the efflux mechanism that is encoded by mef (macrolide efflux) gene [8], which causes resistance to C-14 and C-15 membered macrolides compounds only, and the encoding phenotype is designed M [4].
http://dx.doi.org/10.1016/j.jiph.2016.11.014 1876-0341/© 2017 The Authors. Published by Elsevier Limited. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-ncnd/4.0/).
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2 Table 1 Primers used in the study. Gene
Primer sequence (5 -3 )
Size
Protocol cycle
Reference
erm(A)
F: 5 -AAGCGGTAAACCCCTCTGA-3 R: 5 -TTCGCAAATCCCTTCTCAAC-3
190
30 (30 at 94 ◦ C; 30 s at 52 ◦ C; 1 min at 72 ◦ C)
[15]
erm(B)
F: 5 -CTATCTGATTGTTGAAGAAGGATT-3 R: 5 -GTTTACTCTTGGTTTAGGATGAAA-3
142
Same as erm(A)
[15]
erm(C)
F: 5 -AATCGTCAATTCCTGCATGT-3 R: 5 -TAATCG TGGAATACGGGTTTG-3
299
Same as erm(A)
[15]
lin(A)
F: 5 -GGTGGCTGGGGGGTAGATGTATTAACTGG-3 R: 5 -GCTTCTTTTGAAATACATGGTATTTTTCGATC-3
323
30 (30 s at 94 ◦ C; 30 s at 57 ◦ C; 1 min at 72 ◦ C)
[16]
msr(A)
F: 5 -GGCACAATAAGAGTGTTTAAAGG-3 R: 5 -AAGTTATATCATGAATAGATTGTCCTGTT-3
940
25 (1 min at 94 ◦ C; 1 min at 50 ◦ C; 90sa at 72 ◦ C)
[16]
mef(E)
F: 5 -ATGGAAAAATACAACAATTGGAAACGA-3 R: 5 -TTATTTTAAATCTAATTTTCTAACCTC-3
1218
35 (30 s at 94 ◦ C; 30 s at 50 ◦ C; 90 s at 72 ◦ C)
[17]
mef(A)
F: 5 -AGTATCATTAATCACTAGTGC-3 R: 5 -TTCTTCTGGTACTAAAAGTTG-3
345
Same as mef(E)
[18]
In Streptococcus spp., mef genes include a number of subclasses, of which mef(A) and mef(E) are the most significant [9]. mef(A) gene, originally found in Strepococcus pyogenes [10] and mef(E) gene discovered in S. pneumoniae [11] are very common in S. pneumoniae. In addition, the cotranscription of mef(E) and msr(D), an msr-class gene with homology to msr(A) found in staphylococci, in S. pneumoniae suggested that the products of the two genes may act as a dual efflux system [12]. mef genes provides a low-level of macrolide resistance (Minimum Inhibitory Concentration = 1–32 g/mL) [13]. In order to better understand the epidemiology of macrolide resistance in S. pneumoniae in Lebanon, the main aim of this study was to determine the prevalence of resistance and to identify the genes responsible for this resistance among clinical S. pneumoniae strains isolated in North Lebanon.
geting V3 region was performed using pyrosequencer according to the instructions of the manufacturer [14]. The sequences obtained were aligned using the BioEdit v7.0.1 package (http://www.mbio. ncsu.edu/BioEdit/bioedit.html), then compared with sequences of S. pneumoniae published on the NCBI server (http://www.ncbi.nlm. nih.gov/BLAST/) using the basic local alignment search tool (BLAST) program. Detection of macrolide resistance genes In order to detect the genes known to be responsible for resistance to macrolide (erm(A), erm(B), erm(C), msr(A), mef(A/E) and lin(A) genes) in S. pneumoniae, PCR targeting these genes were performed using primers specific to each gene (Table 1).
Material and methods Results Sample collection This study was conducted in North Lebanon during the period 2010–2015. 132 non-duplicate S. pneumoniae isolates were collected from several clinical specimens including blood, sputum, bronchial wash, cerebrospinal fluid, deep tracheal aspirate and nasal and ear secretions. All isolates were identified by using gram staining and optochin susceptibility followed by latex agglutination testing (PastorexTM Meningitis, Biorad, France). Antimicrobial susceptibility test The susceptibility of different strains to antibiotics was performed by the disk diffusion according to CA-SFM 2015 recommendations. In order to highlight the macrolide resistance we tested the following antibiotics and its concentrations: Erythromycin (15 g), Clindamycin (2 g) and Pristinamycin (15 g) (Biorad, France). We also determined the sensitivity of isolates to penicillin using the oxacillin disk diffusion method (oxacillin disk charged of 1 g). DNA extraction and molecular identification All macrolide resistant S. pneumoniae isolates were used for molecular confirmation of the species identification. DNA was extracted using the QIAmp DNA Mini Kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer’s recommended procedures. The DNA was eluted in 100 L of elution buffer (Qiagen) and stored at −20 ◦ C until use. 16S rRNA sequencing analysis tar-
Molecular identification by pyrosequencing was accomplished and confirmed that all isolates were belonged to S. pneumoniae. Resistance to macrolide was found in 45 strains (34.1%) out of all 132 tested S. pneumoniae. Resistance phenotypes that were determined by disk diffusion susceptibility method with erythromycin, clindamycin and pristinamycin, showed that among 45 macrolideresistant isolates, 32 (71.1%) exhibited the MLSB phenotype: 31 (68.9%) belonged to the cMLSB , and 1 (2.2%) to the iMLSB phenotype. The remaining 13 isolates (28.9%) were confirmed as M phenotype (Table 2). PCR analysis of the 45 macrolide-resistant pneumococcal isolates showed that 37.8% (17/45) harbored the erm(B) gene and 28.9% (13/45) harbored the mef(A/E) gene. 14 isolates (31.1%) carried both erm(B) and mef(A/E) genes and only 1 strain (2.2%) possessed a combination of erm(B), lin(A) and mef(A/E). All the strains belonged to the MLSB phenotype harbored the erm(B) gene, while all the strains with M phenotype had the mef(A/E) gene (Table 3). In the other hand, S. pneumoniae with decreased susceptibility to penicillin (PNSP) is detected by oxacillin disk charged of 1 g (OXA1). Among the 45 strains of S. pneumoniae resistant to macrolide, 28 (62.2%) showed co-resistance to OXA-1 and 17 (37.8%) showed susceptibility to OXA-1. Discussion Nowadays, antibiotic resistance is one of the biggest threats to global health. It can affect anyone, of any age, in any country. This threat is influencing public health and patient safety by decreasing
Please cite this article in press as: El Ashkar S, et al. Molecular detection of genes responsible for macrolide resistance among Streptococcus pneumoniae isolated in North Lebanon. J Infect Public Health (2017), http://dx.doi.org/10.1016/j.jiph.2016.11.014
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Table 2 Distribution of macrolide resistant S. pneumoniae strains according to the nature of the samples. Nature of sample
Macrolide resistant S. pneumoniae strains
S. pneumoniae strains with cMLSb phenotype
S. pneumoniae strains with iMLSb phenotype
S. pneumoniae strains with M phenotype
Blood Sputum Bronchial wash Cerebrospinal fluid Nasal secretions Ear secretions Total
13 5 3 1 14 9 45
10 2 2 1 8 8 31
1 0 0 0 0 0 1
2 3 1 0 6 1 13
Table 3 Macrolide resistance phenotypes and genotypes of 45 erythromycin-resistant isolates. Phenotype
cMLSB iMLSB M
Genotype erm(B)
mef(A/E)
erm(B) and mef(A/E)
erm(B), mef(A/E) and lin(A)
17 – –
– – 13
13 1 –
1 – –
the availability of treatment options and increasing morbidity and mortality. Our study was conducted in North Lebanon, province suffering from a fall in the socio-economic status with a high unemployment rate, crowding and extreme poverty. Globally, resistance to macrolide was found in 45 strains (34.1%) out of all 132 tested S. pneumoniae. This prevalence was in the range to that reported in other neighboring countries, such as Turkey (14.5%) and Saudi Arabia (38.1%) but lower than that reported in East Asian countries, such as Japan (81.9%), Hong Kong (80.6%) and China (81.6%) [19]. There was a substantial geographic variability in the rate of erythromycin resistance with lower prevalence in Colombia (0%), Sweden (6%), Portugal (6.1%) and Brazil (6.2%) [19]. The majority of strains were isolated from several specimen types, including nasal secretions (14/45), blood (13/45), ear secretions (9/45), sputum (5/45), Bronchial wash (3/45), and cerebrospinal fluid (1/45). No statistically significant difference were detected between the prevalence of several resistance phenotypes and the nature of samples. Our data confirmed that S. pneumoniae can be spread from the upper respiratory tract to the sterile regions of the body [20]. In addition, in our study, a predominance of cMLSB phenotype was seen in 68.9% (31/45) of strains followed by the M phenotype that was detected in 28.9% of isolates (13/45). Finally, only one strain of S. pneumoniae (2.2%) exhibited the iMLSB phenotype. In parallel these findings were also supported by an earlier study in Serbia showing that cMLSB (79.8%) had the highest prevalence of resistance phenotypes in macrolide resistant S. pneumoniae, followed by M (16.7%) and iMLSB (3.5%) [21]. Another study conducted in Iran showed that cMLSB (84%) was reported as the predominant phenotype in macrolide resistant S. pneumoniae followed by M phenotype (16%) [22]. PCR analysis of the 45 macrolide-resistant S. pneumoniae isolates in North Lebanon showed that the erm(B) was the prevailing gene present in 37.8% of all strains. Furthermore, 31.1%, 28.9% and 2.2% of strains harbored both erm(B) and mef(A/E) genes, mef(A/E) gene and a combination of erm(B), lin(A) and mef(A/E) genes, respectively. The number of pneumococcal strains with a dual resistance mechanism related to the presence of both erm(B) and mef(A) genes has increased globally. The erm(B) gene was reported as predominant in several regions, such as Belgium (91.5%), France (90%), Spain (88.3%), Serbia (82.4%), Hungary (82.4%), Poland (80.8%), China (76.5%), Japan (58%) and Italy (55.8%) [23]. However, the mef(A) gene was found as more prevalent in UK (70.8%), Greece (66.2%), Australia (59.5%), Finland (55.4%), USA (55.2%), and Germany
(53.2%) [19]. The countries with a high prevalence of the dual resistance mechanism in pneumococci were South Korea and South Africa. The majority of these strains are multiresistant and clonally related [24,25]. On the other hand, lin(A) gene was identified in one strain in combination with erm(B) and mef(A/E) genes. To our knowledge, lin(A) gene conferring resistance only to lincosamide has been reported in many Staphylococcus spp. strains, but it is the first time reported in S. pneumoniae. Moreover, it has been observed that all the strains belonging to the MLSB phenotype harbored the erm(B) gene, while all the strains with M phenotype had the mef(A/E) gene. The presence of both resistance genes was confirmed in 14 strains with MLSB phenotype. In the same context, two recent studies conducted in Lebanon and Iran described the same distribution with predominance of macrolide resistant S. pneumoniae isolates harboring erm(B) gene followed by both erm(B) and mef(A/E) genes and mef(A/E) gene, respectively [22,26]. Furthermore all isolates were screened for Penicillin nonsusceptibility with a 1 g oxacillin disc. It was spotted in our study that 62.2% of macrolide resistant isolates are co-resistant to oxacillin. The prevalence of PNSP strains described in our study was similar to that reported in other neighboring countries [27,28]. These results confirm recent reports showing the dissemination of S. pneumoniae strains resistant to penicillin to several antibiotic classes [29–31]. These findings could be associated with the expansion of antibiotics use without any medical prescription, its misuse in postoperative periods and overuse in the food industry and prophylactic treatment in the Middle Eastern Region [32]. Moreover, our data accord with previous findings of association between inclusion of a macrolide in a -lactam-based empirical antibiotic treatment and mortality among patients with bacteremic S. pneumoniae pneumonia [33]. Conclusion In conclusion, a high rate of macrolide resistant S. pneumoniae (34.1%) was reported in North Lebanon with predominance of cMLSB phenotype and erm(B) gene. Furthermore, to our knowledge, lin(A) gene was reported for the first time in S. pneumoniae. For that, further studies are needed to molecular characterize a probable linkage to mobile genetic elements of this gene. On the other hand, it’s notable that the rate of resistance is rising in Lebanon as well as worldwide. The choice in the treatment against S. pneumoniae is complicated especially when we are
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faced with a strain having co-resistance to both beta-lactams and macrolides (62.2%). Campaigns on behalf of better antibiotic use among prescribers should be organized with more standardized treatment guidelines. Funding This study was financed by the AZM center for research in biotechnology and its applications, Doctoral School of Science and Technology, Lebanese University, Tripoli, Lebanon. Competing interests None declared. Ethical approval Not required. Author contributions Conceived and designed the experiments: MO, RR, HM, FD, MH. Performed the experiments: SA. Analyzed the data: SA, MO, RR, FD, MH. Contributed reagents/materials/analysis tools: MA, MH. Wrote the paper: SA, MO, FD, MH. Acknowledgements We would like to thank Taha Abdou, Sara Amrieh and Mariam Yehya for their excellent technical assistance and Pr. Ghassan Matar from American University of Beirut for kindly providing us control strains. References [1] Klugman KP, Feldman C. Streptococcus pneumoniae respiratory tract infections. Curr Opin Infect Dis 2001;14(2):173–9. [2] Levine OS, O’Brien KL, Knoll M, Adegbola RA, Black S, Cherian T, et al. Pneumococcal vaccination in developing countries. Lancet 2006;367(9526):1880–2. [3] Cornick JE, Bentley SD. Streptococcus pneumoniae: the evolution of antimicrobial resistance to beta-lactams, fluoroquinolones and macrolides. Microbes Infect 2012;14(7–8):573–83. [4] Edelstein PH. Pneumococcal resistance to macrolides, lincosamides, ketolides, and streptogramin B agents: molecular mechanisms and resistance phenotypes. Clin Infect Dis 2004;38(Suppl. 4):S322–7. [5] Matsuoka M, Inoue M, Nakajima Y, Endo Y. New erm Gene in Staphylococcus aureus clinical isolates. Antimicrob Agents Chemother 2002;46(1):211–5. [6] Leclercq R, Courvalin P. Resistance to macrolides and related antibiotics in Streptococcus pneumoniae. Antimicrob Agents Chemother 2002;46(9):2727–34. [7] Charpentier E, Tuomanen E. Mechanisms of antibiotic resistance and tolerance in Streptococcus pneumoniae. Microbes Infect 2000;2(15):1855–64. [8] Zhong P, Shortridge VD. The role of efflux in macrolide resistance. Drug Resist Updat 2000;3(6):325–9. [9] Mingoia M, Morici E, Brenciani A, Giovanetti E, Varaldo PE. Genetic basis of the association of resistance genes mef(I) (macrolides) and catQ (chloramphenicol) in streptococci. Front Microbiol 2014;5:747. [10] Clancy J, Petitpas J, Dib-Hajj F, Yuan W, Cronan M, Kamath AV. Molecular cloning and functional analysis of a novel macrolide-resistance determinant, mefA, from Streptococcus pyogenes. Mol Microbiol 1996;22(5):867–79. [11] Tait-Kamradt A, Clancy J, Cronan M, Dib-Hajj F, Wondrack L, Yuan W. mefE is necessary for the erythromycin-resistant M phenotype in Streptococcus pneumoniae. Antimicrob Agents Chemother 1997;41(10):2251–5.
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Please cite this article in press as: El Ashkar S, et al. Molecular detection of genes responsible for macrolide resistance among Streptococcus pneumoniae isolated in North Lebanon. J Infect Public Health (2017), http://dx.doi.org/10.1016/j.jiph.2016.11.014