Summary
Introduction: Streptococcus pneumoniae is one of the major pathogens responsible for respiratory tract infections, meningitis, and bacteremia. In most countries around the world, an increase in the emergence of macrolide-resistant S. pneumoniae isolates has become a serious problem. Therefore, this study was conducted to investigate the association of erythromycin resistance with the mefA and ermB genes among clinical isolates of S. pneumoniae in Tehran City (Tehran Province, Iran).
Materials and Methods: In this cross-sectional study, 62 erythromycin-resistant S. pneumoniae (ERSP) isolates were obtained from patients in four hospitals in Tehran city during 2013-2015. The macrolide resistance genes mefA and ermB were detected in ERSP isolates using polymerase chain reaction (PCR) assays. Furthermore, the copy number and expression level of the mefA gene were determined by real-time PCR.
Results: In the present study, the overall resistance to erythromycin in S. pneumoniae isolates was 84.93% (62 isolates out of 73). Either mefA or ermB was identified in 10 (16.13%) and 36 (58.06%) ERSP isolates, respectively, and 7 (11.29%) ERSP isolates harbored both genes. Comparison of the mean relative expression of the mefA gene in ERSP isolates with different erythromycin minimal inhibitory concentration (MIC) values indicated that the expression level was increased approximately twofold for each doubling of the MIC value.
Conclusion: Our findings highlighted the dominance of ribosomal methylation encoded by ermB as the most common mechanism of macrolide resistance in clinical S. pneumoniae isolates from Iran. Furthermore, in the present study, the expression levels of the mefA gene showed a significant increase in the ERSP isolates with higher erythromycin MIC values.
Introduction
Streptococcus pneumoniae (Pneumococcus) is an ordinary resident of the human upper respiratory tract, but it may cause a wide variety of infections in children and adults, ranging from pneumonia, meningitis, otitis media, septicemia, sinusitis, and septic arthritis to relatively benign soft-tissue infections[1-3].
Macrolides are commonly used as first-line agents for the empirical treatment of community-acquired pneumonia (CAP), and their combination with a beta-lactam is recommended for severe pneumonia. However, treatment of pneumococcal infections has become challenging due to the ever-increasing emergence of macrolide-resistant S. pneumoniae[4-6].
Resistance of S. pneumoniae isolates to macrolides was identified in the late 1980s. Two main macrolide resistance mechanisms have been described in S. pneumoniae: target-site modification and active efflux of the drug out of the bacterium. Target-site modification is mediated by an rRNA-methylating enzyme and is coded by the ermB gene, resulting in coresistance to macrolide, lincosamide, and streptogramin B antibiotics (MLSB resistance)[7, 8]. The macrolide efflux pump (M phenotype) is encoded by the mefA gene, and bacterial isolates with this phenotype are resistant to erythromycin, clarithromycin, and azithromycin. High-level macrolide resistance is commonly associated with the ermB gene, whereas the mefA gene usually results in a lower level of resistance[9, 10]. It is noteworthy that the prevalence of the two main macrolide resistance mechanisms in pneumococci varies depending on the local epidemiology; thus, ermB-mediated ribosomal methylation commonly occurs in European countries, while macrolide-specific efflux pump-mediated resistance is clinically relevant in the USA[8, 11].
Molecular epidemiological information on antibiotic resistance patterns and mechanisms of resistance can help to prevent and treat infectious diseases, such as multidrug-resistant S. pneumoniae infections[12, 13]. Given that widespread distribution of macrolide and multidrug resistance in clinical S. pneumoniae isolates is a major concern, the need for extensive research is greater than ever. Accordingly, this study was performed to investigate the association of erythromycin resistance with the mefA and ermB genes among clinical isolates of S. pneumoniae in Tehran City, Iran.
Methods
Study Design and Bacterial Isolates
In this cross-sectional study, 62 non-duplicate erythromycin-resistant S. pneumoniae (ERSP) isolates were collected from patients in four hospitals in Tehran City from December 2015 to July 2017. The isolates were obtained from various clinical samples including cerebrospinal fluid (CSF), blood, sputum, eye swabs, nasal discharge, bronchoalveolar lavage (BAL), and other body fluids (brain abscesses, synovial fluid, throat swabs, and abdominal fluid). The study was approved by the Executive Board of the Shahid Beheshti University of Medical Sciences (Tehran Province, Iran). This study was a retrospective, laboratory based study; therefore, there was no need to obtain informed written consent from the patients.
The S. pneumoniae isolates were identified by conventional microbiological methods and biochemical testing (hemolysis, Gram staining, susceptibility to optochin disc [MAST, UK], and solubility in bile)[14]. Polymerase chain reaction (PCR) assays were used to confirm diagnosis of S. pneumoniae infection in isolates by detecting the cpsA target gene.
Erythromycin-Resistant S. pneumoniae Isolates
Erythromycin-resistant S. pneumoniae isolates [minimal inhibitory concentration (MIC) ≥1 μg/ml] were identified using the broth microdilution method according to the clinical and laboratory standards institute guidelines[15]. The MIC values were read visually as the lowest concentration of the antimicrobial agent that prevented visible growth. S. pneumoniae ATCC 49619 was used as a standard strain.
Detection of the mefA and ermB Genes
Polymerase chain reaction assays were used to detect the mefA and ermB genes in ERSP isolates with specific primers (Table 1). The primers were designed in the present study using Primer-BLAST [National Center for Biotechnology Information (NCBI), USA] and primer3 software. Genomic DNA was extracted from pure cultures of the strains using the High Pure PCR Template Preparation Kit according to the manufacturer’s instructions.
Polymerase chain reaction was conducted in a final volume of 25 μl using the HotStar Taq Master Mix kit containing 12.5 μl of 2× HotStar Taq Master Mix (containing 3 mM MgCl2, 0.4 mM of each dNTP, and 0.08 U/μl Taq DNA polymerase in reaction buffer), 1 μl of the DNA template, 1 μl of each primer (20 pmol), and 9.5 μl of ddH2O. DNA amplification was performed in a thermocycler with an initial denaturation step at 95 ºC for 5 minutes; 30 amplification cycles of 30 seconds at 95 ºC, 30 seconds at different temperatures for different genes, and 40 seconds at 72 ºC; followed by an additional extension step of 7 minutes at 72 ºC. The amplified products were separated by electrophoresis through 1% agarose gel and visualized by RedSafe staining.
DNA sequencing of nine S. pneumoniae isolates randomly selected from each of the resistance genotypes (mefA, ermB, and mefA+ermB) was performed by Bioneer Corporation (South Korea), and the obtained sequence data were aligned using the BLAST program and compared with the GenBank database in the NCBIational CenterBiotechnology Information.
Expression Levels of the mefA Gene
The copy number and expression level of the mefA gene were determined in ERSP isolates by real-time PCR.
RNA extraction from the isolates was performed using the High Pure RNA Isolation Kit according to the manufacturer’s instructions. The RNA was treated with DNase using DNase I to remove any remaining genomic DNA, and complete removal of contaminating DNA was confirmed by PCR. Reverse transcription was performed using the PrimeScript RT reagent Kit for 15 minutes at 37 °C, the enzyme was inactivated for 5 seconds at 85 °C, and then the reaction was cooled.
Synthesized complementary DNA was amplified by quantitative real-time PCR (qPCR) using a Rotor-Gene 6000TM Real-Time PCR System. Real-time PCR amplification was performed in a volume of 25 μl containing 12.5 μl of Power SYBR® Green PCR Master Mix, 1 μl of each primer (50 pmol/μl), 2 μl of cDNA, and 8.5 μl of DNase free ddH2O. The amplification conditions for real-time PCR assays were as follows: initial denaturation at 95 °C for 30 seconds, followed by 40 cycles of 95 °C for 7 seconds, 53 °C for 20 seconds, and 60 °C for 25 seconds. Triplicates of a negative control were included in all qPCR runs, and 16s rRNA was used as an endogenous control. The primers used in this study are described in Table 1. Amplification data were analyzed using the software.
Statistical Analysis
The data were entered and analyzed using SPSS statistical software. Frequencies and percentages were calculated, the Pearson chi-square test and analysis of variance were performed as appropriate, and p values of less than 0.05 were regarded as statistically significant.
Results
Erythromycin-Resistant S. pneumoniae Isolates
In the present study, 62 ERSP isolates (MIC ≥1 μg/ml) were collected from various clinical samples including CSF (13 isolates, 21%), blood (12 isolates, 19.4%), sputum (13 isolates, 21%), eye swabs (3 isolates, 4.8%), nasal discharge (2 isolates, 3.2%), BAL (5 isolates, 8.1%), and other body fluids (14 isolates, 22.6%). It is noteworthy that the overall prevalence of resistance to erythromycin in S. pneumoniae isolates was 84.93% (62 isolates out of 73).
The MIC values of erythromycin for the ERSP isolates in the susceptibility test were determined using the broth microdilution method as shown in Table 2.
Frequency of the mefA and ermB Genes
In this study, the mefA and ermB genes were detected in ERSP isolates by PCR assays. Either of the mefA and ermB genes were identified in 10 (16.13%) and 36 (58.06%) ERSP isolates, respectively. It is noteworthy that 7 (11.29%) isolates harbored both genes simultaneously, and 9 (14.52%) isolates had neither of the genes (Table 3).
Finally, the nucleotide sequence data reported here have been submitted to the GenBank sequence database, and the accession numbers KU739787, KU739788, KU739789, and KU739790 for the mefA gene and KT758056 for the ermB gene have been assigned. Statistical analysis showed that there was no significant relationship between the presence of the mefA and ermB genes and the MIC values for erythromycin in clinical ERSP erythromycin-resistant S. pneumoniae isolates (p value = 0.616) (Table 3).
Expression Levels of the mefA Gene
Comparison of mean relative expression of the mefA gene in ERSP isolates with different erythromycin MIC values (ranging between 8 and 32 μg/ml) indicated that expression levels were increased by approximately twofold for each doubling of MIC values (p<0.05) (Figure 1).
MIC: Minimal inhibitory concentration
Discussion
Macrolides are currently recommended as the mainstay of treatment for empirical therapy of CAP. However, the emergence of macrolide-resistant S. pneumoniae isolates has increased over the past decade, and clinical failure of macrolide treatment in pneumococcal infections has been reported in various parts of the world. Notably, studies have shown that macrolide resistance in pneumococcal infections has remained a serious problem in many Asian countries[11-13].
Target modification by a ribosomal methylase encoded by the ermB gene and drug efflux encoded by the mef gene are two major mechanisms mediating resistance to macrolides in S. pneumoniae. The macrolide-specific efflux mechanism is clinically relevant in macrolide-resistant pneumococci in North America, whereas ermB-mediated ribosomal methylation has been found in more than 80% of ERSP isolates in most European countries[11, 17].
In the present study, 62 ERSP isolates (MIC≥1 μg/ml) were collected from various clinical samples. The distribution rates of the mefA and ermB genes in ERSP isolates were 16.13% and 58.06%, respectively. These results are consistent with many reports from Asian countries such as China, Taiwan, Sri Lanka, and Korea, as well as most other studies conducted in Iran, showing that ribosomal methylation encoded by ermB is the most common mechanism of resistance in ERSP isolates[11, 18]. In contrast, in some studies from Hong Kong, Singapore, Thailand, and Malaysia, the macrolide-specific efflux encoded by mefA was more common in erythromycin-resistant isolates[11]. Overall, the dominance of each of the two major macrolide resistance mechanisms in S. pneumoniae isolates from Asian countries seems to differ on the basis of geographical area. Notably, in ERSP isolates lacking the mefA and ermB genes, other genes involved in macrolide resistance may have induced erythromycin resistance.
As mentioned earlier, active macrolide efflux is one of the major mechanisms of macrolide resistance in S. pneumoniae in many parts of the world. The use of relative real-time PCR assay allows for the characterization of macrolide-specific efflux gene expression in macrolide-resistant S. pneumoniae isolates[19, 20]. In this study, the expression level of the mefA gene in ERSP isolates was determined by real-time PCR. A comparison of mean relative expression of the mefA gene in ERSP isolates with different erythromycin MIC values indicated that expression levels were increased approximately twofold for each doubling of the MIC value. Similar findings have also been reported in rare studies conducted elsewhere. Wierzbowski et al.[21] indicated that higher expression levels of the mefE gene were associated with higher MICs of erythromycin in ERSP isolates. Although these results highlight the association between increased expression of macrolide-specific efflux genes and higher levels of macrolide resistance in S. pneumoniae, there is an apparent need for further studies in this area. Considering the sample size limit, one of the limitations of this study was the lack of investigation of high-level erythromycin-resistant isolates (with MICs for erythromycin usually ≥256 μg/ml).
Conclusion
Our findings revealed a dominance of ribosomal methylation encoded by the ermB gene as the most common mechanism of macrolide resistance in clinical S. pneumoniae isolates from Iran. Furthermore, in the present study, increased expression of the mefA gene was associated with higher levels of macrolide resistance in macrolide-resistant S. pneumoniae isolates. Finally, it is suggested that further studies be conducted in order to investigate the role of macrolide-specific efflux pumps in increasing levels of macrolide resistance in S. pneumoniae.
Ethics
Ethics Committee Approval: The study was approved by the executive board of Shahid Beheshti University of Medical Sciences (decision date-number: 2015/10-28).
Informed Consent: Since this was a retrospective laboratory based study, informed consent was not received.
Peer-review: Externally and internally peer-reviewed.
Authorship Contributions
Surgical and Medical Practices: F.F., S.R.T., M.Y., A.H., A.N.A., A.S., Concept: F.F., S.R.T., M.Y., A.H., A.N.A., A.S., Design: F.F., S.R.T., M.Y., A.H., A.N.A., A.S., Data Collection or Processing: F.F., S.R.T., M.Y., A.H., A.N.A., A.S., Analysis or Interpretation: F.F., S.R.T., M.Y., A.H., A.N.A., A.S., Literature Search: F.F., S.R.T., M.Y., A.H., A.N.A., A.S., Writing: F.F., S.R.T., M.Y., A.H., A.N.A., A.S.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: This study was a part of the Ph.D. thesis of the first author and was supported and approved by Shahid Beheshti University of Medical Sciences (grant number: 420).