Shaker, Amr, Aboshanab, Khaled*, Mabrouk Aboulwafa, Mohammad, Hassouna, Nadia
Department of Microbiology and immunology, Faculty of Pharmacy, Ain Shams University (ASU), Cairo, Egypt. Organization of African Unity St., POB: 11566, Abbassia, Cairo, Egypt.
Abstract Title Plasmid-mediated resistance to macrolides in some Staphylococcus isolates recovered from patients with lower respiratory tract infection. Background Staphylococcus aureus and coagulase-negative staphylococci are considered to be among the major causes of nosocomial and community-acquired infections. Macrolides, lincosamides and streptogramin type B (MLS) antibiotics are widely indicated for the treatment of staphylococcal infections. A great concern is the increase in the prevalence of macrolide resistance among Staphylococcus spp. The aim of our study was to detect plasmid-harbored genes responsible for macrolide resistance in staphylococci involved in lower respiratory tract infections (LRIs) in Egypt. Methods and findings A total of 189 bacterial isolates were recovered from 180 sputum and bronchoalveolar lavage specimens obtained from patients with LRIs. The specimens were collected during the period of 2010 to 2012 from Microbiological diagnostic laboratories of two governmental hospitals specialized in the treatment of chest infections, Al-Sadr (Abbassia) and Al-Demerdash hospitals. The antibiogram analysis revealed that 64, out of 189 isolates (33.9%) were resistant to the tested antibiotics (Erythromycin, Clarithromycin, Azithromycin and Clindamycin). Among the resistant isolates, only 8 isolates (12.5%) were identified to be Staphylococcus spp. Among these isolates, four showed constitutive (cMLS) phenotype, while the other isolates exhibited inducible (iMLS) phenotype. The molecular analysis of resistant isolates revealed the presence of plasmids in the eight test isolates. By using PCR, two genes suspected to be responsible for plasmid-mediated resistance could be detected using two specific primer pairs for erythromycin ribosomal methylase (erm) and erythromycin resistance ATP-binding protein (coded as msr). The erm gene was detected in four isolates (50%), while the prevalence of msr gene constituted 25% (detected in two isolates only). Further verification of msr gene was performed by DNA sequencing of the PCR product obtained by using the plasmid extract of one isolate as a template. The nucleotide sequence of msr gene was submitted to the GenBank database, and given the accession code KJ710361. Conclusion Plasmid-mediated resistance to macrolides prevails among Staphylococcus isolates involved in LRIs as the 8 resistant isolates of this genus was shown to harbor plasmids, with prevalence of 50% (erm gene) and 25% (msr gene). Therefore, the reasons behind the prevalence of plasmid-mediated resistance to macrolides have to be studied, and control measures should be undertaken.
Macrolide antibiotics; staphylococci; erm gene; msr gene.
Macrolide antibiotics such as erythromycin, clarithromycin and azithromycin have been widely used in treatment of respiratory tract infections caused by Gram-positive pathogens including Staphylococcus spp. [1-2]. MLS antibiotics exert their antimicrobial action by inhibition of bacterial protein synthesis which is achieved by reversible binding of these agents to the P-site of the 50S subunit of the bacterial ribosome [1-3].
There are three different mechanisms responsible for bacterial resistance to MACs in staphylocooci: (1) Target site modification mediated by erm gene; (2) active efflux mediated by msr A gene; and (3) enzymatic inactivation of antibiotic mediated by esterases (encoded by ere gene) or phosphotransferases (encoded by mph gene) [4-5]. The first mechanism confers resistance not only to MACs, but also to lincosamides and streptogramin type B (MLS phenotype), which is achieved by dimethylation of specific adenine residue in 23S rRNA molecule in the ribosome [4,6-7]. The expression of erm gene can be either constitutive (cMLS phenotype), or inducible (iMLS phenotype) . The second mechanism confers resistance to either MAC antibiotics only (M phenotype) or MACs and streptogramin type B (MS phenotype) . The MAC resistance genes as erm and msr genes are widely distributed in staphylococci of humans, and located on small multicopy plasmids [8-12] erm genes are mostly responsible for erythromycin resistance in different Staphylococcus spp. strains and are borne by plasmids. Also, to date, the only efflux proteins responsible for acquired macrolide resistance characterized in Staphylococccus spp. are ABC transporters encoded by plasmid borne msr genes .
The emergence of antibiotic resistance among pathogenic bacteria, either in hospital or community- acquired infections is considered to be a major public health problem. Plasmids, which are an extrachromosomal pieces of DNA, can replicate independently of the genome, and are considered of great importance in the spread of antibiotic resistance genes, which result in the acquisition of resistance to many antibiotics . Due to their capability of horizontal gene transfer between different species and genera, plasmid-mediated resistance is considered to be a significant problem in the spread of antibiotic resistance among pathogenic bacteria [14-15].
The aim of this study was to detect macrolide target site modification gene (erm gene) and macrolide active efflux gene (msr gene) on plasmids extracted from Staphylococcus spp recovered from patients with lower respiratory tract infections (LRIs) in Egypt. Specific PCR primers were used to detect target site modification gene erm (erythromycin ribosomal methylase), and active-efflux gene msr.
A total of 189 clinical bacterial isolates, recovered from patients with LRIs from the Microbiology diagnostic laboratories of Al-Sadr (Abbassia) and Al- Demerdash Hospitals in Egypt during the period of 2010 to 2012, were studied. The history sheets of all patients were examined, and all patients read and signed an “informed consent” form before the beginning of the study verifying their acceptance for using their data for research purpose.
The collected isolates were obtained from sputum and bronchoalveolar lavage specimens of both pediatric and adult patients by culture on 5% sheep blood agar plates and incubating them overnight at 37°C. Mannitol salt agar plates and coagulase test were used for identification of Staphylococcus aureus. The isolates were preserved in glycerol broth at -20°C till use.
Antimicrobial susceptibility test
The susceptibilities of the collected isolates were determined by disc diffusion method on Mueller hinton agar according to clinical and laboratory standard institute (CLSI). The tested antibiotics (BBL™ Sensi-Disc™, USA) include: erythromycin (15 μg), clarithromycin (15 μg), azithromycin (15 μg) and clindamycin (2 μg). The inducible type of resistance was demonstrated using double-disc diffusion test (D test) by placing clindamycin discs and erythromycin discs at distances of 16-25 mm followed by incubation at 37°C for 24 hrs .
Plasmid profile analysis
Analysis of plasmid-DNA for the resistant bacterial isolates was performed by plasmid DNA extraction followed by direct agarose gel electrophoresis of the extracted plasmids . Plasmids were extracted by alkaline lysis method as described by Birnboim and Doly  using GeneJet Plasmid Miniprep Kit (Fermentas, USA) according to the manufacturer’s manual.
Amplification of the selected resistance genes was performed through PCR, using plasmid-DNA of each resistant strain as a template and the primers outlined in Table 1.
PCR amplification was carried with the cycling parameters as follows: After an initial denaturation step at 95°C for 4 min, 30 cycles of amplification were performed as follows: Denaturation at 95°C for 30s, annealing temperature at 53°C for 45 sand extension temperature at 72°C for 1 min, this was followed by I cycle of final extension at 72°C for 5 min, and finally the reaction was hold at 4°C for 10 min. The sizes of PCR products of these genes were analyzed by 0.8% agarose gel electrophoresis containing ethidium bromide (0.5μg/ml).
Sequencing of PCR products
The obtained PCR products were purified using GeneJET™ PCR Purification kit (Fermentas, USA) at Sigma Scientific Services Company, Egypt. Finally, sequencing was done at GATC Biotech Company (Germany), through Sigma Scientific Services Company (Egypt) by the use of ABI 3730xl DNA Sequencer.
Computer programs used for sequence analysis
The obtained sequence files (forward and reverse) were analyzed using the following programs: Staden-package program version 3  was used for sequence assembly and formation of final contigs; Frameplot  for detection of the ORFs in the final contigs; Clustal W  for amino acid alignment. Finally DNA search in the GenBank database using BLAST (Basal Local Alignment Research tool) to visualize fully and/or partially conserved domains or regions within the macrolide resistance proteins products.
Collection of bacterial isolates from clinical specimens
A total of 189 bacterial isolates were recovered from sputum and bronchoalveolar lavage specimens from patients with LRIs.
Antimicrobial susceptibility tests and detection of Staphylococcus spp. resistant isolates
Using Gram-stain, 47 isolates, out of 189 collected isolates, were found to be Gram-positive (24.9%), 64 isolates (33.9%) were found to be resistant to the tested antibiotics. Among the resistant isolates, 12 isolates (18.7%) were found to be Gram-positive, including 8 isolates (n=8/64; 12.5%) which were identified to be Staphylococcus spp. Out of these 8 isolates, 7 isolates (S34b, S56, S63, S67, S77, S78 and R26) were confirmed to be Staphylococcus aureus based on yellowish growth on mannitol salt agar and positive coagulase test, while one isolate (S26) was identified as coagulase-negative Staphylococcus spp. The results of antibiotic sensitivity test were summarized in Table 2.
Among the 8 resistant Staphylococcus spp isolates, 4 isolates (S26, S34b, S77 and R26) showed complete resistance to all tested antibiotics. While the other 4 isolates (S56, S63, S67 and S78) showed D-shaped inhibition zone around clindamycin discs.
Plasmid profile analysis and PCR
Plasmid extraction, carried out on Staphylococcus spp resistant isolates, revealed the presence of plasmids in the 8 resistant isolates. The extracted plasmids were used as template for PCR using the primers listed in Table 1. The gel electrophoresis of PCR products showed the presence of msr and erm genes (figure 2 and figure 3). The prevalence of the resistance genes among the tested isolates were summarized in Table 3. The correlation between the genotype and phenotype of resistant isolates was shown in Table 4.
Figure 2: Agarose gel electrophoresis of the PCR products of two positive erythromycin resistance ATP-binding protein-coding genes (msr). Lanes: 1, PCR product of isolate S26; 2, PCR product of isolate S34b; 3, PCR product of isolate S56; 4, 1664 bp PCR product of msr gene in isolate S63; 5, PCR product of isolate S78; 6, PCR product of isolate S67; 7, PCR product of isolate S77; 8, PCR product of isolate R26 and M, 1 Kb size marker (Fermentas, USA).
Figure 3: Agarose gel electrophoresis of the PCR products of four positive erythromycin ribosomal methylase coding genes (erm). Lanes: 1, 617 bp PCR product of erm gene in isolate S26; 2, PCR product of isolate S34b; 3, PCR product of isolate S56; 4, PCR product of isolate S63; 5, PCR product of isolate S67; 6, PCR product of isolate S77; 7, PCR product of isolate S78; 8, PCR product of isolate R26 and M, 1 Kb size marker (Fermentas, USA).
Sequencing of PCR products
Further verification of the msr gene was performed by DNA sequencing of the PCR product obtained by using the plasmid extract from isolate S78 as a template for PCR. The sequence analysis revealed the presence of msr gene. The nucleotide sequence of msr gene was submitted to the nucleotide GenBank database, and given accession code KJ710361. The results of multiple sequence alignment of msr gene with its homologous proteins by using Clustal W software were shown in figure 4.
Figure 4: Multiple alignment of amino acid sequence of MSR-query Erythromycin resistance ATP-binding protein of Staphylococcus aureus isolate (S78) (AC=KJ710361) against its homologous in the GenBank database: MSRA-1Sa= Erythromycin resistance protein, Staphylococcus aureus, AC= YP_254220.1; MSRA-2Sa = Erythromycin resistance protein, Staphylococcus aureus; AC= EVR81644.1; MSRA-3Sa = Erythromycin resistance protein, Staphylococcus aureus, AC= EVR51631.1; MSRA-4Sa = Erythromycin resistance protein, Staphylococcus aureus, AC= EUR46906.1. The numbers indicate positions within the corresponding proteins. AC = GenBank accession number.
This study was concerned with the detection of different determinants of macrolide resistance in plasmids extracted from Staphylococcus spp. recovered from sputum and bronchoalveolar lavage specimens from patients with LRIs in Egypt.
There are two main mechanisms of MLS resistance in staphylococci, which are: Target site modification due to erm genes, and active-efflux due to msr A gene [13,23-24]. msr gene affects only MACs (M phenotype) or MACs and streptogramin type B(MS phenotype). While, The expression of erm gene can be constitutive (cMLS phenotype) or inducible (iMLS phenotype) .
In this study, the antimicrobial susceptibility testing of the recovered Staphylococcus spp was carried out using disc diffusion method according to CLSI. 4 isolates (S26, S34b, S77 and R26) were found to be resistant to MACs as well as clindamycin (linosamide), which indicate cMLS resistance phenotype. The other 4 isolates (S56, S63, S67 and S78) showed flattening of the clindamycin inhibition zone towards an adjacent erythromycin disc by using double-disc diffusion test (D test), while, they are resistant to the other MACs, which indicate iMLS resistance phenotype.
The phenotype of MLS resistance, either constitutive or inducible, may show great variations based on the patient groups in different hospitals, and on geographical region. This variation may be attributed to several factors as: The inconsistent use of MACs in different hospitals, patient age and the origin of the tested isolate [26-27]. In our study, it was found that 4 resistant Staphylococcus Spp (50%) showed cMLS resistance. While, the other 4 isolates (50%) showed iMLS resistance. In study carried out in London by Hamilton-Miller et al. iMLS phenotype was the predominant phenotype (43%) followed by cMLS phenotype which was found in 24% of the isolates . In another study conducted by Fiebelkorn et al. in Texas, cMLS phenotype was more prevalent (41.7%) followed by both iMLS and MS phenotypes (3.3% each) . The high incidence of iMLS phenotype in this study may be attributed to the increased concurrent use of MAC and clindamycin antibiotics .
In addition to the phenotypic tests used for screening of MLS resistance, genetic tests were also carried out. It was found that the 8 resistant isolates showed plasmid bands upon plasmid extraction and running the extract on 0.8% agarose gel electrophoresis. PCR was done on all 8 resistant isolates, using plasmid extracts as templates, to amplify the two plasmid-mediated genes: erm and msr. erm gene was more prevalent than msr gene among the 8 resistant isolates. The results revealed the presence of erm gene in 4 isolates (50%), and msr gene in only 2 isolates (25%). It was noticed that erm gene was more prevalent than msr gene. This finding was in accordance with the prevalence results in the study conducted in Turkey by Aktas et al. .
In this study, 3 resistant Staphlococcus spp isolates (S34b, S77 and R26) showed cMLS phenotype, and 1 isolate (S56) showed iMLS phenotype. However, PCR results, using the primers listed in table 1, were negative for erm genes. This study was concerned with plasmid-mediated genes and the PCR was carried out on plasmid extracts of the resistant isolates to detect plasmid-harbored genes. The resistance in the erm-negative isolates may be due to the presence of either erm or msr genes or both on the genomic-DNA. 3 isolates (S63, S67 and S78) were positive for erm genes, and showed iMLS resistance phenotype. While only 1 isolate (S26) was positive for erm gene, and showed cMLS phenotype. In inducible resistance, the bacteria produce inactive mRNA that is unable to encode methylase. The mRNA becomes active only in the presence of a macrolide inducer. While, in constitutive resistance, active methylase mRNA is produced in the absence of an inducer . Induction is related to the presence of an attenuator upstream from the structural erm gene coding for the methylase .
The msr gene, responsible for M phenotype, was found in 2 isolates in this study (S63 and S78). However, these 2 isolates showed iMLS resistance phenotype. This may be due to the presence of erm genes in these isolates. So, the M phenotype of msr gene was masked by the iMLS phenotype of erm gene.
Therefore, results obtained from our study were of great significance regarding the prevalence of plasmid- mediated MAC resistance genes in staphlococci, which may result in horizontal transfer of MAC resistance genes among different gram-positive bacteria causing LRIs. Therefore, the reasons behind the prevalence of these plasmid-mediated genes among staphylococci have to be studied in order to limit the dissemination of macrolide resistance.
erm= Erythromycin ribosomal methylase
LRIs= Lower resoiratory tract infections.
MLS= Macrolides, lincosamides and streptogramin type B..