Abstract: Background and Objective: Staphylococcus is a group of bacteria that can cause a multitude of diseases. Bacteria isolates were collected from different hospitals in Taif governorate in KSA. Out of 89 isolates, eight isolates of Staphylococcus spp. were subjected to fingerprinting based on their resistance/susceptibility to antibiotics which are commonly used in KSA and all over the world. Most of the isolates showed multidrug resistance. Interestingly, out of the eight isolate, Staphylococcus isolate number 6 found to be as Methicillin Resistant Staphylococcus epidermidis (MRSE). Methodology: It was identified using the sequencing of 16S rDNA. Methicillin resistance in Staphylococcus isolates is primarily mediated by the mecA gene, which codes for the modified penicillin-binding protein 2a, mecA is carried on a mobile genetic element that called the Staphylococcal Cassette Chromosome mec (SCCmec). Thus in the present study, the different genes of mecA and SCCmec were verified in the eight isolates using primer specific PCR technique. The mecA I gene (with size about 162 bp long) and mecA III gene (with size about 449 bp long) were found in the eight isolates. Interestingly, mecA II gene (with size about of 540 bp long) was observed in only five isolates. Surprisingly, SCCmec I gene was not found in any of the eight isolates. While, SCCmec II and SCCmec III gene were observed in only six isolates. The repetitive element PCR technique (Rep-PCR) was used to study the genetic similarity between the examined isolates. Six primers were used in this study. Results: The results revealed that approximately 108 different banding patterns were obtained, dividing the isolates into two clusters. Conclusion: Results refer to the PCR assay offers a rapid, simple and accurate identification of antibiotic resistance profiles and could be used in clinical diagnosis as well as for the surveillance of the spread of antibiotic resistance determinants in epidemiological studies.
INTRODUCTION
Antibiotics have been used in clinical practice for about 80 years and throughout that period the problems posed by resistant bacteria have escalated at a pace that has forced near continuous development of new antibacterial drugs. A large variety of antibiotics are currently being used in human and veterinary medicine but their efficacy has been threatened by microbial resistance1-3. Staphylococcus epidermidis is an emerging nosocomial pathogen, In particular biofilm-forming strains of S. epidermidis that are also methicillin-resistant (MRSE) have become a very serious clinical problem4. The prevalence of MRSE has increased in many parts of the world causing serious infections in hospitals that pose a serious burden in terms of medical and socioeconomic costs and cause significant morbidity and mortality5,6. The resistance in MRSE is due to the expression of Penicillin Binding Protein 2a (PBP2a) that encoded by mecA genes7,8. The mecA genes are located on the mobile genetic elements that called Staphylococcal Cassette Chromosome (SCC)9,10. The SCC is a large genetic mobile element which differs in size and genetic composition among different strains of MRSE5,6,11. Different types of SCCmec cassettes were extensively studied by PCR techniques9,12. In addition, other antibiotic resistance genes may also be present in the cassette rendering resistance to multiple antibiotics13,14. Detection of mecA gene by PCR is considered as the gold standard15,16. For methicillin resistance as these genes are highly conserved among staphylococcal species.
In the present study, the PCR method was used for the detection of mecA genes among the Staphylococcus isolates additionally, various classes of repeated DNA sequences also have been used in this study, which described in diverse prokaryotic genomes14,17, as a tool for genetic sub typing of the different Staphylococcus isolates. Repetitive element sequence-based PCR (rep-PCR) is a new typing method that differentiates microbes by using primers complementary to interspersed repetitive consensus sequences that enable amplification of diverse-sized DNA fragments consisting of sequences between the repetitive elements18,19. Multiple amplicons of different sizes can be fractioned by electrophoresis and the resulting DNA fingerprint patterns, specific for individual bacterial clones, can be compared. Numerous studies have shown that the application of rep-PCR using oligonucleotide primers based on the repetitive extragenic palindromic (Rep) elements (Rep-PCR)20 or on the Enterobacterial Repetitive Intergenic Consensus (ERIC) sequences (ERIC-PCR) has been successful in typing a variety of bacteria20,21.
MATERIALS AND METHODS
Sample collection and growth: About 89 clinical samples of urine and stool were collected from hospitalized patients in the Taif hospitals, KSA, about one year period. The swabs of urine and stools samples were taken in aseptic conditions and were transported immediately to the microbial genetics laboratory at Deanship of Scientific Research, Taif University, KSA and processed for direct Gram staining followed by culture on blood agar, mannitol, salt agar and Mac Conkey agar (Himedia) and were incubated at 37°C for 24 h. The β-hemolytic colonies on the blood agar plate, the yellow colored colonies on the mannitol salt agar and pink colored colonies on Mac Conkey agar were subjected for Gram staining as previously reported22. The organisms identified as S. aureus were tested further for β-lactamase production by mecA genes.
Isolation and purification of clinical bacterial isolates: Sterile dry swabs were used for streaking of clinical samples onto sterile petri dishes containing nutrient agar media (Biolife, USA). Inoculated streaked dishes were incubated at 28°C for 48 h. Single colony was picked up by sterile inoculation needles and then sloped into cultures of nutrient agar media.
Antibiotic sensitivity test: About nine types of antibiotics, 1 μg oxacillin (OX), 10 μg gentamicin (GM), 5 μg vancomycin (VA), 30 mg amoxicillin (AMC), 30 μg cefotaxime (CTX), 30 μg amikacin (AN), 30 μg cefazolin (Cz), 15 μg erthromycin (E) and 30 μg tetracycline (Te) were used for disc diffusion bioassay23. Clinical bacterial isolates suspensions were spread by sterile glass rods on the surface of nutrient agar media. Then antibiotic discs (Bioanalyse®) were placed onto the surface of the inoculated nutrient agar plates. The plates were then incubated at 28°C for 48 h and then inhibition zones were observed and measured in mm.
Biochemical characteristics of antibiotic resistant bacteria: The cultural, morphological and biochemical criteria of the eight Staphylococcus antibiotic resistant bacteria isolates were used as a taxonomic criteria. Gram stain was the key step and was carried out as previously reported10.
Genomic DNA extraction: The cell pellets from all isolates were used to extract genomic DNA using extraction kit (Jena Bioscience, Germany) following the manufacturer’s instructions.
Rep-PCR technique: For repetitive sequence analysis, PCR conditions for Staphylococcus isolates in the present investigations were standardized. Seven repetitive sequence primers were used to amplify genomic DNA of the eight isolates according to Hassan et al.24. Out of these, six primers which presented strong band resolution were chosen for the study, BOX A1(5'-CTA CGG CAA GGC GAC GCT GAC G-3'), REP1R-I (5'-III ICG ICG ICA TCI GGC-3') (forward), REP2-I (5'-ICG ICT TAT CIG GCC TAC-3') (reverse), MBO-REP1 (5'-CCG CCG TTG CCG CCG TTG CCG CCG-3'), (GTG)5 (5'-GTG GTG GTGGTG GTG-3'), REP2-II (5'-GAGAGAGAGAGAGAGAA-3') and REP8-I (5'-GAC GAGAGAGAGAGAGAGA-3'). Following the experiments for optimization of component concentrations, PCR amplification of repetitive sequence primers were carried out in 25 μL volume containing 1 μL (about 20 ng) of genomic DNA, 12.5 μL of Go Taq® Green Master Mix (Promega, USA), 1 μL of primer (20 pmol) and deionized distilled water (up to a total volume of 25 μL). For DNA amplification, the C1000TM Thermo Cycler Bio-Rad, Germany, was programmed under the conditions involving denaturation at 94°C for 5 min, 30 cycles of denaturation at 94°C for 1 min, primer annealing at 52°C for 45 sec and primer extension at 72°C for 2.5 min, final extension step at 72°C for 10 min.
Detection of antibiotic resistance genes: The PCR amplification for detection of the antibiotic genes mecA and SCCmec was carried out as previously reported25,26 using specific primers that showing in Table 1. The PCR reactions were carried out in a total volume of 25 μL containing 1X PCR buffer, dNTPs, 1 units of Taq DNA polymerases, 10 pmol of each primer and the DNA template. The PCR amplifications were performed in C1000TM Thermo Cycler (Bio-Rad). The PCR conditions for mecA genes was: 95°C for 5 min, followed by 30 repeated cycles of 30 sec at 94°C, 30 sec of annealing at 54°C and 1 min of extension at 72°C, followed by 7 min as final extension at 72°C.
| Table 1: | Primers name and sequences of antibiotic resistance genes of Staphylococcus |
The PCR conditions for SCCmec genes were: 95°C for 5 min, 30 cycles of denaturation at 95°C, annealing at 58°C and extension at 72°C for 1 min each, followed by a final extension of 10 min at 72°C. Amplification products (10 μL) were analyzed on 1.5% agarose gels stained with ethidium bromide and visualized by UV illumination and were photographed by a Bio-Rad Gel Doc 2000 device.
PCR amplification of 16S-rRNA gene: The DNA (2 μL), approximately 50 ng, was used as template for the polymerase chain reaction assays. The amplifications of 16S rDNA gene amplification were done according to Hassan and Ismail27.
Sequencing of 16S-rRNA gene: About 900 bp 16S rDNA fragments were purified using QIA quick PCR purification kit (QIAGEN, Valencia, CA, USA) according to the manufacturer’s instructions and sequenced with the same primers using the sequencer (Gene analyzer 3121). The bacterial 16S-rDNA sequences obtained were then aligned with known 16S-rDNA sequences in GenBank using the Basic Local Alignment Search Tool (BLAST) at the National Center for Biotechnology Information and percent homology scores were generated to identify bacteria.
Data analysis: In order to determine the genetic relationship among studied bacteria, Rep-PCR data were scored for presence (1) or absence (0) of the bands using Gene Tools software from Syngene. A simple matching coefficient was estimated by means of the Jaccard’s coefficient to construct a similarity matrix. Cluster analysis and dendrogram were produced on the basis of the unweighted average pair group method (UPGMA) using the NTSYS-PC Statistical Package28.
RESULTS AND DISCUSSION
Isolation of antibiotic resistant bacteria: About 89 clinical samples of urine and stool swabs were collected from Saudi Arabian patients in Taif city and were then analyzed for presence of pathogenic bacteria on nutrient agar media. All samples showed obvious bacterial cultures that were surveyed for presence of multidrug resistant bacteria. Out of 89 cultures tested, 8 multidrug resistant bacteria isolates were identified as Staphylococcus spp. and selected for isolating the antibiotic resistance genes.
Biochemical characteristics of multidrug resistant bacteria: Based on molecular, cultural, morphological and biochemical characteristics, eight isolates were belonging to Staphylococcus spp. and they were characterized as Gram positive, short coccui bacteria and non-motile cells. Other biochemical tests showed negative results. Similar results were obtained by El-Amin and Faidah29. They identified as Staphylococcus strains that isolated from hospitals of western region in Saudi Arabia based on Gram stain, catalase and coagulase tests29.
Antibiotic susceptibility testing: Since the introduction of antibiotics in medicine, Staphylococcus strains has shown a frequent and rapid development and spread of antibiotic resistance and has developed resistance to all types of antibiotics such as tetracyclines, which are broad-spectrum antibiotics used in the treatment and prevention of bacterial infections23,27,30. Most tetracycline resistant bacteria have acquired tetracycline resistance genes8. In the present study, antimicrobial susceptibility testing was performed by the Bioanalyse Disc Diffusion method using nutrient agar plates, according to the Clinical and Laboratory Standards Institute guidelines31. The antimicrobial agents tested and their corresponding concentrations were described previously in the material and methods section. After incubating the inoculated plates aerobically at 28°C for 48 h, the susceptibility of the eight Staphylococcus isolates to each antimicrobial agent was detected using Combination Disk Diffusion Test32.
| Table 2: | Antimicrobial resistance profiles of eight Staphylococcus isolates |
Inhibition zones diameter was measured in mm, OX = 1 μg oxacillin, GM =10 μg gentamicin, VA = 5 μg vancomycin, AMC = 30 mg amoxicillin, CTX =30 μg cefotaxime, AN = 30 μg amikacin, Cz = 30 μg cefazolin, E =15 μg erthromycin, TE =30 μg tetracycline and 0.0 = Complete resistance (complete growth) | |
The results were interpreted in accordance with criteria provided by CLSI23,31. Most of all of the eight Staphylococcus isolates were resistant to one or more antimicrobial agent. Interestingly, the Staphylococcus 6 isolate showed resistance to all antibiotics agents tested in this study. This isolate was characterized and identified by sequence analysis of 16S rDNA region. We found it belonging to Staphylococcus epidermidis with highly genetic similarity to Staphylococcus epidermidis strain AU12-03. On the other hand, Staphylococcus 5 isolate was sensitive to all tested antibiotics (Table 2 and Fig. 1). Seven of the Staphylococcus isolates were found to be sensitive to the gentamicin, cefotaxime, amikacin and cefazolin (Table 2). Only two isolates were found to be sensitive to vancomycin, erthromycin and tetracycline (Table 2). Additionally, seven of the Staphylococcus isolates were found to be resistant to oxacillin (Table 2). It was clearly reported that methicillin resistance in staphylococci is mainly due to the expression of the mec-gene23,31. Methicillin Resistant Staphylococcus epidermidis (MRSE) is increasingly important nosocomial pathogen particularly in a Neonatal Intensive Care Unit (NICU)5,8. The high risk factors in new-borns, in addition to the context of intensive care, very low birth weight, prematurity, immaturity of immunological defences, impaired ability to opsonize and kill staphylococci5,10.
PCR analysis for mecA and SCCmec genes: The PCR amplification products of mecA genes producing in Staphylococcus isolates were shown in Fig. 2-4. Results showed that the three genes were identified in some isolates. Eight isolates of Staphylococcus found to be carried both mecA I gene and mecA III gene with size about of 162 and 449 bp long, respectively (Fig. 2 and 4). These results, in some aspects, are similar to the reports that published previously in Saudi Arabia23.
| Fig. 1(a-b): | Combination disk diffusion test of antibiotic resistance in Staphylococcus isolates number (a) Staphylococcus 6 and (b) Staphylococcus 4 |
| Fig. 2: | Amplification of mecA I gene producing in Staphylococcus isolates by single PCR with size about of 162 bp. M: 50 bp DNA ladder |
| Fig. 3: | Amplification of mecA II gene producing in Staphylococcus isolates by single PCR with size about of 540 bp |
| Fig. 4: | Amplification of mecA III gene producing in Staphylococcus isolates by single PCR with size about of 449 bp |
Interestingly, mecA II gene, with size about of 540 bp long, was observed only in five isolates (Staphylococcus 3, 4, 5, 6 and 8) (Fig. 3), mecA gene is responsible for the resistance of Staphylococcus to the β-lactams antibiotic group. And this genetic material is not belonging to the Staphylococcus genome but it has been acquired by Staphylococcus spp. many times over the past 40 years from unknown sources10. The gene product of mecA is a penicillin binding protein 2a (PBP2a). Staphylococcus normally produces four PBPs enzymes that are anchored on the cytoplasmic membrane33.
| Fig. 5: | Amplification of SCCmec II gene producing in Staphylococcus isolates by single PCR with size about of 398 bp |
| Fig. 6: | Amplification of SCCmec III gene producing in Staphylococcus isolates by single PCR with size about of 280 bp |
The functions of these enzymes are the assembly and regulation of the latter stages of the cell wall biosynthesis34. Whereas these four PBPs are susceptible to modification by β-lactam antibiotics, an event that leads to bacterial death, PBP2a is refractory to the action of all available β-lactam antibiotics. The PBP2a is capable of taking over the functions of the four typical PBPs of Staphylococcus in the face of the challenge by β-lactam antibiotics. Based on the classes of the mec gene complex and the ccr gene types, eleven types (I to XI) of SCCmec have been assigned for Staphylococcus35. However, only type I-V is globally distributed while others appear to exist as local strains in the country of origin35. Moreover, the PCR ampilicon of SCCmec genes were producing in Staphylococcus isolates. Obviously, only two genes were identified in some isolates of Staphylococcus (Fig. 5 and 6). Surprisingly, SCCmec I gene was not found in any of the eight isolates. The SCCmec II gene with size about of 398 bp long was observed in all eight Staphylococcus isolates (Fig. 5). While, SCCmec III gene (with size about of 280 bp long) was found in only six isolates (Staphylococcus 1, 2, 3, 4, 6 and 8) (Fig. 6).
| Fig. 7: | Rep-PCR profile of eight antibiotic resistance Staphylococcus isolates generated with six repetitive sequence primers, M: 100 bp DNA ladder |
Staphylococcus 6 isolates was observed as highly resistance to all antibiotics tested included methicillin resistant, It have mecA and SCCmec genes and molecular identified as Staphylococcus epidermidis using 16S rDNA gene so it could identified as Methicillin Resistant Staphylococcus epidermidis (MRSE)5, who reported that Staphylococcus epidermidis is the most common etiologic agent of diseases like bacteremia, osteomyelitis, urinary tract infection and peritonitis caused by ambulatory dialysis with a frequent association with colonization of intravascular catheters and orthopaedic devices. Moreover, methicillin resistant Staphylococcus is resistant to all pencillins, penems, carbapenems and cephalosporis5.
Rep-PCR analysis: Repetitive element sequence-based PCR (Rep-PCR) is a new typing method that differentiates microbes by using primers complementary to interspersed repetitive consensus sequences that enable amplification of diverse-sized DNA fragments consisting of sequences between the repetitive elements20. The Rep-PCR fingerprinting method, utilizing repetitive sequence oligonucleotides, is particularly a powerful tool for genetic studies and it is useful as a screening genotyping method20. The Rep-PCR can generate various fingerprint patterns with unlimited number of fragments24. In this study, six Rep-PCR primers were used for estimating of genetic diversity of Staphylococcus isolates. The Rep-PCR reactions were performed in duplicate and all amplification products were found to be reproducible (Fig. 7 and Table 3). The Rep-PCR results using primer (BOX A1) has showed a total of 26 bands sized ranged from 150-2230 bp long in all eight Staphylococcus isolates. Five common bands were observed in all isolates which exhibited about 19.2% monomorphism, while the other twenty one fragments have showed 80.8% polymorphism (Table 3). In case of (GTG)5 primer, a total of twenty four fragments have showed 96% polymorphism among the eight Staphylococcus isolates. The molecular size of the amplicon products ranged from 150-2000 bp long. Also, this primer recognized different unique fragments at 150, 200, 400, 1500 and 1800 bp specific to isolate No. 8, 1 and 7, respectively. According to genetic similarity and intraspecies differentiation, the eight Staphylococcus isolates were grouped into two main different clusters with about 58% genetic similarity.
| Fig. 8: | Dendrogram analysis among the eight antibiotic resistance Staphylococcus isolates based on the six repetitive sequence primers |
| Table 3: | Polymorphism level detected by the six repetitive sequence primers that have been used for Rep-PCR analysis |
Interestingly, the first cluster was divided to two sub-clusters and Staphylococcus 6 isolate was found to be alone in the first sub-cluster, while, Staphylococcus 1, 2, 3 and 4 were grouped in the second sub-cluster. On the other hand, the other three Staphylococcus isolates were grouped in the second cluster (Fig. 8 and Table 3).
The Rep-PCR technique was proved to be useful genetic markers used for antibiotic resistance bacteria fingerprinting. Although, major bands from Rep-PCR reactions are highly reproducible, minor bands could be difficult to repeat due to repetitive sequence priming nature of this PCR reaction and potential confounding effects associated with co-migration with other markers36,37. The use of multiple primer sets in Rep-PCR analysis can be used as a rapid method for preliminary biotyping of multidrug resistant strains. In a previous study using different repetitive sequence primers, the discriminatory power of Rep-PCR and its ability to characterize strains was demonstrated24,36.
CONCLUSION
In the present study, we characterised Staphylococcus spp. Stains that isolated from different hospitals in Taif region, KSA, using different molecular technique. The results clearly state that the antibiotic resistance genes of mecA and SCCmec are verified in the Staphylococcus isolates using primer specific PCR technique.
Significance statement: The PCR-dependent assay technique is offers a rapid, simple and accurate identification of antibiotic resistance gene profiles. Therefore, it could be used in clinical diagnosis as well as for the surveillance of the spread of antibiotic resistance determinants in epidemiological studies.
ACKNOWLEDGMENT
The authors would like to acknowledge the support provided by Taif University, KSA for funding this study through project No.1-434-2685.