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ORIGINAL ARTICLE
Year : 2020  |  Volume : 23  |  Issue : 7  |  Page : 912-918

Molecular analysis and genotyping of methicillin-resistant Staphylococcus aureus strains isolated from different clinical sources


1 Department of Nutrition and Dietetics, Ondokuz Mayis University, Faculty of Health Science, Samsun, Turkey
2 Department of Microbiology, Ankara University, Faculty of Veterinary Medicine, Ankara, Turkey
3 Department of Microbiology, Health Science University, Samsun Education and Research Hospital, Samsun, Turkey
4 Department of Microbiology, Ondokuz Mayis University, Faculty of Veterinary Medicine, Samsun, Turkey

Date of Submission12-Feb-2019
Date of Acceptance06-Mar-2020
Date of Web Publication3-Jul-2020

Correspondence Address:
Dr. M U Sogut
Ondokuz Mayis University, Faculty of Health Science, 55220 - Samsun
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/njcp.njcp_88_19

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   Abstract 


Background: Investigating genetic relatedness between methicillin-resistant Staphylococcus aureus (MRSA) strains from humans and different animal species may clarify the epidemiological characteristic of MRSA infections together. Aim: The aim of the study was to perform genotypic characterization and type strains of MRSA isolated from different clinical sources, by molecular techniques. Materials and Methods: The molecular characterization of the strains was performed by polymerase chain reaction (PCR), using several specific oligonucleotides. These were as follows: S. aureus species-specific sau gene, mecA gene coding PBP2a responsible for methicillin resistance, femA gene coding for a protein, which influences the level of methicillin resistance of S. aureus, and is universally present in all MRSA strains; spa gene coding for protein A; coa gene coding for coagulase, and blaZ gene coding for the production of beta-lactamase. To determine the genetic diversity of these strains, random amplified polymorphic DNA–polymerase chain reaction (RAPD-PCR) was performed. Results: Among the 415 S. aureus strains, 61 were phenotypically identified as MRSA, and confirmed as S. aureus by amplification of sau gene. However, 90.16% of the strains were mecA positive, while all were negative for femA gene. The presence and polymorphism of coa and spa genes were investigated and 83.60% and 18.03% strains were positive for coa and spa, respectively. While these strains were grouped into six coa-types by PCR, no polymorphism was found for spa gene among strains having only single 190 bp of the band. bla genes were found in 75.40% of strains. These strains were divided into 12 RAPD types. Conclusions: The results showed the relatively high heterogeneity and variation of coa gene among MRSA strains, while further studies on sequencing of these strains may identify which sequence type is predominant in this region.

Keywords: Methicillin-resistant Staphylococcus aureus, molecular typing, RAPD, virulence genes


How to cite this article:
Sogut M U, Bas B, Bilgin M, Sezener M G, Findik A. Molecular analysis and genotyping of methicillin-resistant Staphylococcus aureus strains isolated from different clinical sources. Niger J Clin Pract 2020;23:912-8

How to cite this URL:
Sogut M U, Bas B, Bilgin M, Sezener M G, Findik A. Molecular analysis and genotyping of methicillin-resistant Staphylococcus aureus strains isolated from different clinical sources. Niger J Clin Pract [serial online] 2020 [cited 2020 Oct 26];23:912-8. Available from: https://www.njcponline.com/text.asp?2020/23/7/912/288892




   Introduction Top


Staphylococcus aureus (S. aureus)is a major cause of infections in hospitals and communities. Presently, the increasing antibiotic resistance of S. aureus is widely observed globally, and this complicates the treatment of associated infections, as well as control measures.[1] Since the first report of methicillin-resistant S. aureus (MRSA) in 1961,[2] it has gradually spread all over the world. One of the considerable features of MRSA is its resistance against an antibiotic class named beta-lactams.[3] Though in the past, MRSA almost exclusively caused hospital-associated infections, the advent of community-acquired MRSA has led to infections in people without hospital-related risk factors.[1] Methicillin resistance occurs due to the production of an altered penicillin-binding protein (PBP2a) with a low affinity for all penicillin classes. mecA gene encodes PBP2a [4] and its expression is regulated by associated repressor and inducer genes such as mecR, mecI, ccr, and by various other S. aureus genes like fem (factors essential for methicillin resistance) and aux (auxillary genes).[5] These genes have been reported to also have importance in the expression of methicillin resistance, in addition to mecA.[6] Considering that only mecA detection does not confirm the presence of S. aureus, several constitutive genes such as femA, fem B, and nucA genes are being used. However, it has been reported that there was polymorphism within these genes; therefore, some conflicts occur in confirmation of S. aureus species by polymerase chain reaction (PCR) targeting these genes.[7] Another mechanism for beta-lactam resistance in S. aureus is the production of beta-lactamases and its production is encoded by structural blaZ gene, so, blaZ has been used to determine MRSA profile as well.[8] Coagulase and protein A are important virulence factors and phenotypic determinants in S. aureus; thus, variations in the sequence of genes (coa and spa) coding for these two species-specific proteins have been widely used in PCR typing for S. aureus.[9],[10] DNA-based typing methods are reportedly based on the principle that epidemiologically related bacterial isolates have genetic features that are different from those of other epidemiologically unrelated strains.[11] One of the PCR-based typing methods, random amplified polymorphic DNA (RAPD) was used to type MRSA strains isolated from different human sources.


   Materials and Methods Top


Clinical specimens

A total of 415 S. aureus strains, which had been isolated from various clinical sources, were evaluated between December 2016 and December 2017 in our study in the Microbiology Laboratory of Samsun Education and Research Hospital, Turkey. Clinical samples were acquired from the following sources: blood samples [14 (22.9%)], urine samples [10 (16.3%)], wound samples [17 (27.8%)], sputum samples [8 (13.1%)], throat swab [1 (1.6%)], tracheal aspirates [6 (9.8%)], pleural fluid samples [2 (3.2%)], and abscess sample [1 (1.6%)].

Bacterial isolates

All bacterial strains were isolated by inoculating the clinical samples on Blood Agar base, supplemented with 5% sheep blood, and incubated at 37°C for 18-24 hours. After the incubation period, suspected colonies were identified by conventional methods (colony morphology, Gram staining, catalase and coagulase test, etc) as S. aureus.

Phenotypic determination of methicillin resistance

Methicillin susceptibilities of the S. aureus strains were determined by Kirby-Bauer disc diffusion method, using oxacillin discs (1 μg). Obtained results were evaluated according to the protocol of the Clinical and Laboratory Standards Institute [12] by the following criteria.

Molecular characterization of isolates

DNA extractions from MRSA determined by disc diffusion tests were performed by commercially available DNA extraction kits (PureLink Genomic DNA Kits, Invitrogen, Canada). The extractions were performed according to the manufacturers' instructions.

The molecular characterization of the strains was performed by PCR, using several specific oligonucleotides. The sequences of each primers and expected product (bp) are as presented in [Table 1].
Table 1: Oligonucleotid primer sequences of primer sets

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To identify and confirm S. aureus strains genotypically by PCR, a method, which has been reported by Abd El-Razik et al.[13] was modified and optimized. Amplification was performed in 50 ml reaction volumes containing 1xSPCR reaction buffer (100 mM Tris–HCl, 500 mM KCl, pH 8.3), 3 mM MgCl2, 0.2 μM of each primer, 0.2 mM of each dNTP, 1 U Taq polymerase, and 5 μl of extracted DNA. The reactions were carried out under following conditions: an initial denaturation step of 94°C for 2 min, followed by 35 cycles of 94°C for 45 sec denaturation, 60°C for 45 sec annealing, 72°C for 1 min extension, and a final extension step at 72°C for 10 min. For femA gene, the amplification reaction was performed in 25 μl final volumes containing 1xSPCR reaction buffer, 3 mM MgCl2, 0.4 mM of each primer, 0.2 μM of each dNTP, 1 U Taq polymerase, and 5 μl of template DNA. The reactions were carried out under the following conditions: 95°C for 2 min. initial denaturation step, followed by 30 cycles of 95°C for 2 min denaturation, 54°C for 1 min annealing, 72°C for 1 min extension, and a final extension step at 72°C for 7 min.[14] The amplification of mecA gene was performed by a modified method in 25 μl reaction volumes. The content of the reaction mixture was as follows: 1xSPCR buffer, 2.5 mM MgCl2, 0.8 μM of each primer, 0.2 mM of each dNTP, 1 U Taq polymerase, and 5 μl of template DNA. The reactions were carried out under the following conditions: 94°C for 2 min. initial denaturation step, followed by 30 cycles of 94°C for 1 min denaturation, 55°C for 1 min annealing, 72°C for 2 min extension, and a final extension step at 72°C for 7 min.[14] To determine the presence and polymorphism of coa gene, a PCR protocol, which has been used by Aslantas et al.[15] was modified and optimized. The reaction was carried out in a 50 μl final volume. The reaction mixture was prepared as follows: 1xSPCR buffer, 2.5 mM MgCl2, 1 μM of each primer, 0.2 mM of each dNTP, 2 U Taq polymerase, and 5 μl of template DNA. The reactions were carried out under the following conditions: 95°C for 2 min. initial denaturation step, followed by 30 cycles of 95°C for 30 sec denaturation, 58°C for 2 min annealing, 72°C for 4 min extension, and a final extension step at 72°C for 10 min.

To determine the presence and polymorphism of spa gene, a protocol that has previously been used by Montesinos et al.[11] was modified and optimized. We used primers, which were designed to amplify the polymorphic X region that contains a variable number of 24 bp tandem repeats of the spa gene coding for protein A. The reaction was carried out in a 50 μl final volumes containing 1xSPCR reaction buffer, 2.5 mM MgCl2, 0.25 μM of each primer, 0.2 mM of each dNTP, 1.5 U Taq polymerase, and 5 μl of extracted DNA. The reactions were carried out under following conditions: an initial denaturation step of 95°C for 3 min, followed by 30 cycles of 94°C for 1 min denaturation, 60°C for 1 min annealing, 72°C for 1 min extension, and a final extension step at 72°C for 10 min.

The amplification of blaZ gene was carried out in a 25μl reaction volume containing 1xSPCR reaction buffer, 2,5 mM MgCl2, 0.2 μM of each primer, 0,2 mM of each dNTP, 0,5 U Taq polymerase, and 5 μl of extracted DNA. The reactions were carried out under following conditions: an initial denaturation step of 95°C for 5 min, followed by 30 cycles of 94°C for 1 min denaturation, 54°C for 1 min annealing, 72°C for 1 min extension, and a final extension step at 72°C for 10 min.[16]

The amplification products of all genes were detected by electrophoresis in 1.5% agarose gel, and DNA was visualized by staining with ethidium bromide.

Molecular typing of strains by RAPD-PCR analysis

To determine RAPD-PCR patterns of each MRSA strains, M13 (5′- GAG GGTGGC GGT TCT- 3′) oligonucleotide was used. Amplification was carried out by modifying a method reported by Verselovic et al.[17] The reaction mixture was prepared in a total volume of 25 μl containing 1x PCR reaction buffer, 2.5 mM of MgCl2, 200 μM of each dNTP, 2.5 U Taq DNA polymerase, 25 pmol of universal M13 primer, and 5 μl of template DNA. The amplification program was started with an initial denaturation at 94°C for 5 min. Following the initial denaturation, denaturation at 94°C for 1 min, annealing at 40°C for 1 min, and extension at 72°C for 3 min were repeated 40 times. A final extension was performed at 72°C for 7 min. The amplification products were detected by electrophoresis in 1.5% agarose gel, and DNA was visualized by staining with ethidium bromide.

Similarities between RAPD patterns were determined based on the Dice similarity coefficient. A dendrogram that graphed genetic relatedness between MRSA strains was created using “Unweighted Pair Group Method with Arithmetic Averages (UPGMA)” by Quantity One and Software.

Reproducibility, discriminatory power, and confidence intervals

To determine the reproducibility of RAPD-PCR typing by inter-assay analysis, analysis of isolates was repeated on five consecutive days. To determine the discriminatory indices of RAPD-PCR typing method, the formula described previously [18] was used. The confidence intervals (CIs) of this method were calculated according to the formula described previously.[10]


   Results Top


Phenotypic determination of methicillin resistance

A total of 415 S. aureus strains, which had been isolated from various clinical sources were investigated and 61 (13.5%) of them were found as resistant to oxacillin according to Kirby-Bauer tests.

Molecular characterization of isolates

All 61 S. aureus strains, which were determined as methicillin-resistant, produced 1318 bp band and confirmed as S. aureus by PCR. These isolates were selected to perform initial molecular characterization. Fifty-five (90.16%) of all S. aureus strains were positive for mecA gene (310 bp), which is responsible for intrinsic resistance to methicillin. All (100%) strains were negative for the femA gene occurring naturally in S. aureus, which is essential for the expression of methicillin resistance. Forty-six (75.40%) strains were given 173 bp band and determined as bla-positive. Fifty-one (83.60%) of the strains were coa gene-positive. Polymorphism was found among coa- positive strains and they were grouped into six coa-types (C1-C6). The strains gave one or two bands with eight different sizes, ranging between 180-480 bp. While 32 (62.74%) of the strains gave only one band, the remaining strains produced two bands. coa-types and the band profiles of the strains are shown in [Table 2]. The most predominant coa-type was C6, which included 22 strains having single band. These constituted 43.13% of all coa-positive strains, as other types included nine or less number of strains. Eleven (18.0%) strains produced a single 190 bp band, which were spa gene-positive. No polymorphism was found among these strains.
Table 2: The results of coa genotyping of S. aureus isolates

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Molecular typing of strains by RAPD-PCR analysis

The genetic diversity among MRSA strains isolated from various human sources was investigated by RAPD-PCR using M13 primer. All strains gave bands with M13 primer [Figure 1].
Figure 1: RAPD patterns of S. aureus

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Among the strains, genetic diversity was observed at 70.0% similarity, and strains were grouped into 12 RAPD-types (R1-R12), including three clusters and five unique types. Dendrogram derived from RAPD-PCR data is shown in [Figure 2]. The most predominant RAPD type of strains, R11, included 20 (32.78%) strains of all isolates. Other types included less number of strains than R1. While R6, R7, and R9 included two strains, R4 included only one strain. Similarity matrices of RAPD data are summarized in [Table 3].
Figure 2: RAPD patterns of S. aureus and dendrogram obtained by UPGMA

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Table 3: Dendrogram obtained by UPGMA and phylogenetic closeness of S. aureus

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Reproducibility, discriminatory power, and confidence intervals: The reproducibility of RAPD-PCR was 100%. The discriminatory indices (D) and confidence intervals for coa typing and RAPD-PCR analysis performed in the strains originated from different human sources were 0.802 (78.0-81.0%) and 0.852 (84.0-85.0%), respectively.


   Discussion Top


MRSA was reported in the early 1960s and then ultimately spread worldwide over the next several decades. MRSA is now endemic in healthcare facilities in virtually, although recent data indicate a decrease in the number of invasive MRSA infections.[19] Community-acquired/associated MRSA appeared inexplicably in the 1990s and is currently a major problem in many countries worldwide.[19],[20] Unlike healthcare-associated MRSA infections, which occur in individuals with predisposing risk factors, community-acquired/associated MRSA typically causes disease in otherwise healthy individuals.[20]

Two species-specific proteins (coagulase and protein A), which are also important virulence factors have been widely used to characterize S. aureus strains, based on variations in the sequence of coa and spa genes that code for these proteins.[9] MRSA is conferred by the presence of mecA gene coding for an alternative target (altered) protein PBP2a. Although this gene as a marker for the detection of methicillin resistance is generally used, mecA alone has been reported not to solely confer methicillin resistance. It has been shown that fem (factors essential for methicillin-resistance) or the auxiliary genes like fem A/B/X are also important in the expression of methicillin resistance.[7] In this study, 61 phenotypically resistant S. aureus strains from various clinical cases were investigated for mecA gene, and also for the presence of blaZ gene coding for beta-lactamase, which is responsible for beta-lactam antibiotic resistance. All the phenotypically methicillin-resistant strains were found as 90.16% mecA positive and 75.40% were blaZ positive. However, in the report of Chikkala et al.[8], none of the strains was fem gene-positive, although variations existed, even in the genomic sequences around femA. Also, 22 strains of S. aureus were found negative for both femA and nuc genetic markers, possibly since it has been suggested that likely mutations or deletions in the nuc gene were present. They were also first to report the absence of femA, and also probable variations in the sequences around the femA gene in clinical S. aureus strains in India. In Turkey, Fındık et al.[14] earlier found that no MRSA strain was positive for fem genes. In this study, 9.83% of MRSA strains were negative both for mec gene and femA gene. Although mecA and femA genes are specific markers for methicillin resistance, it has been suggested that methicillin resistance in S. aureus may be significantly regulated by other genes, such as, mecR1 and mecI genes or some other unidentified factors, rather than femA gene.[21]

In this study, MRSA strains identified both phenotypically (oxacillin susceptibility in agar disc diffusion test) and genotypically (mecA and fem gene amplifications) were typed using coa- and spa-PCR analysis. As coa gene was identified in 83.60% of the MRSA strains, a polymorphism among MRSA strains was observed, and six coa-types (C1-C6), which were observed in different band patterns varying between 180 and 480 bp were also identified. The majority of strains (32/51) gave two bands, while 37.25% showed single band pattern. However, the most predominant coa-type was C6, and the strains belonged to this type that showed a single band (180 bp). In several studies, different numbers of coa-types that showed various sizes of band patterns have been identified.[22],[23],[24] In Turkey, four patterns were identified among 120 MRSA strains. The difference in band patterns and coa-types may be considered to depend on the presence of different allelic forms of coa gene and geographical variation.[25]

Some researchers have reported that the X region of the spa gene of S. aureus was polymorphic, due to a variable number of repeats, and the number of these repeats was suggested to be used in strain discrimination.[26] In this study, however, spa gene was selected as a discriminatory marker for the subtyping of MRSA strains. In several studies, various sizes of the PCR products, which were reflecting the number of 24 bp repeat units contained in the spa gene have been reported.[9],[27] Whereas, no band polymorphism was observed among MRSA strains in this study. Furthermore, the majority (82.0%) of the MRSA strains were spa gene negative and 18.0% of the MRSA strains produced a single band (190 bp). Shakeri et al.[27] have reported that 3.4% of MRSA strains had no spa gene (no band), and the majority (83.0%) of MRSA strains gave one band (different sizes) of spa gene. These results were, however, lower than that those found in this study. In addition to the more prevalent strains without spa gene, there was no diversity in spa gene among the MRSA strains identified in this study. It was, therefore, considered that the MRSA strains were typed into strains without spa band but with a single band.

PCR-based methods are simple, cheap, and useful techniques for epidemiological studies, and some of which like RAPD-PCR, is an easy, fast, and economically affordable method, which has been widely used for typing of S. aureus strains.[1],[27]

MRSA strains isolated from different human samples in this study were typed by RAPD-PCR, and all the strains were typeable. According to a good discriminator power (0.852) of RAPD-PCR in this study, a total of 12 RAPD types were grouped into three clusters and five unique types. The most predominant type, R11 included 20 MRSA strains isolated from different sources. A total of 50.0% of the 20 strains of R11 type MRSA in C6 type, were the most predominant coa type. However, there is a need to work with a larger number of MRSA strains, to verify that predominant strains were involved in similar coa and RAPD types. In several studies, S. aureus strains have been subtyped by RAPD using different primers, to investigate the genetic relatedness among the MRSA strains.[28],[29] Generally, it has been found that the MRSA strains isolated from each hospital were grouped into the same cluster.[29],[30] In this study, although the majority of the MRSA strains were grouped into one predominant RAPD type, the remaining were included in other types. There was a genetic diversity in MRSA strains. None of the samples was from in-patients, as they were samples from out-patients admitted to the hospital, from different regions of Samsun. Therefore, MRSA strains had different RAPD profiles, according to their different geographical origin. The same observation may also true for coa types of MRSA in this study.


   Conclusions Top


Recently, identification, monitoring, and controlling of MRSA strains both in hospitals and in the community have become very important. Rapid, easy, cheap, and accurate diagnostic tools and typing methods make it possible to take well-timed measures for prevention against MRSA outbreaks, and also to monitor the spreading of strains and transmission routes. In this study, all MRSA strains were characterized genetically by some PCR-based molecular typing methods. While a genetic diversity was found among these strains using coa-PCR and RAPD-PCR, they could not be typed by spa-PCR, which has previously been reported to be a successful method for this purpose. It was concluded that coa- and RAPD-PCR typing were useful for epidemiological studies of MRSA but the differences in polymorphism of spa gene among MRSA strains should be considered.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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    Tables

  [Table 1], [Table 2], [Table 3]



 

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