Abstract: Gram-positive bacteria are more sensitive to antibiotics than gram-negative bacilli because of the lack of outer membrane which prevent easy access of the drug into the bacterial cells. However, there are many gram-positive organisms with natural, intrinsic resistance to antimicrobials. In addition, these bacteria are able to acquire resistance to frequently used drugs rapidly through selective pressure of the environment and also via the genetic evolution of bacteria. The resistance of those bacteria to antibacterial agents is mediated by antibiotic resistant genes. Therefore, the current study was designed to explore the effect of seaweed extracts on several antibiotic resistant genes in Staphylococcus aureus mec genes mecA, mecR1 and mecI that regulate the expression of methicillin resistance was investigated by PCR.

INTRODUCTION

Staphylococcus aureus which is namely by Sir Alexander Ogston, is an important opportunistic human pathogen and also a part of the normal microbial flora in the upper respiratory tract of human. It is a yellow pigmented species, non-motile, non-spore forming, catalase positive and gram positive cocci that is measure approximately 0.7-1.2 μm in diameter (David et al., 1999; Washington et al., 2006). It has low GC (Michael et al., 2005) content and is resistant to drying and readily dispersed in dust particles through the air. It is also a facultative aerobe bacterium which produces acid from glucose both aerobically and unaerobically. The expression of the mecA gene and the resulting production of PBP2a is regulated by proteins encoded by the penicillinase-associated blaR1-blaI inducer-repressor system and the corresponding genomic mecR1-mecI elements (Hackbarth and Chambers, 1993; Tesch et al., 1990; Sharma et al., 1998). Hiramatsu et al. (1992) identified in Staphylococcus aureus N315 the mecR1-mecI regulator element, which is located upstream of the mecA gene and is divergently transcribed from mecA. The mecI gene codes for a repressor protein and the mecR1 gene for a ß-lactam-sensing transmembrane signalling protein. Methicillin and oxacillin are, however, not good inducers for this system, often resulting in slow induction of methicillin resistance. Phenotypically susceptible strains, known as pre-methicillin-resistant S. aureus (pre-MRSA) and pre-methicillin-resistant coagulase-negative staphylococci (pre-MRCNS), have been discovered, which do not express methicillin resistance, as mecA is fully repressed by mecI (Hiramatsu, 1995; Weller, 1999) The induction of mecA transcription is very slow and might be due to mutations of mecI (Weller, 1999)^. Distribution of mec regulator genes among methicillin-resistant Staphylococcus strains from various countries has already been studied by hybridization and sequencing, which showed that the loss or inactivation of the mecI gene leads to derepression of mecA gene transcription (Weller, 1999; Shimaoka et al., 1994; Suzuki et al., 1993). In this study, we have amplified the antibiotic resistance genes of S. aureus mecA, mecR1 and mecI by Polymerase Chain Reaction after treated and untreated with marine seaweeds. Majority of the seaweeds grow by attaching to the hard surfaces like rocks and shells and can be found as far as 130 feet (40 m). The rich tropical waters surrounding the coast and islands, harbor a variety of seaweeds such as red algae (Rhodopyta), brown algae (Phaeopyta), green algae (Chlorophyta) and blue green algae (Cyanophyta), representing a potential source of useful products. Red algae which are found at where the water is much calmer can be utilized as a source of superfood for centuries. It comes in a variety of colors which gives rise to their variety of uses. In China, Japan and the Indo-Pacific region, several dozen species of red algae are used. This therapeutic superfood provides the body with a full array of nutrients including complete protein, complex carbohydrates, essential fatty acids, fiber, vitamins, minerals, trace elements, enzymes and sulfated polysaccharides. Red algae are capable of working on multiple levels to strengthen the body and solidify the body’s primary defense system. Efficacy of red algae as antibacterial agent is few in mention especially the local red algae found around Malaysian coasters. Therefore, this study is in search for the antibacterial properties in red algae in order to find a new antibacterial agent that can inhibit or reduce the growth of bacteria in human body. Red algae that are used in this study are Gracilaria changii, which grow mild in Pantai Morib, Selangor and Euchema denticulatum which grow mild and cultivated in Pantai Sabah. Several approaches of molecular biology tools through genomic analysis were used to explore and to understand antibacterial mechanism of the red algae extract in this study. In the present study, the advantages of molecular tools through genomic analysis could be used to prove that both seaweed extracts will work very well as a new antibacterial agent in a new decade not only just by prescreening test but also through genomic analysis for understanding the mechanism of inhibition. The recent advances in gene amplification methods have been used in this study such as Polymerase Chain Reaction.

MATERIALS AND METHODS

Genomic study: Propagation of Bacterial Culture Treated with Gracilaria changii and Euchema denticulatum Extract: a single pure colony of S. aureus isolates was inoculated to a 250 mL capacity Enlermeyer flask containing an adequate volume of sterilized Luria Bertani broth (Invitrogen Inc.) containing either 50 mg mL^-1 G. changii or 40 mg mL^-1 E. denticulatum extract. The broth was then incubated 4-5 h at 37°C with constant shaking on the shaker. This was followed by the procedure of the DNA extraction using the GeniSpin^TM Bacterial DNA Kit (BST^Techlab).

DNA extraction: Five isolates of S. aureus including MRSA and non-MRSA strains were extracted using the GeniSpin^TM Bacterial DNA Kit (BSTTechlab). One E. coli strain, which serve as negative control, was extracted by using the same method. The culture was aliquot into 1.5 mL microcentrifuged tube and pelleted until an adequate quantity of bacteria was obtained. Cells then were resuspended in 100 μL TE buffer and the bacterial cell wall was removed by lysozyme (10 mg mL^-1) digestion and followed by buffer BTL and proteinase K (15 mg mL^-1) digestion. RNaseA (25 mg mL^-1) was added to remove the RNA, which normally co-purifies with genomic DNA. Following lysis, binding condition was adjusted and the sample was applied to an I-Spin^TM column after adding the Buffer BDL and absolute ethanol. Two rapid wash steps using Wash Buffer will removed trace salt and protein contaminants and finally DNA was eluted in water or low ionic strength buffer. This DNA can be directly used in downstream applications without the need for further purification. The eluted DNA was then run onto 0.8% agarose gel electrophoresis to check the present of genomic DNA and stained with ethidium bromide to visualize under transilluminator. Lastly, the DNA was stored at-20°C in a refrigerator.

Polymerase Chain Reaction (PCR): All the primers that were utilized in this study were synthesized by Research Biolabs, Singapore. The sequences of primers for the amplification of mecA, mecR1 and mecI genes were in accordance to previously published data (Murakami et al., 1991; Suzuki et al., 1993) (Table 1). Using the Thermal Block Cycler (T-Personal) provided by Biometra, amplification of mecA, mecRI and mecI genes were achieved according to data represented in Table 2 and 3.

Table 1:	Sequences of primers used in detection and amplification of genes

Table 2:	The reaction mixture for each primer

Table 3:	Thermal cycling profile for each primer

RESULTS AND DISCUSSION

The total genomic DNA of high molecular weight were successfully extracted from 5 isolates of untreated and treated S. aureus (Fig. 1), including MRSA and non-MRSA strains and one isolate of Escherichia coli (Fig. 2) using GeniSpin^TM Bacterial DNA Kit (BST^Techlab). The ratio of absorbance at 260-280 nm (A260:A280) ranged from 1.238-2.000. The DNA concentrations were between 40-560 μg mL^-1. Amplification of mecA, mecR1 and mecI, Genes by Polymerase Chain Reaction (PCR). Amplification of various genes through Polymerase Chain Reaction (PCR) with respective specific primers, were sensitive at the DNA template concentration of 100 ng μL^-1 for Staphylococcus aureus genomic DNA. This study demonstrates that the mecA (Fig. 3), mecR1 (Fig. 4) and mecI (Fig. 5) genes have been successfully amplified and isolated from 3 local isolates of MRSA at 533, 310 and 481 bp, respectively. All the isolates treated with either Gracilaria changii or Euchema denticulatum extracts also showed the amplification of these genes whereby a single band corresponding to the respective PCR products were observed.

An increasing number of investigators have employed the tools of molecular biology to facilitate the diagnostic process. In the current study, since the preliminary screening reveals the significant finding of antibacterial activity of Gracilaria changii and Euchema denticulatum extracts against Staphylococcus aureus isolates, the molecular biology tools through genomic analysis was used to explore and to understand this inhibitory activity to predict the antibacterial mechanisms of both extracts on several selected genes of S. aureus including the antibiotic resistance genes, DNA repair gene and cell wall synthesis gene. In this study, a high molecular weight and good quality of DNA without RNA contamination was successfully extracted from all the treated and untreated S. aureus. Good quality of DNA was obtained as evidenced from the agarose gel analysis which is a crucial technique in detecting the presence of genomic DNA. To amplify the gene of interest, the most important component is the primer, a short segment of nucleotides, which have complementary base pairs to the length of the DNA.

Fig. 1:	The genomic DNA extracted from untreated and treated Staphylococcus aureus. Lanes 1-5 are the non-treated isolates, lanes 6-10 are the isolates treated with Gracilaria changii while lanes 11-15 are the isolates treated with Euchema denticulatum extract. Lane M in the Lambda

Fig. 2:	The genomic DNA extracted from untreated and treated Escherichia coli. Lanes 1 is the untreated isolate, lane 2 is the isolate with Gracilaria changii and lane 3 is the isolates treated with Euchema denticulatum extract. Lane M in the Lambda Hind 3 molecular weight marker

The correct identification of S. aureus and the detection of the methicillin resistant genes based on molecular methods have evolved as the method of choice for definitive identification of antibiotic resistance pattern. Antibiotic resistant genes that are chosen in this study were mecA, mecR1 and mecI gene. According to Hiramatsu et al. (1992), mecA encoded a penicillin-binding protein, PBP2a, mecI codes for a repressor protein while mecR1 codes for a β-lactam-sensing transmembrane-signalling protein. These antibiotic resistant genes are believed responsible for the expression of methicillin resistance (Ubukata et al., 1990) and make the treatment of infection by this strains become difficult. S. aureus strains which conferred by these genes known as Methicillin-Resistant S. aureus (MRSA). In this study, 3 isolates of S. aureus obtained from Universiti Malaya Medical Centre (UMMC) were screened for the presence of mecA, mecR1 and mecI gene. The primers used for the detection of the mecA gene have already been published by Murakami et al. (1991) while the primers used for the detection of the mecR1 (membrane spanning part) and mecI gene were those published by Suzuki et al. (1993). The polymerase chain reaction assays then were performed using the template of untreated genomic and genomic treated with either Gracilaria changii or Euchema denticulatum extract.

Fig. 3:

Amplification of mecA gene of MRSA isolates by PCR. Lanes 1-3 are the band of gene of untreated genomic, lane 4 is the gene of genomic treated with G. changii, lane 5 is the gene of genomic treated with E. denticulatum while lane M is the 100 bp molecular weight marker. mecA gene positive isolates have a single band present in the region between the ladders of 500-600 bp. The actual band position is at 533 bp. Lane C is a negative control showing no band indicating that there was no contamination during the process

Fig. 4:

Amplification of mecR1 gene of MRSA isolates by PCR. Lanes 1-6 are the band of gene of untreated genomic, lanes 7-9 are the gene of genomic treated with G. changii, lanes 10-12 are the gene of genomic treated with E. denticulatum while lane M is the 100 bp molecular weight marker. mecR1 gene positive isolates have a single band present in the region between the ladders of 300-400 bp. The actual band position is at 310 bp. Lane C is a negative control showing no band indicating that there was no contamination during the process

Fig. 5:

Amplification of mecI gene of MRSA isolates by PCR. Lanes 1-6 indicate the band of gene of untreated genomic, lanes 7-10 are the gene of genomic treated with G. changii, lanes 11-15 are the gene of genomic treated with E. denticulatum while lane M is the 100 bp molecular weight marker. mecI gene positive isolates have a single band present in the region between the ladders of 300-400 bp. The actual band position is at 481 bp. Lane C is a negative control showing no band indicating that there was no contamination during the process

For detection and amplification of those genes, the experimental conditions were re-optimized from the earliest time to obtain the best possible results whereas the template, primer, MgCl₂ and dNTP mix concentrations are optimized in the present study. For mecA and mecR1 genes, the annealing temperature was increased to 59°C for 30 sec, the primer extension temperature was reduced to 55°C for 30 sec and the number of cycle was reduced to 25 cycles. Since the hot start technique was used, the denaturation time also were re-optimized for all genes.

All the isolates tested including the isolates treated with both seaweed extracts, harbored selected the antibiotic resistance genes as indicated by successful amplification shown by the strong positive signal of single DNA band for mecA, mecR1 and mecI genes through PCR assay.

The successful amplification of the gene of interest is dependent upon the amount and quality of the template DNA. Nevertheless, in this study, the PCR was successfully performed even though the purity and the concentration of the genomic were low since DNA can be amplified even with 30-100 ng μL^-1 of concentration.

CONCLUSION

As a conclusion from the results discussed above, the findings in this study assumed that the supplement of the methanol extract of G. changii and E. denticulatum into the MRSA isolates, will not deleting the antibiotic resistance genes in those isolates since the mecA, mecR1 and mecI genes still were amplified in the treated samples.