Partial Sequencing of 16S RRNA Gene of Selected Staphylococcus Aureus Isolates and Its Antibiotic Resistance

The choice of primer used in 16S rRNA sequencing for identification of Staphylococcus species found in food is important. This study aimed to characterize Staphylococcus aureus isolates by partial sequencing based on 16S rRNA gene employing primers 16sF, 63F or 1387R. The isolates were isolated from milk, egg dishes and chicken dishes and selected based on the presence of sea gene that responsible for formation of enterotoxin-A. Antibiotic susceptibility of the isolates towards six antibiotics was also tested. The use of 16sF resulted generally in higher identity percentage and query coverage compared to the sequencing by 63F or 1387R. BLAST results of all isolates, sequenced by 16sF, showed 99% homology to complete genome of four S. aureus strains, with different characteristics on enterotoxin production and antibiotic resistance. Considering that all isolates were carrying sea gene, indicated by the occurence of 120 bp amplicon after PCR amplification using primer SEA1/SEA2, the isolates were most in agreeing to S. aureus subsp. aureus ST288. This study indicated that 4 out of 8 selected isolates were resistant towards streptomycin. The 16S rRNA gene sequencing using 16sF is useful for identification of S. aureus. However, additional analysis such as PCR employing specific gene target, should give a valuable supplementary information, when specific characteristic is expected.


INTRODUCTION
Staphylococcal food poisoning is occurred when people consume food contaminated by Staphylococcus aureus that produce enterotoxins. Various staphylococcal enterotoxins (SE) incriminated in staphylococcal food poisoning have been reported. They were SEA, SEB, SEC, SED and SEE, and recently new serological types of SEs (SEG, SEH, SEI, SEJ, SEK, SEL, SEM, SEN, SEO, SEP, SEQ, SER and SEU) were also identified (Argudin et al., 2010;Xie et al. 2011, Roussel et al., 2015. Staphylococcal enterotoxins-A (SEA), which is found on most food poisoning by S. aureus, is expressed in the mid-exponential phase, and its gene appears to be transferred by temperate bacteriophage (Argudin et al., 2010). The prevalence and genetic diversity of S. aureus has been investigated in raw and pasteurized milk (Rall et al., 2008), ready-to-eat foods (Huong et al., 2010), milk and food product (Salasia et al., 2011), and street-vend foods (Rohinishree & Negi, 2011). For epidemiological purposes, the accuracy of identification of Staphylococcus species isolated from various food products is critical. In this regard, molecular characterization is reported to be more accurate (Becker et al., 2004) than phenotypic identification (Rohinishree & Negi, 2011). The 16S rRNA gene is extensively used as taxonomic marker molecules during molecular characterization (Janda & Abott, 2007), particularly during the sequence analysis for differentiating species and sub species of bacteria (Rohinishree & Negi, 2011).
Generally, during sequencing analysis, the DNA target is firstly amplified using a pair primers followed by the sequencing using a single primer (SenGupta & Cookson, 2010). Identification based on highly conserved genes such as 16S rRNA usually uses long sequences primers (≥ 500 bp to about 1500 bp) (Janda & Abbott, 2007), although species specific shorter sequences can also be applied. For this purposes, universal primers for amplification of 16S rRNA genes are widely available, such as primers 63F and 1387R (1350bp) (Marchesi et al., 1998) and 27f and 1492r (Frank et al., 2008). Relatively shorter primer, i.e. 16sF and 16sR3 were also used (Lee et al., 2007). Since the choice of primers will affect the diversity of bacterial species that will be detected (Fredriksson et al., 2013), in this study three different primers targeting the 16S rRNA gene, i.e.
16sF, 63F and 1387R, was used separately to sequence DNA of selected S. aureus isolates from milk, egg dishes and chicken dishes. Milk, egg dishes and chicken dishes were reported contaminated by S. aureus in previous study (Handayani et al., 2014).
Furthermore, as information on the spreading of antibiotic resistance strains among S. aureus in food is also important, the antibiotic susceptibility testing of the isolates against antibiotics was also conducted in this study. The antibiotics tested were antibiotics that usually used to control human infections as well as to control and treat infections on farms. Many S. aureus strains that demonstrated resistance to different antibiotics were isolated from hospitals (Schmitz et al., 1999;Brown & Ngeno, 2007;Xie et al., 2011), hospital waste waters (Thompson et al., 2012), as well as from animal based food product, such as raw milk and dairy products (Jamali et al., 2015), poultry retail meat (Teramoto et al., 2016), and goat milk powder (Xing et al., 2016).

Bacterial Strain and Isolates
The wild-type of S. aureus from raw milk (S1, S4 and S10), egg dishes (TB1), sautéed chicken cuts (UA2 and UA13) and chicken cuts satay (SJ1 and SJ4) were isolated in previous study (Handayani et al., 2014). S. aureus ATCC 25923 was used as a reference bacterium. All bacteria were grown in a tryptic soy broth or agar (Difco Laboratory, Detroit, MI, U.S.A.), and incubated at 37 o C for 18h-24h. For confirmation, the isolates were spread onto Baird-Parker Agar (Oxoid Ltd., Hampshire, UK) supplemented with egg-yolk tellurite. Plates were incubated at 37°C for 18-24 h, thereafter the colonies were picked and streaked on Mannitol Salt Agar (Oxoid Ltd., Hampshire, UK). Typical colonies were then tested for production of catalase using Staphylase test kit (Oxoid Ltd., Hampshire, UK) and biochemical identification using API Staph (bioMérieux Inc., North Carolina, USA) according to manufacturer's instructions.

DNA Extraction
Genomic DNA was isolated as described previously by Mason et al. (2001) with slight modification, as reported by Handayani et al. (2014), i.e. lysostaphin (10 mg/mL) was substituted by lysozyme (Bio Basic Canada Inc., Ontario, Canada) solution (10 mg/ml). The concentration of genomic DNA was determined by the Spectrophotometer UV -1800 (Shimadzu, Japan) at 260 nm while the quality was assessed based on the ration of the reading at 260/280 nm. The integrity of the DNA was checked by running in 1.5% agarose gel at 75V for 40 min electrophoresis (Bio-Rad Laboratories Pte. Ltd, Singapore).

Detection of 16S rRNA and Sea Gene by PCR
The amplification of the gene encoding 16S rRNA and sea was performed using primers listed in Table 1 at Thermal Cycler 2720 (Applied Biosystems, California, USA). PCR master mix consisted of 12.5 µL of DreamTaq Green master mix (Thermo Fisher Scientific, Massachusetts, USA), 1 µL of each primer (10 µM), 2 µL of DNA template, and 8.5 µL nuclease free water (Thermo Fisher Scientific, Massachusetts, USA).

Sequence Analysis of 16S rRNA Gene
The genotypic characterization of bacteria isolates was made through partial sequence analysis of 16S rRNA gene, using single primer 16sF, 63F, or 1387R. The PCR products that were amplified with primers 16sF/16sR3 were sequenced using primer 16sF. The PCR products amplified with primers 63F/1387R were sequenced using primer 63F and 1387R separately. The process of DNA sequencing was conducted by BigDye Applied Biosystem sequencer engine model 3730 at Macrogen inc., Singapore. Partial sequence data obtained, in FASTA format, was then submitted to the BLAST process at NCBI (National Center for Biotechnology Information) database for the identification of isolates (http://blast.ncbi.nlm.nih.gov/ Blast.cgi).
The BLAST process was conducted using nucleotide collection searching setting (nr/nt) with Staphylococcus aureus subsp. aureus (taxid:46170) as organism of choice. The sequencing results were then compared to the reported 16S rRNA gene sequences of Staphylococcus species available in the GenBank database. The isolates were identified based on the highest homology percentages to the reported sequences.

Genotypic Characteristic of Isolates
Ten isolates of 78 presumptive S. aureus isolates were positive for S. aureus (Handayani et al., 2014). Eight of these isolates were reconfirmed in this study carrying sea gene that responsible for formation of staphylococcal enterotoxin-A (SEA). The presence of sea gene was indicated by the occurrence of 120 bp amplicon, after PCR amplification using primer SEA1/SEA2 ( Table 2). The reference strain, S. aureus ATCC 25923, did not show this gene. In addition to the work of Handayani et al. (2014), all isolates also demonstrated 1350 bp amplicon after amplification by 63F/1387R primer (universal primer). The amplified PCR products of some isolates are shown in Figure 1.
The BLAST results are listed in Table 3. Sequencing by primer 16sF resulted in high identity percentages (almost all achieved 99%) towards the existing genome of S. aureus strains found in database of NCBI GenBank, in comparison to the sequencing by 63F and 1387R. Sequencing by 63F showed similarity to S. aureus strains in a range between 94% and 99%, while by 1387R in a range of 90% to 99%. In addition, using 16sF also resulted in low E-value. The lower the E-value, or the closer it  is to zero, the match is more significant (Pearson, et al., 2014). The expect value (E) is a parameter that describes the number of hits one can "expect" to see by chance when searching a database of a particular size. As shown in  Table 4. Since all isolates were detected carrying sea gene (Table 2), the isolates were in agreeing to S. aureus subsp. aureus ST288 that able to form enterotoxin-A. The present study has been indicating that sequencing with 16S rRNA as gene target has been successfully identifying the isolates to specific strains. Additional PCR analysis employing SEA1/SEA2 primers increased accuracy of characterization of the isolates.

Antibiotic Resistance Among the Isolates
Four isolates showed resistance to streptomycin (Table 5). All isolates, however, were susceptible to gentamycin and oxytetracycline. Interestingly, the resistant strains to streptomycin were isolated from different food sources, i.e. from milk (S10), egg dishes (TB1), sautéed chicken cuts (UA13) and chicken cuts satay (SJ1). Next to resistance to streptomycin, isolate TB1 showed also intermediate resistance to kanamycin, chloramphenicol and tetracycline.

DISCUSSION
Among the SEs, SEA is reported as the most common cause of staphylococcal food poisoning worldwide, but the involvement of other SEs has been also found. SEA is considered as the main cause of staphylococcal food poisoning, probably due to its high resistance to proteolytic enzymes (Argudin et al., 2010). In the present study, all (eight) isolates were identified carrying sea gene, suggesting the potential risk of these strains due to production of SEA. Another study conducted in Indonesia by Salasia et al. (2011), however, did not find this gene in 11 food isolates (i.e. fermented milk product, sausage, meat ball, cakes and cheese), but found seb, sec, see and seg genes. They found, however, sea gene in 5 isolates of 19 milk isolates.
Regarding the sequencing results, as predicted, the use of 3 different primers resulted in various identity percentages. Sequencing by primer 16sF (5'-CCGCCTGGGGAGTACG-3') resulted in higher identity percentages (almost all achieved 99%) towards the existing genome of S. aureus strains in comparison to the sequencing by 63F (5'-CAGGCCTAACACATGCAAGTC-3') and 1387R (5'-GGGCGGWGTGTACAA GGC-3'). As was presented in Table 3, sequencing by 16sF also resulted in higher query coverage than that achieved by 63F and 1387R, except for isolate SJ4. These results highlighted that 16S rRNA sequencing by short sequence could provide sufficient identification amongst S. aureus strain. The 16sF has been used before to detect 16S rRNA genes of S. aureus isolates from food sample (Lee et al., 2007;Lee & Park 2016).
This study found that all isolates demonstrated 99% homology to the sequence genome of 4 strains of S. aureus by sequencing using primer 16sF. Based on the highest total score of the BLAST results, all isolates showed similarity to the genome of S. aureus strain MSSA476 and S. aureus subsp. aureus ST772-MRSA-V strain DAR4145. Strain MSSA476 is an invasive community acquired methicillin-susceptible S. aureus (Holden et al. 2004). On the other hand strain DAR4145 is a multidrug resistant strain of ST772-MRSA-V (Steinig et  al., 2015). These results suggested that the isolates in this study showed equal likelihood whether they were corresponding to the MRSA or the MSSA. This finding is possible since these strains also occupied many people in Indonesia. Recent epidemiological study showed MRSA carriage rate of 4.3%, and MSSA carriage rate of 1.5% among 1,502 patients in hospitals in Java and Bali (Santosaningsih et al., 2014).
As discused above, when sequenced by 16sF all isolates showed good homology (99%) to some strains found in the NCBI GenBank data base with different characteristics on its antibiotic resistance. Next to this characteristic, the coresponding strains also showed differences on capability to form enterotoxin. Strain MSSA476 and DAR4145, as well as strain DSM 20231 did not produce enterotoxin-A (NCBI, 2016). On the other hand, S. aureus subsp. aureus ST288 isolate 10338,10497,15532,16035,18341,18412 and 18583 are known as enterotoxin-A producers. Therefore, additional information to confirm the presence of a gene that responsible for formation of enterotoxin-A is considerably important. Since all selected isolates were confirmed carrying sea gene, indicated by the presence of 120 bp amplicon after PCR amplification using primer SEA1 and SEA2 as presented in Table 2, all isolates were most in agreeing with S. aureus subsp. aureus ST288. The sea gene is 771 bp in size encoding an enterotoxin A precursor of 257 amino acid residues (Huang et al., 1987). Specific primers SEA1 and SEA2 were frequently used in PCR analysis to detect the presence of sea gene in S. aureus isolates from food, such as in raw and pasteurized milk (Rall et al., 2008); Kérouanton et al., 2007) and ready-to-eat Kimbap (Lee et al., 2007). Specific PCR primers have commonly been employed to confirm the presence or absence of specific characteristics associated with target microorganisms such as virulence factors.
Next to genotypic characteristic, information on antibiotic resistance amongst S. aureus strains found in food is also important for surveillance and epidemiology study. This study found that four isolates from different food sources (from milk, egg dishes, sautéed chicken cuts and chicken cuts satay) were resistant to streptomycin. Streptomycin resistance among S. aureus isolates was also reported in other study. Jamali et al. (2015) found that amongst S. aureus isolates from raw milk and dairy products (n=328), 5.8% demonstrated resistance to streptomycin, 4% to kanamycin, 3.7% to chloramphenicol, and 2.1% to gentamicin. Most isolates were resistant to tetracycline (56.1%) followed by to penicillin (47.3%). The high percentage of resistant isolates to these last two antibiotics could be due to the widespread use of these antibiotics to control and treat infections on dairy farms (Jamali et al., 2015). Moreover, the fact that streptomycin resistant strains were found in milk, egg dishes and chicken dishes indicated possible  occurrence of cross contamination from human or food vendor. Schmitz et al. (1999) found that 21% of the S. aureus isolates (n=699) collected from different hospitals in Europe were resistant to streptomycin. They also found, however, 23% of the S. aureus isolates were resistant to gentamycin, 29% to tobramycin, and 31% to kanamycin. In more recent study, Onwubiko & Sadiq (2011) also found that 55.8% of S. aureus from clinical isolates in a tertiary health institution in North-western Nigeria (n= 129) showed resistance to streptomycin, 68.8% to tetracycline, 38.1% to chloramphenicol, and 7.6% to gentamycin. The spreading of S. aureus strains that resistant to antibiotics has become a global concern. Continued surveillance of S. aureus producing enterotoxin-A in milk, egg and poultry food products at genotypic levels is necessary to understand and limit further increases of staphylococcal food poisoning incidences.

CONCLUSION
This study has demonstrated that in order to increase the accuracy of the identification results, next to the sequencing of S. aureus targeting 16S rRNA gene, PCR analysis using specific primer is considerably important. All eight isolates were carrying sea gene, detected by PCR analysis, indicating that they can produce staphylococcal enterotoxin-A. Genotypic characterization of the selected strains by sequencing using 16sF, showed agreeing to the sequence genome of S. aureus subsp. aureus ST288 that also produce enterotoxin-A. This study also found that 4 of 8 selected isolates were resistant to streptomycin.