**3. Results**

**Figure 3a** shows the temperature gradient for *coa* gene, where the range of 56–60°C was observed a specific amplification and 56°C was selected temperature to annealing specific primers in target gene. **Figure 3b** shows the sensitivity of the method for target *coa* gene in range of genomic DNA concentrations (50, 5 and 0.5 ng/μl; 50, 5 and 0.5 pg/μl; 50, 5 and 0.5 fg/μl); the method has a sensitivity up to 0.5 pg/μl.

The detection by PCR was performed using the pair of primers of *coa* gene [48]. In an Eppendorf PCR tube (0.2 mL), 3 mM MgCl2, 0.8 μM oligonucleotides, 1.6 μM dNTP, 1 unit of Taq DNA polymerase, 10 ng/μl ADN, and buffer 1× were added to make a final volume of 25 μl. A fragment of 674 bp was amplified by the primers *coaf* 5′‐ATA GAG CTG ATG GTA CAG G‐3′ and *coar* 5′‐GCT TCC GAT TGT TCG ATG C‐3′), and the PCR protocol was performed in a SimpliAmp Thermal Cycler (Applied Biosystem Ref A24812): 94°C for 10 min (1 cycle), 35 cycles of: 94°C for 30 s, 56°C for 1 min and 72°C for 1 min, with a final cycle of 72°C for 5 min.

Additionally, sensitivity test was performed with base in the genomic DNA concentration. The procedure was done according to Shree et al. [51]; briefly, from genomic DNA of the ATCC 11632 were done dilutions (50, 5, 0.5 ng/μl; 50, 5, 0.5 pg/μl and 50, 5, 0.5 fg/μl). PCR and electrophoresis gel were carried out as we described previously for *coa* gene. The assay was

The phylogenetic tree was constructed by comparing conserved sequences from the adjacent 16S gene, using the F2C 5′‐AGAGTTTGATCATGGCTC‐3′ and C 5′‐ACGGGCGGTGTGTAC‐3′ primers, in order to obtain a fragment of approximately 1600 bp which was bidirectionally sequenced; the mix reaction was as described above and the PCR conditions were as follows: 95°C for 10 min (1 cycle), 32 cycles of: 95°C for 1 min, 60°C for 1 min and 72°C for 2 min, with

The purification of PCR products was performed according to the Qiaquick PCR Purification Kit (Qiagen, EUA, Ref 28106), and 400 ng was evaporated in a dry bath at 56°C for 12 h for bidirectional sequencing. The sequences of regions were compared with the National Center of Biotechnology Information (NCBI) data (http://www.ncbi.nlm.gov/) using BLAST‐N. The output was grouped such that all members exhibited more than 90% similarity; the alignment of the DNA sequence data was analyzed in Mega 6 software for the phylogenetic tree building

To test the antimicrobial effect, the disk diffusion method (Kirby‐Bauer) was applied, using Mueller‐Hinton agar (MCD Ref 7131) and five antibiotics with different concentrations: tetracycline 30 μg (Oxoid Ref CT0054B), trimethoprim‐sulfamethoxazole 25 μg (Oxoid Ref CT0052B), clarithromycin 15 μg (Oxoid Ref CT0693B), oxacillin 1 μg (Oxoid Ref CT0159B), and penicillin G 10 U (Oxoid Ref CT0043B). Each assay was performed in triplicate and the diameter

**Figure 3a** shows the temperature gradient for *coa* gene, where the range of 56–60°C was observed a specific amplification and 56°C was selected temperature to annealing specific primers in target gene. **Figure 3b** shows the sensitivity of the method for target *coa* gene in range of genomic DNA concentrations (50, 5 and 0.5 ng/μl; 50, 5 and 0.5 pg/μl; 50, 5 and 0.5

done in triplicates.

154 Frontiers in Frontiers in Staphylococcus Aureus *Staphylococcus aureus*

**3. Results**

a final cycle of 72°C for 5 min.

with bootstrap analysis (1000 repeats).

of the inhibition zone was calculated (mm) [52].

fg/μl); the method has a sensitivity up to 0.5 pg/μl.

**Figure 3.** (a) Agarose gel electrophoresis showing temperature gradient for *coa* target gene by PCR with specific pri‐ mers. M, size marker (1 kb plus DNA Ladder, Invitrogen™); lane 1, 50°C; lane 2, 52°C; lane 3, 54°C; lane 4, 56°C; lane 5, 58°C; lane 6, 60°C and lane 7, negative control (ultrapure water, Invitrogen™). (b) Agarose gel electrophoresis showing the sensitivity of detection for *coa* gene using different DNA concentrations. M, size marker (1 kb plus DNA Ladder, Invitrogen™); lane 1, 50 ng; lane 2, 5 ng; lane 3, 0.5 ng; lane 4, 50 pg; lane 5, 5 pg; lane 6, 0.5 pg; lane 7, 50 fg; lane 8, 5 fg and lane 9, 0.5 fg.

The incidence of *S. aureus* was 1.7% (**Figures 2** and **4**). All the isolates were situated in the ATCC 11632 clade in accordance with other reported sequences belonging to this pathogen in the NCBI database with a bootstrap of 98 (**Figure 5**). Similar reports were given by the GenBank sequences D83357.1, D83355.1, JN315147.1, JN390832.1, JN390831.1, JN315154.1, JN315153.1, JN315151.1, JN315150.1, and JN315149.1 being reported in the same operational taxonomic unit (OTU) with similarities in their morphologic, physiologic, and biochemical characteristics.

All the isolates seemed to be resistant to penicillin G 10 U and were susceptible to oxacillin, tetracycline, clarithromycin, and trimethoprim‐sulfamethoxazole (**Figure 6**).

**Figure 4.** Amplification of target pathogen isolated from lettuce by PCR with specific primers. M, molecular marker (1  kb plus DNA Ladder, Invitrogen™); lane 1, positive control ATCC 11632; line 2, isolated 1; line 3, isolated 2 and line 4, negative control (ultrapure water, InvitrogenTM).

**Figure 5.** Phylogenetic tree of *S. aureus* maximum verisimilitude constructed from partial sequences of 16S ribosomal DNA of 2 prokaryotic clones and 17 reference sequences obtained from NCBI.

**Figure 6.** Tests of antibiotic susceptibility; 1: positive control (*Staphylococcus aureus* ATCC11632), 2: isolated 1, 3: isolat‐ ed 2. (a) Oxacillin 1 μg, (b) trimethoprim‐sulfamethoxazole 25 μg, (c) penicillin G 10 U, (d) tetracycline 30 μg, and (e) clarithromycin 15 μg.

## **4. Discussion**

For *coa* gene, the temperature gradient showed that 56°C was the optimal annealing temper‐ ature (*T*a) for oligonucleotides, showing an adequate specificity for the detection of *S. aureus*. The *T*a is defined as the highest temperature where the optimal aligning and amplification occur [53]; this parameter is crucial for the standardization of the method because a low *T*<sup>a</sup> can cause nonspecific amplification, giving undesired PCR products; this is when two or more bands are observed in gel electrophoresis. In this study, the primers features and the correct design lead us to obtain a good and specific amplification in a range of 56–60°C. Likewise, a high *T*a can cause a low or non‐amplification, reducing the possibility to anneal; for this reason, an optimization of priming temperature is necessary [54]. Additionally, annealing was satisfactory at low DNA concentrations (up to 0.5 pg/μl) showing adequate sensitivity. Isolated from lettuce samples were confirmed by amplification of the 674 bp fragment.

For the strategy with the 16S an optimal annealing temperature of 54°C was established for a fragment of approximately 1400 bp; isolates 1 and 2 were aligned in the same clade as the positive control (ATCC 11632) strain. Clinical animal isolates reported at NCBI D83357.1, D83355.1 and isolated from human throats suffering clinical infections JN315147.1, JN390832.1, JN390831.1, JN315154.1, JN315153.1, JN315151.1, JN315150.1, and JN315149.1 show that isolates 1 and 2 are potentially dangerous if the vegetable is not properly sanitized before consuming.

Low incidence of *S. aureus* is directly related to good manufacturing practice of packing companies, mainly because the exposure time of the product in contact with the exterior is very short. Likewise, the product is never in direct contact with the staff due to the use of hairnets, gloves, face masks, aprons ,and boots, as well as all staff washing and disinfecting their hands before entering work and after toileting.

The bacterial counts found in this study were below the health limit of 102 –103 CFU g‐1 of *S. aureus* in food set by the Codex Alimentarius, stabilizing a good quality of lettuce with respect to this pathogen. A study by Viswanathan and Kaur [55] reports the presence of *S. aureus* in 23% of a total of 120 samples from various vegetables in India. This incidence is attributed to postharvest and human contamination due to the management of the foods. These results make evident the permanence of the pathogen in this food group, the proper handling of Mexican producers, and the safety of their food.
