Typical characteristics of
The growth in food demand and production growth of vegetables have led to the development of intensive production systems with the aim of having regular access to enough high‐quality food. The aim is to determine the incidence of Staphylococcus aureus in fresh lettuce by PCR in order to enhance the efficiency for detection and identification process. The Baird‐Parker method was used for isolating pathogens from 54 lettuce samples. Genomic DNA extraction was performed according the Mericon DNA Bacteria Plus Kit. The detection by PCR was performed using the pair of primers: coa gene (5′‐ATAGAGCTGATGGTACAGG‐3′ and 5′‐GCTTCCGATTGTTCGATGC‐3′). 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. To test the antimicrobial effect, we used the disk diffusion method (Kirby‐Bauer) using Mueller‐Hinton agar and five antibiotics with different concentrations. The incidence of S. aureus was 1.7%. All the isolates were situated in the ATCC 11632 clade in accordance with other reported sequences belonging to this pathogen in the NCBI database. All the isolates seemed to be resistant to penicillin (10U). The molecular techniques used in this study are suitable for the identification of S. aureus isolated from lettuce, increasing our capability of detecting this pathogen by improving the process and increasing the efficiency contributing to the safety of this vegetable.
- S. aureus
- fresh produce
- coa gene
They are coagulase variant, non‐spore‐forming, facultative anaerobes (except
S. aureus in food
The presence of pathogens in minimally processed vegetables and their ability to survive and grow has been well documented .
Therefore, it is considered that
1.3. Staphylococcal intoxication
One of the most important foodborne diseases transmitted around the world is Staphylococcal intoxications; of all outbreaks of food poisoning that occur, on average 20% are due to the consumption of food contaminated with enterotoxins produced by bacteria of the genus
In the Middle East, many types of vegetables are eaten raw in salads or used as garnish appetizers, and in traditional meals, they are perceived as healthy food; however, in other parts of the world, these raw vegetables have been major contributors of foodborne diseases in recent years [18, 19]. In the United States, green leafy vegetables have been identified as part of the 10 riskiest foods regulated by the Food and Drug Administration (FDA), representing almost 40% of foodborne outbreaks according to data obtained from the Center for Disease Control and Prevention (CDC) .
The consumption of green leafy vegetables provides numerous health benefits, and there is a direct relationship between consumption of these vegetables and the reduction of chronic diseases such as hypertension, diabetes, atherosclerosis, and cancer . Currently, most fresh‐cut products are washed in chlorinated water (50–200 mg L-1 of active chlorine) to reduce the levels of microorganisms. Sodium hypochlorite (NaCl) is the most widely used disinfectant in the fresh‐cut industry . The lack of thorough cooking in fresh cuisine can result in foodborne diseases if contaminated by pathogens. Despite these foods being ready to eat, it has been reported that their quality is not satisfactory in Vienna, Austria , Johannesburg, South Africa , Korea , and Catalonia, Spain . Reports show that the main pathogens in ready‐to‐eat foods include
In Mexico, staphylococcal intoxications are responsible for 45% of the outbreaks caused by food poisoning. These data partially reflect the incidence of this disease in the country, considering that it only represents the outbreaks that have been reported or studied; however, it is useful to show that on a national level, staphylococcal poisoning is a major foodborne . The main places where the outbreaks were reported to occur are at parties or social gatherings, schools or daycare centers, restaurants, and hospitals, in this order of importance .
In recent years, Mexico has become one of the most dynamic markets for the US horticultural importers, displacing Japan to third place . From January to April 2014, a total of 51,109 tons of lettuce equivalent to 42.289 mdd were exported, the main exporters being: Guanajuato (56.8%), Nuevo Leon (19.9%), Baja California (17%), Sonora (1.9%), other states (4.4%) .
1.4. Identification of
Various methods have been developed for the isolation and quantitative identification of
Processed foods may contain relatively small numbers of debilitated viable cells, whose presence must be demonstrated by appropriate means (BAM, Bacteriological Analytical Manual) .
Among the most important methods used globally are those recommended by the Association of Official Analytical Chemistry (AOAC) (975.55‐1976,
A brief description of the method includes:
1.4.1. Baird‐Parker method
184.108.40.206. Preparation of the samples
Take different portions of the food, transfer 25 g or mL into dilution bottles with 225 mL of phosphate buffer or peptone water to prepare a dilution 1:10, and homogenize for 1 or 2 min in blender or peristaltic homogenizer.
220.127.116.11. Analytical procedure
Transfer 0.1 mL of direct sample with a sterile pipette if liquid, or 0.1 mL of the initial suspension (dilution 10-1) in the case of other products, onto plates with Baird‐Parker agar with addition of egg yolk emulsion. Do this in duplicates and repeat this procedure for subsequent dilutions 10-2, 10-3 if necessary.
Carefully distribute the inoculum on the agar surface as soon as possible, with a sterile glass rod bent at a right angle, using one for each plate and dilution. The plates must be kept with the top upward until the inoculum is fully absorbed by the agar.
Invert and incubate the plates from 44 to 48 h at 36°C and subsequently search for colonies with typical morphology: black in color, circular, bright, convex, flat from 1 to 2 mm in diameter, showing one opaque zone, wet and with a clear halo.
Select the plates having between 15 and 20 typical and atypical colonies for their confirmation. From each sample, select five typical colonies for confirmation or five atypical colonies to perform Gram staining. In the case of observing positives bacilli, the colony will be taken as negative for
When the plates contain <15 typical colonies, a note citing “estimated value” must be added to the report of results.
18.104.22.168. Confirmation procedure
For the procedure, select and inoculate each typical colony in a tube with 0.5 mL brain heart infusion broth (BHI) and tubes with Trypticase soy agar (TSA). Simultaneously use a positive control (
22.214.171.124. Thermonuclease test
The production of this enzyme is inhibited by anaerobiosis and is stimulated by the presence of oxygen, it requires calcium ions for its enzymatic activity, its optimal pH is 8.6 and is precipitated with ammonium sulfate. Its thermal stability (resistant to temperatures of 130°C for 16.6 min) is the only association with the growth of
When interpreting this test, it is essential to consider the existence of enterotoxigenic strains negatives to both tests. Among the negative coagulase strains, some have developed the ability to synthesize enterotoxins. Although
For this test procedure, slides are prepared with 3 mL of toluidine‐DNA blue agar. Using a Pasteur pipette makes equally spaced holes in the agar. In a boiling water bath, heat 0.3 mL of bacterial culture in BHI for 15 min. With the use of a Pasteur pipette transfer a drop of bacterial culture to a hole of toluidine blue agar‐DNA. Repeat for each strain including the positive and negative controls. Incubate at 35 ± 1°C in a humid chamber from 4 to 24 h. The appearance of a pink halo of at least 1 mm qualifies as a positive test.
See Table 1 for the characteristics of
|Anaerobic utilization of glucose||+||+||–|
1.4.2. Molecular methods
The molecular identification is centered of detection and sequencing a specific bacterial DNA and used to identify and classify taxonomically several groups of microorganisms, including bacteria by amplification of specific target region by PCR. Another strategy is amplified by PCR‐specific genes that belong to certain species, based on specific features like virulence factors or antibiotics resistance genes . The development of identification techniques for a clinical rapid diagnosis is necessary. The PCR is a rapid, sensitive, and less time‐consuming than the conventional bacteriological identification methods  and is extensively used to identify bacteria isolated from different kind of samples, including foods , soil , and infected human tissue .
1.4.3. Identification based on 16S ribosomal RNA gene
The 16S rRNA gene is part of all bacteria and is commonly used for taxonomic purposes because it is a highly conserved region; the rate of protection from change is assumed to result from the importance as a serious constituent of cell function . Into the sequence of 16Sr RNA gene are indicated variable regions; Chakravorty et al.  in his study describe nine regions with sufficient diversity that are suitable for taxonomic analysis; their investigation determined that V1 hypervariable region best differentiated among
2. Study case
2.1. Materials and methods
The aim was to determine the incidence of
Study zone. Fifty‐four lettuce samples were obtained from a company of fresh products located in Sonora, Mexico (Figure 1); all the productive process was evaluated, such as cut area, storage, and transportation.
It is important to mention that Mexican vegetable producers strive to be at the top of the market, which involves providing the customers top‐quality products all the time. To achieve this goal, they implemented quality control assurance programs as well as partnered with prestigious external certification labs to help they watch every step of the way.
2.2. Transport and handling of sample
Samples were collected as described by Seow et al. , briefly, personnel of the laboratory was transferred to the production site in the Yaqui Valley and sampled lettuce from the process, were sampled in original package and immediately placed in sterile resealable bags, and later were transported with iceboxes and stored to 4°C until analysis. Product information such as production date, lote, and “best before” were registered in the database of the study. All the samples were analyzed within 24 h after time of collection, in the meantime, keeping them in their original storage conditions.
2.3. Isolation and identification
The Baird‐Parker method was used for isolating pathogens from 54 lettuce samples (Figure 2).
The detection by PCR was performed using the pair of primers of
Additionally, sensitivity test was performed with base in the genomic DNA concentration. The procedure was done according to Shree et al. ; 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
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 a final cycle of 72°C for 5 min.
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 with bootstrap analysis (1000 repeats).
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 of the inhibition zone was calculated (mm) .
Figure 3a shows the temperature gradient for
The incidence of
All the isolates seemed to be resistant to penicillin G 10 U and were susceptible to oxacillin, tetracycline, clarithromycin, and trimethoprim‐sulfamethoxazole (Figure 6).
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
The bacterial counts found in this study were below the health limit of 102–103 CFU g-1 of
The molecular techniques used in this study are suitable for the identification of
AF Chávez‐Almanza gratefully acknowledges the Consejo Nacional de Ciencia y Tecnología (CONACYT) by PhD scholarship: 411992. This research was funded by the Instituto Tecnológico de Sonora (Project PROFAPI‐00359) and by the Consejo Nacional de Ciencia y Tecnología (PDCPN2014: 248160).
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