Staphylococcus aureus is one of the main pathogens associated with foodborne outbreaks in Brazil and food handlers can carry toxigenic and resistant S. aureus strains. The aims of this study were to verify the frequency of virulence genes, to identify the agr groups and to determine the antimicrobial resistance profile of S. aureus strains isolated from food handlers of pilot kitchens located in São Paulo, Brazil. A total of 74 strains of the Staphylococcus genus were detected and 50% were identified as of the species S. aureus. The enterotoxin genes detection, tst and luk-PV detection, agr typing, mecA detection, ccr complex detection and SCCmec typing were performed using PCR. The antimicrobial resistance testing was performed by the disk diffusion method. The enterotoxin genes were identified in 36 S. aureus, including sea (83.8%). The tst gene was detected in 18.92% of the strains and the luk-PV was detected in only one isolate. Agr typing classified 58.3% of the strains as type I. Seven (18.92%) strains were classified as MRSA and the ccr2 complex was detected in six of these isolates. The SCCmec typing characterized strains as type II, III, IV and V. Moreover, there were also a greater number of resistant strains to penicillin (83.78%) and clarithromycin (67.57%). In conclusion, the study revealed a significant prevalence of S. aureus, and the presence of different virulence genes and a worrying resistance profile in S. aureus strains isolated from food handlers in this country.
Foodborne diseases are an important public health problem, and it is estimated that 600 million people get sick annually due to the consumption of contaminated food around the world.1 In general, foods can be contaminated by several microorganisms, including bacteria which are one of the main causes (66%) of foodborne diseases.2,3 The transmission of these pathogens usually occurs due to poor food handling and low sanitation practices during processing.1,4 In Brazil, Staphylococcus aureus is one of the most frequent pathogens associated with foodborne outbreaks.5
S. aureus is an important opportunistic pathogen that colonizes the nasal cavity, oropharynx and skin of approximately 30% of the world population.6 This microorganism invades host tissues and is responsible for causing various diseases.7 To establish and maintain infection, S. aureus has been able to encode several virulence factors during their growth, including superantigenic toxins, such as classical Staphylococcal Enterotoxins (SEA, SEB, SEC and SED) and Toxic Shock Syndrome Toxin-1 (TSST-1).7 Another important toxin group are leukotoxins, such as Panton-Valentine Leukocidin (PVL).8
The expression of these virulence factors is regulated and controlled by genetic regulatory systems, such as the accessory gene regulator (agr) system.9 The agr system is a group with quorum sensing activity and controls expression of different genes involved in tissue colonization and invasion.9,10
Until this moment, there are four agr polymorphism types due to mutation and/or insertion of fragments, resulting in variations of the agrC and agrD genes, denominated agrI, agrII, agrIII and agrIV.9,10 It is important to emphasize that studies have also reported that different agr groups are indirectly linked to disease severity profiles due to relationship between agr group and the genetic background of the strain, although the reasons for this association are not yet clear.9,11,12
Methicillin-Resistant Staphylococcus aureus (MRSA) is one of the main causes of infections in hospital and community settings and has been isolated from food and food handlers in different countries, including in Brazil.5,13,14 In addition, MRSA strains can cause serious foodborne outbreaks due to the presence of numerous toxins in their genome, thus being a potential risk to public health.5,15
S. aureus may cause severe diseases and has been difficult to treat humans with this infection worldwide.5 Food handlers can be a potential reservoir and transmission vehicle of virulent and antimicrobial-resistant S. aureus strains, including MRSA.5
Therefore, the aims of this study were to verify the frequency of virulence genes, to identify the agr groups, and to determine the antimicrobial resistance profile of Staphylococcus aureus strains isolated from nasal cavities and underside of nail of the food handlers of three pilot kitchens located in the West region of São Paulo State, Brazil.
Materials and methodsSocioeconomic, sanitary, and health-related data collectionThe study was approval was obtained at the local research ethics committee (CEP) as Protocol CAAE: 59635316.6.0000.5515.
The study was carried out with the participation of 41 food handlers from three Pilot Kitchens (PK) located in the West region of São Paulo State in Brazil. Each participant answered a standardized questionnaire, comprising 22 objective questions, containing information about the processing way and handling of food. In addition, the socioeconomic, sanitary, and health-related data of the participants were collected.
IsolatesTwo bacterial samples were collected from each participant, one from the nasal cavities and one from the underside of nails. The specimens were collected using a sterile swab, moistened with saline solution (0.85%). The samples were stored at the Microbiology Laboratory located on Campus 1 of UNOESTE, Presidente Prudente - SP, in Brazil.
Phenotypic Staphylococcus identificationThe isolates obtained from samples of nasal cavities and the underside of nails were plated on Baird-Parker medium and were subjected to Gram stain for observation of colony morphology. Subsequently, the catalase and coagulase tests were performed.
DNA extractionBacteria DNA was extracted by using the Illustra tissue and cells prep genomic mini spin kit (GE Healthcare, Little Chalfont, UK), according to the manufacturer´s instructions, following an adapted protocol described by Pereira et al.16 The extracted DNA was stored in a freezer at -20°C.
Species identification and detection of virulence genesThe Polymerase Chain Reaction (PCR) was performed to confirm the S. aureus identification from the sau gene amplification. The PCR technique was also used to detect the sea, seb, sec-1, sed, tst, and luk-PV genes.
The sea, seb, sec-1, sed, and tst genes were amplified using a protocol described by Cunha & Calsolari17 and Johnson et al.18 PCR reactions for the detection of PVL genes (lukS-PV and lukF-PV) were performed as described by Lina et al.19 The amplification was revealed by electrophoresis in a 1% agarose gel and stained with ethidium bromide. International reference toxigenic S. aureus strains were used as positive controls, including, ATCC 13565 (EEA), ATCC 14458 (EEB), ATCC 19095 (EEC) e ATCC 23235 (EED). For negative control, S. xylosus ATCC 29971 was used.
Determination of agr groupThe S. aureus strains that carried one or more virulence genes analyzed were submitted to the agr group typing method by PCR. The amplification occurred from 1.5 µL of extraction DNA in a 13 µL reaction mixture, containing 1.3 U Taq DNA polymerase, 104 µmoL/L of Deoxyribonucleotide Triphosphates (dNTP), 10.4 mmoL/L of Tris-HCl, Ph 8.4, 0.39 mmoL/L MgCl2 and 0.5 µL primers agr1, agr2, agr3, agr4 and pan described by Gilot et al.,20 where the pan was used together with the four agr primers.
The PCR products were submitted to electrophoresis in a 1% agarose gel and stained with ethidium bromide. The amplified products size was compared with the standards: pan and agr1 with 441 bp, pan and agr2 with 575 bp, pan and agr3 with 323 bp, and pan and agr4 with 659 bp. International reference strains were used as positive controls, including R137 (agrI), N315 (agrII) e S. aureus ATCC 25923 (agrIII).
The mecA gene detection and determination of SCCmecThe mecA gene detection was performed by conventional PCR reactions according to the protocol described by Murakami et al.21S. aureus strains ATCC 33591 (positive control) and ATCC 25923 (negative control) were included in all reactions. Staphylococcal Cassette Chromosome mec (SCCmec) was typed in MRSA strains. Reactions were performed by multiplex PCR, as described by Oliveira & De Lencastre.22
Antimicrobial resistance testThe antimicrobial resistance test was performed using the disk diffusion method described in the guidelines of the Clinical Laboratory Standards Institute (CLSI).23 The choice of antimicrobials was based on Staphylococcus infection treatment. The antimicrobials tested were penicillin (10 µg), oxacillin (1 µg), clarithromycin (15 µg), erythromycin (15 µg), cefoxitin (30 µg), tetracycline (30 µg), levofloxacin (5 µg), amoxicillin (10 µg), clindamycin (2 µg), linezolid (10 µg), and vancomycin (30 µg).
Data analysisData analysis was done using software Bioestat 5.3. Chi-Square test was used for comparing the frequencies of the sea, seb, sec-1, sed, tst and mecA genes with the following variables: sex, age, hospitalization/surgery, antibiotic use and medicine use by assuming p-value ≤0.05 as significant difference.
ResultsSocioeconomic, sanitary, and health-related dataForty-one food handlers from three Pilot Kitchens (PK) participated in this study, including 16 from PK1, 11 from PK2, and 14 from PK3. Among the 41 participants, 35 (85.37%) were female and 6 (14.63%) male. Related data to the functions performed and the health of the participants, including hospitalization episodes, surgical procedures, and use of antimicrobials in the last 12 months, as well as use of other medications are shown in Table 1.
Questionnaire data answered by the food handlers of the three Pilot Kitchens.
Participants were also asked about the use of Personal Protective Equipment (PPE) and the drying hands habit during their workday. Of the 41 participants, 33 (80.49%) reported using PPE, such as aprons and gloves, and 8 (19.51%) reported not using them, and all reported using disposable paper towels and cloth towels for drying hands.
Identification of isolatesEighty-two bacterial samples were collected from the underside of nails and nasal cavities of each participant in the pilot kitchens. Specifically, 32 in PK1, 22 in PK2 and 28 in PK3. Of all isolates, 74 (90.24%) were identified as Staphylococcus through Gram method and catalase and coagulase tests, and four samples were discarded due to contamination. In four samples there was no growth of the bacterial genus in question. The sau gene was detected in 37 (50%) isolates, confirming the S. aureus identification.
Detection of virulence genesOf the 37 S. aureus isolates submitted to the PCR method to detect the enterotoxins genes, 31 (83.78%) were positive for the presence of the sea gene, 4 (10.81%) for the seb gene, 18 (48.65%) for the sec-1 gene and 5 (13.51%) for the sed gene. Regarding the tst gene amplification, this occurred in 7 (18.92%) isolates. The luk-PV gene was detected in only one isolate.
Determination of agr groupThe 36 S. aureus strains that carried one or more analyzed virulence genes were submitted to the agr group typing method. After the analysis, amplification of agrI was observed in 21 (58.33%) isolates, agrII in 4 (11.11%) isolates, and agrIII in 15 (41.67%) isolates. In none of the samples there was the amplification of agrIV. In 11 (35.56%) samples, there was no detection of any of the four polymorphisms studied.mecA gene detection and determination SCCmec
Regarding the presence of the mecA gene, there was amplification in seven of the 37 isolates, therefore 18.92% of S. aureus were classified as MRSA and were submitted to the analysis of the ccr gene complex. The ccr2 was detected in 6 (85.71%) isolates, and ccr3 and ccrC in 2 (28.57%) different isolates. In 1 (14.28%) isolate only ccrC was detected. SCCmec typing revealed the presence of one isolate type II, one type III, one type IV, and one type V. Three (42.86%) strains were characterized as Not Typed (NT).
The data about virulence genes, agr groups, mecA detection and SCCmec typing are summarized in Table 2.
Characteristics of the 37 S. aureus isolates from food handlers of Pilot Kitchens.
U, Underside of nails; N, Nasal cavities; NT, Not typed.
The frequency of toxins genes and the mecA gene and the variables sex, age, hospitalization/surgery, antibiotic use, and use of another medication are shown in detail in Table 3 (p-values).
Associations between frequency of toxins genes, mecA and questionnaire data (p-values).
| Genes detected in strains from the m underside of nails | ||||||
|---|---|---|---|---|---|---|
| sea | seb | sec-1 | sed | tst | mecA | |
| Questionnaire data | ||||||
| Sex (female or male) | 0.45 | 0.68 | 0.76 | 0.10 | 0.25 | 0.42 |
| Age (younger or older than 30-years old) | 0.77 | 0.09 | 0.63 | 0.36 | 0.77 | 0.93 |
| Hospitalization/surgery (last year) | 0.02a | 0.60 | 0.29 | 0.45 | 0.33 | 0.24 |
| Antibiotic use (last year) | 1.00 | 0.46 | 1.00 | 0.28 | 0.17 | 0.10 |
| Medicine use (last year) | 1.00 | 0.46 | 0.26 | 0.03a | 1.00 | 0.00a |
| Genes detected in strains from nasal cavities | ||||||
|---|---|---|---|---|---|---|
| sea | seb | sec-1 | sed | tst | mecA | |
| Questionnaire data | ||||||
| Sex (female and male) | 0.11 | 0.56 | 0.89 | 0.12 | 0.00a | 0.55 |
| Age (younger or older than 30-years old) | 0.80 | 0.80 | 0.48 | 0.80 | 0.26 | 0.29 |
| Hospitalization/surgery (last year) | 0.01a | 0.68 | 0.26 | 0.68 | 0.00a | 0.68 |
| Antibiotic use (last year) | 0.64 | 0.31 | 0.46 | 0.31 | 0.90 | 0.64 |
| Medicine use (last year) | 0.16 | 0.16 | 0.00a | 0.16 | 0.13 | 0.16 |
The antimicrobial resistance test showed that 31 (83.78%) S. aureus isolates were resistant to penicillin, 22 (59.46%) to oxacillin, 24 (64.86%) to clarithromycin, and 25 (67.57%) to erythromycin. Seven (18.92%) strains were resistant to tetracycline, 3 (8.11%) strains to cefoxitin, 3 (8.11%) to levofloxacin, 2 (4.41%) to amoxicillin, 1 (2.70%) to clindamycin and none of the isolates were resistant to linezolid and vancomycin.
DiscussionThe present study investigated the presence of important toxins genes, identified the agr groups, determined the antimicrobial resistance profile against 11 antimicrobials and carried out mecA detection and SCCmec typing in Staphylococcus aureus isolated from nasal cavities and underside of nail of the food handlers of three pilot kitchens located in the West region of São Paulo State, Brazil.
Good food handling practices are responsible for ensuring the hygienic-sanitary quality of foods during processing.4 Specifically, from the information obtained by the questionnaires answered, regarding knowledge about the use of PPE, all employees demonstrated to be aware about this topic. In addition, all admitted to use paper and cloth towels that were in common use for drying hands. It is known that food handlers who use cloth towels have increased bacterial colonies counts from hand samples, suggesting that these towels are capable of disseminating S. aureus in a food handling environment.24
In this study, it was observed that S. aureus was isolated in half of the samples of nasal cavities and underside of nails from food handlers. According to Castro et al.,15 Ahmed,25 and Saber et al.26 the presence of S. aureus in the hands and/or noses of food handlers was of 30%, 25%, and 30%, respectively.
The pathogenic potential of S. aureus strains was investigated by the presence of toxins genes, such as classical enterotoxins (SEs). Specifically, SEs are characterized as superantigens, which are proteins that can trigger severe and systemic manifestations, including fever, vomiting, diarrhea, abdominal cramps, and sweating.7
Of all 37 S. aureus isolates, 35 (94.6%) had at least one of the classical enterotoxins genes. The sea gene was detected in 83.78% of S. aureus. This gene is commonly associated with contaminated food and is considered the enterotoxin gene most prevalent and leading cause of staphylococcal food poisoning worldwide.15,25,27-30
The seb and sec-1 genes were detected in 10.81% and 48.65% of S. aureus strains studied, respectively. In contrast, other studies with food handlers demonstrated that the seb gene was more frequent than sec in Iran and Sudan.25,30 Finally, the sed gene was detected in 13.51% in the present study and this gene has been the least described among the other classical SEs genes.25,30,31
It is important to mention that the TSST-1 is also described as a superantigen. Unlike enterotoxins, TSST-1 has not yet been associated with food consumption, but if contaminated hands with TSST-1 producer strains touch foods, cross-contamination can occur, which can lead to Toxic Shock Syndrome (TSS) in humans.32,33 In the present study, the tst gene was found in 7 (18.92%) S. aureus isolates. The presence of toxin-producing S. aureus has been frequently reported in healthy individuals and, generally, these S. aureus strains have high prevalence rates of enterotoxin-producing genes and a lower detection of TSST-1 and Panton-Valentine Leukocidin (PVL) genes.15,31,33,34
PVL is a cytolysin encoded by the lukF-PV and lukS-PV genes, which participate in the pores formation in the leukocyte membrane, leading to cell lysis predisposing skin and soft tissue infections and necrotizing pneumonia.33,35 Aung et al.33 observed that 12.5% of S. aureus strains presented the luk-PV genes, while the TSST-1 gene was the least prevalent among these isolates from food handlers.
In this study, agr typing in the S. aureus strains showed that agrI type was the most prevalent (58.33%). This polymorphism can be involved in food poisoning due to enterotoxins production.36 Similarly to our results, different authors demonstrated that agrI was the most common polymorphism found in strains isolated from food handlers or food products.37-40
The agrII group was found in 11.11% of the S. aureus isolates. This low frequency has been observed in other studies with food handlers or food products and more associate to clinical isolates.27,39-41 The agrIII group, that may be associated with toxic shock syndrome, was detected in 41.67% of the S. aureus strains. In contrast, other authors found a lower frequency of the agrIII group in S. aureus isolated from food handlers and food products (3.1%, 12.84%, 23.5%, and 19.2%) compared to the present study.27,36,37,41,42
Finally, the polymorphism agrIV is associated with the production of exfoliative toxins.36 This polymorphism has rarely been described and was not detected in the present study as well as in other studies carried out in different parts of the world.27,37,38,42
Regarding antimicrobial resistance, 91.89% of the S. aureus isolates were resistant to at least one of the antibiotics tested, including resistance to penicillin (83.78%), erythromycin (67.57%) and clarithromycin (64.86%). Furthermore, a total of 18.92% of the isolates were identified as (MRSA).
Methicillin resistance and β-lactams is associated with the mecA gene, which is transported through a mobile genetic element denominated SCCmec. SCCmec encompasses the mec complex, regulatory genes, ccrAB or/and ccrC, plus other accessory genes encoding for a new specific Penicillin-Binding Protein (PBP2a).5,43,44
MRSA has been recognized as an important nosocomial pathogen and listed by World Health Organization as one of the high-priority antibiotics-resistant pathogens. Furthermore, in 2019 MRSA caused more than 100,000 deaths worldwide.45,46 This bacterium have been isolated from food in different countries, including Brazil, and is also associated with the occurrence of foodborne diseases.5,14,45,47
In Brazil, Rodrigues et al.47 isolated Staphylococcus spp. from cheese processing plants, including food handlers, and the mecA gene was present in six strains (6%). On the other hand, still in Brazil, Ferreira et al.,48 demonstrated that 28.6% of the food handlers of public hospitals had MRSA strains in hands or nostrils. This incidence of MRSA isolated from food and food handlers in the country emphasize the need for better food handling practices, preventing these strains from being transmitted to the community.5
The present study also performed SCCmec typing. Specifically, the SCCmec type IV is more associated with healthy individuals’ colonization, while the types I‒III are related to nosocomial infections.45 This difference was not observed in this data, since the types II, III, IV, and V were detected in S. aureus isolates from healthy food handlers. In addition, three samples were classified as non-typeable, which can be attributed to the diversity of the elements that make up the SCCmec.49
Finally, the data obtained in the present study also indicated that in isolates from the underside of the nails, there was association between the sea gene with hospitalization/surgical process, and the sed and the mecA gene with the use of medicines. In isolates from the nasal cavities, there was association between the sec-1 gene with the use of medicines and the tst gene with sex and hospitalization/surgical process. There is a scarcity of data literature about these associations, suggesting the need for further investigation of the relationship of these genes with the related variables.
ConclusionsIn conclusion, the study revealed a significant prevalence of S. aureus colonizing the nasal cavities and lower nails of food handlers in the São Paulo State in Brazil, as well as the pathogenic potential was corroborated by the presence of important virulence genes and the prevalence of MRSA strains, reinforcing the threat for humans. Thus, biosecurity measures should be prioritized in this environment due to the risk of food contamination.
We thank the São Paulo State Research Foundation for Scientific and Technological Development (FAPESP; Grants 2018/12652-6 and 2018/00056-0) for supporting research development and the Universidade do Oeste Paulista (UNOESTE) for structure and financial support.
