Abstract
The marine clam Anomalocardia brasiliana is a candidate as a sentinel animal to monitor the contamination levels of coliforms in shellfish-harvesting areas of Brazil's northeastern region. The aim of the present study was to search enterotoxin-encoding genes plus the mecA gene among coagulase-negative staphylococci (CNS) isolates from shellfish meats of A. brasiliana. The specimen clam (n=48; 40 clams per sample) was collected during low tide in the bay area of Mangue Seco from April through June 2009, and random samples of chilled and frozen shelled clam meat (n=33; 250 g per sample) were obtained from retail shops from January through March 2012. Seventy-nine CNS isolates were identified, including Staphylococcus xylosus, S. cohnii spp. urealyticus, S. sciuri, and S. lentus. A high percentage of isolates resistant to erythromycin (58.5%), penicillin (51.2%), and tetracycline (43.9%), and the fluoroquinolones levofloxacin (39%) and ciprofloxacin (34.1%) were recorded from those environmental samples. Isolates from retail shops were particularly resistant to oxacillin (55.3%) and penicillin (36.8%). All CNS resistant to oxacillin and/or cefoxitin were positive for the presence of the mecA gene, but phenotypically susceptible to vancomycin. Also, the enterotoxin-encoding genes seg and seh were detected through multiplex–polymerase chain reaction in 77.7% and 88.8% of the isolates from environmental samples, versus 90.5% and 100% of the isolates from retail shops, respectively. The data reveal the risk to public health due to consuming raw or undercooked shellfish containing enterotoxigenic plus methicillin-resistant CNS.
Introduction
Staphylococcal strains producing enterotoxins are of particular concern in seafood products since poisoning can cause episodes of vomiting, nausea, and diarrhea (Le Loir et al., 2003). The classic staphylococcal enterotoxins A, B, C, D, and E (SEA, SEB, SEC, SED, and SEE, respectively) have been reported in dairy products, cakes, meat, and eggs (Argudín et al., 2010). In addition, coagulase-negative staphylococci (CNS) have gained importance due to their association with food poisoning and nosocomial infections in neonatal intensive care units (Udo et al., 1999; Jain et al., 2004). However, in spite of the emergence of CNS in different foods, little is known about their occurrence in shellfish species (Rall et al., 2010). In addition, reports of methicillin-resistant strains among CNS reinforce the need to revise their importance to food safety (Bhargava and Zhang, 2012; Hammad et al., 2012; Moura et al., 2012).
Methicillin resistance is mediated by the mecA gene, which is present on the staphylococcal cassette chromosome mec (SCCmec). This gene encodes the penicillin binding protein PBP2a, which confers resistance to methicillin and several β-lactam antibiotics (Kondo et al., 2007). The integration and excision of SCCmec in specific genomic loci is performed by CCR recombinases, allowing horizontal intra- and interspecies transfer (Ito et al., 1999; Gordon and Lowy, 2008). In general, the isolates harboring SCCmec type IV have community origin, and horizontal transfer of SCCmec between methicillin-resistant CNS strains and S. aureus has been reported (Hassen et al., 2004; Rossney et al., 2007).
In the present study, CNS isolated from A. brasiliana were identified among samples collected in the harvesting area of Mangue Seco and retail shops in Brazil's northeastern region, and characterized for the presence of the mecA gene as well as the sea, seb, sec, sed, see, seg, and seh enterotoxin genes.
Materials and Methods
Collection of samples of A. brasiliana
The environmental samples (n=48; 40 clams per sample) were collected during low tide at Mangue Seco harvesting area (07° 50′03″ S, 34° 54′21″ W), in Brazil's northeastern region, from April through June 2009. Random samples of nonindustrialized chilled and frozen shelled clam meat (n=33; 250 g per sample) were purchased from local retail shops of large companies from January through March 2012 in the metropolitan area of Recife, Pernambuco. All samples were transported in isothermal plastic bags (24°C–26°C) and processed on the same day of collection.
Identification of staphylococci
The methodology was adapted from the official Brazilian Analytical Manual (Brasil, Ministry of Agriculture, 2003). Twenty-five grams of shellfish meat was homogenized in 0.1% sterile saline peptone and submitted to serial dilutions ranging from 10−1 to 10−3. From each dilution, a 0.1-mL aliquot was plated on mannitol agar (Oxoid). Mannitol-fermenting bacteria as well as suspected CNS producing small pink or red colonies with no color change in mannitol agar were subjected to gram staining, catalase testing, and oxidative-fermentative testing using OF media (Himedia) with glucose. After confirmation of the genus Staphylococcus, the enzyme coagulase was characterized among all isolates in tubes using rabbit plasma (Laborclin). Coagulase-negative isolates resistant to oxacillin and/or cefoxitin were subjected to identification to the species level using the API Staph commercial identification system (Biomerieux).
Antimicrobial susceptibility test
The antimicrobial susceptibility of the isolates was measured by the disk-diffusion method, according to the instructions of the document M100-S22 from the Clinical and Laboratory Standards Institute (CLSI, 2012). The following antimicrobials were used (Laborclin, Brazil): penicillin (10 U), oxacillin (1 μg), cefoxitin (30 μg), gentamicin (10 μg), erythromycin (15 μg), tetracycline (30 μg), ciprofloxacin (5 μg), levofloxacin (5 μg), chloramphenicol (30 μg), clindamycin (2 μg), and rifampin (5 μg). All tests were conducted in duplicate. For quality-control standard, the strain S. aureus ATCC No. 25923 was used. Multidrug resistance was defined as resistance to at least one antimicrobial from two or more classes.
Minimum inhibitory concentration of vancomycin
The resistance/susceptibility profile of coagulase-negative staphylococcal strains to vancomycin (Sigma) was tested in 96-well microplates (CLSI, 2003), and classified according to the minimum inhibitory concentration values established by CLSI (2012). The tests were performed in duplicate, with the standard strain S. aureus ATCC 25923 being used for quality control.
Detection of the mecA gene
For molecular analysis, individual colonies were grown in brain heart infusion broth (Oxoid) for 24 h at 37°C. Total DNA was obtained using the traditional phenol-chloroform protocol as described by Freitas et al. (2008). The pellet was suspended in 10 μL of RNase solution (10 mg/mL), quantified in a NanoDrop 2000c spectrophotometer (Thermo Scientific), and stored at−20°C. The detection of the mecA gene was performed using polymerase chain reaction (PCR) with the primers described by Kondo et al. (2007). The reactions were prepared to a final volume of 25 μL containing 20 ng of DNA, 2.5 μL of Green GoTaq reaction buffer (Promega), 200 μM of each dNTP, 1.5 mM of MgCl2, 10 pmol of each primer, and 1U of Taq polymerase (Promega). After initial denaturation at 94°C for 5 min, the thermal cycler (Biometra) was programmed for 30 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 1 min, followed by 72°C for 7 min. An aliquot of 5 μL amplified product was subjected to electrophoresis on 1% agarose gel (tris-borate-EDTA [TBE] buffer) with ethidium bromide and visualized under an ultraviolet (UV) transluminator scanned using the Kodak 1D software version 3.5.2 (Scientific Imaging Systems). The standard strain of S. aureus ATCC No. 33591 was used as positive control.
Detection of staphylococcal enterotoxin genes
The primers used for detection of classic enterotoxins were described by Becker et al. (1998). Multiplex-PCR was performed for the genes sea, seb, sec, sed, and see. The reactions were prepared to a final volume of 25 μL containing 20 ng of DNA, 2.5 μL of Green GoTaq reaction buffer (Promega), 200 μM of each dNTP, 1.5 mM of MgCl2, 10 pmol of each primer, and 1 U of Taq polymerase (Promega). The thermocycler program consisted of denaturation at 95°C for 4 min, and 30 cycles of 95°C for 1 min, 60°C for 1 min, 72°C for 2 min, followed by 72°C for 5 min. The following staphylococcal strains were used as controls (Food Research Institute, Madison, WI): S. aureus FRI 722 for sea, S. aureus FRI S6 for seb, S. aureus FRI 361 for sec, and S. aureus FRI 1151 for sed. A survey of the genes seg and seh was performed by PCR using the primers described by Rosec and Gigaud (2002). The reactions were prepared to a final volume of 25 μL containing 20 ng of DNA, 2.5 μL of Green GoTaq buffer (Promega), 200 μM of each dNTP, 1.5 mM of MgCl2, 20 pmol of each primer, and 1 U of Taq polymerase (Promega). The S. aureus strains FRI 361 and CR6 were used as controls for the genes seg and seh, respectively. The amplification products (amplicons) were separated by gel electrophoresis in 1.5% agarose stained with ethidium bromide, visualized under an UV transluminator.
DNA sequencing
For confirmation of the amplified segments, two amplicons of each gene evaluated were selected and purified with ExoSAP-IT (USB Corporation, Cleveland, OH), according to the manufacturer's instructions and then sequenced. The sequences obtained were aligned using the MEGA program, version 5 (Tamura et al., 2011). We analyzed the similarity of these sequences with reference sequences deposited in the National Center for Biotechnology Information (NCBI) database, using Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1997).
Results
We identified 79 coagulase-negative staphylococcal isolates in the samples of A. brasiliana shellfish meats. In general, there was a high percentage of isolates from environmental samples with antimicrobial resistance (n=41) associated with erythromycin (58.5%), penicillin (51.2%), tetracycline (43.9%), and the fluoroquinolones levofloxacin (39%) and ciprofloxacin (34.1%). These strains were all sensitive to cefoxitin and gentamicin (Table 1). In addition, a high number of isolates from samples from retail shops (n=38) were resistance to oxacillin (55.3%) and penicillin (36.8%) (Table 1). On the other hand, these strains were all susceptible to gentamicin, levofloxacin, chloramphenicol, and rifampicin. Multidrug resistance to two or more classes of antimicrobials was found in 48.8% of the isolates from environmental samples and only 10.5% (B4, B13, B10, and E3) of isolates from retail shops. Among environmental isolates, 8 of 41 were resistant to more than four classes of antimicrobials (S20, S21, S22, S23, S6, S27, S34, and S38). On the other hand, none of the isolates from retail shops were resistant to more than four classes of antimicrobials, and only two of 38 (B10 and E3) were resistant to three classes of antimicrobials. All strains were susceptible to vancomycin.
Percentage of sensitivity (S), intermediate resistance (I), and resistance (R) of the total isolates.
Isolates that showed phenotypic resistance to oxacillin and/or cefoxitin were identified at the species level (n=30) and submitted to molecular characterization. Staphylococci isolated from environmental samples (n=9) included S. xylosus, S. cohnii spp. urealyticus, and S. sciuri. Among the species isolated from shellfish meat sold at retail shops (n=21), S. sciuri was the most prevalent (16 of 21), followed by S. xylosus and S. lentus. Regardless of the species, all strains were positive for the presence of the mecA gene (Table 2). Moreover, the enterotoxin-encoding genes seg and seh were detected in 77.7% and 88.8% of isolates from environmental samples versus 90.5% and 100% of isolates from retail shops, respectively (Table 2). The amplicons containing the presumed seg, seh, and mecA genes were sequenced, and BLAST results showed 100% identity to the seg and seh of S. aureus and to the mecA of S. sciuri in the NCBI GenBank, confirming their identity.
The genes sea, seb, sec, sed, and see were not identified among the isolates.
ICL, identification confidence level of API Staph.
Discussion
To assess methicillin resistance among CNS, the CLSI currently recommends that disk diffusion tests be performed with cefoxitin to replace oxacillin (CLSI, 2012). Nevertheless, we have shown that oxacillin is more sensitive for predicting methicillin resistance, since 100% of isolates containing the mecA gene were phenotypically resistant to oxacillin, whereas only 13.3% were resistant to cefoxitin. Thus, the test using cefoxitin may not accurately reflect the phenotypic resistance of CNS, which has also been observed by other authors (Frigatto et al., 2005; Hung et al., 2011). To avoid misinterpretation, the detection of the mecA gene by PCR is currently considered the criterion standard for identification of methicillin-resistant S. aureus (MRSA) strains (Tokue et al., 1992), but this has not yet been established for CNS.
The multidrug-resistant staphylococcal species identified in this study are rarely associated with human nosocomial- or community-acquired infections. However, the risk of transmission of resistance genes by horizontal interspecies transfer was clear and has been described by others (Bloemendaal et al., 2010). For instance, a previous study showed that S. sciuri harbored a homolog encoding a protein with 88% overall amino acid similarity to that of the S. aureus (Wu et al., 1996). Therefore, the mecA gene of MRSA and its homolog in S. sciuri may share a common ancestor. Moreover, this species may accumulate markers of resistance to multiple classes of antibiotics (Couto et al., 2000), and thus may be an important source of resistance genes.
Multidrug-resistant CNS was less frequent among isolates from samples obtained in retail shops compared to environmental samples. In addition, the contamination levels of environmental samples appear to reflect the microbiological status of seawater in the harvesting area of Mangue Seco Beach. In the present study, we detected a high percentage of CNS resistant to β-lactam antibiotics and also to erythromycin, ciprofloxacin, levofloxacin, and tetracycline. Considering the limited options for the treatment of infections caused by multidrug-resistant staphylococcal species, it is worrisome that CNS resistant to broad-spectrum antibiotics have been introduced in the community through the food chain.
Regarding the coagulase-negative species identified in this study, at least S. sciuri, S. cohnii, S. xylosus, and S. lentus have been reported to produce enterotoxins (Bautista et al., 1988; Valle et al., 1990; Rodriguez et al., 1996; Vernozy et al., 1996; Udo et al., 1999). Recently, Podkowik et al. (2013) provided evidence of CNS causing food poisoning. In particular, the enterotoxins SEG and SEH have been identified in different food sources involved in outbreaks (Pereira et al., 1996; Omoe et al., 2002; Ikeda et al., 2005; Jorgensen et al., 2005). In the present study, we have shown that most CNS harboring the mecA gene also have the seg and seh enterotoxin genes. Previously, Hu et al. (2011) suggested that there is an association between the mobile genetic element SCCmec and enterotoxin-encoding genes, and that such coexistence appears to contribute to the pathogenicity of isolates. Our data reinforce this suspicion, since most CNS isolates carrying mecA (as part of SCCmec) were positive for at least one enterotoxin-encoding gene. The data reveal the risk to public health due to consuming raw or undercooked shellfish containing enterotoxigenic plus methicillin-resistant CNS.
Footnotes
Acknowledgments
The authors thank the PDTIS Fiocruz Program for the support given to the molecular analyses. The authors also thank the National Research Council (CNPq) for funding, and CAPES for the scholarship granted to J. Batista. Dr. Lima-Filho thanks the Tutorial Education Program for his scholarship.
Disclosure Statement
No competing financial interests exist.