Prevalence of enterotoxin-encoding genes and antimicrobial resistance in coagulase-negative and coagulase-positive Staphylococcus isolates from black pudding

Introduction: Staphylococcal species are pathogens that are responsible for outbreaks of foodborne diseases. The aim of this study was to investigate the prevalence of enterotoxin-genes and the antimicrobial resistance profile in staphylococcus coagulase-negative (CoNS) and coagulasepositive (CoPS) isolates from black pudding in southern Brazil. Methods: Two hundred typical and atypical colonies from Baird-Parker agar were inoculated on mannitol salt agar. Eighty-two mannitol-positive staphylococci were submitted to conventional biochemical tests and antimicrobial susceptibility profiling. The presence of coagulase (coa) and enterotoxin (se) genes was investigated by polymerase chain reaction. Results: The isolates were divided into 2 groups: 75.6% (62/82) were CoNS and 24.4% (20/82) were CoPS. The biochemical tests identified 9 species, of which Staphylococcus saprophyticus (37.8%) and Staphylococcus carnosus (15.9%) were the most prevalent. Antimicrobial susceptibility tests showed resistance phenotypes to antibiotics widely administered in humans, such as gentamicin, tetracycline, chloramphenicol, and erythromycin. The coa gene was detected in 19.5% (16/82) of the strains and 4 polymorphic DNA fragments were observed. Five CoNS isolates carrying the coa gene were submitted for 16S rRNA sequencing and 3 showed similarity with CoNS. Forty strains were positive for at least 1 enterotoxin-encoding gene, the genes most frequently detected were sea (28.6%) and seb (27.5%). Conclusions: The presence of antimicrobial resistant and enterotoxin-encoding genes in staphylococci isolates from black pudding indicated that this fermented food may represent a potential health risk, since staphylococci present in food could cause foodborne diseases or be a possible route for the transfer of antimicrobial resistance to humans.

known for their implications in human health and disease.S. aureus is considered to be one of the most common pathogens responsible for the outbreaks in 1994 and 1998 in São Paulo (Brazil); in general, 51.5% of the outbreaks were caused by S. aureus 5 .In addition, the incidence of nosocomial infections caused by CoNS has increased in the last few years.In Brazil 6 and the United States of America (USA), CoNS are the most common cause of nosocomial infections in the intensive care nursery.They are responsible for blood stream infections in neonates, also causing infections of the urinary tract, wounds, bloodstream, and the endocardium in immunocompetent individuals, where S. saprophyticus is the most prevalent species 7 .A prospective study was conducted from June 2001 to May 2002 in a hospital burn unit, with 252 patients; 49 (19.4%) of these developed clinically and microbiologically proven sepsis and the most prevalent bacteria were S. aureus and CoNS 8 .
Perhaps the most notable virulence factors associated with staphylococci are the heat-stable enterotoxins (SEs) produced by certain strains.These toxins are a leading cause of gastroenteritis, including vomiting, abdominal cramping, diarrhea, and malaise, in 3-10 h following the consumption of preformed toxin by susceptible individuals 9 .The SEs are classified into 5 classical serological types: SEA, SEB, SEC 1,2,3 , SED, and SEE, but recently other enterotoxins were described in the literature, including SEG, SEH, SEI, SER, SES, SET and the enterotoxin-like proteins SElJ, SElK, SElL, SElM, SElN, SElO, SE1P, SE1Q, and SElU 10 .Among the CoPS, S. aureus is frequently responsible for outbreaks of food poisoning, due to its ability to express 7 different toxins.However, other CoPS, such as S. intermedius and Staphylococcus hyicus can also express enterotoxins 11 .During the period from 1999 to 2009, 6,349 outbreaks of foodborne diseases were reported in Brazil, and 20.5% of these cases were caused by Staphylococcus spp.Furthermore, enterotoxigenic CoNS have also been isolated from the hands of food handlers and food, demonstrating the importance of CoNS in public health 12,13 .The use of antimicrobial agents in animal husbandry, as a growth promoter, has a selective effect in the emergence and maintenance of resistant bacteria in animals, animal products, and the environment 2 .Staphylococcus spp.have been isolated from poultry-processing plants, chicken carcasses, milk, and dairy products [14][15][16][17] .Evidence suggests that resistant microorganisms or their antibiotic resistance genes can be transferred from food, animals, or the environment to humans 1 .
So far, in Brazil, there have been no studies examining the presence of enterotoxin-encoding genes and antimicrobial resistance in staphylococcal isolates from black pudding.Therefore, the aim of the present study was to investigate the prevalence of enterotoxinencoding sea, seb, sec, sed, and see genes by polymerase chain reaction (PCR) and antimicrobial resistance profiling in coagulasenegative and coagulase-positive isolates from black pudding in southern Brazil.

Bacterial isolates and biochemical characterization
Twenty samples of black pudding purchased in a public market in Pelotas in southern Brazil, in the State of Rio Grande do Sul (RS), during March to November 2008, were analyzed.The first isolation step was to inoculate 25 g of black pudding into sterile buffered peptone water (225ml) with subsequent 10-fold serial dilutions.One milliliter of each suspension was spread over the surface of each of 3 plates containing Baird-Parker agar (BPA) (Merck, Darmstadt, Germany) supplemented with 5% egg yolk-tellurite emulsion.The plates were incubated for 45-48h at 35°C.Two hundred typical circular and atypical colonies of S. aureus were randomly selected from BPA and inoculated on mannitol salt agar (MSA) (Hi-Media, Mumbai, India).Typical colonies were smooth, convex, moist, 2-3 mm in diameter, gray to jet-black, often with a light (off-white)-colored margin, surrounded by an opaque zone, and frequently with a clear outer zone.Eighty-two mannitol-positive isolates were identified to the genus level by gram staining and catalase production.The biochemical characterization of Staphylococcus species was carried out following the method proposed in the literature 2 .The enzymatic activity of staphylocoagulase (free coagulase) was determined in a tube with rabbit plasma (Laborclin-Pinhais, Brazil) according to the manufacturer.The S. aureus strains ATCC 25923 and ATCC 19095 were used as positive controls, and negative controls were inoculated with sterile water instead of bacteria.

Antimicrobial susceptibility test
The antimicrobial susceptibility test was performed by the diskagar diffusion method as recommended by the Clinical and Laboratory Standards Institute 18 .Five antimicrobials commonly used in the treatment of clinical infection and agricultural procedures were tested (concentrations are expressed in µg ml -1 ): erythromycin (ERY; 15µg), tetra-cycline (TET; 30µg), gentamicin (GEN; 10µg), vancomycin (VAN; 30µg), and chloramphenicol (CLO; 30µg).The strain S. aureus ATCC 25923 was used as a control.

Polymerase chain reaction (PCR) amplification of the coagulase (coa) gene
Genomic deoxyribonucleic acid (DNA) extraction and PCR amplification of the coa gene were performed based on previously described protocols 19,20 (Table 1).Reactions were performed in the Eppendorf Mastercycler Thermal Cycler under the following cycle conditions: 5 min at 94°C; followed by 40 cycles of 1 min at 94°C, 1 min at 56.7°C, and 1 min at 72°C; followed by 5 min at 72°C.Staphylococcus aureus strains ATCC 13565, ATCC 14458, ATCC 19095, ATCC 23235, ATCC 25923, and ATCC 27664 were used as positive controls and were kindly provided by Instituto Oswaldo Cruz, Rio de Janeiro, Brazil.

Amplification of 16S rRNA gene and sequencing
Isolates classified as CoNS and coapositive were submitted for 16S ribosomal ribonucleic acid (rRNA) gene amplification and sequencing.The primers and PCR reaction followed the protocols previously described 21 .A 520-bp DNA fragment was purified using the Illustra GFX™ PCR DNA and Gel Band Purification kit (GE Healthcare, Buckinghamshire, UK) and analyzed in an Applied Biosystems (ABI) sequencer model

PCR for the detection of enterotoxin-encoding A (sea) and D (sed) genes
All strains were tested for the presence of sea and sed genes (using the primers described in Table 1).The PCR reactions were performed in a final volume of 25µl containing 1mM MgCl 2 (Invitrogen, Carlsbad, CA), 10 pMol of each primer (Integrated DNA Technologies; IDT, Coralville, IA), 1U Taq DNA polymerase (Invitrogen), 1× PCR buffer (Invitrogen), 200µM deoxynucleoside triphosphates (ABgene, Epsom, UK), and deionized water (Milli Q plus, Millipore, Billerica, MA).Reactions were performed in an Eppendorf Mastercycler Thermal Cycler under the following cycle conditions: 5 min at 94°C; followed by 30 cycles of 45 s at 94°C, 45s at 54°C, and 45 s at 72°C; followed by 5 min at 72°C.The S. aureus ATCC 13565 and ATCC 23235 strains were used as positive controls for the sea and sed genes, respectively.

Multiplex PCR for detection of the enterotoxin-encoding genes B (seb), C (sec), and E (see)
Multiplex PCR was used to determine the frequency of the seb, sec, and see genes in all isolates.The nucleotide sequences of the primers are described in Table 1.The PCR reactions (25µl) contained 1mM MgCl 2 (Invitrogen), 5pMol of each primer (IDT), 1U Taq DNA polymerase (Invitrogen), 1× PCR buffer (Invitrogen), 300µM deoxynucleoside triphosphates (ABgene), and deionized water (Milli Q plus, Millipore) per reaction.Reactions were performed in an Eppendorf Mastercycler Thermo Cycler under the following conditions: 5 min at 94°C; followed by 35 cycles of 45s at 94°C, 45s at 55°C, and Isolation and identification of coagulase-negative and coagulase-positive staphylococci from black pudding A total of 200 colonies from BPA were inoculated on MSA agar.Eighty-two colonies showing mannitol fermentation were selected and divided into 2 groups based on the coagulase test: 75.6% (62/82) CoNS and 24.4% (20/82) CoPS.All isolates were tested for the presence of the coa gene by PCR and 19.5% (16/82) were coapositive (Table 2).Four polymorphic DNA fragments of the coa gene were observed in the strains (Table 2).Among the 16 coa positives, 5 were identified as coagulase-negative and submitted for 16S rRNA gene amplification and sequencing.Three strains showed 99%, 97%, and 90% similarity with Staphylococcus vitulinus (GenBank: AM062694.1),Staphylococcus cohnii (GenBank: HQ154559.1),and Staphylococcus equorum (GenBank: DQ232735.1),respectively.To the best of our knowledge, this is the first time that the coa gene was detected in these CoNS species.Two CoNS isolates showed similarity to Staphylococcus pseudintermedius and S. aureus, and were reclassified as CoPS species.

Antimicrobial susceptibility testing
In the present study, 63.4% of coagulase-negative and coagulase-positive staphylococci strains from black pudding showed antibiotic resistance (Table 3).Resistance to erythromycin (25.6%) and tetracycline (23.2%) were the most frequent profiles detected.All strains were susceptible to vancomycin, 93.9% to chloramphenicol, and 91.5% to gentamycin.Thirteen (15.9%) strains exhibited multidrug resistance, and erythromycin/tetracycline resistance was the profile most commonly observed.

Isolation and identification of coagulase-negative and coagulase-positive staphylococci from black pudding
In this study, we found no correlation between the presence of the coa gene and the detection of coagulase activity among the staphylococci isolates studied, suggesting that the presence of the coagulase gene is not necessarily associated with its expression.Nine CoPS strains showed coa-negative amplification.This behavior can be explained by (1)   Moura TM et al -Enterotoxin genes and antibiotic resistance in staphylococci from food the production of pseudocoagulase, which has been described in CoNS 22 and could lead to false-positive coagulase activity, or (2) the presence of various numbers of degenerate repeat sequences in the coa gene, which gives a polymorphic characteristic in number and sequence, and could generate a mutation in the target DNA region of the coa gene 20 .On the other hand, 5 CoNS strains identified by the coagulase test carried the coa gene.These strains were sequenced, and the DNA sequence was analyzed against the National Center for Biotechnology Information (NCBI) database for similarity with S. vitulinus, S. cohnii, and S. equorum.Previous studies also observed the presence of the coa gene in CoNS isolated from cheese 11 .Other authors have suggested that the presence of coa gene is not necessarily associated with the phenotypic expression of the enzyme 23 .The coagulase test, though specific and sensitive, is subjected to variability in sample conditions.Factors like nutrient availability, environment, and intrinsic physiological conditions of the bacteria can be associated with the non-expression of this gene 24 .The expression of the coa gene usually occurs during the exponential growth phase, and it can be repressed by an accessory gene regulator (agr).Another locus of regulation, called the Staphylococcus accessory regulator (sar), also affects the expression of exoproteins in S. aureus, and mutants at this locus produce low amounts of coagulase 25 .A mutation in one of these loci could theoretically result in a coagulase-negative phenotype, since a small amount of this enzyme does not cause clotting of plasma.PCR amplification of the coa gene showed DNA fragments of 550-900bp between the strains.The same polymorphic DNA fragments have been observed in the coa gene in other studies 20,26 .
The Staphylococcus species identified in the current study have also reported by other authors 27,28 in artisanal morcilla and fermented meat products.S. saprophyticus is considered to be a frequent contaminant of fermented sausages and raw meats and also has been isolated from rectal swabs of cattle carcasses and pigs.In humans, the main reservoir of S. saprophyticus is the gastrointestinal tract 7,10 .The frequency of S. carnosus (15.9%) was relatively elevated in the present study, compared to other studies 27,28 .The presence of S. carnosus in black pudding samples can be justified because this species is often described as a common commercial starter culture for manufacturing sausages 29 .Staphylococcus vitulinus is a member of the Staphylococcus sciuri group and can be isolated from animals and various food products of animal origin, like cheeses and sausages 10,30,31 .The occurrence of different species of staphylococci in black puddings can be explained by the fact that some species are common contaminants of food and others are associated with a lack of hygiene during food manipulation.
The lower prevalence of S. aureus (3.7%) detected in black pudding samples in the present study agree with previously described results 32 , in which S. aureus was not isolated from samples of black pudding analyzed in Buenos Aires, Argentina.The low occurrence of S. aureus demonstrated here, compared to other studies, can be explained by the fact that some laboratories in developing countries screen for presumptive S. aureus based on growth on MSA and/or DNase tests.The MSA medium was developed for the presumptive isolation of S. aureus in a single step for clinical samples, which is convenient for diagnostic laboratories.However, misclassification of the species could occur when the classification is based on mannitol fermentation only.

Antimicrobial susceptibility test in staphylococci strains from black pudding
Antimicrobial resistant staphylococci isolated from food have been identified in previous studies [14][15][16][17] .None of the strains were resistant to vancomycin, consistent with previous studies [14][15][16]33 . In he current study, a high prevalence of erythromycin and tetracycline resistance was observed.One possible reason for this phenotype in staphylococcal isolates from black pudding could be that tetracycline and erythromycin are drugs used in veterinary medicine and as growth promoters 34,35 .Although tetracycline is an antimicrobial that is not approved by the European Union as a food supplement for animals, therapeutic and prophylactic use in veterinary medicine is common, and Staphylococcus spp.resistant to tetracycline are frequently found in cured ham, fermented fish, hard and soft cheese, meat starter cultures, and sausage 14,16,17 .Multidrug-resistant staphylococcal isolates were detected in the present study, consistent with other studies that have isolated multidrug-resistant Staphylococcus spp.from food 14,16,17 .Resistant bacteria isolated from food are an important problem, since food may act as a vehicle for the transfer of antibiotic resistant microorganisms to humans 1,2 .

Prevalence and distribution of enterotoxin-encoding genes (se) in coagulase-negative and coagulase-positive staphylococci
In the present research, 40.2% of the coagulase-negative and coagulase-positive staphylococci isolates from black pudding were positive for one or more enterotoxin-genes.Coagulase-positive staphylococci are important with regard to food hygiene, because of their ability to express enterotoxins.Many studies are conducted to evaluate enterotoxin-encoding genes in CoPS isolated from food or raw materials 13 .The CoPS isolated from black pudding in the present study were positive for enterotoxin-encoding genes.The species Staphylococcus schleiferi has been described as enterotoxigenic in refrigerated raw milk, but enterotoxigenic S. pseudintermedius was not observed in food samples, and was only isolated from dogs 36,37 .Enterotoxin-encoding genes were not identified in 3 isolates of S. aureus.The high prevalence of CoNS (67.5%) that were positive for enterotoxin-encoding genes is an important result.The enterotoxigenicity of CoNS has been described by other authors 10,12,13 , but few studies have been conducted to determine the presence of enterotoxigenic CoNS in food products.Until today, in Brazil, there have been few studies investigating the occurrence of enterotoxinencoding genes in coagulase negative strains; this could be explained by the fact that the Brazilian legislation regarding food contamination does not require the determination of CoNS in animal products.In Minas Gerais, Brazil 13 , evaluation of CoNS isolated from dairy products responsible for outbreaks of food poisoning revealed that some strains carry enterotoxin-encoding genes.The presence of staphylococcal enterotoxin sea and sec genes have also been observed 38 in samples of CoNS isolated from food.These data demonstrate the toxigenic potential of CoNS isolated from black pudding.
In the current study, the sea gene was the most prevalent, followed by the seb, sec, see, and sed genes.This result agrees with previous observations that the staphylococcal enterotoxins SEA, SEB, and SEC are the toxins frequently identified in foodborne outbreaks, and that the staphylococcal enterotoxins SED and SEE are less common 11 .The SEA and SEB toxins are known to occupy the same locus on the chromosome, which may explain why these enterotoxins are commonly found together in outbreaks of food poisoning 39 .

Conclusions
The present study showed that staphylococci isolated from black pudding are predominantly CoNS, and that S. saprophyticus and S. carnosus were the most prevalent isolates.The detection of enterotoxin-encoding genes and resistance in staphylococcal isolates from black pudding indicated that this fermented food may represent a potential health risk, since staphylococci present in food could cause foodborne diseases or be a possible route for the transfer of antimicrobial resistance to humans.This is the first study that describes the detection of antibiotic-resistant staphylococci and the presence of enterotoxin-encoding genes in staphylococcal isolates from black pudding in southern Brazil.

TABLE 2 -Phenotype and genotype analysis of coagulase in Staphylococcus spp. isolates from morcilla. Isolate Size of coa Phenotype n % Species gene by PCR a
using the polymer pop6 and the Big Dye Terminator v3.1 kit (Applied Biosystems, Foster City, CA).The nucleotide sequence was compared with the GenBank database using the Basic Local Alignment Search Tool (BLAST) (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
coa: coagulase; PCR: polymerase chain reaction; CoPS: coagulase-positive staphylococci.CoNS: coagulase-negative staphylococci.aapproximated values in base pairs; b identified by sequencing; c strains originally identified as CoNS based on the coagulase test.3130