Tiane Martin de MouraI,II; Fabrício Souza CamposIII; Pedro Alves d'AzevedoIV; Sueli Teresinha Van Der SandI,II; Ana Cláudia FrancoI,III; Jeverson FrazzonV; Ana Paula Guedes FrazzonI,II
IPrograma de Pós-Graduação em Microbiologia Agrícola e do Ambiente, Departamento de Microbiologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS IILaboratório de Bacteriologia, Departamento de Microbiologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS IIILaboratório de Virologia, Departamento de Microbiologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS IVLaboratório de Cocos Gram-Positivos, Universidade Federal de Ciências da Saúde de Porto Alegre, Porto Alegre, RS VInstituto de Ciência e Tecnologia de Alimentos, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS
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.
Keywords: Staphylococcal enterotoxin. Coagulase. Antimicrobial-resistance.
INTRODUÇÃO: Estafilococos são patógenos responsáveis por surtos de doenças transmitidas por alimentos. O estudo investigou a prevalência de genes de enterotoxinas e o perfil de resistência aos antimicrobianos em estafilococos coagulase-negativo (CoNS) e estafilococos coagulase-positivo (CoPS) isolados de morcilhas no sul do Brasil.
MÉTODOS: Duzentas colônias típicas e atípicas do ágar Baird-Parker foram inoculadas em ágar sal-manitol. Oitenta e dois estafilococos manitol-positivos foram submetidos a testes bioquímicos e perfil de susceptibilidade antimicrobiana. A presença dos genes da coagulase (coa) e enterotoxinas (se) foi investigada por reação em cadeia da polimerase (PCR).
RESULTADOS: Os isolados foram divididos em dois grupos: 75,6% (62/82) CoNS e 24,4% (20/82) CoPS. Através dos testes bioquímicos, 9 espécies foram determinadas, Staphylococcus saprophyticus (37,8%) e Staphylococcus carnosus (15,9%) foram as mais prevalentes. Testes de susceptibilidade demostraram fenótipos de resistência aos antibióticos administrados em humanos, como gentamicina, tetraciclina, cloranfenicol e eritromicina. O gene coa foi detectado em 19,5% (16/82) das cepas e quatro fragmentos de DNA polimórficos foram observados. Cinco CoNS contendo o gene coa foram submetidos ao sequenciamento do 16S rRNA e três mostraram similaridade com CoNS. Quarenta amostras foram positivas para pelo menos um gene se, os mais frequentes foram sea (28,6%) e seb (27,5%).
CONCLUSÕES: A presença de resistência aos antimicrobianos e de genes se nos isolados de morcilha indicou que este alimento pode representar um risco potencial à saúde, já que a presença nos alimentos pode causar doenças de origem alimentar ou ser uma possível rota de transferência de estafilococos resistentes aos humanos.
Palavras-chaves: Enterotoxina estafilocócica. Coagulase. Resistência antimicrobiana.
Black pudding or blood sausage is a type of sausage, made from the blood, fat, and skin of cattle or pig, stuffed into natural or synthetic casing, and tied manually. This kind of sausage is very popular in south Brazil, Argentina, and Uruguay. Animal products are susceptible to microorganism contamination, and bacteria present in food could cause foodborne disease or be a possible route for the transfer of antimicrobial resistance to humans1,2.
Members of the genus Staphylococcus are gram-positive cocci, and are natural inhabitants of the skin and mucous membranes of humans and animals. Currently, according to literature3, this genus comprises 45 species, which are divided into 2 groups: coagulase-positive staphylococci (CoPS) and coagulase-negative staphylococci (CoNS), based on the ability to coagulate rabbit plasma. On the one hand, some CoNS species are components of the natural microbiota of food, and play an important role in the manufacturing processes of diverse meat-derived products; in particular, in dry fermented sausages, they act as starters to ensure the quality and safety of the final products4. On the other hand, CoPS and CoNS species, such as Staphylococcus aureus, Staphylococcus epidermidis, and S. saprophyticus, are well 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. aureus5. In addition, the incidence of nosocomial infections caused by CoNS has increased in the last few years. In Brazil6 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 species7. 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 CoNS8.
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 individuals9. The SEs are classified into 5 classical serological types: SEA, SEB, SEC1,2,3, 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 SElU10. Among the CoPS, S. aureusis 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 enterotoxins11. 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 health12,13.
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 environment2. Staphylococcus spp. have been isolated from poultry-processing plants, chicken carcasses, milk, and dairy products14-17. Evidence suggests that resistant microorganisms or their antibiotic resistance genes can be transferred from food, animals, or the environment to humans1.
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 35ºC. 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 literature2. 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 Institute18. 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 we re p e r fo r m e d i n t h e E p p e n d o r f Mastercycler Thermal Cycler under the following cycle conditions: 5 min at 94ºC; followed by 40 cycles of 1 min at 94ºC, 1 min at 56.7ºC, and 1 min at 72ºC; followed by 5 min at 72ºC. 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 described21. 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 3130 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).
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 MgCl2(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 94ºC; followed by 30 cycles of 45 s at 94ºC, 45s at 54ºC, and 45 s at 72ºC; followed by 5 min at 72ºC. 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 MgCl2 (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 94ºC; followed by 35 cycles of 45s at 94ºC, 45s at 55ºC, and 45s at 72ºC; followed by 5 min at 72ºC. The S. aureus ATCC 14458, ATCC 19095, and ATCC 27664 strains were used as positive controls for the seb, sec, and see genes, respectively.
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 coagene 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.
Table 2 shows the overall distribution of mannitol-fermenting CoNS and CoPS species identified from black pudding. Nine different species were identified; S. saprophyticus was the most prevalent (37.8%), followed by Staphylococcus carnosus (15.9%), S. vitulinus (8.5%), S. pseudintermedius (7.3%), S. cohnii (6.1%), S. aureus (3.7%), S. equorum (2.4%), S. schleiferi(1.2%), and S. intermedius (1.2%). Thirteen isolates (15.9%) could not be identified to the species level and were classified as Staphylococcus spp.
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.
Prevalence and distribution of enterotoxinencoding genes (se) in coagulase-negative and coagulase-positive staphylococci
Forty (48.8%) of 82 strains were positive for 1 or more enterotoxin-encoding genes (Table 4); amongst these, 67.5% (27/40) were coagulase-negative and 32.5% (13/40) were coagulase-positive. The sea gene was the most frequently detected (28.6%), followed by seb (27.5%), sec (20%), see (17.5%), and sed (5%). The Figure 1 shows the amplification profile of the enterotoxin-encoding sea, seb, sec, sed, and see genes in the control. Five strains (3 CoNS and 2 CoPS) exhibited more than 1 gene.
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) the production of pseudocoagulase, which has been described in CoNS22 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 gene20. 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 cheese11. Other authors have suggested that the presence of coa gene is not necessarily associated with the phenotypic expression of the enzyme23. 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 gene24. 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 coagulase25. 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 studies20,26.
The Staphylococcus species identified in the current study have also reported by other authors27,28in 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 tract7,10. The frequency of S. carnosus (15.9%) was relatively elevated in the present study, compared to other studies27,28. 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 sausages29. Staphylococcus vitulinus is a member of the Staphylococcussciuri group and can be isolated from animals and various food products of animal origin, like cheeses and sausages10,30,31. 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 results32, 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 studies14-17. None of the strains were resistant to vancomycin, consistent with previous studies14-16,33. In the 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 promoters34,35. 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 sausage14,16,17. Multidrug-resistant staphylococcal isolates were detected in the present study, consistent with other studies that have isolated multidrug-resistant Staphylococcus spp. from food14,16,17. Resistant bacteria isolated from food are an important problem, since food may act as a vehicle for the transfer of antibiotic resistant microorganisms to humans1,2.
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 materials13. 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 dogs36,37. 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 authors10,12,13, 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, Brazil13, 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 secgenes have also been observed38 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 sedgenes. 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 common11. 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 poisoning39 .
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.
The authors would like to thank the laboratories of Universidade Federal de Pelotas (UFPEL), Universidade Federal do Rio Grande do Sul (UFRGS), and Universidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA) for partnership and for providing infrastructure and resources.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico(CNPq).
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Dra. Tiane Martin de Moura
Av. Sarmento Leite 500/sala 209
90050-170 Porto Alegre, RS, Brasil
Phone: 55 51 3308-3935; Fax 55 51 3308-4111
Received in 12/09/2011
Accepted in 13/03/2012