J. Dairy Sci. 98:4273–4278 http://dx.doi.org/10.3168/jds.2014-9064 © American Dairy Science Association®, 2015.
Prevalence of enterotoxin genes and antimicrobial resistance of coagulase-positive staphylococci recovered from raw cow milk J. G. Rola,1 W. Korpysa-Dzirba, A. Czubkowska, and J. Osek
Department of Hygiene of Food of Animal Origin, National Veterinary Research Institute, Al. Partyzantow 57, 24-100 Pulawy, Poland
ABSTRACT
Raw milk may be contaminated by enterotoxigenic coagulase-positive staphylococci (CPS). Several of these microorganisms show antimicrobial resistance, which poses a potential risk for consumers. The aim of this study was to determine the occurrence of enterotoxin genes and antimicrobial resistance of CPS isolated from cow milk. A total of 115 samples were analyzed for the presence of CPS according to the International Organization for Standardization standard (ISO 6888–2). The genes were identified using 2 multiplex PCR assays. Resistance of the isolates to 10 antimicrobials was determined using the minimum inhibitory concentration method. Overall, 71 samples (62%) were contaminated with CPS and 69 isolates were further analyzed. Among them, 20 (29%) strains harbored the enterotoxin genes. The most commonly detected staphylococcal enterotoxin markers were sed, sej, and ser, whereas none of the analyzed isolates possessed the seb and see genes. Almost one-half of the tested strains (43%) were resistant to one or more antimicrobial agents. Resistance to penicillin was the most common, followed by sulfamethoxazole and chloramphenicol. On the other hand, all strains were susceptible to ciprofloxacin, erythromycin, gentamicin, cefoxitin, and streptomycin. None of the strains was positive for the mecA and mecC (methicillin-resistant Staphylococcus aureus) genes. These results indicate that enterotoxigenic and antimicrobial-resistant CPS strains are present in raw milk, which may be a potential risk for public health. Key words: milk, staphylococci, antimicrobial resistance, enterotoxin genes INTRODUCTION
Staphylococcus aureus is a common microorganism present on the skin and mucosal surfaces of humans
Received November 4, 2014. Accepted March 29, 2015. 1 Corresponding author:
[email protected]
and animals as well as in the environment. Contamination of raw milk with S. aureus may occur from infected dairy animals, but human handling, water, and milking equipment may also be important sources of these bacteria (Bergonier et al., 2003; Jørgensen et al., 2005a,b). Staphylococcus aureus produces a variety of extracellular proteins, including staphylococcal enterotoxins (STE), which cause staphylococcal food poisoning (SFP; Yesim Can and Haluk Celik, 2012). The symptoms of this illness include nausea, vomiting, abdominal cramps, and diarrhea occurring 1 to 8 h after consumption of contaminated food. Five main STE types (A, B, C, D, and E) responsible for the symptoms of SFP have been identified. In addition, several other variants of STE or staphylococcal-like toxins have been described (Lina et al., 2004; Ono et al., 2008). The emergence of antibiotic-resistant S. aureus in farm environment is a potential risk for public health. Antibiotics on dairy farms are used to treat infections such as mastitis and as a preventive measure during dry cow therapy (Haran et al., 2012). The major groups of antimicrobial agents introduced for therapeutic use in food-producing animals are β-lactams, tetracyclines, aminoglycosides, macrolides, and sulfamethoxazole. The discovery of the third generation of fluoroquinolones with a broader spectrum activity has led to interest in their use in animals (Brown, 1996). In the last decade, bacterial isolates from food have shown a considerable increase in resistance against most antibiotics (Yesim Can and Haluk Celik, 2012). Since 1960, methicillin and oxacillin have been used for the effective control of staphylococcal infections; however, methicillin-resistant S. aureus (MRSA) strains are now found with increasing frequency in many countries (Lowy, 2003). Such microorganisms are also often resistant to other antimicrobial agents, including aminoglycosides, macrolides, chloramphenicol, tetracyclines, and fluoroquinolones (Türkyilmaz et al., 2010). Methicillin resistance in MRSA is determined by the chromosomally located mecA gene, which encodes a penicillin binding protein (PBP2c) with a low affinity for β-lactams; therefore, such strains are resistant to all β-lactam antibiotics (Bystron et al., 2009). The MRSA genotypically classified under clonal complex 398 has been detected among
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pig farmers in the Netherlands and other countries (Khanna et al., 2008; Denis et al., 2009) and is known to cause infections in humans and animals (Witte et al., 2007). Such strains were also identified in cattle, and their presence in milk possess a potential risk to people working with cattle such as farm workers and veterinarians as well as milk consumers (Vanderhaeghen et al., 2010). The objective of this study was to determine the prevalence of enterotoxin genes and antimicrobial resistance in coagulase-positive staphylococci isolated from raw cow milk collected in Poland. MATERIALS AND METHODS Milk Samples
A total of 115 samples of raw milk collected between 2009 and 2013 from 15 dairy farms and 15 dairies located in the eastern part of Poland were used in this study. After sampling, milk was transported to the laboratory under refrigeration within 24 h for further analyses. Isolation and identification of CPS
Isolation of CPS was performed using Baird-Parker agar with rabbit plasma fibrinogen (bioMérieux, Marcy l’Etoile, France) at 37 ± 1°C for 48 h (ISO 6888–2, 1999). One suspected colony from each sample was cultured in brain heart infusion broth (Oxoid, Basingstoke, UK) at 37°C for 24 h for further analysis, including catalase reaction, hemolytic properties, and ability to coagulate rabbit plasma (coagulase tube test). DNA Isolation
Staphylococcus aureus isolates were grown in brain heart infusion broth at 37°C for 24 h, and 1 mL of the culture was transferred to Eppendorf tubes and centrifuged at 13,000 × g for 1 min at room temperature. Then, the bacterial cells were treated with 10 μL of lysostaphin (1 mg/mL; Sigma-Aldrich, St. Louis, MO) for 30 min at 37°C and DNA was extracted using the Genomic-Mini kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer’s instructions. Detection of Enterotoxin Genes
Two multiplex PCR (mPCR) assays were used to detect STE-encoding genes. The first (mPCR1), performed with 6 pairs of primers, allowed detection of the following genes: sea, seb, sec, sed, see, and ser (De Buyser et al., 2009a). The second reaction (mPCR2) Journal of Dairy Science Vol. 98 No. 7, 2015
enabled us to identify the seg, seh, sei, sej, and sep genes (De Buyser et al., 2009b). Both mPCR were carried out in a TProfessional Standard Thermocycler (Biometra, Jena, Germany) with the conditions as follows: 94°C for 3 min, then 35 cycles at 94°C for 30 s, 55°C for 40 s (mPCR1), and 52°C for 30 s (mPCR2), 72°C for 90 s with final extension at 72°C for 7 min. The amplified PCR products were visualized by standard gel electrophoresis in a 2% agarose gel stained by ethidium bromide (5 μg/mL). The gels were photographed under UV light using the Gel-Doc 2000 system (Bio-Rad, Hercules, CA). Identification of MRSA
The MRSA were identified by PCR detection of the mecA, nuc, and 16S rRNA genes as described previously (National Food Institute, 2008). The presence of the mecC gene was also analyzed according to the PCR protocol recommended by the European Union Reference Laboratory for Antimicrobial Resistance (National Food Institute, 2012). Briefly, in both PCR DNA was amplified by 30 cycles of denaturation (94°C for 30 s), annealing (55°C and 59°C for 30 s, respectively), and elongation (72°C for 1 min). Determination of Phenotypic Antimicrobial Resistance
Staphylococcus aureus isolates were cultured on Columbia agar supplemented with 5% sheep blood (bioMérieux) at 37°C ± 1°C for 24 h ± 2 h. After incubation, a suspension of 0.5 McFarland density was prepared and 50 μL was transferred to 11 mL of Mueller-Hinton broth (Trek Diagnostic Systems, East Grinstead, UK). Afterward, 50 μL of bacterial suspension in Mueller-Hinton broth was used to inoculate microplates (DKVP, Trek Diagnostic Systems) with the following antimicrobials (μg/mL): chloramphenicol (2–64), ciprofloxacin (0.12–8), erythromycin (0.25–16), gentamicin (0.25–16), penicillin (0.06–16), streptomycin (4–64), sulfamethoxazole (32–512), tetracycline (0.5–32), trimethoprim (0.5–32), and cefoxitin (0.5–32). The reference strain of S. aureus ATTC 25923 was used as a control for each microplate. The plates were incubated for 18 to 24 h at 36°C ± 1°C and MIC, defined as the lowest concentration of antibiotics in which the bacterial growth was totally inhibited, were read using the Vision system (Trek). The cutoff values for the interpretation of the MIC results were in accordance with the European Committee on Antimicrobial Susceptibility Testing and the European Union Reference Laboratory for Antimicrobial Resistance.
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ENTEROTOXIGENIC COAGULASE-POSITIVE STAPHYLOCOCCI IN RAW MILK
Table 1. Antimicrobial resistance of Staphylococcus aureus isolates tested in the study Number (%) of resistant strains Antimicrobial agent
MIC, μg/mL, R>
Chloramphenicol Ciprofloxacin Erythromycin Gentamicin Penicillin Streptomycin Sulfamethoxazole Tetracycline Trimethoprim Cefoxitin
16 1 1 2 0.125 16 128 1 4 4
RESULTS
Coagulase-positive staphylococci were found in 71 of 115 (62%) analyzed milk samples. Among them, 46 (65%) were collected at dairy farms and 25 (35%) in dairies. The level of contamination with CPS ranged from 1.0 × 100 to 1.0 × 105 cfu/mL and from 1.1 × 101 to 4.5 × 103 cfu/mL for the samples collected in dairy farms and dairies, respectively. Further analyses were conducted for 69 out of 71 isolates because the remaining 2 strains could not be isolated from the selective agar medium. All the isolates were coagulase and catalase positive, whereas hemolytic properties were observed in 63 (91%) strains. The PCR amplification of the 16S rRNA and nuc genes confirmed that all of them were S. aureus. Twenty (29%) strains had enterotoxigenic properties with the following genes identified: sea in 1 (5%), sec in 2 (9%), sed in 11 (50%), ser in 11 (50%), seg in 4 (18%), seh in 3 (14%), sei in 4 (18%), sej in 11 (50%), and sep in 3 (14%) isolates, respectively. The sed, sej, and ser genes were only detected together, similar to the seg and sei genes. The genes encoding SEB and SEE toxins were not identified. Determination of antimicrobial resistance showed that all isolates were susceptible to ciprofloxacin, erythromycin, gentamicin, cefoxitin, and streptomycin. The majority of the strains were also susceptible to chloramphenicol, tetracycline, and sulfamethoxazole (Table 1). The highest resistance rate of 44 and 26% was found to penicillin, for the isolates from dairy farms and dairies, respectively. On the other hand, 39 (57%) isolates were susceptible to all 10 antimicrobial agents used in this study (Table 2). No mecA and mecC (MRSA) positive strains were identified among staphylococci tested. Among all tested isolates, the largest group (51%) was susceptible to all antimicrobials used in the study and had no enterotoxin genes. Several analyzed S. aureus (23%) was resistant to at least one antimicrobial
Dairy farms n = 46 3 0 0 0 20 0 5 2 0 0
(7)
(44) (11) (4)
Dairies n = 23
Total n = 69
0 0 0 0 6 (26) 0 1 (4) 0 1 (4) 0
3 0 0 0 26 0 6 2 1 0
(4)
(38) (9) (3) (1)
and had one or more STE genes. Most of them (56%) were resistant to penicillin and harbored the sed, ser, and sej markers. The vast majority of these isolates originated from milk samples collected at dairy farms (Table 3). DISCUSSION
In the present study, 62% of raw milk was positive for CPS at the levels between 1.0 × 100 and 1.0 × 105 cfu/mL. Different contamination with S. aureus was found in other European countries such as Hungary and Switzerland, where the number of bacteria in raw bovine milk was up to 6.0 × 103 and 3.0 × 103 cfu/mL, respectively (Stephan et al., 2002; Peles et al., 2007). Furthermore, the presence of CPS at the levels of 6.3 × 102 to 2.8 × 105 cfu/mL was found in raw milk in Brazil (De Oliviera et al., 2011). In the present survey, 29% of S. aureus harbored the genes encoding STE. Similar results performed on 59 strains isolated from unprocessed cow milk in Hungary were obtained by Peles et al. (2007), where 27.1% of strains were positive for one or more STE Table 2. Phenotypic resistance patterns among coagulase-positive staphylococci tested Number (%) of resistant isolates Antimicrobial resistance phenotype1 Sensitive to all PEN SMX TET PEN SMX PEN CHL PEN TMP SMX PEN CHL SMX
Dairy farms n = 46 22 15 2 2 2 2 0 1
(48) (33) (4) (4) (4) (4) (2)
Dairies n = 23
Total n = 69
17 (74) 5 (22) 0 0 0 0 1 (4) 0
39 20 2 2 2 2 1 1
(57) (29) (3) (3) (3) (3) (1) (1)
1
Antibiotics: chloramphenicol (CHL), penicillin (PEN), sulfamethoxazole (SMX), tetracycline (TET), and trimethoprim (TMP). Journal of Dairy Science Vol. 98 No. 7, 2015
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Table 3. Antimicrobial resistance and staphylococcal enterotoxin (STE) genes among Staphylococcus aureus isolated from dairy farms and dairies1
ID number
Location
No. of CPS positive samples/ total no. of samples
Dairy farm 01 02
Northeast Northeast
3/8 14/16
03
Northeast
2/2
04
Northeast
4/9
05
Northeast
4/10
06
Northeast
5/8
07
Northeast
3/5
08 09 10
Northeast Northeast Northeast
1/3 2/4 3/4
11 12 13
Northeast Northeast Northeast
1/1 1/1 2/5
14 15 Dairy A
Northeast Northeast
1/1 0/5
Southeast
2/3
B
Northeast
4/4
C D E F G H
Northeast Southeast Southeast Southeast Northeast Southeast
2/4 2/2 5/5 1/1 1/2 2/2
I J K L
Southeast Southeast Southeast Southeast
2/2 1/1 1/1 2/2
M N O
Southeast Northeast Northeast
0/1 0/2 0/2
No. of CPS isolates
Antimicrobial resistance profile
STE genes
3 1 1 1 1 10 1 1 2 2 2 1 1 3 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 0
— PEN SMX TET — — PEN — PEN — PEN PEN, PEN PEN PEN — PEN, PEN, — PEN — PEN, PEN, — PEN PEN SMX TET PEN
— — — — sep — sed, ser, — sed, ser, — — — sed, ser, sed, ser, — sed, ser, sep — sed, ser, sed, ser, — — — — — — seh seh seg, sei
1 1 1 1 2 2 2 5 1 1 1 1 2 1 1 No isolates obtained 0 0 0
PEN — PEN, SMX, TMP PEN — — — — — — PEN — — PEN PEN
SMX
CHL CHL, SMX
SMX CHL
sej sej
sej sej sej sej sej
sed, ser, sej, seg, sei, sep — — sea, seh — — — — — sec seg, sei — — sec, seg, sei —
1
CPS = coagulase-positive staphylococci; STE = staphylococcal enterotoxin. Antibiotics: chloramphenicol (CHL), ciprofloxacin (CIP), erythromycin (ERY), gentamicin (GEN), penicillin (PEN), streptomycin (STR), sulfamethoxazole (SMX), tetracycline (TET), trimethoprim (TMP), and cefoxitin (FOX).
genes. However, other authors reported a higher prevalence of enterotoxigenic S. aureus at the level of 68.4% regarding S. aureus isolated from unprocessed and pasteurized milk (Rall et al., 2008). The STEJournal of Dairy Science Vol. 98 No. 7, 2015
positive isolates had mainly the sea gene (41% strains) followed by the sec (20.5%) and sed (12.8%) markers. In the present studies, the sed, ser, and sej genes were present in half (50.0%) of enterotoxigenic S. aureus,
ENTEROTOXIGENIC COAGULASE-POSITIVE STAPHYLOCOCCI IN RAW MILK
whereas the seg and sei genes in 4 (18%) isolates. It is known that genes encoding STE have different genetic location. Among them, the sed, sej, and ser markers are located on plasmid, whereas the enterotoxin gene cluster (egc) carries the seg and sei genes, which are detected together (Argudin et al., 2010). Bystron et al. (2009) found that 35% of S. aureus isolated from bovine unprocessed milk had enterotoxigenic properties, but only one strain harbored the seb, which was not identified during our analysis. According to the European Union legislation on food safety, SEA to SEE should be routinely detected enterotoxins in food. However, 5 to 10% of SFP is considered to be caused by other enterotoxins (Bystron et al., 2009). Gram-positive bacteria are generally susceptible to methicillin, chloramphenicol, and ciprofloxacin (Gundogan et al., 2006); however, 3 isolates resistant to chloramphenicol were identified in the present study. Although all other CPS strains were susceptible to ciprofloxacin, erythromycin, gentamicin, cefoxitin, and streptomycin, it has to be noted that 43% of the isolates demonstrated resistance to one or more antimicrobial agents. In the present study, strains resistant to 2 antimicrobials were present in 4 (6%) out of all strains analyzed and multiresistance (≥3 groups of antibiotics) was observed in 2 (3%) isolates. These results are in agreement with Werckenthin et al. (2001) who suggested that antimicrobial multiresistance in veterinary medicine is reported only occasionally. However, according to Saini et al. (2012), 15% of the resistant S. aureus isolates recovered from milk samples on dairy farms in Canada were multidrug resistant. On the other hand, a higher prevalence (34.8%) of multiresistant of S. aureus was observed among isolates obtained from bovine, sheep, and goat raw milk (Alian et al., 2012). Previous studies (Jørgensen et al., 2005a,b) indicated that the prevalence of penicillin-resistant S. aureus ranged from less than 10% to over 50% depending on the sample’s origin. In the present study, 38% of CPS isolates were resistant to this antimicrobial agent, whereas the resistance rates of 20 and 73% were found among S. aureus isolated from bulk tank milk in Hungary and from various foods in Portugal, respectively (Peles et al., 2007; Pereira et al., 2009). None of CPS isolated in the present study harbored the gene responsible for the resistance to methicillin. However, data from Korea, Italy, and Japan showed that MRSA were isolated from raw milk and other food products at the levels of 2.5, 3.75, and 0.45%, respectively (Kitai et al., 2005; Moon et al., 2007; Normanno et al., 2007). In the present survey, a relatively low percentage (9%) of staphylococcal isolates were resistant to sulfamethoxazole, compared with the results from other European countries, where the resistance rate of bo-
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vine S. aureus was 4.8, 21.2, and 50.0% in Switzerland, Denmark, and Germany, respectively (Werckenthin et al., 2001). The percentages of the isolates resistant to trimethoprim and sulfamethoxazole were at relatively low levels of 0.5% in France, 3.2% in Germany, and 17.4% in Iran (Werckenthin et al., 2001; Alian et al., 2012). Resistance to trimethoprim identified in the present study was at a low level of 1%. Only single isolates demonstrated resistance to tetracycline and chloramphenicol, which is in agreement with the results obtained by Pereira et al. (2009), where the percentage of such strains was not higher than 1.4%. However, a slightly higher resistance rates to tetracycline (9%) was found by Vyletelova et al. (2011) among staphylococci isolated from milk samples of various origin. In the present investigation, all isolates tested were susceptible to ciprofloxacin, erythromycin, gentamicin, cefoxitin, and streptomycin. Pereira et al. (2009) also did not found any S. aureus isolate resistant to ciprofloxacin and only 5 and 2% strains resistant to erythromycin and gentamicin, respectively. However, 7% of staphylococci recovered from milk samples in the Czech Republic were resistant to ciprofloxacin and gentamicin as well as 26% to erythromycin (Vyletelova et al., 2011). In contrast to the data from the present study, S. aureus isolates resistant to streptomycin were observed in Denmark, Brazil, and Germany at levels of 4.1, 12.1, and 29.3%, respectively (Werckenthin et al., 2001). In 53.8% of isolates resistant to penicillin, one or more STE genes were identified. Single enterotoxinencoding genes were also found among strains resistant to chloramphenicol, sulfonamides, and tetracycline. CONCLUSIONS
Contamination of raw milk with S. aureus, especially enterotoxigenic strains, poses a potential public health threat. These bacteria, resistant to one or more antimicrobial agents, may be transmitted to humans by the consumption of raw milk and raw milk products. For this reason, it is important to determine antimicrobial susceptibility of S. aureus and to monitor the spread of resistant strains in the food chain. Our results indicate the need for continuous monitoring and further improvement of hygienic quality of raw milk by controlling animal health and milking hygiene, and ensuring proper conditions of collection and storage of milk. REFERENCES Alian, F., E. Rahimi, A. Shakerian, H. Momtaz, M. Riahi, and M. Momeni. 2012. Antimicrobial resistance of Staphylococcus aureus isolated from bovine, sheep and goat raw milk. Global Vet. 8:111– 114. Journal of Dairy Science Vol. 98 No. 7, 2015
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