Address for Correspondence: Ghassan ISSA • E-mail: ghassanissa@avrupa.edu.tr Received Date: 09.04.2020 • Accepted Date: 21.07.2020 • DOI: 10.5152/actavet.2020.20011 Available online at actavet.org
Abstract
Staphylococcus aureus is a primary cause of food poisoning
and many types of other infections in humans and animals. This study aims to detect methicillin-resistant S. aureus (MRSA) in raw cow milk collected from different farms in Istanbul, using polymerase chain reaction–based phenotyping and genotyping. A total of 406 raw milk samples were collected from 6 different dairy farms located in Istanbul at different times and under appropriate conditions. The samples were transferred to laboratory under cold storage conditions. After
S. aureus had been isolated from the samples by phenotypic
methods, MRSA strains were detected in isolates by both phenotypic and genotypic methods. Of the 406 milk samp-les examined, S. aureus strains were detected in 119 (29.31%). From the 119 isolated S. aureus strains, there was merely 1 (0.84%) strain with MRSA in phenotypic and genotypic terms. Strategies required to control antimicrobial resistance should be developed and implemented in the field of veterinary and human medicine.
Keywords: Milk, PCR, resistance, Staphylococcus aureus
Detection of Methicillin-Resistant Staphylococcus aureus in
Milk by PCR-Based Phenotyping and Genotyping
Ghassan ISSA
1, Harun AKSU
21Kocaeli Health and Technology University Avrupa Vocational School Medical Laboratory Techniques Program, İstanbul, Turkey
2Department of Food Hygiene and Technology, İstanbul University-Cerrahpaşa, Faculty of Veterinary, İstanbul, Turkey
Cite this article as: Issa, G., Aksu, H., 2020. Detection of Methicillin-Resistant Staphylococcus aureus in Milk by PCR-Based Phenotyping and Genotyping. Acta Vet Eurasia 2020; 46: 120-124.
ORCID IDs of the authors: G.İ. 0000-0002-0229-7632; H.A. 0000-0001-5948-2030.
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
Introduction
Staphylococcus aureus (S.aureus) is a Gram-positive, non-motile,
non–spore forming, non-encapsulated, catalase-positive, coc-cus-shaped facultative anaerobic microorganism with diameters of 0.5–1.5 µm. It is resistant to atmospheric conditions and ubiq-uitously present in nature, being a member of the normal flora of humans, animals, and plants. It is naturally found in the nasal and oral cavities, human and animal feces, on the skin, abscesses, and pimples. It causes food poisoning in the first place and many other diseases (pneumonia, postsurgery wound infections, con-junctivitis, osteomyelitis, mastitis, arthritis, abscess, urinary tract infections, etc.) (Erol, 2007; Lee, 2003; Normanno et al., 2007). The rate of invasive S. aureus infections has been significantly con-tained since the use of benzylpenicillin, but the bacterium ac-quired rapid resistance to benzylpenicillin and its semisynthetic derivative, methicillin. As a result, methicillin-resistant S. aureus (MRSA) has become the main cause of hospital-acquired
infec-tions. Lately, community-associated MRSA infections have been a matter of attention (Kwon et al., 2005; Telli et al., 2006; Ünal, 2006). S. aureus acquires resistance to methicillin by produc-ing low-affinity penicillin bindproduc-ing protein, which is encoded by
mecA genes and causes resistance to all beta-lactam antibiotics
(Kwon et al., 2006; Ünal, 2007; Yu et al., 2008; Zhang et al., 2009). This study aimed to detect MRSA by polymerase chain reaction (PCR)–based phenotyping and genotyping in raw milk samples collected from different farms in Istanbul.
Materials and Methods
A total of 406 raw milk samples were collected from 6 differ-ent dairy farms located in Istanbul at differdiffer-ent times and under appropriate circumstances. Collected under aseptic conditions, the samples were transferred to laboratory environment at 4°C. In order to identify the presence of S. aureus, the raw milk sam-ples were inoculated onto 0.5 mL Baird-Parker agar medium (Oxoid, Hampshire, England). The inoculated Petri dishes were
then incubated at 37°C for 24–48 hours. At the end of the in-cubation period, circular, smooth, convex, gray-black, opaque colonies surrounded by a 2.5-mm-wide transparent zone (leci-thinase positive) formed and were considered as S. aureus–sus-pected colonies. In order to identify the beta-hemolysis of the colonies, the suspected S. aureus isolates were inoculated on blood agar (Oxoid, Hampshire, England) plates. After a 24-hour incubation, beta-hemolytic colonies were selected, and other identification tests (Gram staining, DNase, catalase, mannitol fermentation, coagulase, aerobic fermentation of
carbohy-drates, pigmentation, novobiocin sensitivity) were performed (Bilgehan, 2000; Kayser et al., 1997).
The confirmation of isolates, which were accepted as S. aureus by phenotypic methods, was performed by determining the S.
aureus–specific 16S rRNA gene by genotypic (PCR) methods.
MRSA strains were identified by both phenotypic and geno-typic (PCR) methods. To identify methicillin resistance, the Kirby-Bauer disk diffusion method was implemented accord-ing to the Clinical and Laboratory Standards Institute (CLSI,
Figure 1.a-h. (a) S. aureus in Baird-Parker Agar, (b) Beta-Hemolysis Test in Blood Agar, (c) DNase Test, (d) Mannitol Fermentation Test, (e) Aerobic Fermentation Test, (f) Novobiocin and Polymyxin-B Resistance Test, (g) Cefoxitin and Oxacillin Disc Diffusion Test, (h) ORSAB Screening Agar Test
a d g b e h c f
2009) guidelines. Conventionally, cefoxitin disk diffusion (30 μg, Oxoid, Hampshire, England), oxacillin disk diffusion (1 μg, Oxoid, Hampshire, England), and oxacillin resistance screening agar base (ORSAB, Oxoid, Hampshire, England) tests were per-formed. S. aureus strains were investigated in terms of the pres-ence of mecA genes (CLSI, 2009; Wikler et al., 2007).
For the DNA extraction of the bacterium, a genomic DNA isola-tion kit (Roche, Basel, Switzerland) was used. A total of 4–5 colo-nies were selected from the bacterium incubated on Tryptic Soy
Agar medium (Oxoid, Hampshire, England) overnight at 37°C
and suspended in Hank’s balanced salt solution in sterilized mi-crocentrifuge tubes. Before the experiment, these suspensions were mixed by vortex, and the DNA was extracted according to the guidelines. The obtained DNA extract was stored at −20°C. For specific 16S rRNA determination, a 756-bp amplicon twist-ed Staph756F 5’-AAC TCT GTT ATT AGG GAA GAA CA-3’ and Staph750R 5’-CCA CCT TCC TCC GGT TTG TCA CC-3’ primers were used, and when using mecA1, for mecA gene determination, 5’-GTAGAAATGACTGAACGTCCGATAA-3’ and mecA2 5’-CCAAT-TCCACATTGTTTCGGTCTAA-3’ primers were used. PCR was com-pleted by adding 2.5 µL of 10X buffer with (NH4) 2SO4, 1.5 µL of MgCl2 (25 mM), 0.5 µL of dNTP-Lsg (0.2 mM), 0.5 µL of Primer F (25 µM), 0.5 µL of Primer R (25 µM), 0.125 µL of Taq polymerase (5 U/ µL) (500 U), and 14.4 µL of distilled water. To this mixture, 5 µL of the extracted DNA was added. Next, the tubes were capped, and the specimen was inserted in the PCR device (Thermal Cycler, Bio-Rad, Hercules, California, US). In optimal cycling conditions for the 16S rRNA, an initial denaturation step was performed at 94°C for 10 min, followed by the initial 10 cycles of 94°C for 45 s, 55°C for 45 s (annealing), and 72°C for 75 s (extension). The pro-cess was completed by additional extension at 72°C for 10 min. Then, 25 cycles of denaturation at 94°C for 45 s, annealing at 50°C
for 45 s, and extension at 72°C for 75 s were performed. The pro-cess was finalized by additional extension at 72°C for 10 min. For
mecA, the initial denaturation step was performed at 94°C for 4
min, followed by 10 cycles of 94°C for 45 s, 55°C for 45 s (anneal-ing), and 72°C for 75 s (extension). The process was finalized by additional extension at 72°C for 10 min. After the amplification, the PCR products were analyzed using standard horizontal aga-rose gel electrophoresis Bio-Rad, Hercules, California, US). The analysis of the gel revealed 756 base pairs of 16S rRNA and 310 base pairs of mecA strands (Adaleti et al., 2008; Araj et al., 1999; Jonas et al., 2002; McClure et al., 2006).
Results
In total, 406 milk samples were analyzed, of which, 119 (29.31%) S. aureus strains were obtained, each from a different sample. The isolates obtained from the samples were examined in terms of morphology, microscopic morphology (Gram stain-ing), hemolysis on blood agar, and mannitol fermentation, and after the DNase, catalase, and coagulase tests, the PCR exam-ination revealed the specific 16S rRNA gene. The identification results of S. aureus strains are shown in Figures 1 and 2. Of the 119 strains identified as S. aureus, only 1 (0.84%) strain was both phenotypically and genotypically identified to be MRSA. In order to reveal the phenotypic methicillin resistance in S. aureus strains, cefoxitin disk diffusion (30 μg), oxacillin disk diffusion (1 μg), and ORSAB tests were performed, whereas for the genotypic identification of methicillin resistance, PCR was used for mecA gene detection. The results of the tests used to identify the phenotypic methicillin resistance in isolated S.
aureus strains are shown in Figure 1, and the image of mecA
gene detection revealing the genotypic methicillin resistance is shown in Figure 3.
Discussion
S. aureus is the most dangerous bacterium in the genus Staphy-lococcus. It is one of the primary causes of mastitis occurring in
milk cows worldwide. It is a source of nosocomial community and community-acquired infections. In recent years, the rapid surge of MRSA isolates entails the development of effective strategies to offset the antibiotic resistance of microorganisms and contain Staphylococcus infections. This necessitates the comprehension of the epidemiology, pathogenesis and pop-ulation genetics of S. aureus. For a sustainably healthy life, milk and dairy products are ubiquitously preferred as daily nutrition-al and dietary products. Milk and dairy products serve as a sig-nificant daily dietary source for infants and children to support their physical development needs. However, apart from other contaminants, milk includes microbial contaminants, which pose risks to human health. The presence of several danger-ous pathogenic microorganisms in milk and dairy products is a source of concern. S. aureus is one of these pathogenic microor-ganisms, and its presence in raw milk is a risk for public health. There has been substantial research conducted in Turkey re-garding S. aureus isolation in milk. Kuyucuoğlu and Uçar (2001) reported that microbiological analysis of 164 milk samples from 126 cows with clinical and subclinical mastitis revealed the presence of S. aureus in 40.10% of the samples. In anoth-er study, Türütoğlu et al. (1995) examined 1,594 milk samples with mastitis. Their study concluded that out of 1,126 (70.60%) isolated milk samples analyzed, 316 (28.10%) were identified to have S. aureus. According to Ergün et al.’s (2004) microbiologi-cal study, 262 samples collected from 115 cows with subclinimicrobiologi-cal mastitis included 42.40% coagulase-negative Staphylococcus
and 25.10% S. aureus. Similar studies were also performed in other countries. Juhasz-Kaszanyitzky et al. (2007) obtained 375 (63.02%) S. aureus isolates out of 595 milk samples collected from milk cows with subclinical mastitis in Hungary. Virgin et al. (2009) examined 542 milk samples and identified S. aureus in 218 (40.22%) samples. However, after freezing, 190 (87%) out of 218 milk samples were identified to have S. aureus.
In our study, 406 raw milk samples were examined, and from 119 (29.31%) samples, S. aureus strains were isolated. These re-sults reveal similarities and differences compared with many of the studies performed in our country and other countries. Some distinctions are inevitable considering differences such as the geographical region of the samples and conditional changes in farms and animals (subclinical mastitis, mastitis). Differences in the results of national and international studies on the rate of S.
aureus and MRSA strains could occur because of regional
distinc-tions, differences in isolated samples, and consecutive increase in the number of resistant isolates. Seasonal changes could also be understood to pose differences in effective isolates. Hence, Stastkova et al. (2009) reported that S. aureus and MRSA rates dif-fer in milk samples because of seasonal changes.
According to Türkyılmaz et al.’s (2010) study, out of 93 S. aureus strains isolated from milk samples with mastitis, 16 were re-ported to be MRSA strains. Kwon et al. (2005) examined 75,335 milk samples in Korea and identified the presence of MRSA in 14 (0.018%) samples. Vanderhaeghen et al. (2010) stated that out of 118 S. aureus strains isolated from cows with mastitis, 11 (9.30%) of them were MRSA. Huber et al. (2010) isolated 142
S. aureus strains from milk samples with mastitis and reported
that 2 (1.40%) of them were MRSA strains. In a study performed by Virgin et al. (2009), out of 190 S. aureus strains obtained after the frozen storage, 7 (3.68%) contained mecA gene.
Our study concludes that out of 119 S. aureus strains isolated from 406 raw milk samples, only 1 (0.84%) strain was phenotypically and genotypically identified as an MRSA strain. The presence of resistant genes in raw milk is of great concern for public health because milk is consumed mainly by babies and children who are not completely developed physically and are more sensitive to the side effects of resistant genes. MRSA strains isolated from humans and animals show similarities, and it is stated that MRSA transmission could be possible between animals and humans (Kwon et al., 2006). Various studies have shown that animals can act as a reservoir for MRSA cross-transmission between animals and humans (Middleton et al., 2005; Strommenger et al., 2006). A study by Lee (2003) signified that out of 15 MRSA strains isolated from animals, 6 resemble the strains obtained from humans, and it is reported that the consumption of animal products can result in MRSA transmission to humans.
Finally, integrated surveillance systems addressing antimicrobial resistance issue should be developed in the fields of human and ve-terinary medicine. Further molecular studies should be performed to explain the molecular epidemiology of resistance characteristics adequately, and related strategies should be developed and imp-lemented to contain resistance in human and veterinary medicine. Figure 3. Image of mecA gene obtained from
Ethics Committee Approval: N/A. Peer-review: Externally peer-reviewed.
Author Contributions: Concept - G.İ.; Design - G.İ., H.A.; Supervision - H.A.; Resources - G.İ., H.A.; Materials - G.İ.; Data Collection and/or Process-ing - G.İ.; Analysis and/or Interpretation - G.İ., H.A.; Literature Search - G.İ., H.A.; Writing Manuscript - G.İ.; Critical Review - G.İ., H.A.; Other - G.İ., H.A. Conflict of Interest: The authors have no conflicts of interest to declare. Financial Disclosure: This study was supported by the Istanbul Univer-sity Research Fund (Project No.: 2943).
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