Raw agreement indices among conventional culture/PCR method and real-time PCR assay, with two-step enrichment procedure for the detection of
Abstract
Staphylococcus aureus (S. aureus) can cause a wide variety of infections in humans, such as skin and soft tissue infections, bacteremia, pneumonia, and food poisoning. This pathogen could be carried on the nares, skin, and hair of animals and humans, representing a serious problem at the hospital and the community level as well as in the food industry. The pathogenicity of S. aureus is given by bacterial structures and extracellular products, among which are toxins, which could cause staphylococcal diseases transmitted by food (SFD). S. aureus has the ability to develop resistance to antimicrobials (AMR), highlighting methicillin-resistant strains (MRSA), which have resistance to all beta-lactam antibiotics, except to the fifth-generation cephalosporins. Methicillin resistance is primarily mediated by three mechanisms: production of an altered penicillin-binding protein PBP2’ (or PBP2a), encoded by the mecA gene; high production of β-lactamase in borderline oxacillin-resistant Staphylococcus aureus (BORSA); and mutations in the native PBPs, called modified S. aureus (MODSA). Emerging strains have been isolated from meat-producing animals and retail meat, such as MRSA, MRSA ST398 (associated with livestock), multidrug-resistant (MDR) S. aureus, and enterotoxin-producing S. aureus. Therefore, there is a risk of contamination of meat and meat products during the different processing stages of the meat supply chain.
Keywords
- meat-producing animals
- raw meat
- antimicrobial resistance (AMR)
- methicillin-resistant S. aureus (MRSA)
- livestock-associated methicillin-resistant S. aureus (LA-MRSA)
- multidrug-resistant (MDR)
- enterotoxins
- mecA gene
1. Introduction
In animal production, the emergence and the spread of antimicrobial-resistant pathogens have been associated with the misuse or overuse of antibiotics [1]. Those pathogens or the genes associated with antimicrobial resistance (AMR) could enter into the food supply chain through the food-producing animals and food handlers [2] and be transmitted to humans, threatening the effective treatments of infectious diseases [3].
Methicillin resistance is caused primarily by three mechanisms. The classical mechanism implies the production of an altered penicillin-binding protein, PBP2’ (also called PBP2a), which is encoded by the
Different clones of MRSA have been recognized, such as health care-associated MRSA (HA-MRSA) [10], community-associated MRSA (CA-MRSA) [11], and livestock-associated MRSA (LA-MRSA) [12].
This pathogen can cause different diseases, such as skin and soft tissue infections, bacteremia, pneumonia, and food poisoning [13, 14].
Moreover, multidrug-resistant (MDR)
The food poisoning is caused by eating foods contaminated with heat-stable enterotoxins produced by
Therefore, the ability of
The aim of this chapter is to provide information about the detection, prevalence, characteristics, molecular typing, antimicrobial susceptibility, and the mechanisms of antimicrobial resistance of
2. Methods of detection and identification of Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) in animals and meat
Different culture methods have been used to detect
The test API® Staph has been shown to be a reliable method for phenotypic characterization, as other methods have had a lower precision [29]. In addition, the biochemical identification of
According to Kateete et al. [30], there is no only phenotypic test (including the coagulase test) that can guarantee reliable results in the identification of
In the past decades, methodologies, such as phage typing and genotyping were used. However, these techniques have disadvantages since they are time-consuming and can only be performed in specialized laboratories by trained professionals. Nowadays, more simple and precise techniques are being used, such as the detection by PCR technique, which has been used as the “gold standard” method to identify pathogens.
In relation to the detection and identification of MRSA, there are different methods that have been used, mainly, in clinical laboratories. Among these tests one can mention the determination of minimum inhibitory concentrations (MIC) (dilution in agar or dilution in broth and Etest), oxacillin detection agar (OSA) [32, 33, 34], and detection of the protein PBP2’ by the latex agglutination test [32, 35, 36]. This last test has an accuracy as high as the PCR method and greater than susceptibility testing method to confirm MRSA [37]. Currently, cefoxitin, a potent inducer of the
The isolation and identification of
Real-time PCR technology has been used as an alternative to culture methods for the rapid detection of
The real-time PCR assay carried out by Velasco et al. [43] used a primary and a secondary enrichment of samples from meat-producing animals and retail raw meat in order to detect
Table 1 shows the agreement between the detection of
Comparison within each sample type | No. samples | No. positive by culture/PCR method | No. (%) of samples* | |||
---|---|---|---|---|---|---|
Positive agreement (sensitivity) | Negative agreement (specificity) | Total agreement | ||||
Real-time PCR first enrichment | ||||||
Animals | 77 | 32 | 32 (100.0) | 34 (75.6) | 66 (85.7) | 0.72 |
Meat | 112 | 58 | 52 (89.7) | 42 (77.8) | 94 (83.9) | 0.68 |
Deli meat | 45 | 5 | 4 (80.0) | 40 (100.0) | 44 (97.8) | 0.88 |
Real-time PCR second enrichment | ||||||
Animals | 77 | 32 | 32 (100.0) | 36 (80.0) | 68 (88.3) | 0.77 |
Meat | 112 | 58 | 52 (89.7) | 46 (85.2) | 98 (87.5) | 0.75 |
Deli meat | 45 | 5 | 5 (100.0) | 26 (65.0) | 31 (68.9) | 0.29 |
The kappa statistic for detection of
The total agreement on the detection of the
The real-time PCR assay can decrease the total time for detection of
2.1 Prevalence of Staphylococcus aureus strains in the meat supply chain
Figure 1 shows the prevalence of
The type of production system, natural or conventional, did not affect the prevalence (P > 0.05). A higher prevalence of
A higher prevalence of
As expected, non-packaged meat was more contaminated (43.1%) than packaged meat (5.3%) (P ≤ 0.05), since non-packaged meat is more exposed to bacterial contamination, during processing and commercialization in meat counter at supermarkets and retail stores.
A higher prevalence of
In addition, the
In a study carried out in Fargo, ND, USA [20], the overall prevalence of
Other studies have detected a higher prevalence of
In this study, MRSA was not detected in animals; however, a prevalence of MRSA in swine ranging from 6 to 71% has been detected previously [55, 58]. In pork meat, the prevalence of MRSA has also been reported to be less than 10% in other studies [27, 51, 52].
3. Characterization of Staphylococcus aureus isolated from the meat supply chain
3.1 Molecular characterization of Staphylococcus aureus strains in meat-producing animals and retail meat
Different molecular techniques have been used for typing
Different clones of methicillin-susceptible
The SCC
SCC | Structure of | |||
---|---|---|---|---|
I | Class B | Type 1 | ||
II | Class A | Type 2 | ||
III | Class A | Type 3 | ||
IV | Class B | Type 2 | ||
V | Class C2 | Type 5 | ||
VI | Class B | Type 4 | ||
VII | Class C1 | Type 5 | ||
VIII | Class A | Type 4 | ||
IX | Class C2 | Type 1 | ||
X | Class C1 | Type 7 | ||
XI | Class E | Type 8 | ||
XII | Class C2 | Type 9 | ||
XIII | Class A | Type 9 |
The
In the study carried out by Buyukcangaz et al. [20], five pork samples were positive for MRSA, of which three were ST398 and two were ST5. The most common clones in sheep were ST398 and ST133, in pigs and pork both ST398 and ST9, and in chicken ST5. The clustering of isolates obtained by PFGE agreed well with the MLST types, i.e., the identical restriction patterns or patterns that differed at two to six bands had an identical ST. A total of 34
In addition, contamination of meat with
3.2 Antimicrobial resistance in Staphylococcus aureus from meat-producing animals and meat
Methicillin and other β-lactam antibiotics affect the cell wall synthesis in gram-positive bacteria inhibiting the last stage of the peptidoglycan synthesis called transpeptidation. During the transpeptidation the linkage between N-acetylmuramic acid and the cell wall takes place, catalyzed by transpeptidases and carboxypeptidases, called penicillin-binding proteins (PBPs). These proteins are able to bind penicillin in their active sites through a covalent bond between a serine and the β-lactam ring, resulting in the inhibition of the transpeptidation [76].
Methicillin resistance in
Some studies have isolated
In March 2017, Schwendener et al. [83] reported a new
Other
Another mechanism of resistance to β-lactam antibiotics is the production of the enzyme β-lactamase, which hydrolyses the β-lactam ring resulting in the inactivation of the antibiotic. This enzyme is encoded by
Table 3 shows the resistance profiles of
Antimicrobial resistance profile* | No. of subclasses resistant to | No. (%) of all | ||
---|---|---|---|---|
Animal N = 28 | Carcass N = 12 | Meat N = 15 | ||
PEN-KAN-ERY-CIP-TET | 5 | 3 (10.7) | ||
PEN-CEF-KAN-ERY-TET | 4 | 1 (3.6) | ||
PEN-KAN-ERY-TET | 4 | 1 (3.6) | 1 (8.3) | |
PEN-ERY-CIP-TET | 4 | 10 (35.7) | 1 (8.3) | |
PEN-KAN-ERY | 3 | 1 (3.6) | ||
PEN-ERY-CIP | 3 | 1 (3.6) | ||
PEN-GEN-QDA | 3 | 1 (8.3) | ||
PEN-ERY-QDA | 3 | 1 (6.7) | ||
OXA-PEN-CEF-GEN-KAN | 2 | 1 (6.7) | ||
PEN-ERY | 2 | 1 (3.6) | 2 (13.3) | |
PEN-CIP | 2 | 1 (3.6) | 1 (6.7) | |
PEN-QDA | 2 | 1 (6.7) | ||
PEN-TET | 2 | 1 (3.6) | ||
KAN-ERY | 2 | 1 (6.7) | ||
OXA-PEN-CEF | 1 | 1 (8.3) | ||
PEN | 1 | 1 (3.6) | 7 (58.3) | 7 (46.6) |
Susceptible to all tested | 0 | 7 (25.0) | 1 (8.3) | 1 (6.7) |
The less effective antibiotic was penicillin. The low effectiveness of penicillin could be due to the enzyme penicillinase that hydrolyzes the β-lactam ring and inactivates the drug [5].
Two
The use of antimicrobial agents in pigs is an important risk factor for increasing the prevalence of MRSA, promoting the selective pressure, and enhancing the emerging and the spread of MRSA [88]. In Holland, a high prevalence of MRSA was detected in pigs, with a resistance to different antibiotics, suggesting the spread of MRSA strains within animals in the slaughterhouses [1].
Table 4 shows the antimicrobial resistance profiles of the 133
Antimicrobial resistance profile* | No. of subclasses resistant to | No. (%) of all | ||
---|---|---|---|---|
Animal (n = 58) | Raw meat (n = 69) | Deli meat (n = 6) | ||
ERY-PEN-TET-LINC-CHL-GEN-CIP-QUI/DAL | 8 | 1 (1.4) | ||
ERY-PEN-TET-LINC-CHL-CIP-QUI/DAL | 7 | 1 (1.4) | ||
ERY-PEN-TET-LINC-CHL-STR | 6 | 2 (3.4) | ||
ERY-PEN-TET-LINC-KAN | 5 | 1 (1.4) | ||
PEN-TET-LINC-CHL-STR | 5 | 1 (1.7) | ||
PEN-TET-LINC-GEN | 4 | 1 (1.7) | ||
PEN-TET-LINC-KAN | 4 | 1 (1.4) | ||
PEN-TET-LINC-STR | 4 | 2 (3.4) | ||
ERY-PEN-TET-LINC | 4 | 1 (1.7) | 13 (18.8) | |
PEN-TET-LINC | 3 | 22 (37.9) | 1 (1.4) | |
PEN-LINC-STR | 3 | 1 (1.7) | ||
ERY-PEN-LINC | 3 | 2 (2.9) | ||
ERY-TET-LINC | 3 | 5 (7.2) | ||
PEN-LINC | 2 | 4 (6.9) | 1 (1.4) | 1 (16.7) |
PEN-TET | 2 | 12 (20.7) | 2 (2.9) | |
TET-LINC | 2 | 3 (5.2) | ||
ERY-LINC | 2 | 3 (4.3) | ||
ERY-PEN | 2 | 2 (2.9) | ||
LINC | 1 | 1 (1.7) | ||
PEN | 1 | 3 (5.2) | 10 (14.5) | 1 (16.7) |
TET | 1 | 3 (5.2) | 4 (5.8) | |
ERY | 1 | 1 (16.7) | ||
Susceptible to all tested | 0 | 2 (3.4) | 22 (31.9) | 3 (50.0) |
The rate of MDR strains was 41.4%, in animals was 51.7%, and in meat 36.2% (n = 25). The MDR isolates were found in pigs, pork, and sheep. MDR isolates from pork were mainly ST398 (60%) and ST9 (30%). All MDR strains from sheep were ST398.
Five pork samples that were MRSA (three ST398 and two ST5) exhibited penicillin resistance and four MDR. In addition, most of the
The AMR bacteria in animals have increased over time due to the frequent use of antimicrobial agents at the farm level [1, 89]. Therefore, controlling the use of antibiotics in farming could limit the risk of transmission of AMR pathogens among animals and to humans [90].
3.3 Characteristics of pathogenicity of Staphylococcus aureus strains in meat-producing animals and meat
The main regulator of virulence gene expression is the
In dairy, one of the main virulence factors is the formation of biofilms, which are structured consortia of bacterial cells that are immersed in a polymeric matrix consisting of polysaccharides, proteins, extracellular DNA (eDNA), lipids, and other macromolecules. The biofilms allow bacteria to adhere to inert or living surfaces, increasing their growth rate and survival in a hostile environment [97].
Enterotoxin-producing
4. Conclusions
The genetic similarity between
Further research is needed to expand the knowledge and comprehension of the molecular characterization and the different mechanisms of AMR in
Acknowledgments
The results of the studies showed in this book chapter were supported by the Research Project Fondecyt No. 11140379 (Chile) and the Dean’s Office, College of Agriculture, Food Systems and Natural Resources College, North Dakota State University (Fargo, ND, USA).
Conflict of interest
The authors declare that there is no conflict of interest regarding the publication of this book chapter.
References
- 1.
de Neeling AJ, van den Broek MJM, Spalburg EC, van Santen-Verheuvel MG, Dam-Deisz WDC, Boshuizen HC, et al. High prevalence of methicillin resistant Staphylococcus aureus in pigs. Veterinary Microbiology. 2007;122 :366-372 - 2.
Sáenz Y, Zarazaga M, Brias L, Lantero M, Ruiz-Larrea F, Torres C. Antibiotic resistance in Escherichia coli isolates obtained from animals, foods and humans in Spain. International Journal of Antimicrobial Agents. 2001;18 :353-358 - 3.
Smith DL, Harris AD, Johnson JA, Silbergeld EK, Morris JG. Animal antibiotic use has an early but important impact on the emergence of antibiotic resistance in human commensal bacteria. Proceedings of the National Academy of Sciences. 2002; 99 (9):6434-6439 - 4.
Aguayo-Reyes A, Quezada-Aguiluz M, Mella S, Riedel G, Opazo-Capurro A, Bello-Toledo H, et al. Bases moleculares de la resistencia a meticilina en Staphylococcus aureus . Revista chilena de infectología. 2018;35 (1):7-14 - 5.
Peacock SJ, Paterson GK. Mechanisms of methicillin resistance in Staphylococcus aureus . Annual Review of Biochemistry. 2015;84 :577-601 - 6.
Tsubakishita S, Kuwahara-Arai K, Baba T, Hiramatsu K. Staphylococcal cassette chromosome mec-like element in Macrococcus caseolyticus . Antimicrobial Agents and Chemotherapy. 2010;54 (4):1469-1475 - 7.
García-Álvarez L, Holden MTG, Lindsay H, Webb CR, Brown DFJ, Curran MD, et al. Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: A descriptive study. The Lancet Infectious Diseases. 2011;11 (8):595-603 - 8.
MacFadyen AC, Fisher EA, Costa B, Cullen C, Paterson GK. Genome analysis of methicillin resistance in Macrococcus caseolyticus from dairy cattle in England and Wales. Microbial Genomics. 2018;4 :1-8 - 9.
Angeles Argudín M, Roisin S, Nienhaus L, Dodémont M, De Mendonça R, Nonhoff C, et al. Genetic diversity among Staphylococcus aureus isolates showing oxacillin and/or cefoxitin resistance not linked to the presence of mec genes. Antimicrobial Agents and Chemotherapy. 2018;62 (7):1-6 - 10.
Tiemersma EW, Bronzwaer SLAM, Lyytikäinen O, Degener JE, Schrijnemakers P, Bruinsma N, et al. Methicillin-resistant Staphylococcus aureus in Europe, 1999-2002. Emerging Infectious Diseases. 2004;10 :1627-1634 - 11.
Kennedy AD, Otto M, Braughton KR, Whitney AR, Chen L, Mathema B, et al. Epidemic community-associated methicillin-resistant Staphylococcus aureus : Recent clonal expansion and diversification. Proceedings of the National Academy of Sciences. 2008;105 :1327-1332 - 12.
Golding GR, Campbell JL, Spreitzer DJ, Veyhl J, Surynicz K, Simor A, et al. A preliminary guideline for the assignment of methicillin-resistant Staphylococcus aureus to a Canadian pulsed-field gel electrophoresis epidemic type using spa typing. Canadian Journal of Infectious Diseases and Medical Microbiology. 2008;19 :273-281 - 13.
Tong SYC, Davis JS, Eichenberger E, Holland TL, Fowler VG. Staphylococcus aureus infections: Epidemiology, pathophysiology, clinical manifestations, and management. Clinical Microbiology Reviews. 2015;28 :603-661 - 14.
Argudín MÁ, Mendoza MC, Rodicio MR. Food poisoning and Staphylococcus aureus enterotoxins. Toxins. 2010;2 :1751-1773 - 15.
Voss A, Loeffen F, Bakker J, Klaassen C, Wulf M. Methicillin-resistant Staphylococcus aureus in pig farming. Emerging Infectious Diseases. 2005;11 (12):1965-1966 - 16.
(CFSPH) C for FS and PH. Methicillin-Resistant Staphylococcus Aureus MRSA. Iowa State University; 2011. pp. 1-25 - 17.
Friese A, Schulz J, Zimmermann K, Tenhagen BA, Fetsch A, Hartung J, et al. Occurrence of livestock-associated methicillin-resistant Staphylococcus aureus in Turkey and broiler barns and contamination of air and soil surfaces in their vicinity. Applied and Environmental Microbiology. 2013;79 :2759-2766 - 18.
Nemeghaire S, Argudín MA, Haesebrouck F, Butaye P. Epidemiology and molecular characterization of methicillin-resistant Staphylococcus aureus nasal carriage isolates from bovines. BMC Veterinary Research. 2014;10 :153 - 19.
Hanson BM, Dressler AE, Harper AL, Scheibel RP, Wardyn SE, Roberts LK, et al. Prevalence of Staphylococcus aureus and methicillin-resistantStaphylococcus aureus (MRSA) on retail meat in Iowa. Journal of Infection and Public Health. 2011;4 :169-174 - 20.
Buyukcangaz E, Velasco V, Sherwood JS, Stepan RM, Koslofsky RJ, Logue CM. Molecular typing of Staphylococcus aureus and methicillin-resistantS. aureus (MRSA) isolated from animals and retail meat in North Dakota, United States. Foodborne Pathogens and Disease. 2013;10 :608-617 - 21.
Carrel M, Zhao C, Thapaliya D, Bitterman P, Kates AE, Hanson BM, et al. Assessing the potential for raw meat to influence human colonization with Staphylococcus aureus . Scientific Reports. 2017;7 :1-10 - 22.
Guardabassi L, O’Donoghue M, Moodley A, Ho J, Boost M. Novel lineage of methicillin-resistant Staphylococcus aureus , Hong Kong. Emerging Infectious Diseases. 2009;15 :1998-2000 - 23.
Velasco V, Vergara JL, Bonilla AM, Muñoz J, Mallea A, Vallejos D, et al. Prevalence and characterization of Staphylococcus aureus strains in the pork chain supply in Chile. Foodborne Pathogens and Disease. 2018;15 (5):262-268 - 24.
Pan A, Battisti A, Zoncada A, Bernieri F, Boldini M, Franco A, et al. Community-acquired methicillin-resistant Staphylococcus aureus ST398 infection, Italy. Emerging Infectious Diseases. 2009;15 (5):845-846 - 25.
Le Loir Y, Baron F, Gautier M. Staphylococcus aureus and food poisoning. Genetics and Molecular Research. 2003;2 (1):63-72 - 26.
Aydin A, Muratoglu K, Sudagidan M, Bostan K, Okuklu B, Harsa S. Prevalence and antibiotic resistance of foodborne Staphylococcus aureus isolates in Turkey. Foodborne Pathogens and Disease. 2011;8 :63-69 - 27.
De Boer E, Zwartkruis-Nahuis JTM, Wit B, Huijsdens XW, De Neeling AJ, Bosch T, et al. Prevalence of MRSA in meat. International Journal of Food Microbiology. 2009; 134 :52-56 - 28.
Müller A, Seinige D, Jansen W, Klein G, Ehricht R, Monecke S, et al. Variety of antimicrobial resistances and virulence factors in Staphylococcus aureus isolates from meat products legally and illegally introduced to Germany. PLoS One. 2016;11 (12):e0167864 - 29.
Heikens E, Fleer A, Paauw A, Florijn A, Fluit AC. Comparison of genotypic and phenotypic methods for species-level identification of clinical isolates of coagulase-negative staphylococci. Journal of Clinical Microbiology. 2005; 43 :2286-2290 - 30.
Kateete DP, Kimani CN, Katabazi FA, Okeng A, Okee MS, Nanteza A, et al. Identification of Staphylococcus aureus : DNase and mannitol salt agar improve the efficiency of the tube coagulase test. Annals of Clinical Microbiology and Antimicrobials. 2010;9 :23 - 31.
Brakstad OG, Aasbakk K, Maeland JA. Detection of Staphylococcus aureus by polymerase chain reaction amplification of the nuc gene. Journal of Clinical Microbiology. 1992;30 :1654-1660 - 32.
Danial J, Noel M, Templeton KE, Cameron F, Mathewson F, Smith M, et al. Real-time evaluation of an optimized real-time PCR assay versus brilliance chromogenic MRSA agar for the detection of meticillin-resistant Staphylococcus aureus from clinical specimens. Journal of Medical Microbiology. 2011;60 :323-328 - 33.
Kim MH, Lee WI, Kang SY. Detection of methicillin-resistant Staphylococcus aureus in healthcare workers using real-time polymerase chain reaction. Yonsei Medical Journal. 2013;54 (5):1282-1284 - 34.
Nimmo GR, Bergh H, Nakos J, Whiley D, Marquess J, Huygens F, et al. Replacement of healthcare-associated MRSA by community-associated MRSA in Queensland: Confirmation by genotyping. The Journal of Infection. 2013; 67 :439-447 - 35.
Anderson MEC, Weese JS. Evaluation of a real-time polymerase chain reaction assay for rapid identification of methicillin-resistant Staphylococcus aureus directly from nasal swabs in horses. Veterinary Microbiology. 2007;122 :185-189 - 36.
Weese JS, Avery BP, Reid-Smith RJ. Detection and quantification of methicillin-resistant Staphylococcus aureus (MRSA) clones in retail meat products. Letters in Applied Microbiology. 2010;51 :338-342 - 37.
Sakoulas G, Gold HS, Venkataraman L, Degirolami PC, Eliopoulos GM, Qian Q. Methicillin-resistant Staphylococcus aureus : Comparison of susceptibility testing methods and analysis of mecA-positive susceptible strains. Journal of Clinical Microbiology. 2001;39 :3946-3951 - 38.
CLSI. Performance Standards for Antimicrobial Susceptibility Testing. M100 CS. 28th ed. Wayne, PA; 2018 - 39.
Rostami S, Moosavian M, Shoja S, Torabipour M, Farshadzadeh Z. Comparison of mecA gene-based PCR with CLSI cefoxitin and oxacillin disc diffusion methods for detecting methicillin resistance in Staphylococcus aureus clinical isolates. African Journal of Microbiology Research. 2013;7 (21):2438-2441 - 40.
Zhang W, Hao Z, Wang Y, Cao X, Logue CM, Wang B, et al. Molecular characterization of methicillin-resistant Staphylococcus aureus strains from pet animals and veterinary staff in China. Veterinary Journal. 2011;190 :e125-e129 - 41.
Morcillo A, Castro B, Rodríguez-Álvarez C, González JC, Sierra A, Montesinos MI, et al. Prevalence and characteristics of methicillin-resistant Staphylococcus aureus in pigs and pig workers in Tenerife, Spain. Foodborne Pathogens and Disease. 2012;9 (3):207-210 - 42.
Waters AE, Contente-Cuomo T, Buchhagen J, Liu CM, Watson L, Pearce K, et al. Multidrug-resistant Staphylococcus aureus in US meat and poultry. Clinical Infectious Diseases. 2011;52 :1-4 - 43.
Velasco V, Sherwood JS, Rojas-García PP, Logue CM. Multiplex real-time PCR for detection of Staphylococcus aureus , mecA and panton-valentine leukocidin (PVL) genes from selective enrichments from animals and retail meat. PLoS One. 2014;9 (5):e97617 - 44.
Thomas LC, Gidding HF, Ginn AN, Olma T, Iredell J. Development of a real-time Staphylococcus aureus and MRSA (SAM-) PCR for routine blood culture. Journal of Microbiological Methods. 2007;68 :296-302 - 45.
Ryffel C, Tesch W, Birch-Machin I, Reynolds PE, Barberis-Maino L, Kayser FH, et al. Sequence comparison of mecA genes isolated from methicillin-resistant Staphylococcus aureus andStaphylococcus epidermidis . Gene. 1990;94 :137-138 - 46.
Higashide M, Kuroda M, Ohkawa S, Ohta T. Evaluation of a cefoxitin disk diffusion test for the detection of mecA-positive methicillin-resistant Staphylococcus saprophyticus . International Journal of Antimicrobial Agents. 2006;27 :500-504 - 47.
Crombé F, Angeles Argudfn M, Vanderhaeghen W, Hermans K, Haesebrouck F, Butaye P. Transmission dynamics of methicillin-resistant Staphylococcus aureus in pigs. Frontiers in Microbiology. 2013;4 :1-21 - 48.
Edwards SA. Product quality attributes associated with outdoor pig production. Livestock Production Science. 2005; 94 :5-14 - 49.
Agerso Y, Vigre H, Cavaco LM, Josefsen MH. Comparison of air samples, nasal swabs, ear-skin swabs and environmental dust samples for detection of methicillin-resistant Staphylococcus aureus (MRSA) in pig herds. Epidemiology and Infection. 2014;142 :1727-1736 - 50.
O’Connor AM, Gailey J, McKean JD, Hurd HS. Quantity and distribution of salmonella recovered from three swine lairage pens. Journal of Food Protection. 2006; 69 :1717-1719 - 51.
Pu S, Han F, Ge B. Isolation and characterization of methicillin-resistant Staphylococcus aureus strains from louisiana retail meats. Applied and Environmental Microbiology. 2009;75 :265-267 - 52.
O’Brien AM, Hanson BM, Farina SA, Wu JY, Simmering JE, Wardyn SE, et al. MRSA in conventional and alternative retail pork products. PLoS One. 2012; 7 :e30092 - 53.
Tanih NF, Sekwadi E, Ndip RN, Bessong PO. Detection of pathogenic Escherichia coli andStaphylococcus aureus from cattle and pigs slaughtered in abattoirs in Vhembe District, South Africa. Scientific World Journal. 2015;2015 :1-8 - 54.
Mørk T, Kvitle B, Jørgensen HJ. Reservoirs of Staphylococcus aureus in meat sheep and dairy cattle. Veterinary Microbiology. 2012;155 :81-87 - 55.
Khalid KA, Zakaria Z, Ooi PT, McOrist S. Low levels of meticillin-resistant Staphylococcus aureus in pigs in Malaysia. The Veterinary Record. 2009;164 :626-627 - 56.
Lowe BA, Marsh TL, Isaacs-Cosgrove N, Kirkwood RN, Kiupel M, Mulks MH. Microbial communities in the tonsils of healthy pigs. Veterinary Microbiology. 2011; 147 :346-357 - 57.
Atanassova V, Meindl A, Ring C. Prevalence of Staphylococcus aureus and staphylococcal enterotoxins in raw pork and uncooked smoked ham—A comparison of classical culturing detection and RFLP-PCR. International Journal of Food Microbiology. 2001;68 :105-113 - 58.
van de Vijver LPL, Tulinski P, Bondt N, Mevius D, Verwer C. Prevalence and molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) in organic pig herds in the Netherlands. Zoonoses and Public Health. 2014;61 :338-345 - 59.
Rasschaert G, Vanderhaeghen W, Dewaele I, Janež N, Huijsdens X, Butaye P, et al. Comparison of fingerprinting methods for typing methicillin-resistant Staphylococcus aureus sequence type 398. Journal of Clinical Microbiology. 2009;47 (10):3313-3322 - 60.
Tenover FC, Arbeit R, Archer G, Biddle J, Byrne S, Goering R, et al. Comparison of traditional and molecular methods of typing isolates of Staphylococcus aureus . Journal of Clinical Microbiology. 1994;32 (2):407-415 - 61.
Bens CCPM, Voss A, Klaassen CHW. Presence of a novel DNA methylation enzyme in methicillin-resistant Staphylococcus aureus isolates associated with pig farming leads to uninterpretable results in standard pulsed-field gel electrophoresis analysis. Journal of Clinical Microbiology. 2006;44 (5):1875-1876 - 62.
Argudín MA, Rodicio MR, Guerra B. The emerging methicillin-resistant Staphylococcus aureus ST398 clone can easily be typed using the Cfr9I SmaI-neoschizomer. Letters in Applied Microbiology. 2010;50 (1):127-130 - 63.
Van Wamel WJB, Hansenová Maňásková S, Fluit AC, Verbrugh H, De Neeling AJ, Van Duijkeren E, et al. Short term micro-evolution and PCR-detection of methicillin- resistant and -susceptible Staphylococcus aureus sequence type 398. European Journal of Clinical Microbiology & Infectious Diseases. 2010;29 (1):119-122 - 64.
Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed- field gel electrophoresis: Criteria for bacterial strain typing. Journal of Clinical Microbiology. 1995; 33 (9):2233-2239 - 65.
Murchan S, Kaufmann ME, Deplano A, De Ryck R, Struelens M, Zinn CE, et al. Harmonization of pulsed-field gel electrophoresis protocols for epidemiological typing of strains of methicillin-resistant Staphylococcus aureus : A single approach developed by consensus in 10 European laboratories and its application for tracing the spre. Journal of Clinical Microbiology. 2003;41 (4):1574-1585 - 66.
McDougal LK, Steward CD, Killgore GE, Chaitram JM, McAllister SK, Tenover FC. Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: Establishing a National Database. Journal of Clinical Microbiology. 2003;41 (11):5113-5120 - 67.
Deurenberg RH, Vink C, Kalenic S, Friedrich AW, Bruggeman CA, Stobberingh EE. The molecular evolution of methicillin-resistant Staphylococcus aureus . Clinical Microbiology and Infection. 2007;13 :222-235 - 68.
Stenhem M, Örtqvist Å, Ringberg H, Larsson L, Olsson-Liljequist B, Hæggman S, et al. Imported methicillin-resistant Staphylococcus aureus , Sweden. Emerging Infectious Diseases. 2010;16 (2):189-196 - 69.
Krziwanek K, Metz-Gercek S, Mittermayer H. Methicillin-resistant Staphylococcus aureus ST398 from human patients, upper Austria. Emerging Infectious Diseases. 2009;15 (5):766-769 - 70.
van Belkum A, Melles DC, Peeters JK, van Leeuwen WB, van Duijkeren A, Huijsdens XW, et al. Methicillin- resistant and -susceptible Staphylococcus aureus sequence type 398 in pigs and humans. Emerging Infectious Diseases. 2008;14 (3):479-483 - 71.
Wu Z, Li F, Liu D, Xue H, Zhao X. Novel type XII staphylococcal cassette chromosome mec harboring a new cassette chromosome recombinase, CcrC2. Antimicrobial Agents and Chemotherapy. 2015; 59 (12):7597-7601 - 72.
Baig S, Johannesen TB, Overballe-Petersen S, Larsen J, Larsen AR, Stegger M. Novel SCCmec type XIII (9A) identified in an ST152 methicillin-resistant Staphylococcus aureus . Infection, Genetics and Evolution. 2018;61 :74-76 - 73.
Enright MC, Day NPJ, Davies CE, Peacock SJ, Spratt BG. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus . Journal of Clinical Microbiology. 2000;38 (3):1008-1015 - 74.
Wielders CLC, Vriens MR, Brisse S, De Graaf-Miltenburg LAM, Troelstra A, Fleer A, et al. Evidence for in-vivo transfer of mecA DNA between strains of Staphylococcus aureus . Lancet. 2001;357 :1674-1675 - 75.
Velasco V, Buyukcangaz E, Sherwood JS, Stepan RM, Koslofsky RJ, Logue CM. Characterization of Staphylococcus aureus from humans and a comparison with isolates of animal origin, in North Dakota, United States. PLoS One. 2015;10 (10):e0140497 - 76.
Stapleton PD, Taylor PW. Methicillin resistance in Staphylococcus aureus : Mechanisms and modulation. Science Progress. 2002;85 :57-72 - 77.
Alipour F, Ahmadi M, Javadi S. Evaluation of different methods to detect methicillin resistance in Staphylococcus aureus (MRSA). Journal of Infection and Public Health. 2014;7 :186-191 - 78.
Stegger M, Andersen PS, Kearns A, Pichon B, Holmes MA, Edwards G, et al. Rapid detection, differentiation and typing of methicillin-resistant Staphylococcus aureus harbouring either mecA or the new mecA homologue mecALGA251. Clinical Microbiology and Infection. 2012;18 :395-400 - 79.
Ito T, Hiramatsu K, Tomasz A, De Lencastre H, Perreten V, Holden MTG, et al. Guidelines for reporting novel mecA gene homologues. Antimicrobial Agents and Chemotherapy. 2012; 56 (10):4997-4999 - 80.
Petersen A, Stegger M, Heltberg O, Christensen J, Zeuthen A, Knudsen LK, et al. Epidemiology of methicillin-resistant Staphylococcus aureus carrying the novel mecC gene in Denmark corroborates a zoonotic reservoir with transmission to humans. Clinical Microbiology and Infection. 2013;19 :E16-E22 - 81.
Nadarajah J, Lee MJS, Louie L, Jacob L, Simor AE, Louie M, et al. Identification of different clonal complexes and diverse amino acid substitutions in penicillin-binding protein 2 (PBP2) associated with borderline oxacillin resistance in Canadian Staphylococcus aureus isolates. Journal of Medical Microbiology. 2006;55 :1675-1683 - 82.
Banerjee R, Gretes M, Harlem C, Basuino L, Chambers HF. A mecA-negative strain of methicillin-resistant Staphylococcus aureus with high-level ??-lactam resistance contains mutations in three genes. Antimicrobial Agents and Chemotherapy. 2010;54 (11):4900-4902 - 83.
Schwendener S, Cotting K, Perreten V. Novel methicillin resistance gene mecD in clinical Macrococcus caseolyticus strains from bovine and canine sources. Scientific Reports. 2017;7 (43797):1-11 - 84.
Lowy FD. Antimicrobial resistance: The example of Staphylococcus aureus . The Journal of Clinical Investigation. 2003;111 (9):1265-1273 - 85.
Rubin JE, Ball KR, Chirino-Trejo M. Antimicrobial susceptibility of Staphylococcus aureus andStaphylococcus pseudintermedius isolated from various animals. The Canadian Veterinary Journal. 2011;52 (2):153-157 - 86.
Broekema NM, Van Tam T, Monson TA, Marshall SA, Warshauer DM. Comparison of cefoxitin and oxacillin disk diffusion methods for detection of mecA-mediated resistance in Staphylococcus aureus in a large-scale study. Journal of Clinical Microbiology. 2009;47 (1):217-219 - 87.
Stefani S, Chung DR, Lindsay JA, Friedrich AW, Kearns AM, Westh H, et al. Meticillin-resistant Staphylococcus aureus (MRSA): Global epidemiology and harmonisation of typing methods. International Journal of Antimicrobial Agents. 2012;39 :273-282 - 88.
van Duijkeren E, Ikawaty R, Broekhuizen-Stins MJ, Jansen MD, Spalburg EC, de Neeling AJ, et al. Transmission of methicillin-resistant Staphylococcus aureus strains between different kinds of pig farms. Veterinary Microbiology. 2008;126 (4):383-389 - 89.
Nemati M, Hermans K, Lipinska U, Denis O, Deplano A, Struelens M, et al. Antimicrobial resistance of old and recent Staphylococcus aureus isolates from poultry: First detection of livestock-associated methicillin-resistant strain ST398. Antimicrobial Agents and Chemotherapy. 2008;52 (10):3817-3819 - 90.
Huber H, Koller S, Giezendanner N, Stephan R, Zweifel C. Prevalence and characteristics of meticillin-resistant Staphylococcus aureus in humans in contact with farm animals, in livestock, and in food of animal origin, Switzerland, 2009. Eurosurveillance. 2010;15 (16):1-4 - 91.
Pu S, Wang F, Ge B. Characterization of toxin genes and antimicrobial susceptibility of Staphylococcus aureus isolates from louisiana retail meats. Foodborne Pathogens and Disease. 2011;8 (2):299-306 - 92.
Lowy FD. Staphylococcus aureus infections. The New England Journal of Medicine. 1998;339 (8):520-532 - 93.
Foster TJ, Geoghegan JA, Ganesh VK, Höök M. Adhesion, invasion and evasion: The many functions of the surface proteins of Staphylococcus aureus . Nature Reviews Microbiology. 2014;12 :49-62 - 94.
Gomes-Fernandes M, Laabei M, Pagan N, Hidalgo J, Molinos S, Villar Hernandez R, et al. Accessory gene regulator (Agr) functionality in Staphylococcus aureus derived from lower respiratory tract infections. PLoS One. 2017;12 (4):e0175552 - 95.
Robinson DA, Monk AB, Cooper JE, Feil EJ, Enright MC. Evolutionary genetics of the accessory gene regulator (agr) locus in Staphylococcus aureus. Journal of Bacteriology. 2005; 187 (24):8312-8321 - 96.
Wright JS, Traber KE, Corrigan R, Benson SA, Musser JM, Novick RP. The agr radiation: An early event in the evolution of staphylococci. Journal of Bacteriology. 2005; 187 (16):5585-5594 - 97.
Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: A common cause of persistent infections. Science. 1999; 49 :1318-1322