Technical overview on antimicrobial residue and antimicrobial resistance testing, surveillance and control in the dairy sector
Technical overview on antimicrobial residue and antimicrobial resistance testing, surveillance and control in the dairy sector
Liberty Sibanda
Randox Food Diagnostics Ltd, UK
Over 63,151 tons of antibiotics are used in livestock worldwide, annually (Van Boeckel et al., 2015). In livestock production, antibiotics are applied for both therapeutic and prophylactic purposes. Many surveys carried out in most dairy producing countries have shown that antimicrobials are commonly used in dairy herds (Serraino et al., 2013). As a result, antimicrobial residues are frequently detected in milk and milk products. The antimicrobial classes most commonly used are Penicillins, Tetracyclines, Fluoroquinolones, Cephalosporins, Macrolides, Sulphonamides, Lyncosamides, and Aminoglycosides, and Riphaximin. The European Union (EU) has established safe maximum residue limits (MRLs) for pharmacologically active substances in foodstuffs of animal origin Commission Regulation (EU) No 37/2010. The European Regulation No. 178/2002/EU (EC, 2002) states that food processors have the primary legal responsibility for ensuring food safety and are the first line of defence against antimicrobial exposure to consumers. The frequent use of antimicrobials and their persistence into milk and milk products contributes to antimicrobial resistance. Therefore, constant surveillance of raw milk for both antimicrobial residues and antimicrobial resistance.
Lateral Flow Dipsticks and flow-through tests are the main immunological platforms used as the first line of defence against antimicrobial residues in raw milk. Dipstick tests are receptor-based methods,
quick (usually less than 8 min), and specific for one or two families of antibiotics simultaneously. In order to detect most frequently used antimicrobial agents dairy processors have to use more than five kits in order to gather any meaningful milk quality data (Figure 1.).
Figure 1. Lateral Flow Dipstick
|
|
Another immunological platform is the Enzyme-Linked Immunosorbent Assay (ELISA) Figure 2. ELISA provides both high throughput capacity and sensitivity.
Figure 2. Enzyme-Linked Immunosorbent Assay
|
|
These platforms are used for the detection of veterinary drug residues in both environmental matrices and foods of animal origin. Lateral Flow and ELISA tests for veterinary residue testing are available on the market supplied by companies like Charm Sciences, IDEXX, Neogen, Unisensor, R-Biopharm, to name but just a few. Immunological test platforms, especially ELISA, are also used for viral and bacterial disease detection and surveillance.
The persistent presence of antimicrobials in the environment and animals leads to the development of bacterial antimicrobial resistance genes. Traditionally, antibiotic resistance has been detected by bacterial growth inhibition techniques. However, the detection and surveillance of Drug Resistance Genes (DRGs) is now migrating to Polymerase Chain Reaction (PCR) in order to detect and finger-print the gene conferring resistance when present. This requires isolation of bacteria from milk, followed by DNA extraction, and isolation of the specific DRG. The PCR method is then employed to amplify the DRG to a detectable concentration. PCRs are available from Bio-Rad, Ilumina, to name just a few.
New technologies have now been added to antimicrobial residue monitoring and the fight against antimicrobial resistance. One of the most notable ones is the Biochip Array Technology (BAT). The BAT uses a small Biochip on which many antimicrobial residues can be detected all at once. This process is called multiplexing (Figure. 3). The BAT illustrated in Fig 3., is available on the market for the surveillance and monitoring of animal pathogens (BVD, IBR, Tuberculosis, Salmonella, Leptospira, Neospora, Johne’s Disease & Fasciola), antimicrobial drug residues (Sulphonamides, Quinolones, B-Lactams, Phenicols, Aminoglycosides, Macrolides, Tetracyclines & Sulphonamides), as well as drug resistance gens (MRSA, Trimethoprim Resistance 1, 2, 3, 4, 5, & Vancomyxine Resistance A & B). Randox Food Diagnostics is one of the main suppliers of this type of technology.
Figure 3. Illustration of the multiplexing capability of the Biochip Array Technology for milk testing.
|
|
In addition to these molecular techniques Liquid Chromatography Mass Spectrometry (LC-MS/MS) is used in routine testing for antimicrobial drugs. The advantage of the LC-MS/MS platform, just like the Biochip Array Technology, it offers capacity for multiplexed quantitative detection of a lot of veterinary drugs, depending on the number of drugs desired to be detected. A LC-MS/MS screening method for different antibiotic families (Penicillins, Cephalosporins, Sulphonamides, Macrolides, Lincosamides, Aminoglycosides, Tetracyclines and Quinolones) was developed and validated in milk by Gaugain-Juhel et al., 2009. However, MS/MS based Omics methods for the surveillance of animal pathogens and Drug Resistance Genes are less reported on in the dairy industry or if at all used.
References:
Van Boeckel T.P., Brower C., Gilbert M., Grenfell B.T., Levin S.A., Robinson T.P., (2015). Global trends in antimicrobial use in food animals. Proc Natl Acad Sci. 112(18):5649–54.
Serraino, A., Giacometti, F., Marchetti, G., Zambrini, A.V., Zanirato, G., Fustini, M. & Rosmini, R. (2013). Survey on Antimicrobial Residues in Raw Milk and Antimicrobial Use in Dairy Farms in the Emilia-Romagna Region, Italy. Italian Journal of Animal Science. 12(3):422-425.
Gaudin V., Hedou C., Soumet C., & Verdon E., (2018). Multiplex immunoassay based on biochip technology for the screening of antibiotic residues in milk: validation according to the European guideline. Food Additives & Contaminants: Part A: https: //doi.org/ 10.1080/19440049. 2018. 1538572
Gaugain-Juhel M., Delépine B., Gautier S., Fourmond M.P., Gaudin V., Hurtaud-Pessel D., Verdon E., Sanders P. (2009). Validation of a liquid chromatography-tandem mass spectrometry screening method to monitor 58 antibiotics in milk: a qualitative approach. Food Additives & Contaminants: Part A. 26:1459–1471.