A novel platform test to detect beta-lactam residues in raw milk

The aim of this study was to analyze the microbiome of raw milk obtained from three semi-subsistence farms (A, B, and C) located in the Kuyavian-Pomeranian Voivodeship in Poland. The composition of drinking milk was assessed on the basis of 16S rRNA gene sequencing using the Ion Torrent platform. Based on the conducted research, significant changes in the composition of the milk microbiome were found depending on its place of origin. Bacteria belonging to the Bacillus (17.0%), Corynebacterium (12.0%) and Escherichia-Shigella (11.0%) genera were dominant in the milk collected from farm A. In the case of the milk from farm B, the dominant bacteria belonged to the Acinetobacter genus (21.0%), whereas in the sample from farm C, Escherichia-Shigella (24.8%) and Bacillus (10.3%) dominated the microbiome. An analysis was performed using the PICRUSt tool (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) in order to generate a profile of genes responsible for bacterial metabolism. The conducted analysis confirmed the diversity of the profile of genes responsible for bacterial metabolism in all the tested samples. On the other hand, simultaneous analysis of six KEGG Orthologs (KO), which participated in beta-lactam resistance responsible for antibiotic resistance of bacteria, demonstrated that there is no significant relationship between the predicted occurrence of these orthologs and the place of existence of microorganisms. Therefore, it can be supposed that bacterial resistance to beta-lactam antibiotics occurs regardless of the environmental niche, and that the antibiotic resistance maintained in the population is a factor that shapes the functional structure of the microbial consortia.

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Control de residuos de antibióticos en leche cruda en una empresa lechera en Jalisco México: estudio retrospectivo

e-CUCBA ◽

10.32870/ecucba.vi16.190 ◽

2021 ◽

Vol 8 (16) ◽

pp. 1-5

Author(s):

Mario Noa- Pérez ◽

◽

Silvia Ruvalcaba- Barrera ◽

José Pablo Torres- Morán ◽

Ramón Reynoso- Orozco ◽

...

Keyword(s):

Control Measure ◽

Reduction Rate ◽

Raw Milk ◽

Raw Material ◽

First Year ◽

Antibiotic Residues ◽

Beta Lactam ◽

Residue Detection ◽

Average Size ◽

The Impact

The current regulation in Mexico is explicit about the prohibition of the presence of antibiotic residues in raw cow's milk. Due to the impact of the presence of antibiotic residues in milk in the production of dairy products, some dairy companies have implemented their own programs to control antibiotic residues to avoid collection of contaminated material. The aim of this study was to analyze the results of the implementation of a program to control the presence of antibiotic residues in raw cow´s milk collected by a big local dairy industry. During the applications of this program, the frequency of contaminated milk containing the two main groups of antibiotics: β-lactams and tetracyclines, in 9 concentration facilities of a local dairy company, with an average size receiving 800,000- 1,000,000 L/day during 5 years. The antibiotic residue detection was performed using SNAP® Beta-Lactam Test and SNAP® Tetracycline Test simultaneously. During the first year, 222 tanks (2,955,538 L) were tested, of them 93 were positive for antibiotic residues (42.11%). In the fifth year, from 889 tanks containing 5,658,062 L only 13 were positive (1.46%), obtaining a reduction rate of 10.16% annual average. When the companies control the presence of inhibitor residues in the raw milk buy the milk, the farmers discard the contaminated milk; however, they sell it even with antibiotics when the buyers do not carry out controls, so this control measure becomes a solution. The results showed a significant decrease in contamination, being a useful solution to the problem of the appearance of antibiotic residues in milk in the raw material of the plant.

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Validation Study of a Receptor-Based Lateral Flow Assay for Detection of Beta-Lactam Antibiotics in Milk

Journal of AOAC International ◽

10.1093/jaoac/92.3.959 ◽

2009 ◽

Vol 92 (3) ◽

pp. 959-974 ◽

Cited By ~ 5

Author(s):

Mohamed Abouzied ◽

Michael Sarzynski ◽

Aaron Walsh ◽

Heather Wood ◽

Mark Mozola

Keyword(s):

Bovine Milk ◽

Operating Parameter ◽

Raw Milk ◽

Cross Reactivity ◽

Penicillin G ◽

Beta Lactam ◽

Milk Samples ◽

Beta Lactam Antibiotics ◽

Cell Counts ◽

Lactam Antibiotic

Abstract Avalidation study designed tomeet the requirements of the AOAC Research Institute and the U.S. Food and Drug Administration (FDA), Center for Veterinary Medicine, was conducted for a receptor-based, immunochromatographic method (BetaStar US) for detection of beta-lactam antibiotic residues in raw, commingled bovine milk. The assay was found to detect amoxicillin, ampicillin, cephapirin, cloxacillin, and penicillin G at levels below the FDA tolerance/safe levels but above the maximum sensitivity thresholds established by the National Conference on Interstate Milk Shipments. Results of the Part I (internal) and Part II (independent laboratory) dose-response studies using spiked samples were in very close agreement for all five drugs tested, with differences between the Part I and Part II 90/95 sensitivity values ranging from 0 to 1 ppb. The test was able to detect all five drugs at the approximate 90/95 sensitivity levels when present as incurred residues in milk collected from cows that had been treated with the specific drug. Asixth drug, ceftiofur, was found to be undetectable at levels of 500 ppb (as total ceftiofur metabolites from incurred residues in milk samples). The selectivity of the assay was 100, because no false-positive results were obtained in tests of >1000 control milk samples. The assay was found to be applicable to the testing of frozen raw milk samples. Results of ruggedness experiments established the operating parameter tolerances for the BetaStar US assay. Results of cross-reactivity testing established that the assay detects certain other beta-lactam drugs (dicloxacillin and ticarcillin), but it does not cross-react with any of 30 drugs belonging to other classes. Abnormally high bacterial or somatic cell counts in rawmilk produced no interference with the ability of the test to detect beta-lactams at tolerance/safe levels.

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Comparison of Rapid Tests Used to Detect Antibiotic Residues in Milk1

Journal of Food Protection ◽

10.4315/0362-028x-53.2.158 ◽

1990 ◽

Vol 53 (2) ◽

pp. 158-164 ◽

Cited By ~ 19

Author(s):

GARY F. SENYK ◽

JOSEPH H. DAVIDSON ◽

JANICE M. BROWN ◽

ERIC R. HALLSTEAD ◽

JOHN W. SHERBON

Keyword(s):

Bacillus Stearothermophilus ◽

Raw Milk ◽

Penicillin G ◽

Beta Lactam ◽

Rapid Methods ◽

Milk Samples ◽

Beta Lactam Antibiotics ◽

Rapid Tests ◽

Combined Data ◽

Disc Assay

Five rapid methods for detection of antibiotics in milk were compared. The Bacillus stearothermophilus var. calidolactis disc assay was also performed on the same samples. The rapid methods were: Angenics Spot Test, Charm II, Delvotest P, Penzyme Farm, and Penzyme Lab III. Ten antibiotics (penicillin G, cephapirin, cloxacillin, ampicillin, streptomycin, chloramphenicol, erythromycin, novobiocin, tetracycline, and gentamicin) were used individually to spike eight raw milk samples at five levels of antibiotic. Antibiotic levels were chosen that would result in zones of <16 mm, 16 mm, and >16 mm on the disc assay. Only the disc assay, Charm II and Delvotest P were compared on non-beta-lactam antibiotics. A small percentage of milks with no antibiotic added tested positive with the Charm II and Penzyme Lab III. On combined data for penicillin G, cephapirin, and cloxacillin, for which all methods were compared, the percent correctly categorized as pass (below actionable) for the <16 mm zone spiked level, reject or caution at the 16 mm zone level, and reject or caution at the >16 mm zone level were: Angenics 79, 83, 100; Charm II 66, 92, 100; Delvotest P 74, 93, 100; Disc Assay 100, 74, 100; Penzyme Farm 93, 61, 92; Penzyme Lab III 81,78, 100 respectively. In most cases, the rapid methods showed greater apparent sensitivity than the disc assay and did not fail to reject milks spiked with antibiotic in excess of the 16 mm zone level.

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Quantitative Estimates of Beta-Lactam Residues in Raw Milk Around a Reference Standard: Collaborative Study

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/65.6.1407 ◽

1982 ◽

Vol 65 (6) ◽

pp. 1407-1412

Author(s):

Roy E Ginn ◽

Ronald A Case ◽

Vernal S Packard ◽

Sita R Tatini ◽

◽

...

Keyword(s):

Bacillus Stearothermophilus ◽

Collaborative Study ◽

Raw Milk ◽

Assay Method ◽

Zone Size ◽

Reference Standard ◽

Beta Lactam ◽

Quantitative Estimates ◽

The Difference ◽

Disc Assay

Abstract A collaborative study was conducted to determine the reliability of a Bacillus stearothermophilus disc assay method for differentiating various concentrations of penicillin in raw milk. Participating laboratories tested 10 different samples (including one negative) in blind duplicate. Triplicate standards were alternated with triplicate unknowns around the periphery of each of 5 different plates. Zone diameters were measured and the difference in zone size of pairs of adjacent standard and unknown samples were analyzed by a paired t-test. Penicillin concentrations 0.003 IU/mL different from the reference concentrations were consistently distinguishable at a 95% confidence level. Such discriminatory power was determined to be possible with as few as 3 plates (9 replicates) per unknown. The method has been adopted official first action.

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Bacillus stearothermophilus Disc Assay for Detection of Inhibitors in Milk: Collaborative Study

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/65.5.1208 ◽

1982 ◽

Vol 65 (5) ◽

pp. 1208-1214 ◽

Cited By ~ 1

Author(s):

James W Messer ◽

James E Leslie ◽

Gary A Houghtby ◽

James T Peeler ◽

Jerald E Barnett ◽

...

Keyword(s):

Bacillus Stearothermophilus ◽

Collaborative Study ◽

Raw Milk ◽

Penicillin G ◽

Confirmatory Test ◽

Beta Lactam ◽

Aoac Method ◽

Positive Results ◽

Paper Disc ◽

Disc Assay

Abstract A 2-part (A and B) collaborative study was conducted on a Bacillus stearothermophilus paper disc (12.7 mm) method to detect residual inhibitors in milk. The 18 participating collaborators assayed raw milk samples spiked with a beta-lactam (penicillin G). Of the 18 collaborators, 14 participated in part A and 16 in part B. Part A demonstrated that either Antibiotic Medium No. 4 or PM Indicator Agar is suitable for use in the assay. The lowest concentration detectable, not significantly different from 100% at the α = 0.05 level, was 0.008 unit/mL with either medium. Part B demonstrated that the sensitivity of the method is equal to that of the current AOAC method (16.131- 16.136). The concentration of beta-lactam detected by 50% of the analysts was 0.003-0.005 unit/mL in this study, compared with 0.005 unit/mL reported in an earlier collaborative study on the current AOAC method. No false positive results were reported in part A or part B. All samples found positive by the confirmatory test in part B were correctly identified as a beta-lactam with commercial Penase discs. The lowest concentration detectable by the method, not significantly different from 100% at the α = 0.05 level, was 0.008 unit/mL. The method was adopted official first action.

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Quantitative Assay of Beta-Lactam Residues in Raw Milk Using a Disc Assay Method

Journal of Food Protection ◽

10.4315/0362-028x-45.6.571 ◽

1982 ◽

Vol 45 (6) ◽

pp. 571-573 ◽

Cited By ~ 9

Author(s):

R. E. GINN ◽

R. CASE ◽

V. S. PACKARD ◽

S. TATINI

Keyword(s):

Bacillus Stearothermophilus ◽

Raw Milk ◽

Technical Skill ◽

Assay Method ◽

Reference Level ◽

Reference Standard ◽

Beta Lactam ◽

Quantitative Estimates ◽

Fixed Reference ◽

Disc Assay

Numerous methods have been developed to determine presence of antibiotics in raw milk. Until recently, major effort had been placed on qualitative considerations, and primarily for detecting presence of penicillin (beta-lactam) residues. Only one method, the Sarcina lutea Cylinder Plate (CP) procedure, has been modified to provide for quantitative estimates. The CP method is a rather long, tedious test, requiring considerable technical skill. Need for a simpler, faster quantitative method was apparent. This paper describes a method for making quantitative estimates of beta-lactam residues around a fixed reference standard. The method uses Bacillus stearothermophilus in a disc assay test. Quantitative estimates above or below the reference level of antibiotic are computed through a paired-t statistical analysis. The test can be completed within 3 h.

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Evaluation of rapid beta-lactam antibiotic residues detection kits for raw milk

African Journal of Food Science ◽

10.5897/ajfs2021.2145 ◽

2021 ◽

Vol 15 (11) ◽

pp. 353-359

Author(s):

W. Ndungu Teresiah ◽

Omwamba Mary ◽

S. Muliro Patrick

Keyword(s):

Raw Milk ◽

Antibiotic Residues ◽

Beta Lactam ◽

Beta Lactam Antibiotic ◽

Lactam Antibiotic

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Validation of the BetaStar® Advanced for Beta-lactams Test Kit for the Screening of Bulk Tank and Tanker Truck Milks for the Presence of Beta-lactam Drug Residues

Journal of AOAC International ◽

10.5740/jaoacint.18-0075 ◽

2018 ◽

Vol 101 (6) ◽

pp. 1813-1827

Author(s):

Andrew Denhartigh ◽

Lindsay Reynolds ◽

Katherine Palmer ◽

Frank Klein ◽

Jennifer Rice ◽

...

Keyword(s):

Bovine Milk ◽

Operating Parameter ◽

Raw Milk ◽

Cross Reactivity ◽

Beta Lactam ◽

Beta Lactams ◽

Cell Counts ◽

Drug Classes ◽

Test Kit ◽

Control Milk

Abstract A validation study was conducted for an immunochromatographic method (BetaStar® Advanced for Beta-lactams) for the detection of beta-lactam residues in raw, commingled bovine milk. The assay detected amoxicillin, ampicillin, cloxacillin, penicillin, cephapirin, and ceftiofur below the U.S. Food and Drug Administration tolerance levels but above the maximum sensitivity thresholds established by the National Conference on Interstate Milk Shipments. The results of internal and independent laboratory dose-response studies employing spiked samples were in agreement. The test detected all six drugs at the approximate 90/95% sensitivity levels in milk from cows treated with each drug. Selectivity of the assay was 100%, as no false-positive results were obtained in testing 1148 control milk samples. Testing the estimated 90/95% sensitivity level for amoxicillin (8.5 ppb), ampicillin (6.9 ppb), cloxacillin (8.9 ppb), penicillin (4.2 ppb), and cephapirin (17.6 ppb), and at 100 ppb for each antibiotic, resulted in 94–100% positive tests for each of the beta-lactam drugs. The results of ruggedness experiments established the operating parameter tolerances for the assay. Cross-reactivity testing established that the assay detects other certain beta-lactam drugs, but it does not cross-react with any of 30 drugs belonging to seven different drug classes. Abnormally high bacterial or somatic cell counts in raw milk produced no assay interference.

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