Validation of the AnticFast® Beta-Lactams & Tetracyclines Combo Test Kit for Detection of Residues of Beta-Lactams (Penicillins and Cefalosporins) and Tetracyclines in Raw Cows' Milk: AOAC Performance Tested MethodSM 032403.

BACKGROUND: The AnticFast® Beta-lactams & Tetracyclines Combo Test Kit is a qualitative two-step (2 min + 5 min) rapid lateral flow assay to detect ß-lactam (penicillins and cefalosporins) and tetracycline antibiotic residues in raw commingled cows' milk. OBJECTIVE: The method performance was evaluated according to Commission Implementing Regulation 2021/808 and Community Reference Laboratories Residues Guidelines for the Validation of Screening Methods for Residues of Veterinary Medicines and submitted for AOAC Performance Tested MethodsSM certification. METHODS: The AnticFast Beta-lactams & Tetracyclines Combo Test Kit was evaluated for detection capability (CCß), selectivity, false-positive results, repeatability, robustness, suitability for various milk types and milk compositions, milks from various species, and test kit consistency and stability. Samples included milks spiked at concentrations bracketing the EU maximum residue limits (MRLs) for ß-lactams and tetracyclines as well as bulk farm and tanker milks. RESULTS: The AnticFast Beta-lactams & Tetracyclines Combo Test Kit is specific for the detection of residues of ß-lactams and tetracyclines in milk and does not detect residues from other antibiotic families. Interference was seen with clavulanic acid, a ß-lactamase inhibitor, which was expected. The test can detect (minimum a 95% detection) all residues of ß-lactams (penicillins and cepfalosporins) and tetracyclines (parent drugs and their 4-epimers) present on the EU-MRL list for milk at their respective MRL except for desfuroylceftiofur and cefalexin, with a 95% detection only above the MRL. No false positives were detected in 599 blank samples (out of 305 farm and 301 tanker load samples) tested on both channels. Five real positives were detected and confirmed on the tetracycline channel for the farm milk samples, and two positives were detected and confirmed on the ß-lactam channel for the tanker samples. Robustness testing indicated that the detection in high-protein raw cows' milk and heat-treated milk types (UHT, sterilized, and reconstituted milk powder) may be slightly hampered. For substances with a detection capability well below the MRL, this interference does not cause problems since detection at MRL remains guaranteed, but care should be taken for substances with a CCß at or near their MRL. Diminished sample flow was seen for high-fat raw cows' milk and for all other cows' milk types other than raw milk and blank ewes' milk, so sample flow should always be verified for these milk types. CONCLUSIONS: Results of this validation show that the AnticFast Beta-lactams & Tetracyclines Combo Test Kit is a reliable test for rapid screening of raw cows' milk for residues of ß-lactam and tetracycline antibiotics. HIGHLIGHTS: The AnticFast Beta-lactams & Tetracyclines Combo Test Kit is an easy, reliable, robust, and highly specific test for screening of ß-lactam (penicillins and cefalosporins) and tetracycline antibiotic residues in milk with incubation at room temperature. In raw cows' milk, all tetracyclines are detected below 10 µg/kg.

Time-resolved fluorescence microspheres-antibody-penicillin-binding protein assisted construction of immunochromatographic assay for sensitive detection of 22 ß-lactams in milk.

A sensitive immunochromatographic assay (ICA) using time-resolved fluorescence microspheres (TRFMs) coupled with an indirect-labeling mode was developed for simultaneously determining 22 kinds of ß-lactams in milk samples. The TRFMs labeled anti-receptor monoclonal antibodies (mAbs) conjugated to penicillin-binding proteins (PBPs) as ternary TRFMs-mAb-PBPs (TMP) nanoscaffolds provide excellent solubility, brightness, and stability. Thanks to the fact that they not only fully expose the binding sites of PBPs, thereby enhancing the biological affinity of PBPs towards the target, but also generated superb fluorescence signals, the versatile TMP manifested unique possibilities as efficient probes for ICA with remarkable enhancement in sensitivity in ß-lactams screening. The results showed that the standard curves of the 22 varying ß-lactams displayed linearity in their respective concentration ranges (R2 > 0.98), with the cutoff values of 1-100 ng/mL. The constructed TMP-ICA was successfully applied to the analysis of real milk, with consistent results compared with liquid chromatography-tandem mass spectrometry (LC-MS), providing an effective method for sensing ß-lactams in food matrices.

Transfer of ß-lactam and tetracycline antibiotics from spiked bovine milk to Dambo-type cheese, whey, and whey powder.

The aim of this study was to investigate the transfer of residues of five ß-lactam antibiotics (ampicillin, penicillin G, cloxacillin, dicloxacillin and cephalexin) and two tetracyclines (tetracycline and oxytetracycline) in the processing of cheese and whey powder, evaluating the effect of the processes and the final concentration in each product generated. Raw milk was fortified at two concentration levels with the seven antibiotics. The first concentration level (C1) was chosen according to the maximum residue limit (MRL) of each antibiotic (ampicillin and penicillin G: 4 µg kg-1; cloxacillin and dicloxacillin: 30 µg kg-1; cephalexin, tetracycline and oxytetracycline: 100 µg kg-1). The second concentration level (C2) was spiked as follows according to each antibiotic: 0.5 MRL (cloxacillin, dicloxacillin, cephalexin), 0.1 MRL (tetracycline and oxytetracycline) and 3 MRL (ampicillin and penicillin G). The antibiotics were analyzed by LC-MS/MS. No ampicillin or penicillin G residues were found in cheese or whey powder, although they were detected in whey at concentrations similar to those added to raw milk. Cephalexin was mostly distributed in whey between 82% and 96%, being the antibiotic that presented the highest concentration in whey powder (784 ± 98 µg kg-1) when milk was spiked at the MRL. The whey distribution of cloxacillin and dicloxacillin ranged from 57% to 59% for cloxacillin and from 46% to 48% for dicloxacillin, and both concentrated in whey powder. Tetracyclines were the antibiotics that concentrated in cheese, with retentions between 75% and 80% for oxytetracycline and between 83% and 87% for tetracycline. The distribution of antibiotics in the dissimilar stages of the cheese and whey powder production processes, as well as their concentration in the final products, depend on each type of antibiotic. Knowledge of the transfer of antibiotic residues during the process and final disposal is an input for the risk assessment of their consumption.

Validation of the AnticFast® Beta-Lactams Rapid Test Kit for Detection of Beta-Lactams (Penicillins and Cephalosporins) in Raw Cow's Milk: AOAC Performance Tested MethodSM 032303.

BACKGROUND: AnticFast® Beta-Lactams Rapid Test Kit is a qualitative two-step (2 min + 5 min) rapid lateral flow assay to detect ß-lactam (penicillins and cephalosporins) antibiotic residues in raw commingled cow's milk. OBJECTIVE: The method performance was evaluated according to Commission Decision 2002/657/EC, Commission Implementing Regulation 2021/808, and Community Reference Laboratories Residues Guidelines for the Validation of Screening Methods for Residues of Veterinary Medicines. METHODS: The AnticFast Beta-Lactams Rapid Test Kit was evaluated for detection capability, selectivity, false-positive results, repeatability, robustness, suitability for various milk types and milk compositions, milks from various species, and test kit consistency and stability. Samples included milks spiked at concentrations bracketing the EU maximum residue limits (MRLs) for ß-lactams as well as bulk farm and tanker milks. RESULTS: The AnticFast Beta-Lactams Rapid Test Kit is specific for the detection of ß-lactams in milk and does not detect compounds from other antibiotic families. Interference was seen with clavulanic acid, a ß-lactamase inhibitor, which was expected. The test can detect all residues of ß-lactams (penicillins and cephalosporins) present on the EU-MRL list for milk at their respective MRL except for desfuroylceftiofur and cephalexin, which were above the MRL. No false positives were detected in the 602 (300 blank farm and 302 tanker load) samples tested. Robustness testing indicated that the detection in heat-treated milk types may be slightly hampered. For substances with a detection capability well below the MRL, this interference does not cause problems since detection at MRL remains guaranteed, but care should be taken for substances with a CCß at or near their MRL. Diminished sample flow was seen with reconstituted milk powder and blank ewes' milk, so sample flow should always be verified for these milk types. CONCLUSIONS: Results of this validation show that the AnticFast Beta-Lactams Rapid Test Kit is a reliable test for rapid screening of raw cows' milk for residues of ß-lactam antibiotics. HIGHLIGHTS: AnticFast Beta-Lactams Rapid Test Kit is an easy, realiable, robust and highly specific test for screening of raw cows' milk for residues of penicillins and cephalosporins.

Seasonal analysis of commonly prescribed antibiotics in Istanbul city.

Antibiotics are among the most common medicine groups since they are used to treat infectious diseases, as nutritional supplements in livestock breeding, and for preservation in the food industry. Turkey is among the highest antibiotic consumers in the world. In the present study, the most popular 14 antibiotics available in Turkey were monitored in one hospital sewage and two urban wastewater treatment plant influents and effluents seasonally in Istanbul province, the largest metropolitan center in Turkey. The present research aimed to develop a robust analytical method to determine 14 antibiotics, including six chemical groups, in environmental matrices which are considered significant antibiotic pollution sources, namely hospital sewage and urban wastewater. Solid-phase extraction (SPE) and UPLC-MS/MS analysis parameters included optimized column temperature, eluent, mobile phase, and flow rate. Three SPE cartridges were employed in recovery studies. The antibiotic recovery rates varied between 40 and 100%, and all analytes were identified within 3 min with UPLC-MS/MS under optimal conditions. It was determined that method detection limits (MDLs) varied between 0.07 and 2.72 µg/L for the antibiotics. In all seasons, the highest beta-lactam group antibiotic concentrations were identified in hospital sewage. The season with the greatest variety of antibiotics in urban wastewater was spring. Clarithromycin and ciprofloxacin were the antibiotics determined at the highest concentration in the influent and effluent of the wastewater treatment plant in all seasons. This study showed that the most widely used beta-lactam group antibiotics were found in high amounts in hospital sewage wastewater but in low concentrations in the treatment plants, and hence, it is seen that the degradability of beta-lactam group antibiotics was high. The presence of clarithromycin, ciprofloxacin, lincomycin, levofloxacin, and trimethoprim antibiotics in hospital sewage in higher amounts and also in inlet and outlet of wastewater treatment plants proves that those are resistant antibiotics.

Potential reservoirs of antimicrobial resistance in livestock waste and treated wastewater that can be disseminated to agricultural land.

Livestock manure, dairy lagoon effluent, and treated wastewater are known reservoirs of antibiotic resistance genes (ARGs), antibiotic-resistant bacteria (ARB), and virulence factor genes (VFGs), and their application to agricultural farmland could be a serious public health threat. However, their dissemination to agricultural lands and impact on important geochemical pathways such as the nitrogen (N) cycle have not been jointly explored. In this study, shotgun metagenomic sequencing and analyses were performed to examine the diversity and composition of microbial communities, ARGs, VFGs, and N cycling genes in different livestock manure/lagoon and treated wastewater collected from concentrated animal feeding operations (CAFOs) and a municipal wastewater treatment plant along the west coast of the United States. Multivariate analysis showed that diversity indices of bacterial taxa from the different microbiomes were not significantly different based on InvSimpson (P = 0.05), but differences in ARG mechanisms were observed between swine manure and other microbiome sources. Comparative resistome profiling showed that ARGs in microbiome samples belonged to four core resistance classes: aminoglycosides (40-55 %), tetracyclines (30-45 %), beta-lactam-resistance (20-35 %), macrolides (18-30 %), and >50 % of the VFGs that the 24 microbiomes harbored were phyletically affiliated with two bacteria, Bacteroidetes fragilis and Enterobacter aerogenes. Network analysis based on Spearman correlation showed co-occurrence patterns between several genes such as transporter-gene and regulator, efflux pump and involved-in-polymyxin- resistance, aminoglycoside, beta-lactam, and macrolide with VFGs and bacterial taxa such as Firmicutes, Candidatus Themoplasmatota, Actinobacteria, and Bacteroidetes. Metabolic reconstruction of metagenome-assembled genome (MAGs) analysis showed that the most prevalent drug resistance mechanisms were associated with carbapenem resistance, multidrug resistance (MDR), and efflux pump. Bacteroidales was the main taxa involved in dissimilatory nitrate reduction (DNRA) in dairy lagoon effluent. This study demonstrates that the dissemination of waste from these sources can increase the spread of ARGs, ARB, and VFGs into agricultural lands, negatively impacting both soil and human health.

Analysis of a Cell Wall Mutant Highlights Rho-Dependent Genome Amplification Events in Staphylococcus aureus.

In a study of antibiotic resistance in Staphylococcus aureus, specific cell wall mutants were previously generated for the peptidoglycan biosynthesis gene murF, by the insertion of an integrative plasmid. A collection of 30 independent mutants was obtained, and all harbored a variable number of copies of the inserted plasmid, arranged in tandem in the chromosome. Of the 30 mutants, only 3, F9, F20 and F26, with a lower number of plasmid copies, showed an altered peptidoglycan structure, lower resistance to ß-lactams and a different loss-of-function mutation in rho gene, that encodes a transcription termination factor. The rho mutations were found to correlate with the level of oxacillin resistance, since genetic complementation with rho gene reestablished the resistance and cell wall parental profile in F9, F20 and F26 strains. Furthermore, complementation with rho resulted in the amplification of the number of plasmid tandem repeats, suggesting that Rho enabled events of recombination that favored a rearrangement in the chromosome in the region of the impaired murF gene. Although the full mechanism of reversion of the cell wall damage was not fully elucidated, we showed that Rho is involved in the recombination process that mediates the tandem amplification of exogeneous DNA fragments inserted into the chromosome. IMPORTANCE The cell wall of bacteria, namely, peptidoglycan, is the target of several antibiotic classes such as ß-lactams. Staphylococcus aureus is well known for its capacity to adapt to antibiotic stress and develop resistance strategies, namely, to ß-lactams. In this context, the construction of cell wall mutants provides useful models to study the development of such resistance mechanisms. Here, we characterized a collection of independent mutants, impaired in the same peptidoglycan biosynthetic step, obtained through the insertion of a plasmid in the coding region of murF gene. S. aureus demonstrated the capacity to overcome the cell wall damage by amplifying the copy number of the inserted plasmid, through an undescribed mechanism that involves the Rho transcription termination factor.

Antibiotic resistance genes and bacterial diversity: A comparative molecular study of treated sewage from different origins and their impact on irrigated soils.

Present study aims to investigate how is soil affected following irrigation with treated effluents of different origins by analysing the bacterial diversity, metabolic diversity and antibiotic resistance genes (ARGs). Comparative analysis with previously reported ARGs in effluents was performed to understand the mobility of ARGs from treated wastewater to the irrigated soil with respect to the control soil regimen. Acinetobacter, Burkholderia and Pseudomonas were observed as the most abundant genera in all the samples. The metabolic gene abundance of all the samples suggests a prominent contribution to natural mineral recycling. Most abundant ARGs observed encode resistance for clindamycin, kanamycin A, macrolides, paromomycin, spectinomycin and tetracycline. Treated effluent reuse did not appear to enhance the ARG levels in soils in most cases except for institutional treatment site (M), where the ARGs for aminoglycosides, ß-lactams and sulfonamides were found to be abundantly present in both treated effluent and the irrigated soil. This study finds the importance of wastewater treatment from different origins and the impact of treated wastewater reuse in irrigation. This study also emphasises on the better understanding of ARGs mobility from water to soil.

Validation of Delvotest® Fast BT for Detection of ß-Lactams and Tetracyclines in Raw Cow's Milk: Performance Tested MethodSM 102104.

BACKGROUND: Delvotest® Fast BT is a rapid test for qualitative detection of ß-lactams and tetracycline residues in raw, comingled cow's milk. OBJECTIVE: The test kit was validated under the AOAC Performance Tested MethodSM certification program. Detection capabilities of penicillin G and tetracycline residues were determined. Also, the test method ruggedness, cross-reactivities, interference and masking effects by other antibiotic residues, influence of bacterial and somatic cells, product consistency, and stability of the test kit were studied. METHOD: The test cassette is placed in the specially designated incubator (Delvotest® Fast Start III), the milk sample is added to the loading site, and the cassette is incubated for 7 min at 50°C. The test result is read instrumentally using a specially designated reader (Delvotest® Fast Go Max). RESULTS: Measurements in the method developer study and subsequent confirmation during independent laboratory study showed detection capabilities of 2.1 ppb penicillin G and 95 ppb tetracycline. Regarding robustness, no false positives nor false negatives were found in the method ruggedness, cross-reactivity, masking, and interference studies. In addition, the test performance is not affected by a high number of bacterial or somatic cells present in the milk sample. Stability studies demonstrated good performance of the kit during the shelf life of 12 months. CONCLUSIONS: The validation study demonstrates that Delvotest® Fast BT conforms to the product performance claims and confirms the robustness of the test toward small variations in the operation parameters and normally expected variations in milk samples. HIGHLIGHTS: Delvotest® Fast BT provides users in the entire milk flow logistics from grass to glass (e.g. farmers and dairies) with a rapid and easy to use method for qualitative detection of ß-lactams and tetracycline residues in raw cow's milk.

Multi-class, multi-residue determination of 132 veterinary drugs in milk by magnetic solid-phase extraction based on magnetic hypercrosslinked polystyrene prior to their determination by high-performance liquid chromatography-tandem mass spectrometry.

A quantitative multi-class multi-residue analytical method was developed for the determination of veterinary drugs in milk by high-performance liquid chromatography - tandem mass spectrometry (HPLC-MS/MS). A total of 132 veterinary drugs investigated belonged to almost 15 classes including sulfonamides, ß-lactams, tetracyclines, quinolones, macrolides, nitrofurans, nitroimidazoles, phenicols, lincosamides, pleuromutilins, macrocyclic lactones, quinoxaline antibiotics, benzimidazoles, anthelmintics, coccidiostats and some others. A magnetic solid-phase extraction procedure was developed using magnetic hypercrosslinked polystyrene (HCP/Fe3O4) for the sample preparation prior to HPLC-MS/MS without deproteinization step. The results indicated recoveries of 85-107% for 14 sulfonamides, 85-120% for 13 ß-lactams, 89-115% for 4 tetracyclines, 82-119% for 14 quinolones, 82-115% for 8 macrolides, 97-109% for 4 nitrofurans, 84-115% for 10 nitroimidazoles, 89-114% for 3 phenicols, 86-111% for 3 lincosamides, 97-102% for 2 pleuromutilins, 72-88% for 4 macrocyclic lactones, 87-104% for 4 quinoxaline antibiotics, 76-119% for 21 benzimidazoles, 79-115% for 12 anthelmintics, 81-118% for 12 coccidiostats and 75-119 % for 5 unclassified drugs, with relative standard deviations (RSDs) of less than 20%, and the LOQs ranged from 0.05 to 1 µg kg-1. This methodology was then applied to field-collected real milk samples and trace levels of some veterinary drugs were detected.