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.

LC-MS/MS methodology development and validation for the screening and quantification of five antibiotics in water.

The generalized use of antibiotics in veterinary medicine may cause the development of antibiotic resistance, namely, in farmed fish, and subsequently, transmission of such resistance to humans. To overcome this serious problem, it is necessary to improve the efficiency of antibiotic removal processes from water, and a route for attaining that goal is the use of hydrophobic ionic liquids. LC-MS/MS is an analytical technique with proven utility for pharmaceutical identification and quantification. A methodology for the identification and quantification of five antibiotics, namely ampicillin, amoxicillin, chloramphenicol, enrofloxacin and oxytetracycline, from four families, namely ß-lactams, phenicols, quinolones and tetracyclines, was developed and validated using one SPE extraction and clean-up step for detection analysis by LC-MS/MS. The selected linear range for each analyte is as follows: 5-200 ng mL-1 for ampicillin; 0.1-200 ng mL-1 for amoxicillin and chloramphenicol; and 1-200 ng mL-1 for enrofloxacin and oxytetracycline, respectively. The limits of detection for each target analyte vary between 0.01 ng mL-1 and 0.81 ng mL-1, and the limits of quantification for each target analyte vary between 0.1 ng mL-1 and 5 ng mL-1 for the five antibiotics under study. The extraction recovery value range is between 89.91% and 100.33%. The validation proved that the developed methodology is suitable for the identification and quantification of ampicillin, amoxicillin, chloramphenicol, enrofloxacin and oxytetracycline. After validation, this new assay was successfully applied to a liquid-liquid extraction process using a hydrophobic ionic liquid.

Improvement of the QuEChERS extraction step by matrix-dispersion effect and application on beta-lactams analysis in wastewater sludge by LC-MS/MS.

In the last decade, beta-lactams use in veterinary and human medicine increased to represent today about 15% of the overall consumption. Beta-lactams tend to degrade and metabolize in the environment. Therefore, analytical methods must be sensitive enough to quantify low concentrations of the parent molecules and also allow detection of metabolites. This study presents the development of a modified QuEChERS method for the extraction of seven beta-lactams and one degradation product (Amoxicillin, Ampicillin, Cefapirin, Cefoperazone, Cefquinome, Ceftiofur, Cloxacillin, and Amoxicillin-Diketopiperazine) from sewage treatment plant sludge and their analysis by liquid chromatography coupled with tandem mass spectrometry. Before the QuEChERS extraction, a dispersion step of the sample with EDTA-treated sand was optimized and added, allowing to facilitate the exchanges between the matrix and the extraction solvent. Then, to decrease the interferences present in the extract, a fast and efficient pass-through SPE was implemented. The optimized method was validated and showed satisfactory performances, in adequacy with the analysis of beta-lactams in solid environmental matrices. Limits of quantification lower than 20 ng.g-1 for all analytes, high accuracy (96%-114% quantification on spiked samples nominal concentration) and interday precision (2%-12% RSD) were obtained. This method was then applied to eight sludge samples. Cefapirin and amoxicillin-diketopiperazine were detected in four samples each, at concentrations of 10.2-53.3 ng.g-1 and 3.0-9.5 ng.g-1 respectively. Thus, the developed method is very effective for the extraction of beta-lactams from environmental solid matrices.

Antibiotic standards stored as a mixture in water: methanol are unstable at various temperatures irrespective of pH and glass container silanization.

It is well-established that antibiotics stored individually at their optimal pH and in appropriate solvents are stable over time. However, limited information exists on the stability of antibiotics from multiple classes when prepared and stored as a mixture prior to multiresidue analysis by mass spectrometry. This study tested the stability of antibiotic standard mixtures from eight classes [amphenicols, tetracyclines, sulfonamides, quinolones, macrolides, ß-lactams, lincosamides and miscellaneous (i.e., trimethoprim)] in relation to the water:methanol ratio, presence of sodium hydroxide base (to solubilise quinolones), storage temperature, and container type including plain and silanized glass vials. Antibiotics were analysed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry. Several antibiotics, mainly quinolones, tetracyclines and macrolides, were unstable when stored as mixtures for one week regardless of the water:methanol ratio, storage temperature (4, -20 or -80 °C) and presence/absence of sodium hydroxide. Silanization of glassware improved the storage stability of quinolones and macrolides but reduced the stability of tetracyclines and other antibiotics including florfenicol amine, penicillin G, erythromycin and sulfadiazine. Our results show that several antibiotics in water:methanol are unstable when stored as a mixture and suggest a limited advantage of using base or silanized glass vials for the preparation and storage of antibiotic standards mixed together. Freshly prepared antibiotic standard mixtures are recommended for multi-residue quantitation of antibiotics.Abbreviations AMOX: amoxicillin; AMP: ampicillin; AZ: azithromycin; CAP: chloramphenicol; CE: collision energy; CTC: chlortetracycline; CIP: ciprofloxacin; DOX: doxycycline; ENO: enoxacin; ENRO: enrofloxacin; ERYTH: erythromycin; FF: florfenicol; FFA: florfenicol amine; FLU: flumequine; HDPE: high-density polyethylene; LC-MS/MS: liquid chromatography-tandem mass spectrometry; LIN: lincomycin; MRM: multiple reaction monitoring; NOR: norfloxacin; OFL-D3: ofloxacin-D3; OXO: oxolinic acid; OTC: oxytetracycline; PEN-G: penicillin G; PEN-V: penicillin V; ROX: roxithromycin; SDM: sulfadimethoxine; SDZ: sulfadiazine; SMX: sulfamethoxazole; SMZ-D4: sulfamethazine-D4; SSZ: sulfasalazine; TC: tetracycline; TAP: thiamphenicol; TILM: tilmicosin; TRIM: trimethoprim; TL: tolerance limit; VIRG: virginiamycin; UPLC-MS/MS: ultra-high pressure liquid chromatography-tandem mass spectrometry.

Transfer of certain beta-lactam antibiotics from cow's milk to fresh cheese and whey.

The aim of this study was to investigate the transfer of cephalexin, penicillin-G, and ampicillin & cloxacillin from cow's milk to cheese and whey. For this purpose, raw milk was artificially contaminated to different antibiotic levels and then heat-treated to prepare fresh cheese from it. Antibiotic levels of the milk, whey and cheese were measured with LC-MS/MS. The extent of heat degradation was not sufficient to remove the antibiotic residues from milk. Antibiotic concentrations in whey and fresh cheese were in good accordance with the concentration of the same compound in milk suggesting that contamination of the milk will result in contamination of the product. The investigated antibiotics were transferred less into the cheese curd (1.6-12.5% of the original amount), than into the whey (33.2-74.1%). For penicillin-G even 100% (complete removal) was experienced.

Multi-residue method for the detection of 40 ß-lactam-antibiotics in vaccines by LC-MS/MS.

Antibiotics are widely used to treat bacterial infections during the process of vaccine production and storage resulting in antibiotic residues that can cause serious harm. A simple and sensitive method for residue analysis of 40 ß-lactam antibiotics was developed and validated for vaccines including inactivated enterovirus 71 vaccine (Vero cells), recombinant hepatitis B vaccine (Saccharomyces cerevisiae), and live attenuated varicella vaccine using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI- MS/MS). Samples were prepared with acetonitrile as the protein precipitant. LC separation was performed on a C18 column. These analytes were determined by LC-MS/MS operating multiple-reaction monitoring (MRM) scans in positive mode. The ranges for limits of detection (LOD) and quantification (LOQ) were as follows: 0.02-4 ng/dose (S/N ≥ 3) and 0.04-10 ng/dose in inactivated enterovirus 71 vaccine (Vero cells) and recombinant hepatitis B vaccine (Saccharomyces cerevisiae), 0.04-16 ng/dose and 0.2-20 ng/dose in live attenuated varicella vaccine. The ranges of recoveries of all antibiotics were 84.5%-108.2% in inactivated enterovirus 71 vaccine (Vero cells), 73%-108% in recombinant hepatitis B vaccine (Saccharomyces cerevisiae), and mostly 68.2%-107.8% in live attenuated varicella vaccine. This method simultaneously offers qualitative and quantitative analysis of multi-antibiotics in vaccines, which improves vaccine safety.

Cryptic ß-Lactamase Evolution Is Driven by Low ß-Lactam Concentrations.

Our current understanding of how low antibiotic concentrations shape the evolution of contemporary ß-lactamases is limited. Using the widespread carbapenemase OXA-48, we tested the long-standing hypothesis that selective compartments with low antibiotic concentrations cause standing genetic diversity that could act as a gateway to developing clinical resistance. Here, we subjected Escherichia coli expressing blaOXA-48, on a clinical plasmid, to experimental evolution at sub-MICs of ceftazidime. We identified and characterized seven single variants of OXA-48. Susceptibility profiles and dose-response curves showed that they increased resistance only marginally. However, in competition experiments at sub-MICs of ceftazidime, they demonstrated strong selectable fitness benefits. Increased resistance was also reflected in elevated catalytic efficiencies toward ceftazidime. These changes are likely caused by enhanced flexibility of the Ω- and ß5-ß6 loops and fine-tuning of preexisting active site residues. In conclusion, low-level concentrations of ß-lactams can drive the evolution of ß-lactamases through cryptic phenotypes which may act as stepping-stones toward clinical resistance.IMPORTANCE Very low antibiotic concentrations have been shown to drive the evolution of antimicrobial resistance. While substantial progress has been made to understand the driving role of low concentrations during resistance development for different antimicrobial classes, the importance of ß-lactams, the most commonly used antibiotics, is still poorly studied. Here, we shed light on the evolutionary impact of low ß-lactam concentrations on the widespread ß-lactamase OXA-48. Our data indicate that the exposure to ß-lactams at very low concentrations enhances ß-lactamase diversity and drives the evolution of ß-lactamases by significantly influencing their substrate specificity. Thus, in contrast to high concentrations, low levels of these drugs may substantially contribute to the diversification and divergent evolution of these enzymes, providing a standing genetic diversity that can be selected and mobilized when antibiotic pressure increases.

In situ controllable synthesis of Schiff base networks porous polymer coatings for open-tubular capillary electrochromatography.

A uniform Schiff base network (SNW) film was synthesized in situ in a controllable way through continuous flow of reactants inside the capillary. The properties and application of the as-prepared capillary was investigated in capillary electrochromatography. The effects of reaction monomer concentration and reaction time on coating thickness were studied by SEM. The results show that the reaction condition has a significant influence on the morphology and thickness of the SNW films. The thickness of the film can be controlled by changing the concentration of reaction solution and reaction time. Capillaries coated under different conditions were employed to separate four nucleotides by capillary electrochromatography, which demonstrated significant variation of migration time, peak order, and separation efficiency. Analytes containing nitrogen heterocycle structures, such as nucleotides, methylimidazole isomers, and ß-lactam antibiotics, were successfully separated with the prepared open-tubular columns. Under the selected separation conditions, theoretical plate number of four nucleotides is in a range 45,237-104,505 plates·m-1, and the resolutions are 1.98-8.07. A resolution of 1.75 is obtained for methylimidazole isomers. The nucleotides in a real sample, chicken essence seasoning, were determined using the prepared capillary column with satisfactory recoveries in the range 95 to 105%.

Residues of tetracyclines and ß-lactams antibiotics induce carbonylation of chicken breast.

Background: Worldwide, chicken meat is widely consumed due to its low cost, high nutritional value and non-interference with religious or cultural beliefs. However, during animal husbandry chickens are exposed to many chemical substances, including tetracyclines and ß-lactams, which are used to prevent and cure several infections. Some residues of these compounds may bioaccumulate and be present in chicken meat after slaughtering, promoting oxidative reactions. Methods: In order to evaluate in vitro carbonylation induced by tetracyclines and ß-lactams residues, a proteomic approach was used. For this, chicken muscle was individually contaminated with tetracyclines (tetracycline, chlortetracycline, oxytetracycline, and doxycycline) and ß-lactams (ampicillin, benzathine penicillin, dicloxacillin and oxacillin) at 0.5, 1.0 and 1.5 times their maximum residue level (MRL). Then, sarcoplasmic, myofibrillar and insoluble proteins were extracted and their content were measured using the Bradford method. Protein carbonylation was measured using the 2,4-Dinitrophenylhydrazine alkaline method. Results: Residues of tetracyclines and ß-lactams induced in vitro carbonylation on sarcoplasmic, myofibrillar and insoluble proteins even at 0.5MRL concentrations ( p<0.05). When comparing the carbonylation induced by both antibiotics no differences were found ( p>0.05). Variables such as the partition coefficient (log P) and the concentration of these antibiotics showed a high correlation with the oxidative capacity of tetracyclines and ß-lactams on chicken breast proteins. Conclusions: This study shows that the presence of tetracyclines and ß-lactams residues at MRLs concentrations promotes in vitro carbonylation on chicken breast proteins. Our results provide important insights about the impact of antibiotics on the integrity of meat proteins intended for human consumption.

TRIO Method for Detection of Beta-Lactams, Sulfonamides, and Tetracyclines in Raw Commingled Cows' Milk.

A qualitative 3 min one-step assay for detecting beta-lactam, sulfonamide, and tetracycline antibiotics was validated following milk screening test guidelines developed by FDA-CVM, AOAC-RI, and IDF. The validated 90% detection levels with 95% confidence were: penicillin G 2 part per billion (ppb); amoxicillin 4 ppb; ampicillin 9 ppb; ceftiofur plus metabolites 50 ppb; cloxacillin 9 ppb; cephapirin 15 ppb; sulfadimethoxine 8 ppb; sulfamethazine 9 ppb; chlortetracycline 34 ppb; oxytetracycline 53 ppb; and tetracycline 42 ppb. Detection levels were lower than U.S. and Canadian allowable limits for milk and were consistent with most European Maximum Residue Limits. Tests of raw commingled cows' milk indicated a low positive error rate of <0.3% with no interferences demonstrated by 1.08 MM/mL somatic cells, Gram-positive or Gram-negative bacteria < 300 K/mL, freeze/thawing, or non-targeted drugs. Detection of incurred residues were similar to, or more sensitive to, fortified samples. Some cross reactivity across drug families occurred in interference studies and therefore initial positive samples should be confirmed with drug family specific screening methods. The National Conference of Interstate Milk Shipments approval as a bulk tank/tanker screening test was completed in three stages for each drug family, including a tetracycline confirmation procedure to target U.S. tolerance levels. Detection and robustness were found to be appropriate for multiple countries' regulatory requirements for screening tests. The method development, validation, and approval was intended to diversify and increase the verification tools for the control of the major antibiotic drug families used in managing cows' health and welfare.

BL30SEC Method for Detection of Beta-Lactams in Raw Commingled Cow Milk: AOAC Performance Tested MethodSM 061902.

Testing milk for antibiotics before acceptance into dairies is required by the U.S. Pasteurized Milk Ordinance. Technological advances in tests have reduced screening times and improved detection accuracy. This work describes the validation of the Charm Rapid One Step Assay Beta-Lactam 30 Second Test according to the U.S. Food and Drug Administration Center for Veterinary Medicine protocol for raw commingled milk. Milk is added to the lateral flow test strip in an incubator/reader to deliver a 30 second result. Independent laboratory validation followed sensitivity, interference, and incurred residue protocols. Sensitivity, in parts per billion (ppb = µg/kg), using a probit curve determined 90% percent detection with 95% confidence, which met National Conference of Interstate Milk Shipments (NCIMS) specifications. Six U.S. approved beta-lactam drugs were detected below, but within 50% of, target/tolerance levels for penicillin G 2.9 ppb, ampicillin 5.9 ppb, amoxicillin 5.8 ppb, cephapirin 13 ppb, cloxacillin 8.1 ppb, and ceftiofur metabolites 73 ppb. No interferences were observed from 33 animal drugs at 100 ppb, somatic cells at 1.2 million/mL, or bacterial levels of >300 000 CFU/mL. Incurred residue detection levels were similar to levels determined with the spiked parent compound. The data support NCIMS that the BL30SEC method met U.S. criteria for testing milk for beta-lactams.

BADAN-conjugated ß-lactamases as biosensors for ß-lactam antibiotic detection.

ß-Lactam antibiotic detection has significant implications in food safety control, environmental monitoring and pharmacokinetics study. Here, we report the development of two BADAN-conjugated ß-lactamases, E166Cb and E166Cb/N170Q, as sensitive biosensors for ß-lactam antibiotic detection. These biosensors were constructed by coupling an environment-sensitive BADAN probe onto location 166 at the active site of the PenP ß-lactamase E166C and E166C/N170Q mutants. They gave fluorescence turn-on signals in response to ß-lactam antibiotics. Molecular dynamics simulation of E166Cb suggested that the turn-on signal might be attributed to a polarity change of the microenvironment of BADAN and the removal of the fluorescence quenching effect on BADAN exerted by a nearby Tyr-105 upon the antibiotic binding. In the detection of four ß-lactams (penicillin G, penicillin V, cefotaxime and moxalactam), both E166Cb and E166Cb/N170Q delivered signal outputs in an antibiotic-concentration dependent manner with a dynamic range spanning from 10 nM to 1 µM. Compared to E166Cb, E166Cb/N170Q generally exhibited more stable signals owing to its higher deficiency in hydrolyzing the antibiotic analyte. The overall biosensor performance of E166Cb and E166Cb/N170Q was comparable to that of their respective fluorescein-modified counterparts, E166Cf and E166Cf/N170Q. But comparatively, the BADAN-conjugated enzymes showed a higher sensitivity, displayed a faster response in detecting moxalactam and a more stable fluorescence signals towards penicillin G. This study illustrates the potential of BADAN-conjugated ß-lactamases as biosensing devices for ß-lactam antibiotics.