Determination Of Penicillin Research Articles

Capillary electrophoresis-integrated immobilized enzyme microreactor utilizing single-step in-situ penicillinase-mediated alginate hydrogelation: Application for enzyme assays of penicillinase

Rapid, low-cost and efficient assays for penicillinase activity and inhibition are of vital importance for therapeutics and diagnostics of bacterial resistance to antibiotics. Herein we report a novel approach for on-line enzyme assays for penicillinase utilizing capillary electrophoresis-integrated immobilized enzyme reactors (CE-IMERs). The CE-IMERs are fabricated based on penicillinase-mediated alginate hydrogelation, allowing single-step in-situ encapsulation of enzymes without any additional manipulation process. We show that the fabricated CE-IMERs have high enzyme loading capacity with approximately 61.8% of the original penicillinase in the sol mixture being encapsulated in the “egg-box” hydrogel matrix. Excellent intraday and interday stability and batch-to-batch reproducibility are proved, indicating the reliability of our method for accurate on-line enzyme assays for penicillinase. Enzymatic activities and inhibition of immobilized penicillinase are analyzed, the results of which are in good agreement with those using free enzymes. The proposed method is successfully used for determination of penicillin in pork samples, indicating the potential applications for analysis of complicated real samples.

Penicillin allergy – recommendations for diagnostic work up and patient management

Allergy to penicillins or cephalosporins manifests either within a few minutes after intake or infusion in the form of acute anaphylaxis symptoms, or several hours to days later as exanthematous skin reactions. Exanthematous skin eruptions caused by amoxicillin or ampicillin are currently the most frequently diagnosed clinical reaction pattern within the spectrum of penicillin allergy. Certain single cephalosporins such as cefazolin, ceftriaxone, and cefuroxime are gaining in relevance as triggers of β‑lactam antibiotic-induced IgE(Immunglobulin E)-mediated anaphylaxis reactions. This article provides an overview of selected scientific articles and is based on research in PubMed, studies, and specialist databases. Penicillin allergy work-up is based on patient history and documented medical findings; serological IgE determinations, as well as skin and provocation testing, are routinely performed. While the determination of IgE directed to certain penicillin determinants is one of the few reliable laboratory tests for the diagnosis of drug hypersensitivity, the basophil activation test or the lymphocyte transformation test are reserved for experienced laboratories which are able to critically evaluate their test results. Wheal-and-flare reactions in skin prick and intradermal testing suggest IgE-mediated allergy, while infiltrated erythematous or exanthematous plaques in patch or intradermal testing point to delayed-type hypersensitivity. The overall predictive value of combined in vitro and in vivo allergy testing with β‑lactam antibiotics is approximately 90%. However, subsequent controlled provocation testing is recommended in order to reliably exclude allergic hypersensitivity.

Simultaneous Determination of Four Antibiotics in Raw Milk by UPLC-MS/MS Using Protein Precipitation as Sample Preparation: Development, Validation, and Application in Real Samples

In this study, a rapid and simple analytical method was proposed, based on protein precipitation as sample preparation for simultaneous determination of tetracycline, oxytetracycline, penicillin G and ceftiofur in raw milk by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The method was applied to raw milk samples from dairy cows medicated with tetracycline for subclinical mastitis. Samples were collected from the adjacent teat to the teat treated with tetracycline of eight different cows at 24, 48, 72 and 96 h after treatment. The limits of quantification of the proposed method ranged between 1 and 5 ng g-1 and limits of detection ranged between 0.1 and 0.5 ng g-1. The recoveries ranged from 61 to 111% and the linear range was 1 to 2064 ng g-1 for tetracycline and oxytetracycline, and 5 to 2064 ng g-1 for penicillin G and ceftiofur. Approximately 75 and 63% of the treated animals revealed more tetracycline than legally recommended at 72 and 96 h since last treatment, respectively.

P28: ALLERGY TO CEPHALOSPORINS: NO BETA‐LACTAM CROSS‐REACTIVITY

Concepts of cephalosporin allergy have evolved in recent years, with presumptions of beta-lactam related cross-reactivity being overturned. Nevertheless an allergic reaction to one cephalosporin will typically carry a suspicion of allergy to other cephalosporins and to penicillins. We sought to evaluate patterns of cephalosporin allergy through review of our challenge clinic database. We selected subjects presenting to the Immunology Ambulatory Care Day Unit with a history of allergy to a cephalosporin-family antibiotic who were investigated through a stepwise progression of in vitro testing (IVT), skin prick testing (SPT), intradermal testing (IDT) and drug provocation testing (DPT). In the case of a positive result, alternative drug(s) were tested. Fifty-five (61.1%) of the 90 subjects tested had a history consistent with an IgE mediated reaction, of whom 24 (43.6%) were proven to be allergic to their index cephalosporin. Only 5 (14.2%) subjects with a history of a delayed reaction had positive findings. Sixteen (66%) of the 24 allergic subjects were diagnosed through IVT and skin testing. Of the remaining eight diagnosed via DPT, the majority were cephalexin-allergic subjects with negative skin testing to cephalexin. Major and minor penicillin determinants were negative for all cephalosporin-allergic individuals. Two cephalexin allergic subjects had positive testing (IVT and SPT) to amoxicillin, consistent with side-chain cross reactivity, while one ceftriaxone allergic subject was also cephalexin IDT positive. Cephalosporin allergy does not appear to be a class effect, with most cases found allergic only to their index cephalosporin. Co-sensitisation to other cephalosporins or penicillins rarely occurs, and is likely to be due to side chain cross-reactivity. Routine penicillin skin testing in those presenting with allergy to a cephalosporin is of low yield.

Utility of minor determinants for skin testing in inpatient penicillin allergy evaluation

BackgroundMost patients with a history of penicillin allergy can tolerate penicillin. Skin testing can identify tolerant patients, but not all known allergenic determinants are commercially available. Protocols exist that use only available reagents, but the sensitivity and safety of these protocols, particularly for hospitalized patients, are controversial. ObjectiveTo determine the number of hospitalized patients referred for penicillin skin testing who showed unique positivity to the minor determinants penicilloate and penilloate. MethodsA retrospective chart review was conducted of all inpatients who underwent penicillin skin testing at 1 institution. Patients were referred by their treating physician. All patients underwent skin prick testing to benzylpenicilloyl polylysine (major determinant), penicillin G, penicilloate, penilloate (minor determinants), amoxicillin, and positive and negative controls. If the result was negative, then intradermal testing was done with the same penicillin determinants and the negative control. A 4-mm wheal with flare was considered a positive reaction. ResultsInpatient penicillin skin testing was done in 528 subjects. Any positive test reaction was found in 107 subjects (20%). Three subjects (3%) reacted to penilloate only, 25 (23%) reacted to penicilloate only, 2 (2%) reacted to penicillin G only, and 8 (8%) reacted to amoxicillin only. Sixty-eight subjects (64%) reacted to a compound other than the major determinant. ConclusionThis study found a high rate of exclusively positive skin test reactions to the minor determinants penicilloate and penilloate. Because patients with positive test reactions are at increased risk of reaction to drug challenge, these data support the use of these reagents for penicillin skin testing in hospitalized patients.

Determination of Antibiotic Residues in Honey by High-Performance Liquid Chromatography with Electronspray Ionization Tandem Mass Spectrometry

The aim of the present study was to develop a rapid and simple method for the detection and quantification of antibiotic and antibacterial residues in honey using liquid chromatography with electronspray ionization tandem mass spectrometry. Two different extraction methods were used. The first method uses water and 1% formic acid in acetonitrile for the determination of sulfonamides while the second uses phosphate buffer, 10% trichloroacetic acid, and acetonitrile as the extracting solvent for the determination of tetracyclines, amphenicols, fluoroquinolones, penicillin g, trimethoprim, and tiamulin. The multi-residue method was validated in a thyme honey matrix. Thirty-six different antibiotics and residues from four different families (sulfonamides, tetracyclines, amphenicols, fluoroquinolones) and some individual antibiotics (penicillin g, trimethoprim, and tiamulin) were tested in 20 honey samples originating from Cyprus and Greece. The decision limits (CCα) were from 0.1 to 9.2 μg kg−1; the detection capabilities (CCβ) were from 0.3 to 27.6 μg kg−1 while recoveries were from to be between 65.0 and 116.1%. The method was successfully applied to commercial samples from different types of honey from Greece and Cyprus. Among them, oxolonic acid, sulfathiazole, and sulfadimethoxine were found in three honey samples. Finally, proficiency testing was applied to the proposed method while analysis of certified samples showed good method performance characteristics.

De-labelling self-reported penicillin allergy within the emergency department through the use of skin tests and oral drug provocation testing.

Self-reported penicillin allergy is common among patients attending the ED, but is a poor predictor of true immunoglobulin E-mediated hypersensitivity to penicillin. We hypothesise that with a combination of skin testing and drug provocation testing, selected patients can be safely de-labelled of their allergy. This prospective study enrolled a sample of patients presenting to an urban academic ED between 2011 and 2016 with a self-reported allergy to penicillin. Standardised skin prick and intradermal testing with amoxicillin and both major and minor determinants of penicillin was performed in the department. If negative, testing was followed by a graded oral challenge of amoxicillin over 9 days. The primary end point was the allergy status of participants at the end of the study. A total of 100 patients (mean age 42; standard deviation 14 years; 54% women) completed the testing. Of these, 81% (95% confidence interval 71.9-88.2) showed no hypersensitivity to penicillin and were labelled non-allergic. The majority (16/19) of allergies were confirmed by skin testing, with three suspected allergies detected by the oral challenge. Women were more likely than men to have a true penicillin allergy, with odds ratio of 4.0 (95% confidence interval 1.23-13.2). There were no serious adverse events. Selected patients in the ED who self-report an allergy to penicillin can be safely tested there for penicillin allergy, using skin tests and oral drug provocation testing. This testing allows a significant de-labelling of penicillin allergy, with the majority of these patients able to tolerate penicillin without incident.

Virtual mutation and directional evolution of anti-amoxicillin ScFv antibody for immunoassay of penicillins in milk

In this study, an anti-amoxicillin single chain variable fragment (ScFv) antibody was evolved by directional mutagenesis of a contact amino acid residue based on the analysis of virtual mutation. Comparison with its parental ScFv, the mutant showed highly improved affinity for 11 penicillins with up to 6-folds increased sensitivity. Then, its recognition mechanisms for the 11 drugs were studied by using molecular docking. Results showed that the mutant-penicillins intermolecular forces increased and the total binding energies decreased dramatically, which were responsible for the improvement of antibody sensitivity. The ScFv mutant was used to develop an indirect competitive enzyme linked immunosorbent assay for determination of the 11 drugs in milk. The limits of detection were in the range of 0.2–3.0 ng/mL, the crossreactivities were in the range of 31%–132%, and the recoveries from standards fortified blank milk were in the range of 65.7%–92.4%. This is the first study reporting the directional evolution of a ScFv antibody based on virtual mutation and the use of ScFv antibody for determination of penicillins in foods of animal origin.

A coacervative extraction based on single-chain and double-chain cationic surfactants

A coacervative extraction (CAE) with the common cationic surfactants dodecyltrimethylammonium bromide (DTAB) and didodecyldimethylammonium bromide (DDAB) was firstly developed. Notable characteristics of the CAE was the use of low concentration of salt (0.1molL−1 NaBr) at ambient temperature and without the requirement of organic solvent. The CAE was based on phase separation due to the neutralization of the surface charge of the micelle by electrostatic interaction with the predominant common counter ion (bromide). The coacervative phase was subjected to an optimized micellar liquid chromatography with UV (MLC-UV) method without any treatments. The method was applied to the determination of penicillins including amoxicillin, ampicillin, penicillin-G, oxacillin, and cloxacillin in milk samples. Method detection limits (MDLs) from standard were 0.5–2ngmL−1, and 40–80-fold analyte enrichment were obtained. The CAE-MLC-UV has shown to be of high potential for the analysis of five penicillins in milk with recoveries >90%.

The influence of the carrier molecule on amoxicillin recognition by specific IgE in patients with immediate hypersensitivity reactions to betalactams.

The optimal recognition of penicillin determinants, including amoxicillin (AX), by specific IgE antibodies is widely believed to require covalent binding to a carrier molecule. The nature of the carrier and its contribution to the antigenic determinant is not well known. Here we aimed to evaluate the specific-IgE recognition of different AX-derived structures. We studied patients with immediate hypersensitivity reactions to AX, classified as selective or cross-reactors to penicillins. Competitive immunoassays were performed using AX itself, amoxicilloic acid, AX bound to butylamine (AXO-BA) or to human serum albumin (AXO-HSA) in the fluid phase, as inhibitors, and amoxicilloyl-poli-L-lysine (AXO-PLL) in the solid-phase. Two distinct patterns of AX recognition by IgE were found: Group A showed a higher recognition of AX itself and AX-modified components of low molecular weights, whilst Group B showed similar recognition of both unconjugated and conjugated AX. Amoxicilloic acid was poorly recognized in both groups, which reinforces the need for AX conjugation to a carrier for optimal recognition. Remarkably, IgE recognition in Group A (selective responders to AX) is influenced by the mode of binding and/or the nature of the carrier; whereas IgE in Group B (cross-responders to penicillins) recognizes AX independently of the nature of the carrier.

Selection of DNA aptamers against penicillin G using Capture-SELEX for the development of an impedimetric sensor

This paper describes for the first time the selection of aptamers selective to penicillin. Aptamers were selected using a specific process called Capture-SELEX (Systematic Evolution of Ligands by Exponential Enrichment). This technique is based on the selection of DNA aptamers using penicillin G in solution while the ssDNA library is fixed on a support. One aptamer showing a good affinity to penicillin was finally selected and tested in electrochemical sensor configuration, using electrochemical impedance spectroscopy as detection technique. The developed aptasensor allowed the detection of penicillin in a wide concentration range, comprised between 0.4 and 1000µgL−1 Such performance was compatible with milk analysis, as the maximum residue limit tolerated in this matrix is 4µgL−1. The selectivity of the developed sensor was also studied, showing that the sensor was also able to bind other beta-lactam antibiotics, although with a weaker affinity. Finally the sensor was used for detection of penicillin G in milk. It was shown that a simple sample treatment with isopropanol followed by filtration was sufficient to eliminate matrix effects, allowing the determination of penicillin in milk at concentrations compatible with legislation requirements.

Label-Free Photonic Crystal-Based β-Lactamase Biosensor for β-Lactam Antibiotic and β-Lactamase Inhibitor

A simple, label-free, and visual photonic crystal-based β-lactamase biosensor was developed for β-lactam antibiotic and β-lactamase inhibitor in which the penicillinase (a β-lactamase) was immobilized on the pH-sensitive colloidal crystal hydrogel (CCH) film to form penicillinase colloidal crystal hydrogel (PCCH) biosensing film. The hydrolysis of penicillin G (a β-lactam antibiotic) can be catalyzed by penicillinase to produce penicilloic acid, leading to a pH decrease in the microenvironment of PCCH film, which causes the shrink of pH-sensitive CCH film and triggers a blue-shift of the diffraction wavelength. Upon the addition of β-lactamase inhibitor, the hydrolysis reaction is suppressed and no clear blue-shift is observed. The concentrations of β-lactam antibiotic and β-lactamase inhibitor can be sensitively evaluated by measuring the diffraction shifts. The minimum detectable concentrations for penicillin G and clavulanate potassium (a β-lactamase inhibitor) can reach 1 and 0.1 μM, respectively. Furthermore, the proposed method is highly reversible and selective, and it allows determination of penicillin G in fish pond water samples.

Determination of Penicillins in Milk by a Dual-Optrode Biosensor

ABSTRACTPenicillins are the most frequently found antibiotic residues in milk, as they are commonly used for the treatment of bacterial infections in cows. In the present study, we introduce a method for the rapid detection of penicillin residues in raw milk based on the determination of glucose concentration in milk with a dual flow-through biosensor. The molar concentration of glucose in milk is typically over 500 times lower than the concentration of lactose and is highly dependent on the rate of lactose hydrolysis, which is catalyzed by β-galactosidase. Glucose concentrations in milk change with variation in the β-galactosidase activity. β-Galactosidase is an enzyme produced in the microbiota in milk and its activity is inhibited by benzylpenicillin. Spiking milk with benzylpenicillin lowers glucose concentrations in comparison to high-quality milk after short storage intervals. The presence of penicillin in the milk of treated animals resulted in decreased glucose concentrations in comparison with high-quality milk that contained no antibiotics. The glucose concentration in milk samples was followed by the system enabling the elimination of the effects of bacterial respiration in the output with reliable results in less than 1 min.

The Importance of Amoxicillin and Amoxicillin-Clavulanate Determinants in the Diagnosis of Immediate Allergic Reactions to β-Lactams

Background: Immediate allergic reactions to β-lactam antibiotics are considered to be one of the most important drug hypersensitivities. A positive skin test (ST) with a combination of major and minor penicillin determinants is usually sufficient to recommend avoidance of the culprit drug, whereas a negative ST is usually followed by an oral challenge test (OCT). Recently, concern has been raised regarding the role of amoxicillin (AMX) ST in the diagnosis of AMX allergy. Objective: The aim of this study was to examine the additive value of AMX determinants in STs of patients with immediate hypersensitivity reactions to AMX or AMX-clavulanate (AMX-C). Methods: Patients with a history of immediate AMX or AMX-C allergy underwent an ST using a combination of penicilloyl-polylysine (PPL) and minor determinants as well as AMX. An ST with AMX-C was added when appropriate. Results: Thirty-one patients were evaluated. Eight patients, all of them with a history of AMX allergy, had positive reactions only to the AMX component. Two patients with AMX-C allergy had a positive ST reaction only to the AMX-C component. Moreover, only 14 patients (13 with AMX and 1 with AMX-C allergy) had a positive reaction to PPL, whereas most patients (54.8%) had positive reactions to other determinants. One patient, who was positive for AMX, developed several urticarial lesions after the test. Conclusions: Skin testing with AMX and AMX-C is mandatory in patients with immediate allergy to these drugs. Failure to perform it may result in a false-negative ST jeopardizing these patients with anaphylactic reactions during a hazardous OCT.

Draft Genome Sequence of Neisseria gonorrhoeae Strain NG_869 with Penicillin, Tetracycline and Ciprofloxacin Resistance Determinants Isolated from Malaysia

Gonorrhea is a sexually transmitted infection caused by Neisseria gonorrhoeae and the increasing reports of multidrug-resistant gonococcal isolates are a global public health care concern. Herein, we report the genome sequence of N. gonorrhoeae strain NG_869 isolated from Malaysia which may provide insights into the drug resistance determinants in gonococcal bacteria.

A retrospective comparison of false negative skin test rates in penicillin allergy, using pencilloyl-poly-lysine and minor determinants or Penicillin G, followed by open challenge.

BackgroundA history of penicillin allergy in patients is common, but only 10–15 % are truly allergic. While the gold standard for diagnosing penicillin allergy is challenge, it is not recommended that this be done without first carrying out diagnostic skin testing. This is carried out with the major determinant benzylpenicilloyl (PPL) and the minor determinant mixture (MDM), consisting of penilloate, penicilloate and Penicillin G. However, since availability of the MDM is limited, Penicillin G alone has been used.MethodsA retrospective chart review was carried out on patients tested for penicillin allergy in the Clinical Immunology and Allergy Clinic at the Health Sciences Centre, Winnipeg, Canada between 2005 and 2013. A total of 521 patients charts were reviewed, of whom 240 had skin testing, ImmunoCap® for IgE to Penicillin G and V and had oral challenges with penicillin, amoxicillin or cloxacillin.Results17/240 (7.5 %) were skin test positive, 8 to PPL, 4 to MDM and 5 to Penicillin G. One was also positive on ImmunoCap® testing. Three patients had negative skin tests but weakly positive ImmunoCap®. 222 patients with negative skin tests and serological tests were challenged. Of these, 12 patients reacted to challenge. Three of the challenges were equivocal. Of the nine patients with definite positive challenges, three were tested with Penicillin G and six with MDM. Therefore the false negative rates for testing were 2.3 % with PPL and Penicillin G and 6.97 % for PPL and MDM. The difference was not significant (p = 0.0856).ConclusionsIn this group of patients with a history of penicillin allergy tested with the major determinant of benzyl penicillin and either MDM or Penicillin G, there was no difference in the rate of false negative testing, based on oral penicillin challenges. Therefore, Penicillin G can be safely used as an alternative to MDM in diagnosing penicillin allergy.

Hantzsch Reaction as a Method for Spectrofluorimetric Analysis of Penicillin G

A sensitive, selective, and validated spectrofluorimetric method is being described for the determination of penicillin G. The method utilizes this drug (as a source of the amine moiety) to react with acetylacetone and formaldehyde through the Hantzsch reaction. The faint yellow product of dihydropyridine derivatives was measured at an emission wavelength of 481 nm (after excitation at 416 nm). Linear calibration graphs were obtained in the range of 0.2-200 μg/mL. The experimental limits of detection and quantitation were 0.170 and 0.515 μ/mL, respectively. The relative standard deviation (n=5) at the 100 μg/mL level were 6.11 for intra-day precision and 9.42 for inter-day precision. The method was successfully applied in the analysis of pharmaceutical formulation; the recovery was quantitative, and the result obtained agreed with those obtained by another reported method.

High-performance liquid chromatography–high-resolution time-of-flight mass spectrometry in the identification and determination of penicillin and tetracycline group antibiotics in food

A method of the identification and determination of 27 antibiotics of penicillin (15) and tetracycline (12) groups in food using high-resolution time-of-flight mass spectrometry combined with high-performance liquid chromatography and a simple and fast sample preparation procedure have been developed. The analytical range for penicillins is 3 (50)–300 (1000) μg/kg and for tetracyclines, 0.3 (1.5)–100 (200) μg/kg. A scheme of the identification and determination of antibiotics using the standard addition method has been proposed. The relative standard deviation of the results of analysis does not exceed 10%. The time of sample screening is 30–40 min, the time of the determination of the detected antibiotics is 2–3 h.

A novel fluorescence polarization assay for determination of penicillin G in milk

Detection of penicillin G in milk is of interest because of the wide use of this antibiotic in livestock. Current analytical methods used to quantify the penicillin G in milk are based on HPLC, mass spectrometry and electrophoresis. These methodologies are time-consuming and require trained personnel. In addition, it is not easy to envisage the development of a portable device for in situ analyses based on these methods. We present a novel sensing approach for detecting the presence of penicillin G in milk. The proposed method is based penicillin G conjugate labeled with red-emitting dye with properly produced anti-penicillin G antibodies. The results obtained suggest our method could be applied directly in milk without interference from other substances. The limit of detection of the method was 1.0nmol/L, which is much less than the required MRL in EU regulations (12.0nmol/L).

Involvement of aph(3')-IIa in the formation of mosaic aminoglycoside resistance genes in natural environments.

Intragenic recombination leading to mosaic gene formation is known to alter resistance profiles for particular genes and bacterial species. Few studies have examined to what extent aminoglycoside resistance genes undergo intragenic recombination. We screened the GenBank database for mosaic gene formation in homologs of the aph(3′)-IIa (nptII) gene. APH(3′)-IIa inactivates important aminoglycoside antibiotics. The gene is widely used as a selectable marker in biotechnology and enters the environment via laboratory discharges and the release of transgenic organisms. Such releases may provide opportunities for recombination in competent environmental bacteria. The retrieved GenBank sequences were grouped in three datasets comprising river water samples, duck pathogens and full-length variants from various bacterial genomes and plasmids. Analysis for recombination in these datasets was performed with the Recombination Detection Program (RDP4), and the Genetic Algorithm for Recombination Detection (GARD). From a total of 89 homologous sequences, 83% showed 99–100% sequence identity with aph(3′)-IIa originally described as part of transposon Tn5. Fifty one were unique sequence variants eligible for recombination analysis. Only a single recombination event was identified with high confidence and indicated the involvement of aph(3′)-IIa in the formation of a mosaic gene located on a plasmid of environmental origin in the multi-resistant isolate Pseudomonas aeruginosa PA96. The available data suggest that aph(3′)-IIa is not an archetypical mosaic gene as the divergence between the described sequence variants and the number of detectable recombination events is low. This is in contrast to the numerous mosaic alleles reported for certain penicillin or tetracycline resistance determinants.