Benzylpenicillin-producing Trichophyton erinacei and methicillin resistant Staphylococcus aureus carrying the mecC gene on European hedgehogs - A pilot-study.

BACKGROUND: A high carriage rate of methicillin-resistant Staphylococcus aureus with the mecC gene (mecC-MRSA) has been described among Wild European hedgehogs (Europeaus erineaus). Due to this frequent occurrence, it has been suggested that hedgehogs could be a natural reservoir for mecC-MRSA. However, the reason why hedgehogs carry mecC-MRSA remains unknown, but it has been hypothesized that mecC-MRSA could have evolved on the skin of hedgehogs due to the co-occurrence with antibiotic producing dermatophytes. The aim of this pilot-study was therefore to investigate if hedgehogs in Sweden carry Trichophyton spp. and to provide evidence that these dermatophytes are able to produce penicillin or similar substances. In addition, the study aimed to identify if dermatophytes co-occurred with mecC-MRSA. METHODS: Samples were collected from hedgehogs (Europeaus erineaus) that were euthanized or died of natural causes. All samples were screened for dermatophytes and mecC-MRSA using selective cultivation methods. Suspected isolates were characterized using PCR-based methods, genome sequencing and bioinformatic analyses. Identification of penicillin was performed by ultra-high-performance liquid chromatography-tandem mass spectrometry. RESULTS: In total 23 hedgehogs were investigated, and it was shown that two carried Trichophyton erinacei producing benzyl-penicillin, and that these hedgehogs also carried mecC-MRSA. The study also showed that 60% of the hedgehogs carried mecC-MRSA. CONCLUSION: The pilot-study demonstrated that Trichophyton erinacei, isolated from Swedish hedgehogs, can produce benzylpenicillin and that these benzylpenicillin-producing T. erinacei co-occurred with mecC-MRSA. The study also reconfirmed the high occurrence of mecC-MRSA among hedgehogs.

Dissipation and residue determination of penicillin G and its two metabolites in citrus under field conditions by DSPE/UPLC-MS/MS.

A rapid determination method of residual penicillin G and its two metabolites in citrus was developed and validated by dispersive solid-phase extraction and ultra-high performance liquid chromatography-tandem mass spectrometry (DSPE/UPLC-MS/MS). The samples were extracted with 80% acetonitrile and purified with octadecylsilane. High linearity was obtained with correlation coefficients (r2 ) >0.9981. The limits of quantification were 0.005-0.01 mg/kg. The recoveries of penicillin G and its metabolites spiked in blank citrus were within 76.7-107%, with relative standard deviations of 1.3-9.6%. The dissipation dynamics and distribution of penicillin G in citrus followed first-order kinetics, with half-life of 1.7-2.7 days. Penicillin G degraded easily in citrus and the metabolite was mainly penilloic acid, which can exist stably for long time. The terminal residues of penicillin G in pulp, whole citrus and peels were 0.015-0.701, 0.047-7.653 and 0.162-13.376 mg/kg, respectively. The hazard indexes for risk assessment of citrus were significantly <1, suggesting that the health risks to humans after consumption of citrus were insignificant and negligible. These results could provide necessary data for evaluating the safe and proper use of penicillin G in citrus.

Degradation of Penicillin G by heat activated persulfate in aqueous solution.

We used Heat Activated of Persulfate (HAP) to decompose Penicillin G (PEN G) in aqueous solution. The effect of pH (3-11), temperature (313-353 K), and initial concentration of Sodium Persulfate (SPS) (0.05-0.5 mM) on the decomposition level of PEN G were investigated. The residue of PEN G was determined by spectrophotometry at the wavelength of 290 nm. Also, the Chemical Oxygen Demand (COD) was measured in each experiment. The Total Organic Carbon (TOC) analysis was utilized for surveying the mineralization of PEN G. In addition, based on Arrhenius equation, the activation energy of PEN G decomposition was calculated. The results indicated that the maximum PEN G removal rate was obtained at pH 5 and by increasing the doses of SPS from 0.05 to 0.5 mM, the PEN G decomposition was enhanced. It was found that an increase in temperature is accompanied by an increase in removal efficiency of PEN G. The activation energy of the studied process was determined to be 94.8 kJ mol-1, suggesting that a moderate activation energy is required for PEN G decomposition. The TOC measurements indicate that the HAP can efficiently mineralize PEN G. Besides, the presence of the scavengers significantly suppressed the HAP process to remove the PEN G. Overall, the results of this study demonstrate that using HAP process can be a suitable method for decomposing of PEN G in aqueous solutions.

LC-MS/MS Method for the Determination and Quantitation of Penicillin G and Its Metabolites in Citrus Fruits Affected by Huanglongbing.

In this study, we developed and validated a method for the extraction, identification, and quantitation of penicillin G and its metabolites (penilloic acid and penillic acid) in a variety of citrus fruits by employing sequential liquid/liquid and solid-phase extraction techniques in conjunction with UHPLC-MS/MS. Two product ion transitions per analyte were required for identification, which contributes to a high degree of selectivity. Corrected recoveries of penicillin G using an isotopically labeled internal standard were 90-100% at fortification levels of 0.1, 0.25, 1, and 10 ng/g. Absolute recoveries for penillic acid and penilloic acid were 50-75% depending on the matrix used. The limit of detection (LOD) of penicillin G and its metabolites was found to be 0.1 ng/g when 2 g of citrus was extracted. This method is useful in determining residue levels of penicillin G and its metabolites in citrus trees infected with huanglongbing bacteria after antibiotic treatment.

Development and evaluation of a molecularly imprinted polymer for the detection and cleanup of benzylpenicillin in milk.

A molecularly imprinted polymer (MIP) was designed for benzylpenicillin via suspension polymerization. The specific absorption of benzylpenicillin to the MIP, applied in a molecularly imprinted solid-phase extraction (MISPE), was compared to the nonspecific binding using a NIP (nonimprinted polymer without a target molecule) in a non-molecularly imprinted solid-phase extraction. This validation was performed successfully in acetonitrile solutions and milk extracts spiked with benzylpenicillin. Significant differences in absorption were observed. In acetonitrile, the recoveries using MISPE (90-95%) were a fraction higher than those in milk extracts (70-80%). The validation revealed the limit of detection and the limit of quantitation for the MISPE application in milk samples to be 0.51 and 1.02 µg/kg, respectively. In addition, comparing the results of the analysis of positive milk samples using MISPE with those using a classic sample preparation step showed a Pearson correlation of 0.989. Finally, cross reactivity tests using other antibiotics showed a certain cross reactivity, but non-ß-lactams were barely bound.

Bacterial microbiota present in the gallbladder of cattle and antimicrobial resistance of Staphylococcus isolates / Microbiota bacteriana presente na vesícula biliar de bovinos e resistência antimicrobiana de isolados de Staphyloccocus

Pathogenic microorganisms can reside transiently or permanently in the gallbladder of cattle. Thus, during slaughter, more attention should be given to the gastrointestinal tract, especially to the accessory organ, the gallbladder. The main aim of this study was to characterize the bacterial microbiota present in bile and gallbladder epithelium of cattle slaughtered in a slaughtering plant under sanitary conditions and to evaluate the antimicrobial resistance in strains of the genus Staphylococcus. Thirty intact gallbladders were collected and the in bile and epithelium were researched for the presence of Aerobic Mesophilic Heterotrophic Bacteria (AMHB), Staphylococcus spp., total Enterobacteriaceae, Enterococcus spp. and Salmonella spp.The frequency of isolation of the microorganism mentioned above were, respectively: 23.02 percent, 14.39 percent, 13.67 percent, 24.46 percent, 0 percent and 24.46 percent. Concerning both gallbladder environments, the frequency of isolation of the microorganisms in the epithelium was 64.03 percent, and in the bile 35.97 percent, with no statistical difference, but with significant difference between the population averages. In antimicrobial susceptibility testing, strains of Staphylococcus from both bile and gallbladder epithelium showed sensitivity to the antimicrobials: penicillin G, ceftriaxone, chloramphenicol and gentamicin. The observation that the gallbladder supports a high frequency of microorganisms brings us to the possible fact that cattle might be a persistent carrier of pathogens of great importance to public health...

Microrganismos patogênicos podem residir temporariamente ou permanentemente na vesícula biliar de bovinos. Assim, durante o abate, maior atenção deve ser dada ao trato gastrointestinal, especialmente para o órgão acessório, a vesícula biliar. O principal objetivo deste estudo foi caracterizar a microbiota bacteriana presente na bile e no epitélio de vesículas biliares de bovinos abatidos em matadouro frigorífico sob inspeção sanitária e avaliar a resistência antimicrobiana de estirpes do gênero Staphyloccocus. Foram coletadas 30 vesículas biliares íntegras e foi pesquisada na bile e no epitélio do órgão a presença de bactérias heterotróficas aeróbias mesófilas (BHAM), Staphylococcus spp. e Enterobacteriaceae totais, Escherichia coli, Enterococcus spp. e Salmonella spp. A frequência de isolamento dos microrganismos citados acima foi, respectivamente: 23,02 por cento, 14,39 por cento, 13,67 por cento, 24,46 por cento, 0por cento e 24,46 por cento. Em relação aos dois ambientes da vesícula, a frequência de isolamento dos microrganismos no epitélio foi de 64,03 por cento, e na bile 35,97 por cento, não sendo diferente estatisticamente, mas com diferença significativa entre as médias populacionais.No teste de susceptibilidade aos antimicrobianos, as estirpes de Staphylococcus isoladas a partir da bile e do epitélio da vesícula biliar apresentaram sensibilidade a: penicilina G, ceftriaxona, cloranfenicol e gentamicina. A observação de que a vesícula biliar comporta microrganismos em elevadas frequências atenta para o fato de que o bovino possa ser um portador persistente de patógenos de grande importância em saúde pública...

Experiments and ANFIS modelling for the biodegradation of penicillin-G wastewater using anaerobic hybrid reactor.

The performance of an anaerobic hybrid reactor (AHR) for treating penicillin-G wastewater was investigated at the ambient temperatures of 30-35°C for 245 days in three phases. The experimental data were analysed by adopting an adaptive network-based fuzzy inference system (ANFIS) model, which combines the merits of both fuzzy systems and neural network technology. The statistical quality of the ANFIS model was significant due to its high correlation coefficient R(2) between experimental and simulated COD values. The R(2) was found to be 0.9718, 0.9268 and 0.9796 for the I, II and III phases, respectively. Furthermore, one to one correlation among the simulated and observed values was also observed. The results showed the proposed ANFIS model was well performed in predicting the performance of AHR.

Simultaneous extraction and concentration of penicillin G by hollow fiber renewal liquid membrane.

In this article, hollow fiber renewal liquid membrane (HFRLM) technique was used for recovery of penicillin G from aqueous solution. The organic solution of 7 vol % di-n-octylamine (DOA) + 30 vol % iso-octanol + kerosene was used as liquid membrane phase, and Na(2)CO(3) aqueous solution was used as stripping phase. Experiments were performed as a function of carrier concentration in the organic phase, organic/aqueous volume ratio, pH, and initial penicillin G concentration in the feed phase, pH in the stripping phase, flow rates, etc. The results showed that the HFRLM process was stable and could carry out simultaneous extraction and concentration of penicillin G from aqueous solutions. As a carrier facilitated transport process, the addition of DOA in organic phase could greatly enhance the mass transfer rate; and there was a favorable organic/aqueous volume ratio of 1:20 to 1:30 for this system. The mass transfer flux and overall mass transfer coefficient increased with decreasing pH in the feed phase and increasing pH in the stripping phase, because of variation of the mass transfer driving force caused by pH gradient and distribution equilibrium. The flow rate of the shell side had significant influence on the mass transfer performance, whereas the effect of flow rate of lumen side on the mass transfer performance was slight because of the mass transfer intensification of renewal effect in the lumen side. The results indicated that the HFRLM process was a promising method for the recovery of penicillin G from aqueous solutions.

Facilitated transport of penicillin G by bulk liquid membrane with TBP as carrier.

The facilitated transport of penicillin G from aqueous solutions to the stripping phase through bulk liquid membrane (BLM) containing TBP in 3% iso-octanol and n-butyl acetate was studied. Na(2)CO(3) solution was used as the stripping phase. Experiments were performed as a function of stirring rate, TBP concentration and type of diluent in the liquid membrane phase, pH, and initial penicillin G concentration in the feed phase, Na(2)CO(3) concentration in the stripping phase, etc. The results showed that the BLM process could carry out the simultaneous separation and concentration of penicillin G from dilute aqueous solutions, and arise "up-hill" effect due to the characteristic of non-equilibrium mass transfer. The diffusion of penicillin G complex in the liquid membrane phase played an important role in BLM process. The mass transfer mechanism of BLM for this system was also discussed.

Direct extraction of penicillin G and derivatives from aqueous samples using a stoichiometrically imprinted polymer.

A molecularly imprinted polymer (MIP) prepared using penicillin G procaine salt as the template (PENGp) and a stoichiometric quantity of urea-based functional monomer to target the single oxyanionic species in the template molecule has been applied to the development of a molecularly imprinted solid-phase extraction (MISPE) procedure for the selective preconcentration of beta-lactam antibiotics (BLAs) from environmental water samples. Various parameters affecting the extraction efficiency of the polymer have been evaluated to achieve the selective preconcentration of the antibiotics from aqueous samples and to reduce nonspecific interactions. This resulted in an MISPE-HPLC method allowing the direct extraction of the analytes from the sample matrix with a selective wash using just 10% (v/v) organic solvent. On the basis of UV detection only, the method showed good recoveries and precision, ranging between 93% and 100% (RSD 3.8-8.9%, n = 3) for tap water and between 90% and 100% (RSD 4.2-9.1%, n = 3) for river water fortified with 30 or 60 microg L-1 (50 mL samples) penicillin G, penicillin V, nafcillin, oxacillin, cloxacillin, and dicloxacillin, suggesting that this MIP can be successfully applied to the direct preconcentration of BLAs in environmental water samples.

Physical and reactive extraction equilibria of penicillin G in a hydrogen-bond acceptor solvent system.

Physical and reactive extraction equilibria of penicillin G were investigated experimentally and theoretically in the existence of n-butyl acetate as a hydrogen-bond acceptor solvent. Physical extraction equilibrium experiments were carried out varying the pH of aqueous phase and overall penicillin concentration. We compared the experimental data with the calculated results from four physical extraction equilibrium models suggested here and obtained the most reasonable model. Also, penicillin G was reactively extracted using Amberlite LA-2 in n-butyl acetate. The experimental variables were pH of the aqueous phase, overall amine concentration, and overall penicillin concentration. A combined equilibrium model including our physical extraction equilibrium expression and the reactive extraction equilibrium expression suggested by Reschke and Schügerl was used so as to analyze the current reactive extraction equilibrium system. The calculated results from the reactive extraction equilibrium model were in good agreement with the experimental data.

Separation and purification of benzylpenicillin produced by fermentation using coupled ultrafiltration and nanofiltration technologies.

The purpose of this study was to evaluate the capacity of using coupled ultrafiltration-nanofiltration technologies for separation and purification of benzylpenicillin (BP). More specifically, we verified the efficiency of three ultrafiltration (UF) membranes (cut-off of 5000, 30,000 and 100,000 Da) to remove impurities that cause stable emulsion during the chemical extraction of the antibiotic. We also tested the effectiveness of a nanofiltration (NF) membrane (cut-off of 300 Da) to concentrate the benzylpenicillin recovered from permeates and to decrease the osmotic pressure by reducing the ionic charge of the broth. Results have shown that high recovery (89.0-91.0%) can be obtained in permeate generated by the 30,000 and 100,000 UF membranes, but a slight emulsion will be formed during phase separation. With the 5000 UF membrane, lower recovery is obtained (81.0%) but no emulsion is produced, leading to a high solvent extraction yield (94.6%). The nanofiltration of 30,000 and 100,000 UF permeates leads to very high recovery (98.0%), but stable emulsions are formed, reducing the chemical extraction yield (80.0-82.6%). For the nanofiltration of 5000 UF permeate, excellent recovery of the antibiotic is noted (97.4%) leading to high extraction yield (92.4%) with no emulsion formed. Diafiltration step should be applied during UF procedure in order to increase the antibiotic recovery in the generated permeates.

Equilibrium modeling of extractive enzymatic hydrolysis of penicillin G with concomitant 6-aminopenicillanic acid crystallization.

In the present downstream processing of penicillin G, penicillin G is extracted from the fermentation broth with an organic solvent and purified as a potassium salt via a number of back-extraction and crystallization steps. After purification, penicillin G is hydrolyzed to 6-aminopenicillanic acid, a precursor for many semisynthetic beta-lactam antibiotics. We are studying a reduction in the number of pH shifts involved and hence a large reduction in the waste salt production. To this end, the organic penicillin G extract is directly to be added to an aqueous immobilized enzyme suspension reactor and hydrolyzed by extractive catalysis. We found that this conversion can exceed 90% because crystallization of 6-aminopenicillanic acid shifts the equilibrium to the product side. A model was developed for predicting the equilibrium conversion in batch systems containing both a water and a butyl acetate phase, with either potassium or D-p-hydroxyphenylglycine methyl ester as counter-ion of penicillin G. The model incorporates the partitioning equilibrium of the reactants, the enzymatic reaction equilibrium, and the crystallization equilibrium of 6-aminopenicillanic acid. The model predicted the equilibrium conversion of Pen G quite reasonably for different values of pH, initial penicillin G concentration and phase volume ratio. The model can be used as a tool for optimizing the enzymatic hydrolysis.

Reaction equilibrium of penicillin G with amberlite LA-2 in a nonpolar organic solvent.

Studies have been made of the reactive extraction of penicillin G by Amberlite LA-2, a secondary amine, dissolved in kerosene. On the basis of the previous works about extraction equilibria of monocarboxylic acids by some secondary amines in low polar organic solvents, four equilibrium models were suggested to describe the reaction equilibrium of penicillin G in the liquid-liquid extraction system. The calculated results from the models were compared with the experimental data of 96 runs, and only two equilibrium models seemed to be probable. Ultimately, the most reasonable extraction equilibrium model was chosen through spectroscopic studies on organic solutions obtained by five specific extraction equilibrium experiments.

Microwave-enhanced dehydration and solvent washing purification of penicillin G sulfoxide.

In the present study, a new microwave-enhanced dehydration and solvent washing purification of penicillin G sulfoxide technique has been developed. The results show that microwave irradiation can dehydrate penicillin G sulfoxide from a water content of 14-26 to below 0.5% in 40-60 min in N2 or air. After washing with ethyl acetate to remove impurities and residual water, the penicillin G sulfoxide can be used to synthesize cephalosporanic acid. The recovery of cephalosporanic acid was equal to and the purity of cephalosporanic acid was higher by 2% than that of the current dehydration technique. FTIR spectroscopy was used to study the process of microwave-enhanced dehydration and solvent washing purification of penicillin G sulfoxide.

Reactive extraction of penicillin G in hollow-fiber and hollow-fiber fabric modules.

Penicillin G (Pen G) can be rapidly extracted in hollow-fiber liquid-liquid contactors using N-lauryl-N-trialkylmethylamine (Amberlite LA-2) as the extractant. n-Butylacetate is much better than decanol as a diluent for such an extraction, although decanol can give a partition coefficient four times larger. The overall mass transfer coefficient found is a function of aqueous flow on the lumen side of the fiber, and is less dependent on shell-side flow. In backextraction, the overall mass transfer coefficient determined is only one tenth that of the forward extraction, primarily because the hydrophobic hollow fibers used have a high mass transfer resistance under these conditions. The mass transfer experiments show that hollow-fiber extraction of Pen G is competitive with centrifugal extraction. The prospects for extraction of other fermentation products with hollow fibers can be estimated based on the present study.

Synergistic extraction of penicillin G with aliphatic alcohols and butyl acetate.

A solvent mixture of 1-hexanol and butyl acetate has been successfully used for the extraction of penicillin G. This solvent mixture may not only synergistically and effectively extract penicillin G, but also inhibit emulsion formation during the extraction of penicillin G from the filtrate of the fermentation broth. The equilibrium constants of the extraction, the molar ratios of 1-hexanol and penicillin G in the extracted species and the synergistic extraction of penicillin G have been studied by chemical analysis and by monitoring the absorbency shift in the FT-IR spectra of the functional groups in the solvents.

Penicillin G extraction from model media using an emulsion liquid membrane: a theoretical model of product decomposition.

To confirm the applicability for the extraction of penicillin G by an emulsion liquid membrane (ELM), the degree of decomposition of penicillin G during extraction was theoretically calculated. Decomposition was less than 1% provided that the initial sodium carbonate concentration in the internal phase was correctly determined, which proved the applicability of the ELM process. The procedure to determine the initial carbonate concentration in the internal phase was also described in order that the pH in the internal phase should be within the relatively stable range for penicillin G at the end of the extraction.

Penicillin G extraction from model media using an emulsion liquid membrane: determination of optimum extraction conditions.

Penicillin G extraction by an emulsion liquid membrane (ELM) was investigated. The effects of surfactants, diluents, and carrier mixtures, together with their combined effects on the initial extraction rate and the emulsion stability were examined. Surfactants, diluents, and carriers used were Span80 (sorbitan monooleate)/ECA4360J (nonionic polyamine), n-butyl acetate/kerosene, and DOA (dioctylamine)/Amberlite LA-2 (secondary amine), respectively. The optimum extraction conditions were found to be 20% (v/v) of Span80 in ECA4360J as a surfactant, kerosene as a diluent and Amberlite LA-2 as a carrier.

Immobilization of penicillin acylase on copolymer of butyl acrylate and ethylene glycol dimethacrylate.

The effects of glutaraldehyde, enzyme concentrations and reactants volumes, ionic strength, pH value and carrier particle diameter on immobilization of penicillin acylase onto acrylic carriers were studied. The activity of immobilized enzyme preparations was also studied over a range of pH values and temperatures and thermal and pH stabilities were determined. The use of the immobilized preparation for penicillin G hydrolysis in a batch reactor was investigated. The immobilized enzyme gave a significant reduction in hydrolysis time compared to hydrolysis by the native enzyme.