Activity of imipenem/relebactam against Enterobacterales and Pseudomonas aeruginosa in Spain. SMART 2016-2020

Objectives: To determine susceptibility to the novel β-lactam/β-lactamase inhibitor combination imipenem/relebactam in clinical isolates recovered from intra-abdominal (IAI), urinary (UTI), respiratory (RTI) and bloodstream (BSI) infections in the SMART (Study for Monitoring Antimicrobial Resistance Trends) study in SPAIN during 2016 – 2020.Methods: Broth microdilution MICs for imipenem/relebactam and comparators were determined by a central laboratory against isolates of Enterobacterales and Pseudomonas aeruginosa. MICs were interpreted using EUCAST-2021 breakpoints.Results: In total, 5,210 Enterobacterales and 1,418 P. aeruginosa clinical isolates were analyzed. Imipenem/relebactam inhibited 98.8% of Enterobacterales. Distinguishing by source of infection susceptibility was 99.1% in BSI, 99.2% in IAI, 97.9% in RTI, and 99.2% in UTI. Of intensive care unit isolates (ICU) 97.4% were susceptible and of non-ICU isolates 99.2% were susceptible. In Enterobacterales, activity against Class A, Class B and Class D carbapenemases was 96.2%, 15.4% and 73.2%, respectively. In P. aeruginosa, imipenem/relebactam was active in 92.2% of isolates. By source of infection it was 94.8% in BSI, 92.9% in IAI, 91.7% in RTI, and 93.1% in UTI. An 88.7% of ICU isolates and 93.6% of non-ICU isolates were susceptible to imipenem/relebactam. Imipenem/relebactam remained active against P. aeruginosa ceftazidime-resistant (76.3%), cefepime-resistant (73.6%), imipenem-resistant (71.5%) and piperacillin-resistant (78.7%) isolates. Of all multidrug-resistant or difficult-to-treat resistance P. aeruginosa isolates, 75.1% and 46.2%, respectively, were susceptible to imipenem/relebactam. (AU)

Objetivos: Determinar la sensibilidad a la nueva combinación de β-lactámico e inhibidor de β-lactamasas imipenem/relebactam en aislados clínicos procedentes de infecciones intraabdominales (IIA), urinarias (ITU), respiratorias (ITR) y bacteriemias del estudio SMART (Study for Monitoring Antimicrobial Resistance Trends) en ESPAÑA durante 2016 - 2020. Métodos. Se determinó la CMI mediante microdilución en caldo de imipenem/relebactam y antibióticos comparadores frente a aislados de Enterobacterales y Pseudomonas aeruginosa. Las CMI se analizaron empleando los puntos de corte EUCAST-2021. Resultados: En total, se incluyeron 5.210 aislados de Enterobacterales y 1.418 aislados de P. aeruginosa. Imipenem/ relebactam fue activo frente al 98,8% de los Enterobacterales. Distinguiendo por foco de infección, la sensibilidad fue del 99,1% en bacteriemia, del 99,2% en IIA, del 97,9% en ITR y del 99,2% en ITU. El 97,4% de los aislados procedentes de unidades de cuidados intensivos (UCI) fueron sensibles, y el 99,2% de los aislados no procedentes de UCI. En Enterobacterales, la sensibilidad frente a carbapenemasas de clase A, clase B y clase D fue del 96,2%, 15,4% y 73,2%, respectivamente. En P. aeruginosa,imipenem/relebactam fue activo en el 92,2% de los aislados. Distinguiendo por foco de infección, la sensibilidad frente a P. aeruginosa fue del 94,8% en bacteriemia, 92,9% en IIA, 91,7% en ITR y 93,1% en ITU. El 88,7% de los aislados de la UCI y el 93,6% de los aislados no procedentes de UCI fueron sensibles a imipenem/relebactam. Imipenem/relebactam fue activo frente a aislados de P. aeruginosa resistentes a ceftazidima (76,3%), cefepima (73,6%), imipenem (71,5%) y piperacilina/tazobactam (78,7%). Frente a los aislados de P. aeruginosa clasificados como MDR o DTR, el 75,1% y el 46,2%, respectivamente, fueron sensibles a imipenem/relebactam. (AU)

Molecular Mechanisms of Drug Resistance and Epidemiology of Multidrug-Resistant Variants of Neisseria gonorrhoeae.

The paper presents various issues related to the increasing drug resistance of Neisseria gonorrhoeae and the occurrence and spread of multidrug-resistant clones. One of the most important is the incidence and evolution of resistance mechanisms of N. gonorrhoeae to beta-lactam antibiotics. Chromosomal resistance to penicillins and oxyimino-cephalosporins and plasmid resistance to penicillins are discussed. Chromosomal resistance is associated with the presence of mutations in the PBP2 protein, containing mosaic variants and nonmosaic amino acid substitutions in the transpeptidase domain, and their correlation with mutations in the mtrR gene and its promoter regions (the MtrCDE membrane pump repressor) and in several other genes, which together determine reduced sensitivity or resistance to ceftriaxone and cefixime. Plasmid resistance to penicillins results from the production of beta-lactamases. There are different types of beta-lactamases as well as penicillinase plasmids. In addition to resistance to beta-lactam antibiotics, the paper covers the mechanisms and occurrence of resistance to macrolides (azithromycin), fluoroquinolones and some other antibiotics. Moreover, the most important epidemiological types of multidrug-resistant N. gonorrhoeae, prevalent in specific years and regions, are discussed. Epidemiological types are defined as sequence types, clonal complexes and genogroups obtained by various typing systems such as NG-STAR, NG-MAST and MLST. New perspectives on the treatment of N. gonorrhoeae infections are also presented, including new drugs active against multidrug-resistant strains.

New evidence in severe pneumonia: imipenem/cilastatin/relebactam

Imipenem combined with beta-lactamase inhibitor relebactam (IMI/REL) has an extensive bactericidal activity againstGram-negative pathogens producing class A or class C beta-lactamases, not active against class B and class D. The phase3 clinical trial (RESTORE-IMI-2), double-blind, randomized,evaluated IMI/REL vs. piperacillin-tazobactam (PIP/TAZ) fortreatment of hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP), demonstrated non-inferiorityat all-cause mortality at 28 days (15.9% vs 21.3%), favorable clinical response at 7-14 days end of treatment (61% vs59.8%) and with minor serious adverse effects (26.7% vs 32%).IMI/REL is a therapeutic option in HAP and VAP at approveddosage imipenem 500 mg, cilastatin 500 mg and relebactam250 mg once every 6h, by an IV infusion over 30 min (AU)

Survey of Staphylococcus aureus carriage by free-living red deer (Cervus elaphus): Evidence of human and domestic animal lineages.

Staphylococcus aureus is a pathogen that can affect multiple host species. Evidence of transmission between humans and animals and among different animal species has been reported in recent years. In this study, we investigated 284 free-living red deer (Cervus elaphus) in the Central Italian Alps to assess the prevalence and molecular characteristics of S. aureus in nasal and intestinal samples in relation to host features and environmental factors. A prevalence of 90%, 26.2% and 10.7% of S. aureus was detected in nasal rectal swabs and faeces, respectively. Calves had a higher probability of being S. aureus intestinal carriers than adults, especially in females when considering faecal samples. Clonal complex (CC) 425 was the most prevalent lineage (61.5%). This is a lineage known to be widespread in both domestic and free-living animals. It was followed by CC2671 (15.4%) and CC350 (6.4%). A high rate of the phage-borne virulence factor lukM/lukF-P83 was detected in CC425 and CC350. Further lineages, which are known to occur in both humans and animals, were detected sporadically in red deer faeces only, that is, CC7, CC9, CC121 and CC707, harbouring the genes of the penicillinase operon and a gene for macrolide resistance (CC9 and CC121). Methicillin resistance genes mecA and mecC were not found. Our results suggest that free-living red deer may be reservoir for S. aureus in Alpine habitats.

Graphene Oxide Modulating the Bioelectronic Properties of Penicillinase Immobilized in Lipid Langmuir-Blodgett Films.

In this paper, graphene oxide was incorporated in penicillinase-lipid Langmuir monolayers and transferred to solid supports as Langmuir-Blodgett (LB) films so that the enzyme catalytic properties could be evaluated. Adsorption of penicillinase and graphene oxide on dimyristoylphosphatidic acid (DMPA) monolayers at the air-water interface was investigated by tensiometry, vibrational spectroscopy, and Brewster angle microscopy. The LB films were characterized by quartz crystal microbalance, infrared spectroscopy, luminescence spectroscopy, and atomic force microscopy. Enzyme activity was studied with UV-vis spectroscopy, and the feasibility of the supramolecular device nanostructured as ultrathin films was essayed as an optical sensor device. The presence of graphene oxide in the enzyme-lipid LB film not only tuned the catalytic activity of penicillinase but also helped conserve its enzyme activity after weeks. These results may be related not only to the molecular architecture provided by the film but also to the synergism between the compounds on the active layer, leading to a molecular architecture that allowed a fast analyte diffusion owing to a suitable molecular accommodation which also preserved the penicillinase activity. This work then demonstrates the feasibility of employing LB films composed of lipids, graphene oxide, and enzymes as optical devices for biosensing applications as a proof-of-concept experiment.

Penicillinase plasmid Australia type in Neisseria gonorrhoeae isolated in Poland.

PURPOSE: Neisseria gonorrhoeae is an etiological agent of gonorrhea which remains a major public health problem the mechanisms that determine resistance to drugs of the beta-lactam class, which are recommended for the treatment of gonorrhea, are currently the most important problem in its treatment. Chromosomal mutations are responsible for resistance to ceftriaxone and cefepime. The possibility of mutations in the gene encoding beta-lactamase (blaTEM) in the penicillinase plasmid may also turn out to be a serious threat. METHODS: The occurrence of resistance encoded on penicillinase plasmid has been investigated. For this purpose, the susceptibility of bacteria was determined and the gene for resistance to beta-lactams as well as the plasmids themselves was typed. RESULTS: Of the 333 strains tested, 21 (6.3%) had the beta-lactamase gene and produced penicillinase. Two of the beta-lactamase: TEM-1 and TEM-135 occurred among the tested strains of N. gonorrhoeae. Most of the known penicillinase plasmid types of N. gonorrhoeae were demonstrated: the Asian, the African, the Toronto/Rio plasmids and Australian variants. CONCLUSIONS: In the first 3 years, TEM-1 beta-lactamases dominated in N. gonorrhoeae, which were replaced by TEM-135 in the following years of the study. Not all molecular methods are capable of varying the types of penicillinase plasmids. A particularly noteworthy observation is the fact that the Australia-type of penicillinase plasmid (3270 bp) was identified for the first time in Europe, and the second time in the world.

Towards Multi-Analyte Detection with Field-Effect Capacitors Modified with Tobacco Mosaic Virus Bioparticles as Enzyme Nanocarriers.

Utilizing an appropriate enzyme immobilization strategy is crucial for designing enzyme-based biosensors. Plant virus-like particles represent ideal nanoscaffolds for an extremely dense and precise immobilization of enzymes, due to their regular shape, high surface-to-volume ratio and high density of surface binding sites. In the present work, tobacco mosaic virus (TMV) particles were applied for the co-immobilization of penicillinase and urease onto the gate surface of a field-effect electrolyte-insulator-semiconductor capacitor (EISCAP) with a p-Si-SiO2-Ta2O5 layer structure for the sequential detection of penicillin and urea. The TMV-assisted bi-enzyme EISCAP biosensor exhibited a high urea and penicillin sensitivity of 54 and 85 mV/dec, respectively, in the concentration range of 0.1-3 mM. For comparison, the characteristics of single-enzyme EISCAP biosensors modified with TMV particles immobilized with either penicillinase or urease were also investigated. The surface morphology of the TMV-modified Ta2O5-gate was analyzed by scanning electron microscopy. Additionally, the bi-enzyme EISCAP was applied to mimic an XOR (Exclusive OR) enzyme logic gate.

Ceftazidime-avibactam: effectiveness and safety in the clinical practice. A third hospital level experience / Ceftazidima-avibactam: efectividad y seguridad en práctica clínica habitual. Experiencia de un hospital de tercer nivel

Objectives: Ceftazidime/avibactam (CZA) is a third generation cephalosporin and the first non-beta-lactam beta-lactamase inhibitor combination. The main outcome was to assess the effectiveness and safety of CZA in the clinical practice.Methods: It was a retrospective observational study. The inclusion criteria were age >18 years and receipt of >24 hours of CZA between January 2016 and October 2018. Variables studied included demographic, clinical, and treatment.Results: 63 inpatients in treatment with CZA were included, 39 (61.9 %) were male and the mean (SD) age was 64.3 (15.8) years. Thirty-eight (60.3%) patients presented bacteremia and 28 (44.4%) were admitted in Intensive Care Unit (ICU). Klebsiella pneumoniae were isolated in 43 (68.3) patients, and OXA-48 carbapenemase in 51 (81.0%). Concomitant antibiotic was used in 40 (63.5%) patients. Mortality at 14 and 30 days were 6 (9.5%) and 4 (6.3%) patients, respectively.Thirty-five (55.6%) patients reached microbiological cure and 47 (74.6%) clinical cure. Infection recurrence evaluated at 90 days was achieved in 23 (36.5%) patients. ICU admission and bacteremia showed decreased in clinical cure (p=0.023 and p=0.01, respectively). Only ICU admission had a diminution in microbiological cure (p=0.035) and bacteremia a higher recurrence evaluated at 90 days (p=0.003). Only 3 (4.8%) patients interrupted treatment because of the adverse events.Conclusions: ICU admission had demonstrated a microbiological and clinical cure decreasing. Recurrence evaluated a 90 days was statically significant higher in patients with bacteremia. CZA was a security antibiotic, with a very low incidence of treatment interruptions. (AU)

Objetivo: Ceftazidima/avibactam (CZA) es una cefalosporina de tercera generación y el primer inhibidor de beta-lactamasas no beta-lactámico. El objetivo principal fue evaluar su efectividad y seguridad en la práctica clínica.Métodos: Se realizó un estudio observacional retrospectivo. Los criterios de inclusión fueron: edad >18 años y administración de >24 horas de CZA entre enero de 2016 y octubre de 2018. Las variables estudiadas incluyeron demograficas, clínicas y de tratamiento.Resultados: Se incluyeron 63 pacientes con CZA, 39 (61,9 %) fueron hombres, media (SD) de edad de 64,3 (15,8) años. 38 (60,3%) pacientes presentaron bacteriemia y 28 (44,4%) fueron ingresados en la Unidad de Cuidados Intensivos (UCI). Klebsiella pneumoniae se aisló en 43 (68,3) pacientes y OXA-48 carbapenemasa en 51 (81,0%). 40 (63,5%) pacientes recibieron antibiótico concomitante. La mortalidad a los 14 y 30 días fue de 6 (9,5%) y 4 (6,3%), respectivamente.Treinta y cinco (55,6%) alcanzaron curación microbiológica y 47 (74,6%) curación clínica. Recurrencia de la infección a los 90 días sucedió en 23 (36,5%). El ingreso en UCI y la bacteriemia demostraron una disminución de la curación clínica (p-0,023 y p-0,01, respectivamente). El ingreso en UCI tuvo una disminución en curación microbiológica (p-0,035) y la bacteriemia en una mayor recurrencia a los 90 días (p-0,003). 3 (4,8%) interrumpieron el tratamiento por toxicidad.Conclusiones: El ingreso en UCI se relacionó con disminución de curación microbiológica y clínica. La recurrencia a los 90 días fue mayor en pacientes con bacteriemia. CZA presenta una incidencia baja de interrupciones del tratamiento. (AU)

Epidemiologic and molecular characterization of Beta-lactamase-producing multidrug-resistant uropathogenic Escherichia coli isolated from asymptomatic hospitalized patients / Caracterización epidemiológica y molecular de Escherichia coli uropatógena multirresistente productora de Beta-lactamasa aislada de pacientes hospitalizados asintomáticos

Uropathogenic Escherichia coli (UPECs) are the predominant cause of asymptomatic bacteriuria (ABU) and symptomatic UTI. In this study, multidrug-resistant (MDR) ABU-UPECs from hospitalized patients of Kolkata, India, were characterized with respect to their ESBL phenotype, acquisition of β-lactamase genes, mobile genetic elements (MGEs), phylotype property, ERIC-PCR profile, sequence types (STs), clonal complexes (CCs) and evolutionary and quantitative relationships and compared to the symptomatic ones to understand their epidemiology and evolutionary origin. Statistically significant incidence of ESBL producers, β-lactamase genes, MGEs and novel phylotype property (NPP) among ABU-UPECs similar to the symptomatic ones indicated the probable incidence of chromosomal plasticity on resistance gene acquisition through MGEs due to indiscriminate drug usage. ERIC-PCR typing and MLST analysis showed clonal heterogeneity and predominance of ST940 (CC448) among asymptomatic isolates akin to symptomatic ones along with the evidence of zoonotic transmissions. Minimum spanning tree analysis showed a close association between ABU-UPEC with known and unidentified STs having NPPs with isolates that belonged to phylogroups clade I, D, and B2. This is the first study that reported the occurrence of MGEs and NPPs among ABU-UPECs with the predominance of ESBL production which displayed the deleterious effect of MDR among this pathogen demanding alternative therapeutic interventions. Moreover, this study for the first time attempted to introduce a new approach to ascertain the phylotype property of unassigned UPECs. Withal, increased recognition, proper understanding and characterization of ABU-UPECs with the implementation of appropriate therapeutic measures against them when necessary are the need of the era which otherwise might lead to serious complications in the vulnerable population.(AU)

Opening of a cryptic pocket in ß-lactamase increases penicillinase activity.

Understanding the functional role of protein-excited states has important implications in protein design and drug discovery. However, because these states are difficult to find and study, it is still unclear if excited states simply result from thermal fluctuations and generally detract from function or if these states can actually enhance protein function. To investigate this question, we consider excited states in ß-lactamases and particularly a subset of states containing a cryptic pocket which forms under the Ω-loop. Given the known importance of the Ω-loop and the presence of this pocket in at least two homologs, we hypothesized that these excited states enhance enzyme activity. Using thiol-labeling assays to probe Ω-loop pocket dynamics and kinetic assays to probe activity, we find that while this pocket is not completely conserved across ß-lactamase homologs, those with the Ω-loop pocket have a higher activity against the substrate benzylpenicillin. We also find that this is true for TEM ß-lactamase variants with greater open Ω-loop pocket populations. We further investigate the open population using a combination of NMR chemical exchange saturation transfer experiments and molecular dynamics simulations. To test our understanding of the Ω-loop pocket's functional role, we designed mutations to enhance/suppress pocket opening and observed that benzylpenicillin activity is proportional to the probability of pocket opening in our designed variants. The work described here suggests that excited states containing cryptic pockets can be advantageous for function and may be favored by natural selection, increasing the potential utility of such cryptic pockets as drug targets.

Enzyme-based sensing on nanohybrid film coated over FTO electrode for highly sensitive detection of antibiotics.

Cefepime and Meropenem are the new class of antibiotics, which are particularly used as last potent defender or the antibiotics of the last resort against multi-resistant bacterial species. In this paper, an impedance-based electrochemical biosensor was fabricated for identifying antibiotics of last resort in the forensic samples including gastric lavage and other body fluids. The sensor was developed using platinum nanoparticles (PtNPs) and electrodeposited zinc oxide- zinc hexacyanoferrate hybrid film (ZnO/ZnHCF) on the surface of a fluorine-doped glass electrode (FTO). Further, penicillinase was immobilized onto the modified electrode using penicillinase enzyme. The developed biosensor exhibits a good analytical response for the detection of antibiotics evaluated using electrochemistry studies. The linear response of the fabricated electrode was observed from 0.1 to 750 µM and the electrode limit of detection (LOD) was observed as 0.1 µM. The sensor confirms good accuracy, is highly selective, and sensitive for the target. While storing the modified electrode at 4 °C, the stability of biosensor was evaluated for 45 days, and activity loss of 30-40% was observed. The highly sensitive interface of Penicillinase@CHIT/PtNP-ZnO/ZnHCF/FTO electrode shows a promising future in forensic studies.

Penicillin biosensor based on rhombus-shaped porous carbon/hematoxylin/penicillinase.

In this experiment, we designed an electrochemical sensor using penicillinase (Pen X)-rhombus porous carbon (RPC) as the detection element and hematoxylin as the indicator to detect low concentrations of penicillin sodium (Pen G). A differential pulse voltammetry (DPV) method was used to detect Pen G in the concentration range of 10-8 -10-5 mg·mL-1 under optimal experimental conditions. The results showed that the peak current value and the logarithm of Pen G concentration showed a good linear relationship (R2 = 0.9915), and the LOD was 2.68 × 10-7 mg·mL-1 (S/N = 3). The actual milk samples were detected by the addition method and compared with the high-performance liquid phase method; no significant difference was found in the detection results. The working electrode prepared by cross-linking method not only extends the service life of the sensor, but also improves the sensitivity and reproducibility of the sensor. It can also be used to detect the Pen G residue in the actual milk samples repeatedly. PRACTICAL APPLICATION: In this study, an electrochemical sensor for the rapid detection of penicillin sodium in milk was prepared, which has good sensitivity and fast detection speed.

Light-Addressable Actuator-Sensor Platform for Monitoring and Manipulation of pH Gradients in Microfluidics: A Case Study with the Enzyme Penicillinase.

The feasibility of light-addressed detection and manipulation of pH gradients inside an electrochemical microfluidic cell was studied. Local pH changes, induced by a light-addressable electrode (LAE), were detected using a light-addressable potentiometric sensor (LAPS) with different measurement modes representing an actuator-sensor system. Biosensor functionality was examined depending on locally induced pH gradients with the help of the model enzyme penicillinase, which had been immobilized in the microfluidic channel. The surface morphology of the LAE and enzyme-functionalized LAPS was studied by scanning electron microscopy. Furthermore, the penicillin sensitivity of the LAPS inside the microfluidic channel was determined with regard to the analyte's pH influence on the enzymatic reaction rate. In a final experiment, the LAE-controlled pH inhibition of the enzyme activity was monitored by the LAPS.

Molecular characteristics and antimicrobial susceptibility of penicillinase-producing Neisseria gonorrhoeae isolates in Fukuoka, Japan, 1996-2018.

OBJECTIVES: The objective of this study was to investigate the molecular characteristics and antimicrobial susceptibility of penicillinase-producing Neisseria gonorrhoeae (PPNG) isolates collected in Fukuoka, Japan, from 1996-2018. METHODS: Antimicrobial susceptibility to seven antibiotics was determined by the agar dilution method. Molecular characteristics were determined by Sanger sequencing of the blaTEM allele, plasmid typing and N. gonorrhoeae multiantigen sequence typing (NG-MAST). Furthermore, full sequences of the penA gene, encoding penicillin-binding protein 2 (PBP2), of PPNG isolates with reduced susceptibility to cefixime were analysed. RESULTS: Among 50 PPNG isolates, 17 and 33 were collected during 1996-2006 and 2007-2018, respectively. In 1996-2006, blaTEM-1 in African plasmid was most frequent (64.7%), followed by blaTEM-1 in Asian plasmid (29.4%) and blaTEM-135 in Toronto/Rio plasmid (5.9%). In 2007-2018, blaTEM-135 in Toronto/Rio plasmid was predominant (54.5%), followed by blaTEM-1 in African plasmid (36.4%) and blaTEM-135 in Asian plasmid (6.1%). Among isolates with the blaTEM-135-carrying Toronto/Rio plasmid in 2007-2018, a novel genogroup G15576 was predominant (66.7%). Isolates with the TEM-135 ß-lactamase were more resistant to ciprofloxacin but were more susceptible to ceftriaxone and tetracycline than isolates with TEM-1. Seven PPNG isolates less susceptible to cefixime possessed the plasmidic blaTEM-1 allele and had mosaic or non-mosaic alterations within PBP2. CONCLUSION: The proportion of PPNG with the blaTEM135-carrying Toronto/Rio plasmid increased during the last 12 years. The increase in PPNG carrying the blaTEM-135 allele is of particular concern as it is considered a possible direct precursor of an extended-spectrum ß-lactamase (ESBL).

A two-dimensional photonic crystal hydrogel biosensor for colorimetric detection of penicillin G and penicillinase inhibitors.

A simple penicillinase functionalized two-dimensional photonic crystal hydrogel (2DPPCH) biosensor was developed for colorimetric detection of penicillin G and penicillinase inhibitors. The penicillinase can specifically recognize penicillin G and catalyze it to produce penicilloic acid, which decreases the pH of the hydrogel microenvironment and shrinks the pH-sensitive hydrogel. The particle spacing decrease of the 2D photonic crystal array induced by the hydrogel shrinkage further causes a blue-shift in the diffraction wavelength. While the hydrolysis reaction is repressed upon treatment with clavulanate potassium (a kind of penicillinase inhibitor), no significant change in the diffraction wavelength is found. The detection of targets can be achieved by measuring the Debye diffraction ring diameter or observing the structural color change in the visible region. The lowest detectable concentrations for penicillin G and clavulanate potassium are 1 µM and 0.1 µM, respectively. Moreover, the 2DPPCH is proved to exhibit high selectivity and an excellent regeneration property, and it shows satisfactory performance for penicillin G analysis in real water samples.

Minimal agreement between basophil activation test and immunoassay in diagnosis of penicillin allergy

INTRODUCTION: Basophil activation test (BAT) and immunoassays are the most widely used in vitro tests to diagnose IgE-mediated allergic reactions to penicillin. However, studies to determine if one test is interdependent from another are limited. OBJECTIVE: The present study aimed to measure the agreement between BAT and immunoassay in diagnosis of penicillin allergy. METHOD: BAT was performed using penicillin G (Pen G), penicillin V (Pen V), penicilloyl-polylysine (PPL), minor determinant mix (MDM), amoxicillin (Amx) and ampicillin (Amp) in 25 patients. Immunoassay of total IgE (tIgE) and specific IgE (sIgE) antibodies to Pen G, Pen V, Amx and Amp were quantified. Skin prick test (SPT) using PPL-MDM, Amx, Amp and Clavulanic acid were also performed. RESULTS: Minimal agreement was observed between BAT and immunoassay (k = 0.25). Of two BAT-positive patients, one patient is positive to Amx (59.27%, SI = 59) and Amp (82.32%, SI = 82) but sIgE-negative to all drug tested. This patient is also SPT-positive to both drugs. Another patient is BAT-positive to Pen G (10.18%, SI = 40), Pen V (25.07%, SI = 100) and Amp (19.52%, SI = 79). In sIgE immunoassay, four patients were sIgE-positive to at least one of the drugs tested. The sIgE level of three patients was between low and moderate and they were BAT-negative. One BAT-positive patient had a high level of sIgE antibodies (3.5-17.5kU/L) along with relatively high specific to total IgE ratio ≥ 0.002 (0.004-0.007). CONCLUSIONS: The agreement between BAT and immunoassay is minimal. Performing both tests provides little increase in the sensitivity of allergy diagnosis work-up for immediate reactions to penicillin

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Performance of penicillinase detection tests in Staphylococcus epidermidis: comparison of different phenotypic methods.

BACKGROUND: Staphylococcus epidermidis is the leading coagulase negative staphylococci (CoNS) species associated with healthcare associated infections. In order to de-escalate antimicrobial therapy, isolates of S. epidermidis lacking the blaZ gene should be eligible for targeted antimicrobial therapy. However, testing the susceptibility of coagulase negative staphylococci (CoNS) to penicillin G is no longer recommended by EUCAST, given the low performances for penicillinase detection in CoNS. The objective of this work was to determine a phenotypic method with high performance for detecting penicillinase production in S. epidermidis. RESULTS: Four techniques for the detection of penicillinase production (disk diffusion, zone edge test, nitrocefin test, Minimal Inhibitory Concentration (MIC) by automated system Vitek2®) were evaluated on 182 S. epidermidis isolates, using identification of blaZ gene by PCR as the reference method. The performance of the methods for penicillinase detection was compared by the sensitivity, the specificity, the negative predictive value and the positive predictive value, and with Cohen's kappa statistical test. Among the 182 S. epidermidis included in this study, 55 carried the blaZ gene. The nitrocefin test, characterized by a poor sensitivity (91%), was therefore excluded from S. epidermidis penicillinase detection. The algorithm proposed here for the penicillinase detection in S. epidermidis involved two common antimicrobial susceptibility techniques: disk diffusion method and MIC by Vitek2® system. Disk diffusion method, interpreted with a 26 mm breakpoint for penicillin G, was associated with a high sensitivity (98%) and specificity (100%). This method was completed with zone edge test for S. epidermidis with penicillin G diameter from 26 to 35 mm (sensitivity of 98%). The Vitek2® system is associated with a low sensitivity (93%) and a high specificity (99%) This low sensitivity is associated with false negative results, in isolates with 0.12 mg/L Penicillin G MIC values and blaZ positive. Thus for penicillin G MIC of 0.06 mg/L or 0.12 mg/L, a second step with disc diffusion method is suggested. CONCLUSIONS: According to our results, the strategy proposed here allows the interpretation of penicillin G susceptibility in S. epidermidis isolates, with an efficient detection of penicillin G resistance.

Magnetic mesoporous carbon material based electrochemical sensor for rapid detection of penicillin sodium in milk.

In recent years, to increase growth rate and prevent infectious diseases, an excessive use of antibiotics in livestock breeding processes has resulted in the presence of antibiotic residues in animal foods. In this experiment, a new kind of electrochemical sensor is prepared based on magnetic mesoporous hollow carbon microspheres (MHM) as a penicillinase (Pen X) adsorption carrier to rapidly detect penicillin sodium (Pen G), named Pen X/MHM/MGCE. The MHM-adsorbed penicillinase can be separated from the solution by magnetic attraction and fixed on a magnetic glassy carbon electrode by physical adsorption, which is easy to operate and avoids the interference of a crosslinking agent at the active site of the enzyme. A differential pulse voltammetry (DPV) method is used to immerse the working electrode in test samples containing different concentrations of penicillin sodium solution with 0.5 mg/mL oxidized hematoxylin. According to the quantitative relationship between the current value and the concentration of penicillin sodium, the concentration of penicillin sodium in the tested samples can be determined. The detection range of the sensor is 10-8 to 10-2 mg/mL, the linear relationship is good (R2 = 0.9983), and the detection limit (LOD) is 2.655 × 10-7 mg/mL (S/N = 3). The detection of penicillin sodium in milk using a standard addition method shows good recovery. Furthermore, the proposed method has the advantages of a wide detection range, good enzyme immobilization capacity, good precision and stability, convenient cleaning, and recycling of solid materials. Thus, it can successfully achieve rapid detection in milk samples. PRACTICAL APPLICATION: The sensor provides a low detection limit and high recovery rate of electrode material; therefore, the sensor can realize the rapid and quantitative detection of penicillin sodium in milk.

Modularized Field-Effect Transistor Biosensors.

Field-effect transistors (FETs), when functionalized with proper biorecognition elements (such as antibodies or enzymes), represent a unique platform for real-time, specific, label-free transduction of biochemical signals. However, direct immobilization of biorecognition molecules on FETs imposes limitations on reprogrammability, sensor regeneration, and robust device handling. Here we demonstrate a modularized design of FET biosensors with separate biorecognition and transducer modules, which are capable of reversible assembly and disassembly. In particular, hydrogel "stamps" immobilizing bioreceptors have been chosen to build biorecognition modules to reliably interface with FET transducers structurally and functionally. Successful detection of penicillin down to 0.25 mM has been achieved with a penicillinase-encoded hydrogel module, demonstrating effective signal transduction across the hybrid interface. Moreover, sequential integration of urease- and penicillinase-encoded modules on the same FET device allows us to reprogram the sensing modality without cross-contamination. In addition to independent bioreceptor encoding, the modular design also fosters sophisticated control of sensing kinetics by modulating the physiochemical microenvironment in the biorecognition modules. Specifically, the distinction in hydrogel porosity between polyethylene glycol and gelatin enables controlled access and detection of larger molecules, such as poly-l-lysine (MW 150-300 kDa), only through the gelatin module. Biorecognition modules with standardized interface designs have also been exploited to comply with additive mass fabrication by 3D printing, demonstrating potential for low cost, ease of storage, multiplexing, and great customizability for personalized biosensor production. This generic concept presents a unique integration strategy for modularized bioelectronics and could broadly impact hybrid device development.

Infecciones urinarias por Escherichia coli productor de betalactamasas de espectro extendido en un hospital terciario ¿existe un aumento de las resistencias a los betalactámicos? / Extended spectrum beta-lactamase producing Escherichia coli in a tertiary hospital. Is there an increase of resistances to beta lactams?

Objetivo: El aumento de aislamiento de cepas Escherichia coli resistentes a las betalactamasas (ECRB) dificulta su tratamiento. El objetivo de este estudio es evaluar la situación en nuestro entorno. Material y método: Estudio retrospectivo entre enero 2012 y diciembre de 2015, incluyendo pacientes que presentaban cultivos de orina positivos para E. coli en el Departamento de Salud de Castellón. Analizamos variables como: edad, sexo, procedencia del paciente (medio ambulatorio u hospitalario) y otros factores de riesgo. Se ha realizado un análisis descriptivo para estudiar la prevalencia de ECRB. Resultados: Se obtuvieron 9113 cultivos positivos para E. coli, de estos 273 (2,9%) fueron ECRB. El porcentaje por año de ECRB fue entre un 1,7% y un 3,4% sin observarse incremento en los últimos años. La edad media fue 70 años, sin diferencias entre sexos. 247 cultivos procedían de pacientes ambulatorios (90%), correspondiendo el 96% de estos a mujeres. Los factores de riesgo más asociados a ECBR fueron presentar ITU el último año y haber recibido tratamiento antibiótico en los 3 meses previos, de estos un 50% recibió un betalactámico. Conclusiones: En nuestro medio el aislamiento de ECRB es similar a otras series. La mayoría de pacientes fueron procedentes de medio ambulatorio, tratados previamente con antibióticos y con episodios de ITU recurrentes

Objective: The increase of isolation of beta-lactamase resistant Escherichia coli (BREC) strains makes treatment difficult. The objective of this study is to evaluate the situation in our environment. Methods: Retrospective study including patients who presented positive urine cultures for E. coli in the Department of Health of Castellon between January 2012 and December 2015. We analyzed variables such as age, gender, patient`s origin (outpatient or hospital) and other risk factors. We performed a descriptive analysis to study the prevalence of BREC. Results: 9113 cultures were positive for E Coli, 273(2.9%) of them were BREC. The annual percentage of BREC ranged from 1.7% to 3,4% with no increase over the last years. Mean age was 70 years, without gender differences. 247 cultures came from outpatient patients (90%), being 96% of them women. The factors most frequently associated with BREC were to present UTI over the last year and have received antibiotic treatment the previous 3 months; 50% of these received a beta lactam. Conclusions: In our environment, the isolation of BREC is similar to other series. Most patients come from the outpatient environment, were previously treated with antibiotics and had recurrent UTIs