A penicillinase-modified poly(N-isopropylacrylamide-co-acrylamide) smart hydrogel biosensor with superior recyclability for sensitive and colorimetric detection of penicillin G.

Antibiotics are widely used for treating bacterial infections. However, excessive or improper use of antibiotics can pose a serious threat to human health and water environments, and thus, developing cost-effective, portable and effective strategies to analyze and detect antibiotics is highly desired. Herein, we reported a responsive photonic hydrogel (RPH)-based optical biosensor (PPNAH) with superior recyclability for sensitive and colorimetric determination of a typical ß-lactam antibiotic penicillin G (PG) in water. This sensor was composed of poly(N-isopropylacrylamide-co-acrylamide) smart hydrogel with incorporated penicillinase and Fe3O4@SiO2 colloidal photonic crystals (CPCs). The sensor could translate PG concentration signals into changes in the diffraction wavelength and structural color of the hydrogel. It possessed high sensitivity and selectivity to PG and excellent detection performances for other two typical ß-lactam antibiotics. Most importantly, due to the unique thermosensitivity of the poly(N-isopropylacrylamide) moieties in the hydrogel, the PG-responded PPNAH sensor could be facilely regenerated via a simple physical method at least fifty times while without compromising its response performance. Besides, our sensor was suitable for monitoring the PG-contaminated environmental water and displayed satisfactory detection performances. Such a sensor possessed obvious advantages of superior recyclability, highly chemical stability, low production cost, easy fabrication, wide range of visual detection, simple and intuitive operation for PG detection, and environmental-friendliness, which holds great potential in sensitive and colorimetric detection of the PG residues in polluted water.

Treatment outcomes with benzylpenicillin and non-benzylpenicillin antibiotics, and the performance of the penicillin zone-edge test versus molecular detection of blaZ in penicillin-susceptible Staphylococcus aureus (PSSA) bacteraemia.

OBJECTIVES: The blaZ gene encodes penicillinase, which inactivates penicillin. As there were reports on suboptimal sensitivity for the penicillin zone-edge test, a phenotypic method for blaZ detection, we investigated treatment outcomes in patients with penicillin-susceptible Staphylococcus aureus (PSSA) bacteraemia (phenotypically negative for penicillinase), subjecting isolates to molecular testing for blaZ retrospectively. PATIENTS AND METHODS: A retrospective cohort study was conducted on 121 patients with a first episode of PSSA bacteraemia from 1 January 2012 to 31 October 2015 at Tan Tock Seng Hospital (TTSH), Singapore. Patients were grouped into IV benzylpenicillin and non-benzylpenicillin groups. The primary outcome was overall treatment failure, defined as either 30 day all-cause mortality and/or 90 day relapse. The penicillin (P10) zone-edge test was repeated on archived PSSA isolates, concurrently with penicillin MIC determination via gradient diffusion and PCR for blaZ. RESULTS: Among 121 patients, 57 patients (47.1%) received IV benzylpenicillin as the predominant antibiotic. There was no significant difference in overall treatment failure between treatment with the benzylpenicillin [7/57 (12.3%)] versus non-benzylpenicillin groups [12/64 (18.8%)] (P = 0.33) or cloxacillin/cefazolin [6/37 (16.2%)] (P = 0.59). For 112 PSSA isolates available for testing, repeat penicillin zone-edge testing was negative for penicillinase production, corroborating previous results. A single PSSA isolate with a negative penicillin zone-edge test was found to be positive for blaZ. CONCLUSIONS: We found no differences in overall treatment failure between patients with PSSA bacteraemia treated with benzylpenicillin, anti-staphylococcal ß-lactams cefazolin/cloxacillin and other antimicrobials, when using the penicillin zone-edge test as the phenotypic method for blaZ screening.

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)

Temoneira-1 ß-lactamase is not a metalloenzyme, but its native metal ion binding sites allow for purification by immobilized metal ion affinity chromatography.

ß-lactamases protect bacteria from ß-lactam antibiotics. Temoneira (TEM) is a class A serine ß-lactamase and its coding sequence is designed into DNA vectors, such as pET-21a (+), to provide antibiotic resistance. TEM-1 ß-lactamase was overexpressed efficiently from this vector upon inducing protein expression by IPTG in BL21(DE3) cells. Immobilized metal ion affinity chromatography (IMAC) was used based on the three native putative metal ion binding sites of TEM-1 ß-lactamase, each consisting of a pair of histidine sidechains. Elution was achieved at low concentrations of imidazole (∼15-200 mM). Two steps of IMAC and a subsequent anion exchange purification produced highly pure TEM-1 ß-lactamase with a yield of 1.9 mg/g of wet bacterial pellet weight. Mass spectrometry revealed that the mature form of ß-lactamase (without the signal sequence) was obtained. The secondary structure composition, calculated from the circular dichroism spectrum, showed that the target protein was folded similar to the published crystal structure. Ni(II) binding to the enzyme was also investigated. Increasing amounts of Ni(II) ions had only a small effect on the protein structure. Mass spectrometry detected up to three bound metal ions at 10:1 Ni(II):protein molar ratio, but the major peak was assigned to the monometallated ß-lactamase indicating the presence of a paramount metal ion binding site formed by the H151/H156 pair.

Molecular Epidemiology of Penicillinase-Producing Neisseria gonorrhoeae Isolates and Their blaTEM-135 Gene Variant in Bangkok, Thailand, 2015-2017.

Penicillinase-producing Neisseria gonorrhoeae (PPNG) possessing blaTEM-135 is a serious public health threat. With only a single change in the amino acid sequence, blaTEM-135 could evolve into a TEM-type extended-spectrum beta-lactamase (ESBL), which hydrolyzes extended-spectrum cephalosporins, including ceftriaxone and cefixime. We investigated the molecular epidemiological characteristics, types of plasmids in PPNG isolates, and prevalence of PPNG clinical isolates producing TEM-135 beta-lactamases. N. gonorrhoeae multi-antigen sequence typing (NG-MAST) was used to determine the molecular epidemiological characteristics of 99 PPNG isolates collected from 2015 to 2017. A mismatch amplification mutation assay was used to examine the blaTEM-135 gene prevalence. Of the 89 identified NG-MAST sequence types, 65 (73.0%) were novel. Only 17.7% (43/243) of PPNG isolates belonged to 16 genogroups. The most frequent plasmid was African, followed by Rio/Toronto, and Asian. The blaTEM-135 allele was found in Rio/Toronto plasmids. The blaTEM-135 allele was present in 23.2% (23/99) of the PPNG isolates. PPNG isolates expressing TEM-135 beta-lactamase exhibited significantly higher penicillin MIC (minimum inhibitory concentration) values than TEM-1 PPNG isolates. The PPNG isolates showed high genetic diversity and a high proportion of blaTEM-135 alleles. Mutation of the blaTEM-135 allele is worrisome as only one mutation could cause TEM-1 to evolve into an ESBL variant that degrades ceftriaxone. Ongoing surveillance of blaTEM-135 and new PPNG isolates is imperative.

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.

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)

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)

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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