Sulbactam/Durlobactam: First Approval.

Sulbactam/durlobactam (XACDURO®), is a co-packaged antibacterial product that has been developed by Entasis Therapeutics Inc. for the treatment of infections caused by Acinetobacter baumannii-calcoaceticus complex (ABC). Coadministration of durlobactam (a ß-lactamase inhibitor with potent activity against a broad range of serine ß-lactamases) with sulbactam (an established class A ß-lactamase inhibitor with antibacterial activity against A. baumannii) prevents sulbactam degradation by ABC-produced ß-lactamases. In May 2023, sulbactam/durlobactam was approved in the USA for use in patients 18 years of age and older for the treatment of hospital-acquired bacterial pneumonia and ventilator-associated bacterial pneumonia (HABP/VABP) caused by susceptible isolates of ABC. This article summarizes the milestones in the development of sulbactam/durlobactam leading to this first approval for the treatment of infections caused by ABC.

Structural Insight into the Mechanism of Inhibitor Resistance in CTX-M-199, a CTX-M-64 Variant Carrying the S130T Substitution.

The smart design of ß-lactamase inhibitors allowed us to combat extended-spectrum ß-lactamase (ESBL)-producing organisms for many years without developing resistance to these inhibitors. However, novel resistant variants have emerged recently, and notable examples are the CTX-M-190 and CTX-M-199 variants, which carried a S130T amino acid substitution and exhibited resistance to inhibitors such as sulbactam and tazobactam. Using mass spectrometric and crystallographic approaches, this study depicted the mechanisms of inhibitor resistance. Our data showed that CTX-M-64 (S130T) did not cause any conformational change or exert any effect on its ability to hydrolyze ß-lactam substrates. However, binding of sulbactam, but not clavulanic acid, to the active site of CTX-M-64 (S130T) led to the conformational changes in such active site, which comprised the key residues involved in substrate catalysis, namely, Thr130, Lys73, Lys234, Asn104, and Asn132. This conformational change weakened the binding of the sulbactam trans-enamine intermediate (TSL) to the active site and rendered the formation of the inhibitor-enzyme complex, which features a covalent acrylic acid (AKR)-T130 bond, inefficient, thereby resulting in inhibitor resistance in CTX-M-64 (S130T). Understanding the mechanisms of inhibitor resistance provided structural insight for the future development of new inhibitors against inhibitor-resistant ß-lactamases.

New Conformations of Acylation Adducts of Inhibitors of ß-Lactamase from Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (TB), is naturally resistant to ß-lactam antibiotics due to the production of the extended spectrum ß-lactamase BlaC. ß-Lactam/ß-lactamase inhibitor combination therapies can circumvent the BlaC-mediated resistance of Mtb and are promising treatment options against TB. However, still little is known of the exact mechanism of BlaC inhibition by the ß-lactamase inhibitors currently approved for clinical use, clavulanic acid, sulbactam, tazobactam, and avibactam. Here, we present the X-ray diffraction crystal structures of the acyl-enzyme adducts of wild-type BlaC with the four inhibitors. The +70 Da adduct derived from clavulanate and the trans-enamine acylation adducts of sulbactam and tazobactam are reported. BlaC in complex with avibactam revealed two inhibitor conformations. Preacylation binding could not be observed because inhibitor binding was not detected in BlaC variants carrying a substitution of the active site serine 70 to either alanine or cysteine, by crystallography, ITC or NMR. These results suggest that the catalytic serine 70 is necessary not only for enzyme acylation but also for increasing BlaC affinity for inhibitors in the preacylation state. The structure of BlaC with the serine to cysteine mutation showed a covalent linkage of the cysteine 70 Sγ atom to the nearby amino group of lysine 73. The differences of adduct conformations between BlaC and other ß-lactamases are discussed.

Plasma and Intrapulmonary Concentrations of ETX2514 and Sulbactam following Intravenous Administration of ETX2514SUL to Healthy Adult Subjects.

ETX2514 is a novel ß-lactamase inhibitor that broadly inhibits Ambler class A, C, and D ß-lactamases. ETX2514 combined with sulbactam (SUL) in vitro restores sulbactam activity against Acinetobacter baumannii ETX2514-sulbactam (ETX2514SUL) is under development for the treatment of A. baumannii infections. The objective of this study was to determine and compare plasma, epithelial lining fluid (ELF), and alveolar macrophage (AM) concentrations following intravenous (i.v.) ETX2514 and sulbactam. Plasma, ELF, and AM concentrations of ETX2514 and sulbactam were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in 30 healthy adult subjects following repeated dosing (ETX2514 [1 g] and sulbactam [1 g] every 6 h [q6h], as a 3-h i.v. infusion, for a total of 3 doses). A bronchoalveolar lavage (BAL) was performed once in each subject at either 1, 2.5, 3.25, 4, or 6 h after the start of the last infusion. Penetration ratios were calculated from area under the concentration-time curve from 0 to 6 h (AUC0-6) values for total plasma and ELF using mean and median concentrations at the BAL fluid sampling times. Respective ELF AUC0-6 values, based on mean and median concentrations, were 40.1 and 39.4 mg · h/liter for ETX2514 and 34.7 and 34.5 mg · h/liter for sulbactam. Respective penetration ratios of ELF to total/unbound plasma concentrations, based on mean and median AUC0-6 values, of ETX2514 were 0.37/0.41 and 0.36/0.40, whereas these same ratio values were 0.50/0.81 and 0.50/0.80 for sulbactam. ETX2514 and sulbactam concentrations in AM were measurable and fairly constant throughout the dosing interval (median values of 1.31 and 1.01 mg/liter, respectively). These data support further study of ETX2514SUL for the treatment of pneumonia caused by multidrug-resistant A. baumannii (This study has been registered at ClinicalTrials.gov under identifier NCT03303924.).

Acinetobacter baumannii OmpA Is a Selective Antibiotic Permeant Porin.

OmpAAb is a conserved, abundantly expressed outer membrane porin in Acinetobacter baumannii whose presumed role in antibiotic permeation has not been clearly demonstrated. In this report, we use a titratable heterologous expression system to express OmpAAb in isolation and demonstrate selective passage of small molecule antibiotics through OmpAAb. ETX2514, a recently discovered broad-spectrum ß-lactamase inhibitor, in combination with sulbactam, is currently in clinical testing for the treatment of drug-resistant A. baumannii infections. We demonstrate that ETX2514 permeates OmpAAb and potentiates the activity of sulbactam in an OmpAAb-dependent manner. In addition, we show that small modifications in the structure of ETX2514 differentially affect its passage through OmpAAb, revealing unique structure-porin-permeation relationships. Finally, we confirm the contribution of OmpAAb to bacterial fitness using a murine thigh model of A. baumannii infection. These results, combined with the high sequence homology of OmpA across Acinetobacter spp., suggest that optimization of antibiotic entry through OmpAAb may prove to be a feasible medicinal chemistry design strategy for future antibacterial discovery efforts.

Kinetics of Sulbactam Hydrolysis by ß-Lactamases, and Kinetics of ß-Lactamase Inhibition by Sulbactam.

Sulbactam is one of four ß-lactamase inhibitors in current clinical use to counteract drug resistance caused by degradation of ß-lactam antibiotics by these bacterial enzymes. As a ß-lactam itself, sulbactam is susceptible to degradation by ß-lactamases. I investigated the Michaelis-Menten kinetics of sulbactam hydrolysis by 14 ß-lactamases, representing clinically widespread groups within all four Ambler classes, i.e., CTX-M-15, KPC-2, SHV-5, and TEM-1 for class A; IMP-1, NDM-1, and VIM-1 for class B; Acinetobacter baumannii ADC-7, Pseudomonas aeruginosa AmpC, and Enterobacter cloacae P99 for class C; and OXA-10, OXA-23, OXA-24, and OXA-48 for class D. All of the ß-lactamases were able to hydrolyze sulbactam, although they varied widely in their kinetic constants for the reaction, even within each class. I also investigated the inactivation kinetics of the inhibition of these enzymes by sulbactam. The class A ß-lactamases varied widely in their susceptibility to inhibition, the class C and D enzymes were very weakly inhibited, and the class B enzymes were essentially or completely unaffected. In addition, we measured the sulbactam turnover number, the sulbactam/enzyme molar ratio required for complete inhibition of each enzyme. Class C enzymes had the lowest turnover numbers, class A enzymes varied widely, and class D enzymes had very high turnover numbers. These results are valuable for understanding which ß-lactamases ought to be well inhibited by sulbactam. Moreover, since sulbactam has intrinsic antibacterial activity against Acinetobacter species pathogens, these results contribute to understanding ß-lactamase-mediated sulbactam resistance in Acinetobacter, especially due to the action of the widespread class D enzymes.

Spectroscopy analysis and molecular dynamics studies on the binding of penicillin V and sulbactam to beta-lactamase II from Bacillus cereus.

The molecular recognition and interaction of beta-lactamase II from Bacillus cereus (Bc II) with penicillin V (PV) and sulbactam (Sul) especially conformational changes of Bc II in the binding process were studied through spectroscopy analysis in combination with molecular dynamics (MD) simulation. The results show that in the binding process, a new coordination bond is observed between the Zn2 of Bc II and the carboxyl-O of PV or Sul by replacing His204. Electrostatic interaction between Zn2 and the ligand provide main driving force for the binding affinity. Compared with apo Bc II, there are mainly four loops showing significant conformational changes in ligand-bound Bc II. A weak conformational transformation from ß-sheets to random coils is observed in the loop2 of ligand-bound Bc II. The conformational transformation may depend on the functional group and binding pose of the ligand, giving the binding pocket greater flexibility and accordingly allowing for an induced fit of the enzyme-ligand binding site around the newly introduced ligand. The change in the loop2 of ligand-bound Bc II may lead to the opening of the binding pocket of Bc II. Therefore, loop2 can be considered a gate for control of ligand access in Bc II, hence its dynamic response should be considered in new drug design and development.

Molecular recognition and binding of beta-lactamase II from Bacillus cereus with penicillin V and sulbactam by spectroscopic analysis in combination with docking simulation.

The molecular recognition and binding interaction of beta-lactamase II from Bacillus cereus (Bc II) with penicillin V (PV) and sulbactam (Sul) at 277 K were studied by spectroscopic analysis and molecular docking. The results showed that a non-fluorescence static complex was separately formed between Bc II and two ligands, the molecular ratio of Bc II to PV or Sul was both 1:1 in the binding and the binding constants were 2.00 × 106 and 3.98 × 105 (L/mol), respectively. The negative free energy changes and apparent activation energies indicated that both the binding processes were spontaneous. Molecular docking showed that in the binding process, the whole Sul molecule entered into the binding pocket of Bc II while only part of the whole PV molecule entered into the pocket due to a long side chain, and electrostatic interactions were the major contribution to the binding processes. In addition, a weak conformational change of Bc II was also observed in the molecular recognition and binding process of Bc II with PV or Sul. This study may provide some valuable information for exploring the recognition and binding of proteins with ligands in the binding process and for the design of novel super-antibiotics.

LN-1-255, a penicillanic acid sulfone able to inhibit the class D carbapenemase OXA-48.

OBJECTIVES: Carbapenemases are the most important mechanism responsible for carbapenem resistance in Enterobacteriaceae. Among carbapenemases, OXA-48 presents unique challenges as it is resistant to ß-lactam inhibitors. Here, we test the capacity of the compound LN-1-255, a 6-alkylidene-2'-substituted penicillanic acid sulfone, to inhibit the activity of the carbapenemase OXA-48. METHODS: The OXA-48 gene was cloned and expressed in Klebsiella pneumoniae and Escherichia coli in order to obtain MICs in the presence of inhibitors (clavulanic acid, tazobactam and sulbactam) and LN-1-255. OXA-48 was purified and steady-state kinetics was performed with LN-1-255 and tazobactam. The covalent binding mode of LN-1-255 with OXA-48 was studied by docking assays. RESULTS: Both OXA-48-producing clinical and transformant strains displayed increased susceptibility to carbapenem antibiotics in the presence of 4 mg/L LN-1-255 (2-32-fold increased susceptibility) and 16 mg/L LN-1-255 (4-64-fold increased susceptibility). Kinetic assays demonstrated that LN-1-255 is able to inhibit OXA-48 with an acylation efficiency (k2/K) of 10 ±â€Š1 × 10(4) M(-1) s(-1) and a slow deacylation rate (koff) of 7 ±â€Š1 × 10(-4) s(-1). IC50 was 3 nM for LN-1-255 and 1.5 µM for tazobactam. Lastly, kcat/kinact was 500-fold lower for LN-1-255 than for tazobactam. CONCLUSIONS: In these studies, carbapenem antibiotics used in combination with LN-1-255 are effective against the carbapenemase OXA-48, an important emerging mechanism of antibiotic resistance. This provides an incentive for further investigations to maximize the efficacy of penicillin sulfone inhibition of class D plasmid-carried Enterobacteriaceae carbapenemases.

Detecting a quasi-stable imine species on the reaction pathway of SHV-1 ß-lactamase and 6ß-(hydroxymethyl)penicillanic acid sulfone.

For the class A ß-lactamase SHV-1, the kinetic and mechanistic properties of the clinically used inhibitor sulbactam are compared with the sulbactam analog substituted in its 6ß position by a CH2OH group (6ß-(hydroxymethyl)penicillanic acid). The 6ß substitution improves both in vitro and microbiological inhibitory properties of sulbactam. Base hydrolysis of both compounds was studied by Raman and NMR spectroscopies and showed that lactam ring opening is followed by fragmentation of the dioxothiazolidine ring leading to formation of the iminium ion within 3 min. The iminium ion slowly loses a proton and converts to cis-enamine (which is a ß-aminoacrylate) in 1 h for sulbactam and in 4 h for 6ß-(hydroxymethyl) sulbactam. Rapid mix-rapid freeze Raman spectroscopy was used to follow the reactions between the two sulfones and SHV-1. Within 23 ms, a 10-fold excess of sulbactam was entirely hydrolyzed to give a cis-enamine product. In contrast, the 6ß-(hydroxymethyl) sulbactam formed longer-lived acyl-enzyme intermediates that are a mixture of imine and enamines. Single crystal Raman studies, soaking in and washing out unreacted substrates, revealed stable populations of imine and trans-enamine acyl enzymes. The corresponding X-ray crystallographic data are consonant with the Raman data and also reveal the role played by the 6ß-hydroxymethyl group in retarding hydrolysis of the acyl enzymes. The 6ß-hydroxymethyl group sterically hinders approach of the water molecule as well as restraining the side chain of E166 that facilitates hydrolysis.

Why tazobactam and sulbactam have different intermediates population with SHV-1 ß-lactamase: a molecular dynamics study.

The imine intermediates of tazobactam and sulbactam bound to SHV-1 ß-lactamase were investigated by molecular dynamics (MD) simulation respectively. Hydrogen bond networks around active site were found different between tazobactam and sulbactam acyl-enzymes. In tazobactam imine intermediate, it was observed that the triazolyl ring formed stable hydrogen bonds with Asn170 and Thr167. The results suggest that conformation of imine determined the population of intermediates. In imine intermediate of tazobactam, the triazolyl ring is trapped in Thr_Asn pocket, and it restricts the rotation of C5-C6 bond so that tazobactam can only generate trans enamine intermediate. Further, conformational cluster analyses are performed to substantiate the results. These findings provide an explanation for the corresponding experimental results, and will be potentially useful in the development of new inhibitors.

Theoretical investigation on reaction of sulbactam with wild-type SHV-1 ß-lactamase: acylation, tautomerization, and deacylation.

Molecular dynamics (MD) simulation and quantum mechanical (QM) calculations were used to investigate the reaction mechanism of sulbactam with class A wild-type SHV-1 ß-lactamase including acylation, tautomerization, and deacylation. Five different sulbactam-enzyme configurations were investigated by MD simulations. In the acylation step, we found that Glu166 cannot activate Ser70 directly for attacking on the carbonyl carbon, and Lys73 would participate in the reaction acting as a relay. Additionally, we found that sulbactam carboxyl can also act as a general base. QM calculations were performed on the formation mechanism of linear intermediates. We suggest that both imine and trans-enamine intermediates can be obtained in the opening of a five-membered thiazolidine ring. By MD simulation, we found that imine intermediate can exist in two conformations, which can generate subsequent trans- and cis-enamine intermediates, respectively. The QM calculations revealed that trans-enamine intermediate is much more stable than other intermediates. The deacylation mechanism of three linear intermediates (imine, trans-enamine, cis-enamine) was investigated separately. It is remarkably noted that, in cis-enamine intermediate, Glu166 cannot activate water for attacking on the carbonyl carbon directly. This leads to a decreasing of the deacylation rate of cis-enamine. These findings will be potentially useful in the development of new inhibitors.

Inhibitor resistance in the KPC-2 beta-lactamase, a preeminent property of this class A beta-lactamase.

As resistance determinants, KPC beta-lactamases demonstrate a wide substrate spectrum that includes carbapenems, oxyimino-cephalosporins, and cephamycins. In addition, clinical strains harboring KPC-type beta-lactamases are often identified as resistant to standard beta-lactam-beta-lactamase inhibitor combinations in susceptibility testing. The KPC-2 carbapenemase presents a significant clinical challenge, as the mechanistic bases for KPC-2-associated phenotypes remain elusive. Here, we demonstrate resistance by KPC-2 to beta-lactamase inhibitors by determining that clavulanic acid, sulbactam, and tazobactam are hydrolyzed by KPC-2 with partition ratios (kcat/kinact ratios, where kinact is the rate constant of enzyme inactivation) of 2,500, 1,000, and 500, respectively. Methylidene penems that contain an sp2-hybridized C3 carboxylate and a bicyclic R1 side chain (dihydropyrazolo[1,5-c][1,3]thiazole [penem 1] and dihydropyrazolo[5,1-c][1,4]thiazine [penem 2]) are potent inhibitors: Km of penem 1, 0.06+/-0.01 microM, and Km of penem 2, 0.006+/-0.001 microM. We also demonstrate that penems 1 and 2 are mechanism-based inactivators, having partition ratios (kcat/kinact ratios) of 250 and 50, respectively. To understand the mechanism of inhibition by these penems, we generated molecular representations of both inhibitors in the active site of KPC-2. These models (i) suggest that penem 1 and penem 2 interact differently with active site residues, with the carbonyl of penem 2 being positioned outside the oxyanion hole and in a less favorable position for hydrolysis than that of penem 1, and (ii) support the kinetic observations that penem 2 is the better inhibitor (kinact/Km=6.5+/-0.6 microM(-1) s(-1)). We conclude that KPC-2 is unique among class A beta-lactamases in being able to readily hydrolyze clavulanic acid, sulbactam, and tazobactam. In contrast, penem-type beta-lactamase inhibitors, by exhibiting unique active site chemistry, may serve as an important scaffold for future development and offer an attractive alternative to our current beta-lactamase inhibitors.

Role of E166 in the imine to enamine tautomerization of the clinical beta-lactamase inhibitor sulbactam.

Mechanism-based inhibitors of class A beta-lactamases, such as sulbactam, undergo a complex series of chemical reactions in the enzyme active site. Formation of a trans-enamine acyl-enzyme via a hydrolysis-prone imine is responsible for transient inhibition of the enzyme. Although the imine to enamine tautomerization is crucial to inhibition of the enzyme, there are no experimental data to suggest how this chemical transformation is catalyzed in the active site. In this report, we show that E166 acts as a general base to promote the imine to enamine tautomerization.

Why the extended-spectrum beta-lactamases SHV-2 and SHV-5 are "hypersusceptible" to mechanism-based inhibitors.

Extended-spectrum beta-lactamases (ESBLs) are derivatives of enzymes such as SHV-1 and TEM-1 that have undergone site-specific mutations that enable them to hydrolyze, and thus inactivate, oxyimino-cephalosporins, such as cefotaxime and ceftazidime. X-ray crystallographic data provide an explanation for this in that the mutations bring about an expansion of the binding pocket by moving a beta-strand that forms part of the active site wall. Another characteristic of ESBLs that has remained enigmatic is the fact that they are "hypersusceptible" to inhibition by the mechanism-based inactivators tazobactam, sulbactam, and clavulanic acid. Here, we provide a rationale for this "hypersusceptibility" based on a comparative analysis of the intermediates formed by these compounds with wild-type (WT) SHV-1 beta-lactamase and its ESBL variants SHV-2 and SHV-5, which carry the G238S and G238S/E240K substitutions, respectively. A Raman spectroscopic analysis of the reactions in single crystals shows that, compared to WT, the SHV-2 and SHV-5 variants have relatively higher populations of the stable trans-enamine intermediate over the less stable and more easily hydrolyzable cis-enamine and imine co-intermediates. In solution, SHV-2 and SHV-5 also form larger populations of an enamine species compared to SHV-1 as detected by stopped-flow kinetic experiments under single-turnover conditions. Moreover, a simple Raman band shape analysis predicts that the trans-enamine intermediates themselves in SHV-2 and SHV-5 are held in more stable, rigid conformations compared to their trans-enamine analogues in WT SHV-1. As a result of this stabilization, more of the trans-enamine intermediate is formed, which subsequently lowers the K(I) values of the mechanism-based inhibitors up to 50-fold in SHV-2 and SHV-5.

[Therapeutical and inflammatory response in rats with secondary peritonitis and topical use of ampicilin/sulbactam]. / Resposta terapêutica e inflamatória de ratos com peritonite secundária submetidos ao uso tópico de ampicilina/sulbactam.

OBJECTIVES: The acute peritonitis is an important cause of sepsis and death on intensive care units and surgery. The treatment must include: systemical use of antibiotics, drainage of abscess and restauration of gastrointestinal integrity. The topical use of antibiotics in the peritoneal cavity is controversial. The aim of this study was to evaluate the use of topical use of ampicilin/ sulbactam in the treatment of peritonitis. METHODS: We measured the plasmatic levels of nitric oxide, count of eosinophils, lymphocytes, monocytes, and neutrophils in blood and peritoneal cavity, using a model of peritonitis in rats (transfixation and ligature of cecum). Twenty four Wistar rats were divided in 4 groups (n = 6 each). group A: induction of peritonitis with ligature of cecum and topical treatment with saline; group B: induction of peritonitis with ligature of cecum and topical treatment with ampicilin/sulbactam; group C: transfixation of cecum; group D: laparotomy and peritoneal exsudate + blood sample. The transfixation-ligture of cecum remained for 24 hs before treatment. A relaparotomy was performed in 18 rats and peritoneal exsudate/blood were collected. Dosage of Nitric oxide, count of eosinophil, lymphocytes, monocytes, and neutrophils in blood and peritoneal exsudte were done. RESULTS: The difference was not significant in the levels of nitric oxide, eosinophil, lymphocytes, monocytes, and neutrophils in blood and peritoneal exsudate (p > 0,05) among the studied groups. CONCLUSION: The use of ampicilin associated to sulbactam via intraperitoneal in rats with fecal peritonitis did not change survival.; the levels of plama nitric oxide, count of eosinophil, lymphocytes, monocytes, and neutrophils in blood and peritoneal exsudate were not affected.

Resposta terapêutica e inflamatória de ratos com peritonite secundária submetidos ao uso tópico de ampicilina/sulbactam / Therapeutical and inflammatory response in rats with secondary peritonitis and topical use of ampicilin/sulbactam

OBJETIVOS: A peritonite aguda representa uma importante causa de sepsis e óbito nas unidades de terapia intensiva e cirurgia. Classicamente o seu tratamento deve incluir: a administração sistêmica de antibióticos, a remoção mecânica dos contaminantes e a restauração da integridade gastrintestinal. A utilização de antibióticos diretamente na cavidade peritoneal é controversa. Estudo com o objetivo de avaliar o uso terapêutico, intraperitoneal da ampicilina associada ao sulbactam. MÉTODOS: foram mensurados os níveis plasmáticos do óxido nítrico, bem como a contagem de eosinófilos, linfócitos, monócitos e neutrófilos no sangue e no lavado peritoneal, utilizando-se modelo de peritonite em ratos (ligadura-transfixação cecal). Vinte quatro ratos Wistar, machos, foram divididos em quatro grupos de seis animais, assim distribuídos: grupo A: método de indução de peritonite - soltura da ligadura + tratamento com soro fisiológico; grupo B: método de indução de peritonite + soltura da ligadura + tratamento com soro fisiológico acrescido de ampicilina / sulbactam; grupo C: método de indução de peritonite + soltura da ligadura-transfixação cecal; e grupo D: laparatomia para realização de lavado peritoneal mais coleta de sangue. A ligadura-transfixação do cecum permaneceu por 24 horas, antes do tratamento instaurado. Foi realizada uma relaparotomia nos 18 ratos com coleta de líquido de lavado peritoneal e sangue. Foram dosados os níveis plasmáticos de óxido nítrico e determinado o número de eosinófilos, linfócitos, monócitos e neutrófilos no sangue e no lavado peritoneal. RESULTADOS: Não ocorreu diferença estatisticamente significante (p > 0,05) nos níveis de óxido nítrico, bem como no número de eosinófilos, linfócitos, monócitos e neutrófilos no sangue e no lavado peritoneal, entre os grupos. CONCLUSÃO: Neste estudo, concluiu-se que: a utilização de ampicilina associada a sulbactam por via intraperitoneal nos ratos com peritonite fecal: não modificou a sobrevida; não alterou os níveis plasmáticos de óxido nítrico; não alterou a contagem de eosinófilos, linfócitos, monócitos e neutrófilos tanto no sangue como no lavado peritoneal.

Biological treatability of raw and ozonated penicillin formulation effluent.

In the present study, oxidative pre-treatment of pharmaceutical wastewater originating from the formulation of the penicillin Sultamycillin Tosylate Diydrate via ozonation at varying pH and ozone feed rates was investigated. Biological treatability studies were performed with a synthetic wastewater alone and supplemented with raw and ozonated penicillin formulation effluents. The highest COD (34%) and TOC (24%) removal efficiencies were obtained at pH 11.0, whereas the BOD5 value increased from 16 mg l(-1) to 128 mg l(-1) after 40 min of ozonation, corresponding to an applied ozone dose of 1670 mg l(-1) and 33% relative ozone absorption. The studies showed that no degradation of raw penicillin fraction (30% of total COD) occurred, and degradation of the synthetic wastewater being completely treatable without penicillin addition, was inhibited by 7%. Upon 40 min ozonation, the synthetic wastewater could be completely oxidized and at the same time 35% of ozonated penicillin wastewater removal was obtained. Respirometric studies were conducted in parallel and produced results indicating a 22% decrease in the total oxygen consumption rate established for raw penicillin formulation effluent compared to the results obtained from the aerobic batch reactor. No inhibition of the synthetic fraction was observed for the 40 min-ozonated penicillin formulation effluent, biodegradability of the 60 min-ozonated penicillin effluent decreased possibly due to recalcitrant oxidation product accumulation. The modeling study provided experimental support and information on inhibition kinetics in activated sludge model no. 3 (ASM3) by means of respirometric tests for the first time.

Following the reactions of mechanism-based inhibitors with beta-lactamase by Raman crystallography.

The reactions between three clinically relevant inhibitors, tazobactam, sulbactam, and clavulanic acid, and SHV beta-lactamase (EC 3.5.2.6) have been followed in single crystals using a Raman microscope. The data are far superior to those obtained for the enzyme in aqueous solution and allow us to identify species on the reaction pathway and to measure the rates of the accumulation and decay of these species. A key intermediate on the reaction pathway is an acyl enzyme formed between Ser70 and the lactam ring's C=O group. By using the E166A deacylation deficient variant of the enzyme, we were able to focus on the process of acyl enzyme formation. The Raman data show that all three inhibitors form an enamine-type acyl enzyme reaching maximal populations at 10, 22, and 29 min for sulbactam, clavulanic acid, and tazobactam, respectively. The enamine intermediate exhibits a characteristic and relatively intense band near 1595 cm(-1) due to a stretching motion of the O=C-C=C-NH moiety that shifts to lower frequency upon NH <--> ND exchange. This feature was used to follow the kinetics of enamine buildup and decay in the crystal. Quantum mechanical calculations support the assignment of the 1595 cm(-1) band, as well as several other bands, to a trans-enamine species. The Raman data also demonstrate that the lactam ring opens prior to enamine formation since the lactam ring carbonyl (C=O) peak disappears prior to the appearance of the enamine 1595 cm(-1) band. Tazobactam appears to form approximately twice as much enamine intermediate as sulbactam and clavulanic acid, which correlates with its superior performance in the clinic, a finding that may bear on future drug design.