A novel plasm id-mediated β-lactamase that hydrolyzes broad-spectrum cephalosporins in a clinical isolate ofKlebsiella pneumoniae

A new extended-spectrum beta-lactamase with an isoelectric point (pI) of 6.2 was detected in Klebsiella pneumoniae F161 that was isolated from a patient with infection. This strain was highly resistant to the third or fourth generation cephalosporins such as ceftazidime, ceftriaxone, cefoperazone, and cefpirome. Analysis of this strain by the double disk diffusion test showed synergies between amoxicillin-clavulanate (AMX-CA) and cefotaxime, and AMX-CA and aztreonam, which suggested that this strain produced a extended-spectrum beta-lactamase (ESBL). Genetic analysis revealed that the resistance was due to the presence of a 9.4-kb plasmid, designated as pKP161, encoding for new beta-lactamase gene (bla). Sequence analysis showed that a new bla gene of pKP161 differed from bla(TEM-1) by three mutations leading to the following amino acid substitutions: Val84 --> Ile, Ala184 --> Val, and Gly238 --> Ser. These mutations have not been reported previously in the TEM type beta-lactamases produced by clinical strains. The novel beta-lactamase was overexpressed in E. coli and purified by ion exchange chromatography on Q-Sepharose and CM-Sepharose, and then further purified by gel filtration on Sehadex G-200. The catalytic activity of the purified beta-lactamase was confirmed by the nitrocefin disk.     

Pharmacodynamic evaluation of meropenem,cefepime, or aztreonam combined with a novel β-lactamase inhibitor,nacubactam, against carbapenem-resistant and/or carbapenemase-producing Klebsiella pneumoniae and Escherichia coli using a murine thigh-infection model

BackgroundCarbapenem-resistant Enterobacterales (CRE) and carbapenemase-producing Enterobacterales (CPE) are difficult to treat and are a serious public health threat. Nacubactam (NAC) is a novel non-β-lactam diazabicyclooctane β-lactamase inhibitor with in vitro activity against some Enterobacterales expressing classes of β-lactamases.MethodsThe antimicrobial efficacy of meropenem (MEM), cefepime (FEP), and aztreonam (ATM), each in combination with NAC, were assessed in vitro and in vivo against Klebsiella pneumoniae and Escherichia coli. Ten isolates, including CRE and/or CPE with β-lactamase genes, were used in this study. The relationship between phenotype and in vivo efficacy was assessed in a murine neutropenic thigh-infection model. Efficacy was determined by the change in bacterial quantity.ResultsThe results of the in vitro study showed the minimum inhibitory concentrations of the combination of NAC with either MEM, FEP, or ATM in a 1:1 ratio were 2 to >128-fold lower than those of MEM, FEP, or ATM alone against CRE+ isolates. In addition, combinations of β-lactams and NAC administered in the murine thigh-infection model showed greater efficacy against CRE+/CPE+, CRE+/CPE-, and CRE-/CPE+ isolates harboring various β-lactamase genes (IMP-1, IMP-6, KPC, DHA-1, or OXA-48) compared with MEM, FEP, ATM, and NAC alone.ConclusionMEM, FEP, or ATM in combination with NAC showed potent in vivo antimicrobial activity in a murine thigh-infection model caused by K. pneumoniae and E. coli, including CRE and/or CPE isolates. These findings indicate that these combinations of β-lactams and NAC are potential candidates for the treatment of CRE and/or CPE infections.     

Antimicrobial activity of pomegranate fruit constituents against drug-resistant Mycobacterium tuberculosis and β-lactamase producing Klebsiella pneumoniae

Abstract

Context: The global surge in multi-drug resistant bacteria and the imminence of tuberculosis pandemic necessitate alternative therapeutic approaches to augment the existing medications. Pomegranate, the fruit of Punica granatum Linn. (Punicaceae), widely recognized for potency against a broad spectrum of bacterial pathogens, deserves further investigation in this respect.

Objective: This study determines the therapeutic potential of pomegranate juice, extracts of non-edible peel prepared with methanol/water, and its four polyphenolic constituents, namely caffeic acid, ellagic acid, epigallocatechin-3-gallate (EGCG) and quercetin, against drug-resistant clinical isolates.

Materials and methods: Phenotypic characterisation of Mycobacterium tuberculosis, extended-spectrum β-lactamase (ESBL) and KPC-type carbapenemase producing Klebsiella pneumoniae was performed by biochemical and molecular methods. Resistance profiles of M. tuberculosis and K. pneumoniae were determined using LJ proportion and Kirby–Bauer methods, respectively. Pomegranate fruit extracts, and the compounds, were evaluated at a dose range of 1024–0.5?µg/mL, and 512–0.25?µg/mL, respectively, to determine minimum inhibitory (MIC) and bactericidal concentrations (MBC) against the drug-resistant isolates by the broth micro-dilution method.

Results: The peel extracts exhibited greater antimycobacterial activity (MIC 64–1024?μg/mL) than the potable juice (MIC 256?-?>?1024?μg/mL). EGCG and quercetin exhibited higher antitubercular (MIC 32–256?μg/mL) and antibacterial (MIC 64–56?μg/mL) potencies than caffeic acid and ellagic acid (MIC 64–512?μg/mL).

Discussion and conclusion: The pomegranate fruit peel and pure constituents were active against a broad panel of M. tuberculosis and β-lactamase producing K. pneumoniae isolates. EGCG and quercetin need further investigation for prospective application against respiratory infections.     

Inactivation of a class A and a class C β-lactamase by 6β-(hydroxymethyl)penicillanic acid sulfone

β-Lactamase inhibitors (clavulanic acid, sulbactam, and tazobactam) contribute significantly to the longevity of the β-lactam antibiotics used to treat serious infections. In the quest to design more potent compounds and to understand the mechanism of action of known inhibitors, 6β-(hydroxymethyl)penicillanic acid sulfone (6β-HM-sulfone) was tested against isolates expressing the class A TEM-1 β-lactamase and a clinically important variant of the AmpC cephalosporinase of Pseudomonas aeruginosa, PDC-3. The addition of the 6β-HM-sulfone inhibitor to ampicillin was highly effective. 6β-HM-sulfone inhibited TEM-1 with an IC(50) of 12 ± 2 nM and PDC-3 with an IC(50) of 180 ± 36 nM, and displayed lower partition ratios than commercial inhibitors, with partition ratios (k(cat)/k(inact)) equal to 174 for TEM-1 and 4 for PDC-3. Measured for 20 h, 6β-HM-sulfone demonstrated rapid, first-order inactivation kinetics with the extent of inactivation being related to the concentration of inhibitor for both TEM-1 and PDC-3. Using mass spectrometry to gain insight into the intermediates of inactivation of this inhibitor, 6β-HM-sulfone was found to form a major adduct of +247 ± 5 Da with TEM-1 and +245 ± 5 Da with PDC-3, suggesting that the covalently bound, hydrolytically stabilized acyl-enzyme has lost a molecule of water (HOH). Minor adducts of +88 ± 5 Da with TEM-1 and +85 ± 5 Da with PDC-3 revealed that fragmentation of the covalent adduct can result but appeared to occur slowly with both enzymes. 6β-HM-sulfone is an effective and versatile β-lactamase inhibitor of representative class A and C enzymes.     

Molecular basis of extended-spectrum beta-lactamase production in nosocomial isolates of Klebsiella pneumoniae from Mérida, Venezuela

Twelve Klebsiella pneumoniae isolates resistant to expanded-spectrum cephalosporins and aztreonam, from patients with nosocomial septicaemia at the intensive care unit of the Andes University Hospital, Mérida, Venezuela, were studied for production of extended-spectrum beta-lactamase (ESbetaL) activity. All were also resistant to gentamicin, kanamycin, tetracycline and chloramphenicol but sensitive to cefoxitin, imipenem, amikacin and tobramycin. Production of ESbetaL activity was confirmed by restoring susceptibility to ceftazidime in the presence of clavulanic acid. All isolates carried an identical plasmid of approximately 87 kb. Resistance to beta-lactams, aminoglycosides, tetracycline and chloramphenicol was lost en bloc after plasmid curing by treatment with acridine orange and was transferable en bloc to Escherichia coli by conjugation. Transconjugants always showed the same plasmid profile as that of Klebsiella donors. Isoelectric focusing analysis of the crude extracts of transconjugants showed in all cases, the presence of two beta-lactamases of pI 5.4 and 8.2. Analysis of the plasmid carried by one of the transconjugants by means of hybridization assays, revealed the presence of both bla(TEM) and bla(SHV) determinants. Cloning and sequencing of each determinant identified them as bla(TEM-1) and bla(SHV-5), respectively, the latter being responsible for the ESbetaL activity. Results of this study indicate that ESbetaL determinants of the SHV-type carried by transferable elements, are spreading among nosocomial isolates of K. pneumoniae in Mérida, Venezuela.     

In vitro activity of avibactam (NXL104) in combination with β-lactams against Gram-negative bacteria, including OXA-48 β-lactamase-producing Klebsiella pneumoniae

The objective of this study was to investigate the in vitro antibacterial activity of avibactam (formerly NXL104) in combination with imipenem, cefepime or ceftazidime against Gram-negative bacteria. Bacterial isolates included: Pseudomonas aeruginosa harbouring PER-1 β-lactamase (n = 14); Acinetobacter baumannii harbouring PER-1, OXA-51 and OXA-58 (n = 20); carbapenem-non-susceptible Klebsiella pneumoniae (n = 25) and Escherichia coli (n = 1) harbouring OXA-48; carbapenem-non-susceptible E. coli (n = 1) harbouring both IMP-1 metallo-β-lactamase and extended-spectrum β-lactamase (ESBL); carbapenem-non-susceptible Serratia marcescens (n = 1); and carbapenem-susceptible E. coli (n = 20) and K. pneumoniae isolates (n = 12) with CTX-M-15 ESBL. Minimum inhibitory concentrations (MICs) of imipenem, cefepime and ceftazidime were determined in combination with 4 mg/L avibactam by the Clinical and Laboratory Standards Institute (CLSI) method on Mueller-Hinton agar. Imipenem/avibactam and ceftazidime/avibactam displayed limited potency against A. baumannii isolates, whereas cefepime/avibactam and ceftazidime/avibactam were active against P. aeruginosa. Klebsiella pneumoniae isolates with OXA-48 β-lactamase were resistant to imipenem [MIC for 90% of the organisms (MIC₉₀) ≥4 mg/L]. MIC₉₀ values for the combination of avibactam 4 mg/L with imipenem, cefepime and ceftazidime were in the susceptible range for all strains (MIC₉₀ ≤ 0.5 mg/L). All E. coli and K. pneumoniae isolates with CTX-M-15 β-lactamase were inhibited at ≤1 mg/L for combinations with avibactam and 100% were susceptible by CLSI breakpoint criteria to imipenem, cefepime and ceftazidime. In conclusion, combinations of imipenem, cefepime and ceftazidime with avibactam may present a promising therapeutic strategy to treat infections due to K. pneumoniae with OXA-48 enzyme as well as K. pneumoniae and E. coli with CTX-M-15 enzyme.     

Anti-anaerobic activity of a new β-lactamase inhibitor NXL104 in combination with β-lactams and metronidazole

NXL104 is a new β-lactamase inhibitor (BLI) that inhibits class A and class C β-lactamase enzymes. In this study, the activity of NXL104 in combination with the third-generation cephalosporins ceftazidime (CAZ) and ceftriaxone (CRO) or with piperacillin (PIP) was evaluated against 316 anaerobic bacteria. Minimum inhibitory concentrations (MICs) were determined using an agar dilution method. The BLIs NXL104 or tazobactam (TAZ) were added to the β-lactams at a fixed concentration of 4 mg/L. A triple combination of NXL104 with an 8:1 ratio of CAZ and metronidazole (MTZ) was also tested. The activities of CAZ, CRO and PIP in combination with NXL104 were enhanced against many of the bacteria. MIC(50) values (MIC for 50% of the organisms) for CAZ+NXL104 were 8-16-fold lower than those of CAZ against Gram-negative anaerobes. Antibiotic resistance rates against all anaerobic strains were: CAZ, 37.7%; CRO, 31%; CAZ+NXL104, 15.2%; CRO+NXL104, 5.4%; and MTZ, 4.1%. No resistant strains could be observed with PIP+TAZ, PIP+NXL104 or the triple combination MTZ+CAZ+NXL104. In conclusion, the triple combination of MTZ+CAZ+NXL104 demonstrated potent antibacterial activity against anaerobes representing most clinical species. It appears appropriate for the treatment of polymicrobial infections, since CAZ+NXL104 also exhibits potent activity against β-lactamase-producing Enterobacteriaceae and Pseudomonas aeruginosa. It is currently being tested in phase 2 clinical trials for the treatment of complicated intra-abdominal infections.     

Prevalence of plasmid-mediated quinolone resistance determinants qnr and aac(6')-Ib-cr in Escherichia coli and Klebsiella pneumoniae producing extended-spectrum β-lactamases in Spain

The presence of the plasmid-mediated quinolone resistance determinants qnrA, qnrB, qnrS and aac(6')-Ib-cr was evaluated in a collection of 382 isolates of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae collected between February and March 2006 for the nationwide Spanish GEIH-ESBL 2006 project. In total, 14 isolates (3.7%) were positive for qnr genes (3 qnrA1, 5 qnrB-like and 6 qnrS1) and 62 isolates (16.2%) were positive for the mutant variant of aac(6')-Ib-cr. The Aac(6')-Ib-cr enzyme was the most prevalent plasmid-mediated mechanism of quinolone resistance in Spain. Most of the Aac(6')-Ib-cr-producing E. coli isolates (94.2%) carried two mutations in gyrA and two in parC, whilst only 57.2% of K. pneumoniae harbouring this enzyme were gyrA and/or parC mutants. Most qnr plasmids were transferable, but only four were conjugative. Plasmid incompatibility groups were identified for only four plasmids, belonging to FIA, HI2 and I1γ. The most prevalent ESBLs associated with qnr plasmids belonged to the SHV and CTX-M families. The present study highlights the broad geographical spread of qnr-like determinants in Spain and their association with the SHV-12 and CTX-M-9 ESBLs in human clinical isolates.     

Pharmacogenetic characterization of indacaterol,a novel β2-adrenoceptor agonist

Background and purpose:

Indacaterol is a novel β₂-adrenoceptor agonist in development for the treatment of chronic obstructive pulmonary disease. The aim of this study was to investigate the comparative pharmacology of indacaterol in recombinant cells expressing the common polymorphic variants of the human β₂-adrenoceptor and in human primary airway smooth muscle (ASM) cells.

Experimental approach:

Chinese hamster ovarian-K1 cell lines expressing high and low levels of the common human β₂-adrenoceptor variants were generated [Gly16-Glu27-Val34-Thr164(GEVT), RQVT, GQVT] and also the rare GQVI variant. Human primary ASM cells were isolated from explants of trachealis muscle. Adenosine-3′,5′-cyclic-monophosphate production was used as an outcome measure.

Key results:

In both the low- and high-expression recombinant GEVT ‘wild type’ cell lines indacaterol is a high-efficacy agonist. Salmeterol and formoterol were identified as low- and high-efficacy agonists, respectively, and showed similar potencies to indacaterol irrespective of the β₂-adrenoceptor genotype. The I164 variant cell line was associated with a reduced capacity to generate adenosine-3′,5′-cyclic-monophosphate in response to β₂-adrenoceptor agonist. In the human primary ASM cells indacaterol gave a maximal response intermediate between that of salmeterol and formoterol.

Conclusions and implications:

These data demonstrate that indacaterol is a high-efficacy agonist in recombinant cell systems but acts with lower efficacy in human primary ASM cells. No marked genotype-dependent effects were observed for common variants; however, changes in I164 receptor activity were identified, which were dependent on the level of expression of β₂-adrenoceptors.     

Effect of mirabegron, a novel β3-adrenoceptor agonist, on bladder function during storage phase in rats

Mirabegron, a selective β₃-adrenoceptor agonist, facilitates urine storage function by exerting a relaxing effect on bladder smooth muscle. Here, we investigated the effect of mirabegron on bladder function during the storage phase. We assessed the effect of mirabegron on the resting intravesical pressure in anesthetized rats and also tested antimuscarinics (oxybutynin and tolterodine) under the same experimental conditions. Mirabegron dose-dependently decreased the resting intravesical pressure, while oxybutynin and tolterodine showed no statistically significant effects on resting intravesical pressure. We also investigated the effect of mirabegron on bladder function using cystometry technique in conscious rats with bladder outlet obstruction. While mirabegron dose-dependently decreased the frequency of nonvoiding contractions, considered an index of abnormal response in bladder storage, no significant effects were noted on the amplitude of nonvoiding contractions, micturition pressure, threshold pressure, voided volume, residual volume, or bladder capacity. Neither oxybutynin nor tolterodine affected the frequency of nonvoiding contractions; however, oxybutynin increased residual volume and tended to decrease voided volume in a dose-dependent manner, and tolterodine dose-dependently decreased voided volume. Taken together, these results shed light on the suggestion of mirabegron as a therapeutic agent, compared with antimuscarinics, with its most prominent effect being the facilitation of bladder storage.     

CHF5074, a novel γ-secretase modulator,attenuates brain β-amyloid pathology and learning deficit in a mouse model of Alzheimer's disease

Background and purpose:

We evaluated the effects of 1-(3′,4′-dichloro-2-fluoro[1,1′-biphenyl]-4-yl)-cyclopropanecarboxylic acid (CHF5074), a new γ-secretase modulator, on brain β-amyloid pathology and spatial memory in transgenic mice expressing the Swedish and London mutations of human amyloid precursor protein (hAPP).

Experimental approach:

Sixty 6-month-old hAPP mice were treated for 6 months with CHF5074 or ibuprofen (375 ppm in the diet) or standard diet. Twenty-one wild-type mice received standard diet.

Key results:

Compared with transgenic controls, CHF5074 treatment significantly reduced the area occupied by plaques in cortex (P = 0.003) and hippocampus (P = 0.004). The number of plaques were also reduced by CHF5074 in both cortex (P = 0.022) and hippocampus (P = 0.005). Plaque-associated microglia in CHF5074-treated animals was lower than in transgenic controls in cortex (P = 0.008) and hippocampus (P = 0.002). Ibuprofen treatment significantly reduced microglia area in cortex and hippocampus but not β-amyloid burden. On the last day of the Morris water maze, transgenic controls performed significantly worse than the non-transgenic animals and the CHF5074-treated transgenic mice, on the swimming path to reach the hidden platform. Ibuprofen-treated animals did not perform significantly better than transgenic controls.

Conclusions and implications:

Chronic CHF5074 treatment reduced brain β-amyloid burden, associated microglia inflammation and attenuated spatial memory deficit in hAPP mice. This novel γ-secretase modulator is a promising therapeutic agent for Alzheimer''s disease.     

Synthesis, characterization, and in vitro antiproliferative activity of novel β-elemene monosubstituted derivatives

A series of β-elemene monosubstituted ester, carbamate, acylamide, and carbamidine derivatives were synthesized via intermediates, β-elemene alcohol and β-elemene amine, which were synthesized from the traditional Chinese medicine, β-elemene. The structures of all the new compounds were characterized by NMR, IR, and HRMS. Their in vitro antiproliferative activities on HeLa cell line were tested through the WST-1 assay. The results show that the in vitro antiproliferative activities of the novel compounds are improved compared to that of the parent β-elemene.     

ETME,a novel β-elemene derivative,synergizes with arsenic trioxide in inducing apoptosis and cell cycle arrest in hepatocarcinoma cells via a p53-dependent pathway

Arsenic trioxide (ATO) has been identified as an effective treatment for acute promyelocytic leukemia (APL) but is much less effective against solid tumors such as hepatocellular carcinoma (HCC). In the search for ways to enhance its therapeutic efficacy against solid tumors, we have examined its use in combination with a novel derivative of β-elemene, N-(β-elemene-13-yl)tryptophan methyl ester (ETME). Here we report the effects of the combination on cell viability, apoptosis, the cell cycle and mitochondria membrane potential (MMP) in HCC SMMC-7721 cells. We found that the two compounds acted synergistically to enhance antiproliferative activity and apoptosis. The combination also decreased the MMP, down-regulated Bcl-2 and pro-proteins of the caspase family, and up-regulated Bax and BID, all of which were reversed by the p53 inhibitor, pifithrin-α. In addition, the combination induced cell cycle arrest at the G2/M phase and reduced tumor volume and weight in an xenograft model of nude mice. Overall, the results suggest that ETME in combination with ATO may be useful in the treatment of HCC patients particularly those unresponsive to ATO alone.Key words: Hepatocarcinoma, β-Elemene derivative, As₂O₃, Apoptosis, p53     

Anti-inflammatory potential of β-amyrin,a triterpenoid isolated from Costus igneus

Costus igneus, common name Fiery Costus or Spiral Flag, is a species of herbaceous plant in the Costaceae family. It is cultivated in India for its use in traditional medicine especially for diabetes. The present study was carried out to determine the mechanism of anti-inflammatory action of β-amyrin isolated from the leaves of Costus igneus (C. igneus) using carrageenan-induced rat model and LPS-induced human peripheral blood mononuclear cells (hPBMCs) in vitro model. The differential fractionation of leaves of Costus igneus showed maximum percentage inhibition of paw edema at a dose of 100 mg/kg body weight in methanolic extract (MEC). MEC elicited significant anti-inflammatory effect by inhibiting cyclooxygenase (COX), lipoxygenase (LOX), myeloperoxidase (MPO) and nitric oxide synthase (NOS) activities in monocytes when compared to carrageenan control. The effect of MEC was more pronounced than standard drug Diclofenac (20 mg/kg body weight). After fractionation of MEC using various solvents such as chloroform, hexane, ethyl acetate and butanol, the mechanism of anti-inflammatory effect of chloroform extract (CEC) of MEC was evaluated since it showed maximum beneficial effect at a dose of 50 mg/kg BW Treatment of carrageenan-induced rats with CEC exerted significantly decreased COX-2, MPO, and NOS activities when compared to carrageenan-induced rats. By the partial purification of CEC by liquid–liquid partition chromatography, TLC, mass, IR and NMR spectroscopy, the active component β-amyrin was isolated. Significant decrease in edema was observed by the administration of β-amyrin in a dose-dependent manner and 100 µg of β-amyrin showed 97 % in carrageenan-induced paw edema in rats. Treatment with β-amyrin significantly inhibited PGE2, IL-6 secretion, and NF-κB activation in a concentration-dependent manner on LPS-induced hPBMCs. Thus, β-amyrin, an active component isolated from C. igneus, serves as a promising and expanding platform for treatment of various inflammatory disorders.     

Protective effects of compound FLZ,a novel synthetic analogue of squamosamide,on β-amyloid-induced rat brain mitochondrial dysfunction in vitro

Aim:

The aim of the present study was to assess the effects of N-[2-(4-hydroxyphenyl)ethyl]-2-(2,5-dimethoxyphenyl)-3-(3-methoxy-4-hydroxyphenyl) acrylamide (compound FLZ), a novel synthetic analogue of squamosamide, on the dysfunction of rat brain mitochondria induced by Aβ_25–35 in vitro.

Methods:

Isolated rat brain mitochondria were incubated with aged Aβ_25–35 for 30 min in the presence and absence of FLZ (1–100 μmol/L). The activities of key mitochondrial enzymes, the production of hydrogen peroxide (H₂O₂) and superoxide anion (O₂^·-), and the levels of glutathione (GSH) in mitochondria were examined. Mitochondrial swelling and the release of cytochrome c from mitochondria were assessed by biochemical and Western blot methods, respectively.

Results:

Incubation of mitochondria with aged Aβ_25–35 inhibited the activities of α-ketoglutarate dehydrogenase (α-KGDH), pyruvate dehydrogenase (PDH) and respiratory chain complex IV. It also resulted in increased H₂O₂ and O₂^·- production, and decreased the GSH level in mitochondria. Furthermore, it induced mitochondrial swelling and cytochrome c release from the mitochondria. The addition of FLZ (100 μmol/L) prior to treatment with Aβ_25–35 significantly prevented these toxic effects of Aβ_25–35 on the mitochondria.

Conclusion:

FLZ has a protective effect against Aβ_25–35-induced mitochondrial dysfunction in vitro.