In-depth analysis of the treatment effect and synergistic mechanism of TanReQing injection on clinical multi-drug resistant Pseudomonas aeruginosa.

Antibiotic resistance is a recognized and concerning public health issue. Gram-negative bacilli, such as Pseudomonas aeruginosa (P. aeruginosa), are notorious for their rapid development of drug resistance, leading to treatment failures. TanReQing injection (TRQ) was chosen to explore its pharmacological mechanisms against clinical multidrug-resistant P. aeruginosa (MDR-PA), given its antibacterial and anti-inflammatory properties. We revealed the expression of proteins and genes in P. aeruginosa after co-culture with TRQ. This study developed an assessment method to evaluate clinical resistance of P. aeruginosa using MALDI-TOF MS identification and Biotyper database searching techniques. Additionally, it combined MIC determination to investigate changes in MDR-PA treated by TRQ. TRQ effectively reduced the MICs of ceftazidime and cefoperazone and enhanced the confidence scores of MDR-PA as identified by mass spectrometry. Using this evaluation method, the fingerprints of standard P. aeruginosa and MDR-PA were compared, and the characteristic peptide sequence (Seq-PA No. 1) associated with flagellum was found. The phenotypic experiments were conducted to confirm the effect of TRQ on the motility and adhesion of P. aeruginosa. A combination of co-immunoprecipitation and proteome analysis was employed, and 16 proteins were significantly differentially expressed and identified as potential candidates for investigating the mechanism of inhibiting resistance in P. aeruginosa treated by TRQ. The candidates were verified by quantitative real-time PCR analysis, and TRQ may affect these core proteins (MexA, MexB, OprM, OprF, OTCase, IDH, and ASL) that influence resistance of P. aeruginosa. The combination of multiple methods helps elucidate the synergistic mechanism of TRQ in overcoming resistance of P. aeruginosa.IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen closely associated with various life-threatening acute and chronic infections. The presence of antimicrobial resistance and multidrug resistance in P. aeruginosa infections significantly complicates antibiotic treatment. The expression of ß-lactamase, efflux systems such as MexAB-OprM, and outer membrane permeability are considered to have the greatest impact on the sensitivity of P. aeruginosa. The study used a method to assess the clinical resistance of P. aeruginosa using matrix-assisted laser desorption ionization time of flight mass spectrometry identification and Biotyper database search techniques. TanReQing injection (TRQ) effectively reduced the MICs of ceftazidime and cefoperazone in multidrug-resistant P. aeruginosa (MDR-PA) and improved the confidence scores for co-cultured MDR-PA. The study found a characteristic peptide sequence for distinguishing whether P. aeruginosa is resistant. Through co-immunoprecipitation and proteome analysis, we explored the mechanism of TRQ overcoming resistance of P. aeruginosa.

Efficient Inhibition of Bacterial Biofilm Through Interference of Protein-Protein Interaction of Master Regulator Proteins: a Proof of Concept Study with SinR- SinI Complex of Bacillus subtilis.

Biofilm-associated microbial growth is a major cause of environmental, industrial, and public health concern. Therefore, there is a pressing need to discover and develop efficient antibiofilm strategies. Regulatory proteins vital for biofilm formation might be ideal targets for developing novel antibiofilm therapeutics. Their activities often depend on protein-protein interactions. Therefore, such targets present unique opportunities and challenges to drug discovery. In Bacillus subtilis, a model organism for studying biofilms, SinR acts as the master regulator of the biofilm formation cascade. Under favourable growth conditions, it represses the epsA-O and tapA-sipW-tasA operons, which encode for essential structural components of biofilms. Under unfavourable growth conditions, SinI, an agonist protein, inactivates SinR by forming a heterotrimeric complex. This results in derepression of epsA-O and tapA-sipW-tasA operons and leads to the phenotypic switch from planktonic to biofilm-associated form. We hypothesized that inhibiting SinR-SinI interaction might warrant repression of epsA-O and tapA-sipW-tasA operons and inhibit biofilm formation. To evaluate this hypothesis, we carried out a drug repurposing study for identifying potential inhibitors of SinI. Cefoperazone and itraconazole were identified as potential inhibitors with virtual screening. The stability of their interaction with SinI was assessed in extended MD performed over 100 ns. Both cefoperazone and itraconazole showed stable interaction. In in vitro studies, cefoperazone hindered the interaction of purified recombinant SinI and SinR. In the whole cell-based biofilm inhibition assays also cefoperazone was found to efficiently inhibited biofilm formation. These results provide proof of concept for targeting protein-protein interaction of master regulators as potential target for discovery and development of antibiofilm therapeutics. We propose that similar drug repurposing studies targeting key regulators of biofilm formation cascade could be an efficient approach for discovering novel anti-biofilm therapeutics against priority pathogens.

Repurposing of FDA approved drugs and their validation against potential drug targets for Salmonella enterica through molecular dynamics simulation.

Salmonella is a widely distributed pathogen causing infection of intestinal tract, typhoid, and paratyphoid fever. Number of drugs was developed against salmonella, but in the last few decades due to the emergence of drug resistant strains, most of these drugs became dormant. As a result Salmonellosis emerges as a trivial cause of human mortality worldwide; therefore, there is an urgent need for unexploited drug targets and drugs to treat Salmonellosis. As development of new drug molecules is very time consuming and costly, drug repurposing is in consideration as a better alternative. With the aim to identify a new drug molecule against the Salmonella through repurposing approach, we utilized 14 well reported druggable targets known to play a vital role in the life cycle of pathogens. These targets were used to screen DrugBank and got 53 FDA approved drugs against them. To find the interaction between considered target proteins and screened drugs, molecular docking was performed. Fourteen docked drug-target complexes with reasonable binding affinities were subjected to Molecular Dynamics Simulation (MDS) at 150 ns, using Amber18. At the end MMPBSA and MMGBSA calculations were performed for all stable complexes and finally, got 3 precise and favourable complexes, i.e. ArcB-Cefpiramide, MrcB-Cefoperazone, and PhoQ-Carindacillin. Rigorous structural and energetic analysis for these complexes validates the potential of drug molecules to act as therapeutic drugs against Salmonella enterica. With this study we hypothesize that the drugs Cefpiramide (DB00430), Cefoperazone (DB01329) and Carindacillin (DB09319) will be the good repurposed-drugs for the treatment of Salmonellosis. Communicated by Ramaswamy H. Sarma.

Peptidomimetic-based identification of FDA-approved compounds inhibiting IRE1 activity.

Inositol-requiring enzyme 1 (IRE1) is a bifunctional serine/threonine kinase and endoribonuclease that is a major mediator of the unfolded protein response (UPR) during endoplasmic reticulum (ER) stress. Tumour cells experience ER stress due to adverse environmental cues such as hypoxia or nutrient shortage and high metabolic/protein-folding demand. To cope with those stresses, cancer cells utilise IRE1 signalling as an adaptive mechanism. Here, we report the discovery of the FDA-approved compounds methotrexate, cefoperazone, folinic acid and fludarabine phosphate as IRE1 inhibitors. These were identified through a structural exploration of the IRE1 kinase domain using IRE1 peptide fragment docking and further optimisation and pharmacophore development. The inhibitors were verified to have an impact on IRE1 activity in vitro and were tested for their ability to sensitise human cell models of glioblastoma multiforme (GBM) to chemotherapy. We show that all molecules identified sensitise glioblastoma cells to the standard-of-care chemotherapy temozolomide (TMZ).

Acinetobacter baumannii detected on modified charcoal-cefoperazone-deoxycholate agar in a waste stabilization pond.

Campylobacter is a recommended reference pathogen for the verification and validation of water recycling schemes in Australia and globally. In a larger study investigating the efficacy of pathogen removal in waste stabilization ponds (WSP), we cultivated bacteria from wastewater samples on modified charcoal-cefoperazone-deoxycholate agar (mCCDA) targeting the growth of Campylobacter. A high number of colonies characteristic of Campylobacter grew on this selective medium, but this did not correlate with qPCR data. Using primers targeting the 16S rRNA gene, and additional confirmatory tests to detect VS1, ompA, blaOXA-51-like, blaOXA-23-like genes, we tested 80 random colonies from 10 WSP samples. All 80 were identified as Acinetobacter baumannii. Wastewater grab samples taken three times over 6 months throughout the WSP system showed removal of A. baumannii in the WSP at rates similar to that of Escherichia coli. Our study suggests that mCCDA agar is not a suitable medium for isolating Campylobacter from environmental samples and that A. baumannii can be used as an indicator for removal of pathogens in WSPs.

Development of a Novel Chromogenic Medium for Improved Campylobacter Detection from Poultry Samples.

The presence of expanded-spectrum ß-lactamase (ESBL)-producing Escherichia coli is a common problem in the isolation of Campylobacter from poultry samples using conventional cefoperazone-based selective media. A novel chromogenic medium (CM-HT), based on modified charcoal cefoperazone deoxycholate agar (mCCDA), has been developed as a solution for improved Campylobacter detection from poultry samples. Although the basic components of CM-HT are the same as mCCDA, CM-HT uses both granular charcoal and sodium cefoxitin to enhance viewability and inhibit ESBL-producing bacteria. All tested Campylobacter jejuni (n = 31) and Campylobacter coli (n = 6) strains grew and formed purple-colored colonies on CM-HT. In contrast, the growth of all other tested microorganisms, including ESBL-producing E. coli strains, was suppressed by this medium. Additionally, 84 poultry samples were examined for the presence of Campylobacter using the ISO 10272-1 method (enrichment with Bolton broth) and the NIHSJ-02 method (enrichment with Preston broth) with mCCDA and CM-HT media for the isolation. The numbers of samples from which Camplylobacter was detected on CM-HT using Preston and Bolton broth were 22 and 18, whereas the numbers on mCCDA were 22 and 13, respectively. Only Campylobacter was detected on CM-HT using both enrichment broths; however, there were 5 and 19 samples from which ESBL-producing E. coli was detected on mCCDA using Preston and Bolton broth, respectively. Thus, there was a significant difference between CM-HT and mCCDA in selectivity for ESBL-producing E. coli regardless of which enrichment broth was used. The results obtained demonstrated that CM-HT is a possible solution for the improved isolation of Campylobacter from poultry samples.

Isolation and characterization of Aeromonas schubertii from diseased snakehead, Channa maculata (Lacepède).

Pure bacterial cultures were isolated from diseased snakeheads, Channa maculata (Lacepède), suffering high mortality in a farm in Zhongshan, southern China. Three isolates, namely ZS20100725, ZS20100725-1 and ZS20100725-2, were identified as Aeromonas schubertii. All the isolates showed high 16S rRNA sequence similarities with A. schubertii. The isolates exhibited strong virulence to snakeheads in experimental challenges with LD(50) ranging between 1.4 × 10(4) and 6.4 × 10(6) CFU g(-1). Two of the isolates were positive for haemolysin, elastase, lipase and lecithinase by phenotypic determination, which was further confirmed by PCR amplification of the haemolysin and elastase genes. In sterile liquid medium, the best growth conditions of strain ZS20100725 were 30 °C, pH 7 and 0.5% salinity (w/v). Antibiotic susceptibility tests showed that strain ZS20100725 was susceptible to cefoxitin, cefoperazone and chloramphenicol. Furthermore, histopathology of diseased snakeheads infected with A. schubertii showed necrosis and congestion in liver, kidney and spleen and also damage to the cardiac muscle, intestine and gills.

Improvement of modified charcoal-cefoperazone-deoxycholate agar by supplementation with a high concentration of polymyxin B for detection of Campylobacter jejuni and C. coli in chicken carcass rinses.

Modified charcoal-cefoperazone-deoxycholate agar (mCCDA) was improved by supplementation with a high concentration of polymyxin B. The ability of the supplemented medium to isolate Campylobacter jejuni and C. coli from chicken carcass rinses was compared to that of Campy-Cefex agar and mCCDA. Modification of mCCDA with increased polymyxin B yielded a significantly (P < 0.05) higher isolation rate and greater selectivity than those achieved using Campy-Cefex agar and mCCDA.

Restoring the selectivity of Bolton broth during enrichment for Campylobacter spp. from raw chicken.

AIMS: When isolating Campylobacter spp. from retail raw chicken using BS EN ISO 10272-1:2006, contaminants frequently cause overgrowth on mCCDA plates. Therefore, these organisms proliferate in the enrichment medium, Bolton broth, indicating a lack of selectivity in this medium. This study sought to characterize the contaminant flora and to devise a modified Bolton broth to inhibit their growth. METHODS AND RESULTS: Contaminants (n=30) from separate samples were identified and antibiotic resistances determined. Most (93%) were extended spectrum ß-lactamase (ESBL) producing Escherichia coli, able to hydrolyse the cefoperazone present in Bolton broth and mCCDA. To inhibit these organisms, original formulation Bolton broth was supplemented with potassium clavulanate, at three concentrations, and recoveries of campylobacters from raw chicken were determined. Using standard Bolton broth, only 49% of samples (n=104) yielded campylobacters, but supplementation with 2 mg l(-1) potassium clavulanate increased this significantly (P<0.05), with 91% of samples positive. CONCLUSIONS: Potassium clavulanate can restore the selectivity of Bolton broth when isolating Campylobacter spp. from raw chicken. SIGNIFICANCE AND IMPACT OF THE STUDY: Raw chicken is often contaminated with the pathogen Campylobacter, but the ISO methodology for its detection is becoming compromised by the increasing presence of antibiotic-resistant bacteria. A simple modification ensures effective detection of this pathogen.

[The reaction mechanism between cefoperazone and human serum albumin].

The reaction mechanism between cefoperazone and human serum albumin (HSA) and the affinity between cefoperazone and beta-lactamase were investigated by spectrometry and spectrofluorimetry. The interaction dissociation constants of human serum albumin and cefoperazone have been determined from a double reciprocal Lineweaver-Burk plot. The binding distance and the transfer efficiency between cefoperazone and HSA were also obtained according to the theory of Förster' non-radiation energy transfer. The result suggested that the main binding force between cefoperazone and HSA is electrostatic force interaction. The high beta-lactamase stability of cefoperazone may be correlative with its molecular structure. The antibiotic activity and valency connect with transfer efficiency and dissociation constant. The effect of cefoperazone on the conformation of HSA was also analyzed using synchronous fluorescence spectrometry.

Calorimetric analysis of cephalosporins using an immobilized TEM-1 beta-lactamase on Ni2+ chelating sepharose fast flow.

Two beta-lactamases, penicillinase type I from Bacillus cereus and TEM-1 beta-lactamase from Haemophilus ducreyi, were immobilized on a Chelating Sepharose Fast Flow column loaded with Ni2+ in an active form. Flow-injection analysis of beta-lactams was performed by using an enzyme column reactor fitted into the enzyme thermistor. With both enzymes it was possible to monitor both penicillins and cephalosporins. Moreover, Michaelis constants of the TEM-1 beta-lactamase were markedly increased upon immobilization for all substrates, especially carbenicillin, cephaloridine, and cefoperazone.

Granulocyte colony-stimulating factor enhances endotoxin-induced decrease in biliary excretion of the antibiotic cefoperazone in rats.

We have recently reported that endotoxin (lipopolysaccharide [LPS]) derived from Klebsiella pneumoniae dramatically decreased the biliary excretion of the beta-lactam antibiotic cefoperazone (CPZ), which is primarily excreted into the bile via the anion transport system, in rats. The present study was designed to investigate the effect of human recombinant granulocyte colony-stimulating factor (G-CSF), which is reported to be beneficial in experimental models of inflammation, on the pharmacokinetics and biliary excretion of CPZ in rats. CPZ (20 mg/kg of body weight) was administered intravenously 2 h after the intravenous injection of LPS (250 microgram/kg). G-CSF was injected subcutaneously at 12 microgram/kg for 3 days and was administered intravenously at a final dose of 50 microgram/kg 1 h before LPS injection. Peripheral blood cell numbers were also measured. LPS dramatically decreased the systemic and biliary clearances of CPZ and the bile flow rate. Pretreatment with G-CSF enhanced these decreases induced by LPS. The total leukocyte numbers were increased in rats pretreated with G-CSF compared to the numbers in the controls, while the total leukocyte numbers were decreased (about 3,000 cells/microliter) by treatment with LPS. Pretreatment with G-CSF produces a deleterious effect against the LPS-induced decrease in biliary secretion of CPZ, and leukocytes play an important role in that mechanism.

A comparative study of the binding characteristics of ceftriaxone, cefoperazone and cefsulodin to human serum albumin.

The binding to human serum albumin of three cephalosporins of pharmacological interest: cefoperazone, ceftriaxone and cefsulodin was studied by ultrafiltration and differential scanning calorimetry methods. The identification of the binding sites in albumin was also performed using probes for the so-called sites I, II, bilirubin and fatty acids binding sites. Albumin showed two types of binding sites for cefoperazone and ceftriaxone, while for cefsulodin it showed a single type of binding site. The affinity values were: 5.6 10(4) M-1 and 3.1 10(4) M-1 for cefoperazone and ceftriaxone respectively, while cefsulodin showed low affinity (3.8 10(2) M-1). It was found that only cefoperazone interacted in a slight way with site I on serum albumin, while site II and the bilirubin binding site have capacity of binding the three cephalosporins assayed. Ceftriaxone and cefoperazone showed capacity to bind to the fatty acids binding site on albumin. These cephalosporins increased the thermal stability of the protein, suggesting that these ligands are favouring the compact structure of the native form of the protein more than the unfolded form.

NMR study of whole rat bile: the biliary excretion of cefoperazone and benzyl chloride by an isolated perfused rat liver.

1H NMR spectroscopy at 400 MHz has been applied to the analysis of whole bile samples produced by the isolated perfused rat liver. Using relatively simple NMR experiments biliary excretory products of cefoperazone and benzyl chloride were identified as cefoperazone itself and a benzyl-glutathione conjugate, respectively. Our use of 13C isotopic labelling demonstrates how 1H/13C heteronuclear NMR techniques can be used to produce uncrowded and informative spectra from whole bile. From the use of a HMQC-COSY experiment the structure of a benzyl-glutathione conjugate contained in whole bile was confirmed.

Molecular correlation between in vitro and in vivo activity of beta-lactam and beta-lactamase inhibitor combinations against methicillin-resistant Staphylococcus aureus.

Beta-Lactam resistance in Staphylococcus aureus is associated with beta-lactamase production, with the presence of a new penicillin binding protein (PBP) called PBP2a, with reduced affinity for beta-lactam antibiotics, and with modifications of normal PBPs. We have studied these mechanisms of resistance, in vivo and in vitro, for several beta-lactam antibiotics against both beta-lactamase-producing and non-producing methicillin-resistant S. aureus organisms (MRSA). Our results showed that all tested agents inhibited binding of labeled penicillin G to many PBPs. The combination of cefoperazone and sulbactam was the best combination, and it inhibited radiolabeled penicillin G binding to PBP2a at a lower concentration than that needed for cefoperazone alone. In vivo, the regimen of cefoperazone plus sulbactam was also more effective than cefoperazone alone. For beta-lactamase-negative strains this correlated with an increased binding affinity of cefoperazone plus sulbactam to PBP2a and PBP4. The improved efficacy of cefoperazone plus sulbactam versus cefoperazone with a beta-lactamase producing strain was closely related to cefoperazone hydrolysis by beta-lactamase that was inhibited by sulbactam. This study demonstrates that there is more than one effect of beta-lactamase inhibitors when they are combined with beta-lactam antimicrobial agents, and also that there may be a role for these agents in therapy for MRSA infections.

First and second derivative spectrophotometric determination of cefoperazone and sulbactam in injections.

A simple, spectrophotometric assay to measure the concentrations of cefoperazone and sulbactam in injectable formulations is described. Since zero-order spectra are subject to interference, derivative spectrophotometry was used to enhance the spectral details. A linear relationship between derivative amplitudes and the concentrations of the compounds was found. Beer's law is obeyed up to 75 and 80 micrograms ml-1 of cefoperazone in the first and second derivative modes, respectively, and up to 75 micrograms ml-1 of sulbactam in the second derivative mode. Detection limits were 0.64 and 0.88 microgram ml-1, respectively for cefoperazone in the first and second derivative modes and 0.30 micrograms ml-1 for sulbactam in the second derivative mode. The method is rapid, simple, does not require a separation step and has successfully been applied to the assay of commercial injections containing cefoperazone and sulbactam.

[beta-lactamase activity in sputum of patients with community-acquired lower respiratory tract infections].

beta-Lactamase production and activities in sputa of patients with community-acquired lower respiratory tract infections (LRTI) were determined and following results were obtained: 1) Suspected causative organisms frequently isolated were H. influenzae and Streptococcus pneumoniae. Similar results were previously reported. 2) Various beta-lactamase producing indigenous bacteria were detected. In many cases these indigenous beta-lactamase producing strains were isolated even when suspected causative bacteria were not beta-lactamase producers. 3) beta-Lactamase activities were detected from 61.5% of the sputa tested. Remaining activities of antibiotics added to the sputa were highly correlated with detection of beta-lactamases produced by suspectedly causative and indigenous strains and with presence of beta-lactamase activities in the sputa. Sulbactam/cefoperazone was stable in sputa than other antibiotics tested. 4) We concluded that the beta-lactamase produced by indigenous strains can be one of the factors of indirect pathogenicity in the community-acquired LRTI.

Efficacy of cefoperazone in combination with sulbactam in experimental Staphylococcus aureus endocarditis in rabbits.

The activity of cefoperazone with and without sulbactam was studied in vitro and in vivo against strains of methicillin-resistant and methicillin-susceptible Staphylococcus aureus. Cefoperazone with or without sulbactam was inactive in vitro against the methicillin-resistant strain and was bound by penicillin-binding protein 2a with an IC50 of 190 mg/L (the concentration that reduced radio-labelling with 3H-penicillin by 50%). Cefoperazone was hydrolysed by beta-lactamase in vitro but sulbactam improved cefoperazone activity in a rabbit model of aortic valve endocarditis caused by a beta-lactamase producing methicillin-susceptible strain.

Comparison of N-methylthiotetrazole dispositions in healthy volunteers following single intravenous doses of moxalactam, cefoperazone, and cefotetan.

The N-methylthiotetrazole side chain (NMTT) that is present on several cephalosporins has been implicated in the development of antibiotic-associated hypoprothrombinemia. A randomized three-way crossover trial was conducted to compare the release of the NMTT side chain from three NMTT-containing antibiotics. Single 2-g doses of moxalactam, cefoperazone, and cefotetan were given, followed by serial blood and urine sampling. The concentrations of the parent compound and the NMTT side chain in plasma, urine, and the reconstituted antibiotic solution were determined by high-pressure liquid chromatography. Peak NMTT concentrations ranged from 0.42 to 16.50 micrograms/ml and were significantly higher after moxalactam administration than after cefoperazone or cefotetan administration (P less than 0.01). The NMTT trough concentrations (12.5 h) ranged from nondetectable to 2.47 micrograms/ml and tended to be greater following cefoperazone administration. The amounts of NMTT administered (e.g., the amount in the reconstituted antibiotic solution) were 25.8 +/- 1.4, 15.2 +/- 0.9, and 22.1 +/- 3.0 mg following moxalactam, cefoperazone, and cefotetan administration, respectively (P less than 0.01). In contrast, urinary recoveries of NMTT were 57.4 +/- 26.2, 73.6 +/- 44.3, and 29.7 +/- 22.9 mg following moxalactam, cefoperazone, and cefotetan, respectively. The amount of NMTT formed in vivo and excreted unchanged, as assessed by subtracting in vitro NMTT formation from NMTT urinary recovery, was significantly higher after cefoperazone than after moxalactam or cefotetan administration (P less than 0.05). The discrepancy between in vitro NMTT production (moxalactam > cefotetan > cefoperazone) and the amount of NMTT formed in vivo and excreted unchanged (cefoperazone > moxalactam > cefotetan) demonstrated that the in vivo production of NMTT is dependent on the disposition of the parent cephalosporin.

Influence of protein binding on therapeutic efficacy of cefoperazone.

The effect of protein binding of cefoperazone (89.3% bound to rabbit serum) on antibacterial activity in serum was tested in a model that simulated a closed-space infection in a neutropenic host. Four gram-negative bacilli were tested in the model with cefoperazone doses of 20 and 200 mg/kg administered intramuscularly every 6 h for 16 doses. Cefoperazone efficacy was measured at 92 h by determining the log10 decrease in bacterial count from that of the control for five paired studies with three isolates. A significantly better response was demonstrated when the free (non-protein-bound) drug concentration exceeded the MICs and MBCs for the infecting microorganisms at the infection site at all times (P less than 0.005). This supports the concept that free (unbound) drug is the active component in treating bacterial infections.