Development of a rapid colorimetric time-kill assay for determining the in vitro activity of ceftazidime and tobramycin in combination against Pseudomonas aeruginosa

It is standard clinical practice to use a combination of two or more antimicrobial agents to treat an infection caused by Pseudonionas aeruginosa. The antibiotic combinations are usually selected empirically with methods to determine the antimicrobial effect of the combination such as the time-kill assay rarely used as they are time-consuming and labour intensive to perforin. Here, we report a modified time-kill assay, based on the reduction of the tetrazolium salt, 2,3-bis[2-methyloxy-4-nitro-5-sulfopheny1]-2H-tetrazolium-5-carboxanilide (XTT), that allows simple, inexpensive and more rapid determination of the in vitro activity of antibiotic combinations against P aeruginosa. The assay was used to determine the in vitro activity of ceftazidime and tobramycin in combination against P. aertiginosa isolates from cystic fibrosis patients and the results obtained compared with those from conventional viable count time-kill assays. There was good agreement in interpretation of results obtained by the XTT and conventional viable count assays, with similar growth curves apparent and the most effective concentration combinations determined by both methods identical for all isolates tested. The XTT assay clearly indicated whether an antibiotic combination had a synergistic, indifferent or antagonistic effect and could, therefore, provide a useful method for rapidly determining the activity of a large number of antibiotic combinations against clinical isolates. (C) 2004 Elsevier B.V. All rights reserved.

In vitro activity of tea-tree oil against clinical skin isolates of meticillin-resistant and -sensitive Staphylococcus aureus and coagulase-negative staphylococci growing planktonically and as biofilms

The susceptibility of Staphylococcus aureus [meticillin-resistant (MRSA) and meticillin-sensitive (MSSA)] and coagulase-negative staphylococci (CoNS), which respectively form part of the transient and commensal skin flora, to tea-tree oil (TTO) was compared using broth microdilution and quantitative in vitro time-kill test methods. MRSA and MSSA isolates were significantly less susceptible than CoNS isolates, as measured by both MIC and minimum bactericidal concentration. A significant decrease in the mean viable count of all isolates in comparison with the control was seen at each time interval in time-kill assays. However, the only significant difference in the overall mean log(10) reduction in viable count between the groups of isolates was between CoNS and MSSA at 3 h, with CoNS isolates demonstrating a significantly lower mean reduction. To provide a better simulation of in vivo conditions on the skin, where bacteria are reported to grow as microcolonies encased in glycocalyx, the bactericidal activity of TTO against isolates grown as biofilms was also compared. Biofilms formed by MSSA and MRSA isolates were completely eradicated following exposure to 5 % TTO for 1 h. In contrast, of the biofilms formed by the nine CoNS isolates tested, only five were completely killed, although a reduction in viable count was apparent for the other four isolates. These results suggest that TTO exerts a greater bactericidal activity against biofilm-grown MRSA and MSSA isolates than against some biofilm-grown CoNS isolates.

Protein kinase B/Akt activity is involved in renal TGF-beta 1-driven epithelial-mesenchymal transition in vitro and in vivo

The molecular pathogenesis of diabetic nephropathy (DN), the leading cause of end-stage renal disease worldwide, is complex and not fully understood. Transforming growth factor-beta (TGF-beta1) plays a critical role in many fibrotic disorders, including DN. In this study, we report protein kinase B (PKB/Akt) activation as a downstream event contributing to the pathophysiology of DN. We investigated the potential of PKB/Akt to mediate the profibrotic bioactions of TGF-beta1 in kidney. Treatment of normal rat kidney epithelial cells (NRK52E) with TGF-beta1 resulted in activation of phosphatidylinositol 3-kinase (PI3K) and PKB/Akt as evidenced by increased Ser473 phosphorylation and GSK-3beta phosphorylation. TGF-beta1 also stimulated increased Smad3 phosphorylation in these cells, a response that was insensitive to inhibition of PI3K or PKB/Akt. NRK52E cells displayed a loss of zona occludins 1 and E-cadherin and a gain in vimentin and alpha-smooth muscle actin expression, consistent with the fibrotic actions of TGF-beta1. These effects were blocked with inhibitors of PI3K and PKB/Akt. Furthermore, overexpression of PTEN, the lipid phosphatase regulator of PKB/Akt activation, inhibited TGF-beta1-induced PKB/Akt activation. Interestingly, in the Goto-Kakizaki rat model of type 2 diabetes, we also detected increased phosphorylation of PKB/Akt and its downstream target, GSK-3beta, in the tubules, relative to that in control Wistar rats. Elevated Smad3 phosphorylation was also detected in kidney extracts from Goto-Kakizaki rats with chronic diabetes. Together, these data suggest that TGF-beta1-mediated PKB/Akt activation may be important in renal fibrosis during diabetic nephropathy.

Use of breakpoint combination sensitivity testing as a simple and convenient method to evaluate the combined effects of ceftazidime and tobramycin on Pseudomonas aeruginosa and Burkholderia cepacia complex isolates in vitro

An in vitro method of determining the activity of antibiotics in combination which is simple and convenient to perform and which could be used routinely in clinical microbiology laboratories is desirable. We investigated the activity, against Pseudomonas aeruginosa and Burkholderia cepacia complex clinical isolates, of ceftazidime and tobramycin in combination using a broth macrodilution sensitivity method based on breakpoint minimum inhibitory concentrations and compared the results obtained using this method with those obtained using the microtitre checkerboard method. There was good agreement in interpretation of results between the two methods for both P. aeruginosa (90%) and B. cepacia complex isolates (70%) with tobramycin and for P. aeruginosa isolates (70%) with ceftazidime. As the breakpoint combination sensitivity testing method employs only four tubes and does not require initial determination of individual antibiotic minimum inhibitory concentrations, it is simpler and more convenient for determining the activity of antibiotics in combination than the microtitre checkerboard method. The use of this method in routine microbiology laboratories to determine the activity of antibiotic combinations against clinical isolates should optimise treatment of infection by ensuring that appropriate antibiotic combinations are prescribed. (C) 2004 Elsevier B.V. All rights reserved.

Phosphonoacetate biosynthesis: In vitro detection of a novel NADP(+)-dependent phosphonoacetaldehyde-oxidizing activity in cell-extracts of a marine Roseobacter

A novel phosphonoacetaldehyde-oxidizing activity was detected in cell-extracts of the marine bacterium Roseovarius nubinhibens ISM grown on 2-aminoethylphosphonic acid (2-AEP; ciliatine). Extracts also contained 2-AEP transaminase and phosphonoacetate hydrolase activities. These findings indicate the existence of a biological route from 2-AEP via phosphonoacetaldehyde for the production of phosphonoacetate, which has not previously been shown to be a natural product. The three enzymes appear to constitute a previously-unreported pathway for the mineralization of 2-AEP which is a potentially important source of phosphorus in the nutrient-stressed marine environment.

Nematode neuropeptide modulation of the vagina vera of Ascaris suum: in vitro effects of PF1, PF2, PF4, AF3 and AF4

Ascaris suum possesses a large number of FMRFamide-related peptides (FaRPs) of which KNEFIRFamide (AF1), KHEYLRFamide (AF2) and KSAYMRFamide (AF8/PF3) have been shown to modulate the intrinsic, rhythmic activity of the vagina vera of A. suum in vitro. In the present study, the effects of the nematode FaRPs, SDPNFLRFamide (PF1), SADPNFLREamide (PF2) and KPNFIRFamide (PF4) (from Panagrellus redivivus) and AVPGVLRFamide (AF3) and GDVPGVLRFamide (AF4) (from A. suum) on the in vitro activity of the vagina vera were examined. The effects of each of the peptides were qualitatively and quantitatively distinct. All 3 FaRPs from P. redivivus were inhibitory, causing a cessation of contractions. PF2 was 3 times more potent than PF1, with a threshold of 1 nM. Although PF4 was the least potent (threshold, 10 nM), its effects at greater than or equal to 10 nM were quantitatively the greatest. Both AF3 and AF4 (1 mu M) induced complex, multiphasic responses consisting of an initial contraction and spastic paralysis followed by a return of contractile activity of increased amplitude. AF3 was 3 times more potent than AF4. The effects of these peptides had some similarities to those observed on A. suum somatic body wall muscle in vitro, with PF1, PF2 and PF4 being inhibitory and AF3 and AF4 being excitatory.

Growth Inhibitory Activity of Extracted Material and Isolated Compounds from the Fruits of Kigelia pinnata

A study of the components of the fruits of Kigelia pinnata was undertaken to identify compounds with potential growth inhibitory activity against human melanoma cells, since extracts from the fruits of this plant have been described in traditional medicine to have application in the treatment of skin cancer and other skin ailments. A bioactivity-guided fractionation process yielded a number of crude fractions, which demonstrated cytotoxicity in vitro against human melanoma cells. Compounds isolated and identified included the isocoumarins, demethylkigelin (1) and kigelin 2), fatty acids, oleic (3) and heneicosanoic acids (4), the furonaphthoquinone, 2-(1-hydroxyethyl)-naphtho[2,3-b]furan-4,9-dione (5), and ferulic acid (6). A number of structurally related synthetic compounds were also tested using the MTT assay. The most potent series of these compounds, the furonaphthoquinones, also demonstrated a cytotoxic effect in two human breast cancer cell lines tested.

In vitro bactericidal activity of diterpenoids isolated from Aframomum melegueta K.Schum against strains of Escherichia coli, Listeria monocytogenes and Staphylococcus aureus

Ethnopharmacological relevance: The ethnobotanical use of Aframomum melegueta in the treatment of urinary tract and soft tissue infection suggested that the plant has antimicrobial activity.

Materials and methods: To substantiate the folkloric claims, an acetone, 50:50 acetone:methanol and 2:1 chloroform:methanol extracts were tested against Escherichia coli K12; acetone extract and the fractions of acetone extracts were tested against Listeria monocytogenes. Bioassay-guided fractionation was performed on the extract using L. monocytogenes as the test organism to isolate the bioactive compounds which were then tested against all the other organisms.

Results: Four known labdane diterpenes (G3 and G5) were isolated for the first time from the rhizomes of A. melegueta and purified. These were tested against E. coli, L. monocytogenes, methicillin resistant Staphylococus aureus (MRSA) and S. aureus to determine antibacterial activity. The result showed that two compounds G3 and G5 exhibited more potent antibacterial activity compared to the current clinically used antibiotics ampicillin, gentamicin and vancomycin and can be potential antibacterial lead compounds. The structure of the labdane diterpenes were elucidated using nuclear magnetic resonance (NMR) spectroscopy and Mass spectrometry. A possible mode of action of the isolated compound G3 and its potential cytotoxicity towards mammalian cells were also discussed.

Conclusion: The results confirmed the presence of antibacterial compounds in the rhizomes of A. melegueta with a favourable toxicity profile which could be further optimized as antibacterial lead compounds.

The In Vitro Susceptibility of Biofilm Forming Medical Device Related Pathogens to Conventional Antibiotics

Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum biofilm eradication concentration (MBEC) and kill kinetics were established for vancomycin, rifampicin, trimethoprim, gentamicin, and ciprofloxacin against the biofilm forming bacteria Staphylococcus epidermidis (ATCC 35984), Staphylococcus aureus (ATCC 29213), Methicillin Resistant Staphylococcus aureus (MRSA) (ATCC 43300), Pseudomonas aeruginosa (PAO1), and Escherichia coli (NCTC 8196). MICs and MBCs were determined via broth microdilution in 96-well plates. MBECs were studied using the Calgary Biofilm Device. Values obtained were used to investigate the kill kinetics of conventional antimicrobials against a range of planktonic and biofilm microorganisms over a period of 24 hours. Planktonic kill kinetics were determined at 4xMIC and biofilm kill kinetics at relative MBECs. Susceptibility of microorganisms varied depending on antibiotic selected and phenotypic form of bacteria. Gram-positive planktonic isolates were extremely susceptible to vancomycin (highest MBC: 7.81 mg L−1: methicillin sensitive and resistant S. aureus) but no MBEC value was obtained against all biofilm pathogens tested (up to 1000 mg L−1). Both gentamicin and ciprofloxacin displayed the broadest spectrum of activity with MIC and MBCs in the mg L−1 range against all planktonic isolates tested and MBEC values obtained against all but S. epidermidis (ATCC 35984) and MRSA (ATCC 43300).