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Aims: To investigate the antimicrobial efficacy of an alkaloid, harmaline alone and in combination with chlorhexidine digluconate (CHG) against clinical isolates of Staphylococcus aureus (S. aureus) grown in planktonic and biofilm cultures. Methods: Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined for each micro-organism grown in suspension and in biofilm using microbroth dilution method. Chequerboard assays were used to determine synergistic, indifferent or antagonistic interactions between harmaline and CHG, and the some of results were verified by confocal laser scanning microscopy. Results: Harmaline and CHG showed effective antimicrobial activity against suspensions and biofilm cultures of S. aureus, respectively. As determined by fractional inhibitory concentration index (FICI), synergistic antimicrobial effects between harmaline and CHG were observed in nine and 11 of the 13 S. aureus strains when in suspension and in biofilm, respectively. FICI values were from 0·375 to 1·25 when in suspension and from 0·25 to 1·25 when in biofilm. Conclusions: Synergistic activity of harmaline and CHG against clinical isolates of S. aureus (in suspension and in biofilm) was observed in vitro. Significance and Impact of the Study: This study might provide alternative methods to overcome the problem of drug-resistance of S. aureus both in suspension and in biofilm.

Mycobacterium tuberculosis (M. tuberculosis) remains one of the world’s most prevalent and serious pathogens, and multiple resistant strains are increasingly widespread. The recent emergence of multidrug-resistant and extensively drug-resistant (XDR-TB) strains has increased the importance of searching for alternative targets to develop new antimycobacterial drugs. Plumbagin (PL), a natural plant product, has been reported to have potential antimicrobial activity against M. tuberculosis, but the response mechanisms of M. tuberculosis to PL are still poorly understood. In this study, we used a commercial oligonucleotide microarray to determine the overall transcriptional response of M. tuberculosis H37Rv triggered by exposure to subinhibitory concentrations of PL. A total of 274 genes were found to be differentially regulated by PL. Of these, 103 genes were upregulated, and 171 genes were downregulated. A number of important genes were significantly regulated that are involved in various pathways, such as chaperonins, PE/PPE multigene families, and genes involved in the cell envelope, virulence, and 50S ribosomes. To our knowledge, this genome-wide transcriptomics approach has produced the first insights into the response of M. tuberculosis to a PL challenge. These results will pave the way for exploring the mechanisms of PL action against M. tuberculosis.

Aims: To evaluate the interaction of 1-(1-naphthylmethyl)-piperazine (NMP) and ciprofloxacin (CPFX) in vitro against fluoroquinolone (FQ)-resistant clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). Methods and Results: The in vitro interaction of NMP and CPFX in 12 FQ-resistant clinical isolates of MRSA was assessed using a checkerboard microdilution method. In the study, a synergistic antimicrobial effect between NMP and CPFX was observed in all 12 FQ-resistant strains tested, as determined by the fractional inhibitory concentration index (FICI), and in 10 strains using ΔE models. No antagonistic activity was observed in any of the strains tested. These positive interactions were also confirmed using the time–killing test and agar diffusion assay for the selected strain, MRSA 1862; synergistic activity was observed when NMP was combined with the first-line antimicrobial agent CPFX against Staph. aureus. Conclusions: Synergistic activity between NMP and CPFX against clinical isolates of FQ-resistant Staph. aureus was observed in vitro. Significance and Impact of the Study: This report might provide alternative methods to reduce the resistance of Staph. aureus to CPFX.

A checkerboard microdilution method, performed according to the recommendations of the National Committee for Clinical Laboratory Standards, was used to study the in vitro interaction of fluconazole and allicin in 24 fluconazole-resistant clinical isolates of Candida albicans, one experimentally induced strain S-1, and one ATCC type strain 10231. The interaction intensity was determined by spectrophotometric methods and visual reading of the checkerboard assay, and the nature of the interactions was assessed using two nonparametric approaches [fractional inhibitory concentration index (FICI) and DeltaE models]. Synergism was observed in 23 strains using FICI, and in 22 strains using DeltaE. The DeltaE model gave results consistent with FICI, but no antagonistic action was observed. The positive interactions were also confirmed by the time-killing test and agar diffusion in the selected strains. Moreover, the in vivo experiment showed that a combination of fluconazole and allicin exhibited a good synergism against C. albicans.

8-Methoxypsoralen (8-MOP), a naturally occurring furocoumarin found in many plant species, has been reported to have antimycobacterial activity against Mycobacterium tuberculosis strain H37Rv (ATCC 27294). In the present study, we further test the in vitro synergistic activity of 8-MOP and ethambutol (EMB), isoniazid (INH), or rifampin (RMP) against M. tuberculosis. This study showed that 8-MOP has antimycobacterial activity against two drug-sensitive and six drug-resistant clinical isolates of M. tuberculosis, with the minimum inhibitory concentrations of 100–200 and 200–400 μg/mL, respectively. A synergistic antimycobacterial effect between 8-MOP and EMB, INH, or RMP against six drug-resistant strains was observed, with the fractional inhibitory concentration indices (FICIs) of 0.093–0.156, 0.138–0.285 and 0.093–0.262, respectively. The combination of 8-MOP/EMB, 8-MOP/INH, and 8-MOP/RMP displayed either synergistic activity or had no interaction when tested against the two clinical drug-sensitive strains and the standard strain. No antagonism was observed for any drug combination against any of the strains tested. To our knowledge, this is first report that 8-MOP has synergistic activity with first-line antimycobacterial agents.

The formation of extracellular traps (ETs) has recently been recognized as a novel defense mechanism in several types of innate immune cells. It has been suggested that these structures are toxic to microbes and contribute significantly to killing several pathogens. However, the role of ETs formed by macrophages (METs) in defense against microbes remains little known. In this study, we demonstrated that a subset of murine J774A.1 macrophage cell line (8% to 17%) and peritoneal macrophages (8.5% to 15%) form METs-like structures (METs-LS) in response to Escherichia coli and Candida albicans challenge. We found only a portion of murine METs-LS, which are released by dying macrophages, showed detectable killing effects on trapped E. coli but not C. albicans. Fluorescence and scanning electron microscopy analyses revealed that, in vitro, both microorganisms were entrapped in J774A.1 METs-LS composed of DNA and microbicidal proteins such as histone, myeloperoxidase and lysozyme. DNA components of both nucleus and mitochondrion origins were detectable in these structures. Additionally, METs-LS formation occurred independently of ROS produced by NADPH oxidase, and this process did not result in cell lysis. In summary, our results emphasized that microbes induced METs-LS in murine macrophage cells and that the microbicidal activity of these METs-LS differs greatly. We propose the function of METs-LS is to contain invading microbes at the infection site, thereby preventing the systemic diffusion of them, rather than significantly killing them.

Tuberculosis (TB) is still one of the most common causes of death in the world. The emergence of multidrug-resistant and extensively drug-resistant (XDR-TB) Mycobacterium tuberculosis (M. tuberculosis) strains has increased the importance of searching for alternative targets to develop new antimycobacterial drugs. Linezolid, the first of oxazolidinones, is active in vitro against M. tuberculosis, but the response mechanisms of M. tuberculosis to linezolid are still poorly understood. To reveal the possible mechanism of action of linezolid against M. tuberculosis, commercial oligonucleotide microarrays were used to analyze the genome-wide transcriptional changes triggered by treatment with subinhibitory concentrations of linezolid. Quantitative real-time RT-PCR was performed for selected genes to verify the microarray results. A total of 729 genes were found to be differentially regulated by linezolid. Among these, 318 genes were upregulated, and 411 genes were downregulated. A number of important genes were significantly regulated that are involved in various pathways, such as protein synthesis, sulfite metabolism, and genes involved in the cell envelope and virulence. This genome-wide transcriptomics approach produced the first insights into the response of M. tuberculosis to a linezolid challenge.

Many clinical isolates of Staphylococcus aureus (S. aureus) are resistant to numerous antimicrobials, including the fluoroquinolones (FQs). Flavonoids such as biochanin A (BCA) are compounds that are naturally present in fruits, vegetables, and plant-derived beverages. The goal of this investigation was to study the possible synergy between the antimicrobial agents BCA and ciprofloxacin (CPFX) when used in combination; CPFX was chosen as a representative FQ compound. We used S. aureus strain ATCC 25923 and 11 fluoroquinolone (FQ)-resistant methicillin-resistant S. aureus (MRSA) strains. Results from the drug susceptibility testing and checkerboard assays show that the minimum inhibitory concentration (MIC) of BCA ranged from 64 µg/mL to 512 µg/mL. When BCA was combined with CPFX, the fractional inhibitory concentration index (FICI) data showed that there was synergy in all 12 of the S. aureus strains tested. No antagonistic activity was observed in any of the strains tested. The results of time-kill tests and agar diffusion tests confirm that there was synergy between BCA and CPFX against S. aureus strains. These results suggest that BCA can be combined with FQs to produce a powerful antimicrobial agent.

To investigate effective new rabies vaccines, a fusion protein consisting of the rabies virus (RV) glycoprotein and the heat-labile enterotoxin B subunit of Escherichia coli (LTB) was successfully constructed and delivered in a live attenuated Salmonella strain LH430. Mice were immunised with LH430 carrying pVAX1-G, pVAX1-G-LTB or pVAX1-ori-G-LTB. The antibody titres of mice immunised with oral LH430 carrying pVAX1-G-LTB or pVAX1-ori-G-LTB were significantly higher than those of pVAX1-G-immunised mice. The results of the challenge with the rabies virus standard strain (CVS-11) showed that the LH430 strain carrying the G-LTB gene induced immunity and elevated IL-2 levels in immunised mice ((∗∗)P

Piperine, a major plant alkaloid found in black pepper (Piper nigrum) and long pepper (Piper longum), has shown potential for inhibiting the efflux pump (EP) of Staphylococcus aureus. In this study, a modulation assay showed that piperine could decrease the MIC of ethidium bromide (EtBr) twofold at 32 μg ml−1 and fourfold at 64 μg ml−1 against Mycobacterium smegmatis mc2 155 ATCC 700084. A real-time, 96-well plate fluorometric method was employed to evaluate the EP inhibition ability of piperine in M. smegmatis. Reserpine, chlorpromazine, verapamil and carbonyl cyanide m-chlorophenylhydrazone were used as positive controls. Piperine significantly enhanced accumulation and decreased the efflux of EtBr in M. smegmatis, which suggests that it has the ability to inhibit mycobacterial EPs.

Chelerythrine (a natural quaternary benzophenanthridine alkaloid) is an extract from the roots of Chelidonium majus with potential antimycobacterial activity. To reveal the possible mechanism of action of chelerythrine against Mycobacterium tuberculosis (M. tuberculosis), commercial oligonucleotide microarrays were used to analyze the genome-wide transcriptional changes triggered by treatment with subinhibitory concentrations of chelerythrine. Quantitative real-time RT-PCR was performed for selected genes to verify the microarray results. We interpreted our microarray data using Agilent software. Analysis of the microarray data revealed that a total of 759 genes were differentially regulated by chelerythrine. Of these, 372 genes were upregulated, and 387 genes were downregulated. Some of the important genes that were significantly regulated are related to different pathways (such as urease), methoxy-mycolic acid synthase, surface-exposed lipids, the heat shock response, and protein synthesis. This genome-wide transcriptomics approach produced the first insights into the response of M. tuberculosis to a chelerythrine challenge.

Reports have shown that oleanolic acid (OA), a triterpenoid, exists widely in food, medicinal herbs and other plants, and that it has antimycobacterial activity against the Mycobacterium tuberculosis strain H37Rv (ATCC 27294). In this study it was found that OA had antimycobacterial properties against eight clinical isolates of M. tuberculosis and that the MICs of OA against drug-sensitive and drug-resistant isolates were 50–100 and 100–200 μg ml−1, respectively. The combination of OA with isoniazid (INH), rifampicin (RMP) or ethambutol (EMB) showed favourable synergistic antimycobacterial effects against six drug-resistant strains, with fractional inhibitory concentration indices of 0.121–0.347, 0.113–0.168 and 0.093–0.266, respectively. The combination treatments of OA/INH, OA/RMP and OA/EMB displayed either a synergistic interaction or did not show any interaction against two drug-sensitive strains. No antagonism resulting from the OA/INH, OA/RMP or OA/EMB combination was observed for any of the strains tested. OA exhibited a relatively low cytotoxicity in Vero cells. These results indicate that OA may serve as a promising lead compound for future antimycobacterial drug development.

Thymol (THY) was found to have in vitro antifungal activity against 24 fluconazole (FLC)-resistant and 12 FLC-susceptible clinical isolates of Candida albicans, standard strain ATCC 10231 and one experimentally induced FLC-resistant C. albicans S-1. In addition, synergism was observed for clinical isolates of C. albicans with combinations of THY–FLC and THY–amphotericin B (AMB) evaluated by the chequerboard microdilution method. The interaction intensity was determined by spectrophotometry for the chequerboard assay, and the nature of the interactions was assessed using two non-parametric approaches [fractional inhibitory concentration index (FICI) and ΔE models]. The interaction between THY–FLC or THY–AMB in FLC-resistant and -susceptible strains of C. albicans showed a high percentage of synergism by the FICI method and the ΔE method. The ΔE model gave results consistent with FICI, and no antagonistic action was observed in the strains tested.

Staphylococcus aureus(S. aureus) strains with multiple antibiotic resistances are increasingly widespread, and new agents are required for the treatment ofS. aureus. Cryptotanshinone (CT), a major tanshinone of medicinal plantSalvia miltiorrhizaBunge, demonstrated effective in vitro antibacterial activity against all 21S. aureusstrains tested in this experiment. Affymetrix GeneChips were utilized to determine the global transcriptional response ofS. aureusATCC 25923 to treatment with subinhibitory concentrations of CT. Transcriptome profiling indicated that the antibacterial action of CT may be associated with its action as active oxygen radical generator;S. aureusundergoes an oxygen-limiting state upon exposure to CT.