Killing Curve Assays Research Articles

A new N-acylhydrazone oxadiazole derivative with activity against mycobacteria.

Aim: To evaluate the anti-Mycobacterium tuberculosis (Mtb) potential of the hybrid oxadiazol-4-methoxynaphthalene (6n) derived from N-acylhydrazone (4k).Materials & methods: The study determined the minimal inhibitory concentration of (6n) against Mtb H37Rv and Mtb clinical isolates, potential combination of (6n) with anti-tuberculosis drugs and carried out time kill curve assay of Mtb H37Rv. Additional contribution for the analysis of (6n) was explored by in silico pharmacokinetics, and in vitro and in vivo cytotoxicity determinations.Results: The newly synthesized molecule (6n) demonstrated anti-Mtb activity, low cytotoxicity and selectivity for Mtb.Conclusion: The derivative (6n) emerges as a potential anti-TB drug candidate.

Coevolutionary analysis of Pseudomonas syringae-phage interactions to help with rational design of phage treatments.

Treating plant bacterial diseases is notoriously difficult because of the lack of available antimicrobials. Pseudomonas syringae pathovar syringae (Pss) is a major pathogen of cherry (Prunus avium) causing bacterial canker of the stem, leaf and fruit, impacting productivity and leading to a loss of trees. In an attempt to find a treatment for this disease, naturally occurring bacteriophage (phage) that specifically target Pss is being investigated as a biocontrol strategy. However, before using them as a biocontrol treatment, it is important to both understand their efficacy in reducing the bacterial population and determine if the bacterial pathogens can evolve resistance to evade phage infection. To investigate this, killing curve assays of five MR phages targeting Pss showed that phage resistance rapidly emerges invitro, even when using a cocktail of the five phages together. To gain insight to the changes occurring, Pss colonies were collected three times during a 66-h killing curve assay and separately, Pss and phage were also coevolved over 10 generations, enabling the measurement of genomic and fitness changes in bacterial populations. Pss evolved resistance to phages through modifications in lipopolysaccharide (LPS) synthesis pathways. Bacterial fitness (growth) and virulence were affected in only a few mutants. Deletion of LPS-associated genes suggested that LPS was the main target receptor for all five MR phages. Later generations of coevolved phages from the coevolution experiment were more potent at reducing the bacterial density and when used with wild-type phages could reduce the emergence of phage-resistant mutants. This study shows that understanding the genetic mechanisms of bacterial pathogen resistance to phages is important for helping to design a more effective approach to kill the bacteria while minimizing the opportunity for phage resistance to manifest.

Investigation of the Antibacterial Synergistic Activity of Fosfomycin-Teicoplanin Combination in Methicillin-Resistant Staphylococcus aureus Strains Isolated from Various Clinical Sample

The aim of this study was to investigate the detection of teicoplanin and fosfomycin antibiotic susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) strains by different methods and to evaluate the antibacterial synergistic effect of teicoplanin-fosfomycin combination by using checkerboard assay and time kill curve assay. Forty-five MRSA strains isolated from clinical samples in routine medical microbiology laboratory of Göztepe Prof. Dr. Süleyman Yalçın City Hospital were included in the study. In the first stage of the combination study, minimum inhibitory concentrations (MIC) were investigated by broth microdilution for teicoplanin and by both broth microdilution and agar dilution methods for fosfomycin. The combination of teicoplanin and fosfomycin was tested by the checkerboard method in 45 MRSA strains and combination effect was determined according to fractional inhibitory concentration index (ΣFIC) calculation. The synergistic effect and bactericidal activity of antibiotic combination were studied against a randomly selected strain from the strains used in the study by using time-kill method for 24 hours. As a result of teicoplanin and fosfomycin antibiotic susceptibility studies, all isolates were found to be susceptible to both antibiotics according to the susceptibility breakpoints determined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST). A synergistic effect was found in 22 (49%), additive effect in 22 (49%) and indifferent effect in one (2%) of the 45 strains studied with the checkerboard method. The mean ΣFIC of 45 isolates was found to be 0.5. In the combination study of the antibiotics of the isolate that was studied with time-kill method, synergism was detected for 1/8 MIC concentrations at 12th hour and 24th hour and synergism at 1/4 MIC concentration at sixth hour, 12th hour and 24th hour. In the combination study of 1/4 MIC concentrations of antibiotics, bactericidal effect was detected at sixth hour and this effect was observed to disappear at 12th and 24th hours. High rate of synergistic antibacterial effect of teicoplanin-fosfomycin combination on MRSA isolates was demonstrated as a result of in vitro tests. Such studies conducted on antibiotic-resistant bacterial infections will provide clinicians different treatment options and will contribute to increasing survival. As a result of this study, provided that it is supported by future clinical studies, it can be stated that the teicoplanin-fosfomycin combination may be an effective treatment option in community and hospital-acquired infections caused by MRSA.

Facile synthesis and optical characterization of selenium nanoparticles synthesized using Clitoria ternatea and Zingiber officinale: in vitro biomedical evaluation of antioxidant potential and antibacterial activity against caries-causing microbes

ABSTRACT In this study, selenium nanoparticles will be produced with Clitoria ternatea and Zingiber officinale, and their efficacy as a natural anti-cariogenic agent will be assessed. To do this, a variety of analysis and evaluation techniques have been used, such as UV-Vis spectroscopy to characterize the synthesized selenium nanoparticles that has absorption band at 440 nm. XRD of the Se nanoparticle were analysed and were found to be crystalline in nature with particles size of 36 nm. SEM images confirmed that Se nanoparticles were flaky structured. The antibacterial activity test to assess the selenium nanoparticles activity against dental caries causing bacteria and the zone of inhibition of the nanoparticles were found. Cytoplasmic leakage assay was done to govern the range of damage instigated by the synthesized selenium nanoparticles to bacterial cell membranes and time kill curve assay represents the time taken by the synthesized selenium nanoparticles to kill the cariogenic bacteria.

Evaluation Of Antimicrobial Effects Of Orthodontic Band Cement Incorporated With Zirconia, Gold, Copper Biosynthesized Nanoparticles- An In-Vitro Study

OBJECTIVE: To evaluate the antimicrobial effects of orthodontic band cement incorporated with various biosynthesized nanoparticles. MATERIALS AND METHODS: Zirconia, gold, copper Nanoparticles were green synthesized using white tea extract and dry ginger which are incorporated into orthodontic band cement, characterized using FTIR analysis. Antimicrobial activity against Streptococcus mutans and Lactobacillus acidophilus was tested in vitro by disc diffusion and Time Kill Curve Assay. RESULTS: Zirconia nanoparticles incorporated GIC exhibited better antimicrobial activity against streptococcus mutans, and gold nanoparticles incorporated GIC against lactobacillus acidophilus at varying concentrations at different time period. CONCLUSION: The antibacterial effect against streptococcus mutans and lactobacillus acidophilus were distinctly noted in Zirconia nanoparticles, gold nanoparticles followed by copper nanoparticles. Zirconia and gold nanoparticles produced more efficient antibacterial property and eventually would be effective in curtailing White Spot Lesions.

Evaluation of Antimicrobial Effect of Biologically Safe Selenium Nanoparticles-coated Clear Aligner—An In vitro Study

Objective To synthesize selenium nanoparticles (SeNPs) from plant extract, characterize them, coat them on clear aligners, and evaluate their cytotoxicity and antimicrobial activity. Materials and Methods The green synthesis of SeNPs utilized the leaves of Withania somnifera. The biosynthesized nanoparticles are incorporated in clear aligners using sol-gel thin film dip coating method to check for antimicrobial effect against Streptococcus mutans and Lactobacillus species using time kill curve assay. The cytotoxicity of the material was checked using brine shrimp lethality assay. Results The optical density values indicated that the SeNPs-coated clear aligners effectively inhibited the growth of Lactobacillus sp. and S. mutans. Time kill curve assessed in varying concentration of 25 µg/mL, 50 µg/mL, and 100 µg/mL revealed greater antibacterial activity at a lower concentration with increasing time. Conclusion Clear aligners coated with SeNPs had positive antibacterial effects on suspensions of Lactobacillus sp. and S. mutans and are biocompatible.

Effect of Klebsiella-specific phage on multidrug-resistant Klebsiella pneumoniae- an experimental study

BackgroundThe objective of this study was to study the effect of Klebsiella-specific phage isolated from sewage with and without the combination of antibiotics (imipenem) on the growth of clinical isolates of multidrug-resistant Klebsiella pneumoniae by time-kill assay and also to study the effect of bacteriophage and bacteriophage-antibiotic (imipenem) combination on biofilm production. MethodsA total of 40 MDR K. pneumoniae isolates were used. Klebsiella-specific phage was isolated using K. pneumoniae subspp. pneumoniae ATCC 33495 as the host from sewage. In vitro time kill curve assays were performed to evaluate the effect of Klebsiella-specific phage with and without the combination of antibiotics on the viable cell counts of MDR K. pneumoniae isolates. Microtiter plate method of O'Toole and Kolter was used to study the effect of Klebsiella-specific phage with and without the combination of antibiotics on biofilm production. For the Time kill assay, results were analyzed for significant differences using Friedman test. Tests for significant differences between the different groups were found using the Mann-Whitney U test. The correlation between the formation of biofilm was analyzed using Karl Pearson's coefficient of correlation. P value of <0.05 was considered to be statistically significant. ResultsIn vitro time-kill assay showed a 0.4 log decline and a 0.5 log decline in K. pneumoniae colony counts at 4 h, when phage was administered individually and in combination with imipenem, respectively (p < 0.001). Phage and phage-imipenem combinations reduced the ability of K. pneumoniae to produce biofilm by 38 % and 53 %, respectively. ConclusionIn conclusion, this study suggests that phage therapy has inhibiting activity against MDR K. pneumoniae. It is found to reduce bacterial cell count and biofilm formation but does not have a total cidal effect. However, in order to get a notable result, a phage cocktail or combination of phage with other antibiotic(s) is suggested.

Synergistic activity of Thymus capitatus essential oil and cefotaxime against ESBL-producing Klebsiella pneumoniae

ABSTRACT The objective of the current study was to evaluate the interaction between Tunisian Thymus capitatus essential oil (EO) and cefotaxime against Extended-Spectrum Beta-lactamases (ESBLs) producing Klebsiella pneumoniae hospital strains. GC-MS revealed that the major component of EO was found to be carvacrol (69.28%). The EO exerts an advanced bactericidal effect against all strains. Synergy between EO and cefotaxime was obtained by combined disk diffusion and checkerboard techniques. Combined use of EO and cefotaxime reduced the MIC of imipenem by 8- to 128-fold for all strains (fractional inhibitory concentration index ˂ 0.5, synergy). The time kill curve assay confirmed the advanced activity of combinatory effects of EO and cefotaxime, with total reduce of bacterial number (CFU/mL) after 6 h of culture. Synergistic activity of the combination between EO and cefotaxime constitute an important strategy as therapeutical option to combat infections caused by ESBLs producing Klebsiella pneumoniae.

Tea Tree Essential Oil Kills Escherichia coli and Staphylococcus epidermidis Persisters.

Persister cells are a small subpopulation of non-growing bacteria within a population that can survive long exposures to antibiotic treatment. Following antibiotic removal, persister cells can regrow and populate, playing a key role in the chronic reoccurrence of bacterial infections. The development of new molecules and methods to kill bacterial persisters is critical. Essential oils and other natural products have long been studied for their antimicrobial effects. Here, we studied the effectiveness of tea tree essential oil (TTO), a common component in many commercial care products, against Escherichia coli and Staphylococcus epidermidis persister cells. Using biphasic kill curve assays, we found that concentrations of 0.5% and 1.0% TTO for E. coli and S. epidermidis, respectively, completely eradicated persister cells over a period of 24 h, with the component terpinen-4-ol responsible for most of the killing. Using a colorimetric assay, it was determined that the TTO exhibited its anti-persister effects through a membrane disruption mechanism.

Isolation of Clavibacter michiganensis subsp. michiganenesis-specific bacteriophages from tomato fields in Turkey and their biocontrol potential

BackgroundClavibacter michiganensis subsp. michiganensis (Cmm) is causing wilt and canker and significant economic losses in tomato production. Current control methods with conventional chemicals or antibiotics lost their effectiveness due to the development of bacterial resistance to this agent. Bacteriophages, viruses of bacteria, have been attracting increasing attention as an environmentally friendly means of controlling bacterial diseases. In this study, Cmm and Cmm-specific lytic bacteriophages were isolated from rhizosphere soil and phyllospheres of diseased tomato plants, and the biocontrol potential of bacteriophages was investigated.ResultsEight isolates that showed typical symptoms of Cmm infection in biochemical and pathogenicity tests contained the virulence gene pat-1 in the genome. Twenty-two native bacteriophages were isolated from 90 samples. As a result of the host range tests performed on the local Cmm isolates obtained in the study, temperature and pH tolerance tests were performed with ten phages with a wide host range. Phage24, Phage33 and Phage41, which have higher temperature and pH tolerance than other phages, were used to kill curve assay. Although the combined applications of phages were performed to increase efficacy, it was found that the application of Phage33 alone was more effective.ConclusionPhage33 may prove to be a suitable agent to be used as part of the microbial control strategy of Cmm. In further studies, Phage33 should be formulated to protect it from adverse environmental conditions and tested under field conditions to confirm the results obtained in this study.

Stephania suberosa Forman extract synergistically inhibits ampicillin- and vancomycin-resistant Enterococcus faecium

Increasing antibiotic resistance in enterococci is among the most serious public health problems worldwide. The new naturally occurring antibacterial agents were explored. This study, therefore, investigated the antibacterial potential of Stephania suberosa extract (SSE) and its synergism with ampicillin (AMP) or vancomycin (VAN) against AMP- and VAN-resistant Enterococcus faecium. Disc diffusion assay revealed that SSE inhibited E. faecium DMST 12829, 12852, 12970, and a reference strain of Enterococcus faecalis ATCC 29,212 in a dose-dependent manner. The minimum inhibitory concentration (MIC) of SSE against all E. faecium isolates was 0.5 mg/mL. E. faecium DMST 12,829 and 12,852 were highly resistant to AMP, as indicated by high MIC values, and E. faecium DMST 12,829 and 12,970 were resistant to VAN. Enterococcus spp. were killed by SSE at the minimum bactericidal concentrations (MBCs) ranging from 0.5 to 4 mg/mL. Checkerboard determination showed that SSE plus AMP and SSE plus VAN combinations exhibited synergistic interaction against E. faecium isolates. The killing curve assay of E. faecium isolates confirmed the antibacterial and synergistic activities of combined agents by dramatically reducing the viable counts compared to a single agent. Scanning electron microscope elucidated the cell damage and abnormal cell division. Enterococcal proteases were also inhibited by SSE. These findings support that SSE could reverse the activity of AMP and VAN. Moreover, it can synergistically inhibit AMP- and VAN-resistant E. faecium. Our combined agents could be attractive candidates for developing new combinatorial agents to resurrect the efficacy of antibiotics for treating AMP- and VAN-resistant E. faecium infections.

Molecular docking and proteomics reveals the synergistic antibacterial mechanism of theaflavin with β-lactam antibiotics against MRSA.

Recurrent epidemics of methicillin-resistant Staphylococcus aureus (S. aureus) (MRSA) have illustrated that the effectiveness of antibiotics in clinical application is rapidly fading. A feasible approach is to combine natural products with existing antibiotics to achieve an antibacterial effect. In this molecular docking study, we found that theaflavin (TF) preferentially binds the allosteric site of penicillin-binding protein 2a (PBP2a), inducing the PBP2a active site to open, which is convenient for β-lactam antibiotics to treat MRSA infection, instead of directly exerting antibacterial activity at the active site. Subsequent TMT-labeled proteomics analysis showed that TF treatment did not significantly change the landscape of the S. aureus USA300 proteome. Checkerboard dilution tests and kill curve assays were performed to validate the synergistic effect of TF and ceftiofur, and the fractional inhibitory concentration index (FICI) was 0.1875. The antibacterial effect of TF combined with ceftiofur was better than that of single-drug treatment in vitro. In addition, TF effectively enhanced the activity of ceftiofur in a mouse model of MRSA-induced pneumonia. Our findings provide a potential therapeutic strategy to combine existing antibiotics with natural products to resolve the prevalent infections of multidrug-resistant pathogens.

Antibiotics targeting bacterial protein synthesis reduce the lytic activity of bacteriophages

Combination therapy of bacteriophages and antibiotics requires careful selection of specific antibiotics as it is crucial towards determining the success of phage therapy to treat multiple drug-resistant bacterial infections. So, we examined how different antibiotics can affect phage lytic activity when used in combination against targeted bacteria. Various antibiotics targeting bacterial protein synthesis pathways were tested for their bactericidal action in combination with bacteriophages of Acinetobacter baumannii (φAB145, φAB182), Staphylococcus aureus (φSA115, φSA116) and Salmonella Typhimurium (φST143, φST188). The phages displayed highly significant antagonism with most of the protein/ribosomal machinery targeting antibiotics: φSA115 (13/13); φSA116 (13/13); φST143 (11/13); φAB145 (11/13); φST188 (9/13); φAB182 (7/13). To validate this antagonistic effect, synergy assessment of these phages with gentamicin (GEN) and tetracycline (TE) was performed using time kill curve assays and counting the remaining viable bacterial cells at the end of the experiment. An increase in bacterial turbidity in phage-antibiotic combination groups was observed as compared to the treatment with phages individually. Also, GEN exhibited 4.22, 5.90, 2.02, 3.15, 2.68, and 2.60 log proliferation in viable cell count, respectively, for φSA115, φSA116, φST145, φAB182, φST143 and φAB188 in combination group in comparison to their individual actions. TE supplementation also led to 2.40, 4.90, 1.61, 2.73, 3.93, and 1.81 log increments in viable bacterial count when combined with φSA115, φSA116, φST145, φAB182, φST143 and φAB188, respectively. This study concludes that antibiotics targeting the bacterial protein biosynthetic machinery may lead to a reduction in the lytic activity of bacteriophages, thus lowering their therapeutic potential. Hence, such compounds must be carefully screened before their employment in combination treatment regimens.

Metformin Reverses tmexCD1-toprJ1- and tet(A)-Mediated High-Level Tigecycline Resistance in K. pneumoniae.

Tigecycline (TIG) is one of the last effective options against multidrug resistance bacteria. Recently, the RND (resistance–nodulation–division) efflux pump gene cluster, tmexCD1-toprJ1, and the tetracycline-efflux pump tet(A) mutation were reported to mediate high level resistance to TIG in clinically important pathogens, weakening the efficacy of TIG. In this study, we report the potent synergistic effect of the antidiabetic drug metformin in combination with TIG against tet(A) mutant and tmexCD1-toprJ1 positive K. pneumoniae. The fractional inhibitory concentration index (FICI) of TIG and metformin were less than 0.05 for all the tested isolates. The time–kill curve assay showed that the combination of TIG and metformin exhibited much better antimicrobial effect than TIG alone. The synergistic effect was also confirmed in vivo using a well-studied Galleria mellonella larvae model. Mechanistic studies demonstrated that metformin disrupted the important component of proton motive force, the electric potential (Δψ) and the function of efflux pump, thereby increasing the intracellular concentration of TIG. This finding revealed that metformin might be a possible adjuvant of TIG for combating with superbugs carrying the tet(A) mutant and tmexCD1-toprJ1 genes.

Development of Oxadiazole-Sulfonamide-Based Compounds as Potential Antibacterial Agents.

In this work, substituted 1,2,4-oxadiazoles (OX1–OX27) were screened against five bacterial strains, identified to be OX7 and OX11 as growth inhibitors with minimum inhibitory concentration (MIC) values of 31.25 and 15.75 μg/mL, respectively. The growth inhibitory property of OX7 and OX11 was further validated by disk diffusion, growth curve, and time kill curve assays. Both disrupted biofilm formation with 92–100% reduction examined by the XTT assay were further visualized by scanning electron microscopy analysis. These compounds in combination with ciprofloxacin also exhibit synergy against Escherichia coli cells. With insignificant cytotoxic behavior on HEK293 cells, human red blood cells, and Galleria mellonella larvae, OX11 was tested against 28 multidrug resistant environmental isolates of bacteria and showed inhibition of Kluyvera georgiana and Citrobacter werkmanii strains with 32 and 16 μg/mL MIC values, respectively. The synergistic behavior of OX11 with ampicillin showed many fold reductions in MIC values against K. georgiana and Klebsiella pneumoniae multidrug resistant strains. Further, transmission electron microscopy analysis of OX11-treated E. coli cells showed a significantly damaged cell wall, which resulted in the loss of integrity and cytosolic oozing. OX11 showed significant changes in the secondary structure of human serum albumin (HSA) in the presence of OX11, enhancing HSA stability. Overall, the study provided a suitable core for further synthetic alterations and development as an antibacterial agent.

Metal Sulfide Nanoparticles Based Phytolectin Scaffolds Inhibit Vulvovaginal Candidiasis Causing Candida albicans

Vulvovaginal candidiasis (VVC) is a most common infection caused by Candida species especially, Candida albicans. Herein, we report that phytoprotein based nano scaffolds mitigates the standard antifungal drug (fluconazole) resistant C. albicans. Copper sulfide nanoparticles (CuS NPs) and the phytolectin, jacalin functionalized copper sulfide nanoparticles (JCuS NPs) were evaluated against C. albicans cells. The minimum inhibitory concentration of JCuS NPs (0.1 µM) was tenfold lower than CuS NPs (1 µM). Kill curve assay showed that the NPs exhibit anticandidal activity in a time dependent manner. Standard antifungal drugs like Amphotericin B (AMP B) and Micafungin (MF) were evaluated individually against VVC causing Candida sp. and also combined with NPs to determine their synergistic effect. Interestingly, MF which belongs to echinocandins group of antifungals exhibhited synergistic activity and AMP B which belongs to polyenes group displayed an additive effect. Molecular level studies revealed that JCuS NPs in combination with micafungin inhibits the drug resistant efflux pump of C. albicans by down-regulating the efflux genes. The results revealed that JCuS NPs and its combination with standard drugs have a better anti-candidal effect, suggesting that the lectins has the potential to serve as a drug carrier by targeting the cell surface glycans of Candida.

Antibacterial and Antibiofilm Activities of Novel Antimicrobial Peptides against Multidrug-Resistant Enterotoxigenic Escherichia Coli

Post-weaning diarrhea due to enterotoxigenic Escherichia coli (ETEC) is a common disease of piglets and causes great economic loss for the swine industry. Over the past few decades, decreasing effectiveness of conventional antibiotics has caused serious problems because of the growing emergence of multidrug-resistant (MDR) pathogens. Various studies have indicated that antimicrobial peptides (AMPs) have potential to serve as an alternative to antibiotics owing to rapid killing action and highly selective toxicity. Our previous studies have shown that AMP GW-Q4 and its derivatives possess effective antibacterial activities against the Gram-negative bacteria. Hence, in the current study, we evaluated the antibacterial efficacy of GW-Q4 and its derivatives against MDR ETEC and their minimal inhibition concentration (MIC) values were determined to be around 2~32 μg/mL. Among them, AMP Q4-15a-1 with the second lowest MIC (4 μg/mL) and the highest minimal hemolysis concentration (MHC, 256 μg/mL), thus showing the greatest selectivity (MHC/MIC = 64) was selected for further investigations. Moreover, Q4-15a-1 showed dose-dependent bactericidal activity against MDR ETEC in time–kill curve assays. According to the cellular localization and membrane integrity analyses using confocal microscopy, Q4-15a-1 can rapidly interact with the bacterial surface, disrupt the membrane and enter cytosol in less than 30 min. Minimum biofilm eradication concentration (MBEC) of Q4-15a-1 is 4× MIC (16 μg/mL), indicating that Q4-15a-1 is effective against MDR ETEC biofilm. Besides, we established an MDR ETEC infection model with intestinal porcine epithelial cell-1 (IPEC-1). In this infection model, 32 μg/mL Q4-15a-1 can completely inhibit ETEC adhesion onto IPEC-1. Overall, these results suggested that Q4-15a-1 may be a promising antibacterial candidate for treatment of weaned piglets infected by MDR ETEC.

Trans-Cinnamaldehyde Exhibits Synergy with Conventional Antibiotic against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen worldwide and has acquired multiple resistance to a wide range of antibiotics. Hence, there is a pressing need to explore novel strategies to overcome the increase in antimicrobial resistance. The present study aims to investigate the efficacy and mechanism of plant-derived antimicrobials, trans-cinnamaldehyde (TCA) in decreasing MRSA’s resistance to eight conventional antibiotics. A checkerboard dilution test and time–kill curve assay are used to determine the synergistic effects of TCA combined with the antibiotics. The results indicated that TCA increased the antibacterial activity of the antibiotics by 2-16-fold. To study the mechanism of the synergism, we analyzed the mecA transcription gene and the penicillin-binding protein 2a level of MRSA treated with TCA by quantitative RT-PCR or Western blot assay. The gene transcription and the protein level were significantly inhibited. Additionally, it was verified that TCA can significantly inhibit the biofilm, which is highly resistant to antibiotics. The expression of the biofilm regulatory gene hld of MRSA after TCA treatment was also significantly downregulated. These findings suggest that TCA maybe is an exceptionally potent modulator of antibiotics.

Antibiofilm and synergistic antibacterial effect of Garlic (Allium sativum) against Methicillin resistant Staphylococcus aureus (MRSA)

Background: Methicillin resistant Staphylococcus aureus (MRSA) is one of main bacterial pathogens that is associated with difficult to treat nosocomial and community infections with high mortality. Shiga toxin producing E. Coli (STEC) infections result in bloody diarrhea, vomiting and mild fever. Biofilm formation has been linked with pathogenicity and virulence of these bacterial pathogens. Garlic (Allium sativum) has been traditionally used as an antibacterial agent in India and South East Asia. We have demonstrated that garlic exhibits antibiofilm and antibacterial activity against drug resistant STEC clinical isolates. In addition, organosulphide compounds of garlic have been reported to exhibit antibacterial activity against MRSA. In view of these observations, our laboratory wanted to investigate if crude garlic extract has any antibiofilm activity against MRSA. Further, we wanted to test if for synergistic effect of garlic when used in combination with antibiotics. Methods and materials: The study was performed with three MRSA isolates. Standard disc diffusion, serial dilution and kill curve assays were performed to access the antibacterial and synergistic activity of garlic against MRSA. In vitro microtiter plate assay was performed to quantify the formation of biofilm in the presence and absence of garlic crude extract. In addition, the effect of garlic on biofilm formation was also evaluated using scanning electron microscopy (SEM). Results: The results indicated that crude garlic extract exhibited significant dose dependent antibacterial activity against MRSA and the activity was much better in terms of clarity of zones of inhibition compared to pure organosulphide compounds found in garlic. It was interesting to note that sub-MIC concentrations of garlic were able to sensitize MRSA to methicillin. Garlic at sub-MIC concentrations reduced MRSA biofilm formation by more than 60%. We conclude that crude garlic extract has more antibacterial activity than its active ingredients. This is the first study to report its antibiofilm property against MRSA to best of our knowledge. Conclusion: Garlic organosulphide compounds have the potential to be used in combination with standard antibiotics to treat drug resistant bacterial infections. Garlic organosulphide compounds based novel antibiotic and antibiofilm drugs can be developed to tackle the problem of antibacterial drug resistance.

The Mechanism of Bisdemethoxycurcumin Enhances Conventional Antibiotics against Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infection has posed a serious threat to public health, therefore, the development of new antibacterial drugs is imperative. Bisdemethoxycurcumin (BDMC) is a curcumin analog that exists in nature and possesses extensive pharmacological actions. This review focuses on investigating the antibacterial activity of BDMC alone or in combination with three antibiotics against MRSA. We determined the minimal inhibitory concentration of BDMC, with a broth microdilution assay, and the value against all six strains was 7.8 μg/mL. The synergistic effect of BDMC combined with the antibiotics was determined using a checkerboard dilution test and a time–kill curve assay. The results showed that the antimicrobial effect of BDMC combined with antibiotics was superior to treatment with that of a single agent alone. We examined the antibacterial activity of BDMC in the presence of a membrane-permeabilizing agent and an ATPase-inhibiting agent, respectively. In addition, we analyzed the mecA transcription gene and the penicillin-binding protein 2a (PBP2a) level of MRSA treated with BDMC by quantitative RT-PCR or Western blot assay. The gene transcription and the protein level were significantly inhibited. This study demonstrated that BDMC has potent antibacterial activity, and proved that BDMC may be a potential natural modulator of antibiotics.