Methods for in vitro evaluating antimicrobial activity: A review

In modern times, microbial resistance is a global threat to health and development. The misuse and inappropriate use of antimicrobials is the main cause of developing drug-resistant pathogens. It requires multiple areas in direction to attain sustainable advance goals. As a result of microbial resistance, the necessity for costly medications and expenses are obstructed worldwide. Due to rising attention in the research of new antimicrobial medicaments from a variety of natural or synthetic sources to fight microbial resistance. Thus, natural antimicrobial agents have been used to a great extent nowadays because plant-derived antimicrobial agents are considered to be safer alternatives for health as compared to those synthetic antimicrobial agents. Overall, the active ingredients, water, essential oils, and ethanolic extracts from selected plants and the mixture of a variety of these natural extracts have been used for centuries, because they possess antimicrobial activity which inhibits the growth of microbes. Natural plants as an antimicrobial agent, like extracts of <i>Curcuma longa L., Piper nigrum L., Vachellia leucophloea, Eclipta prostrata, Ocimum sanctum L., Terminalia arjuna, Manihot esculenta Crantz, Lawsonia inermis L., Zingiber officinale Roscoe, Camellia sinensis</i> (L.) Kuntze, <i>Coriandrum sativum</i> L., <i>Carica papaya</i> L., Cinnamomum tamala, and many others have been preferred and used for ages because they are easily available worldwide. They are usually of low cost and have little or no side effects. Several antimicrobial screening approaches like the disk-diffusion method, well diffusion method, micro broth dilution assay, sterile disk method, and agar diffusion method are generally cast off for measurement of reproducibility and standardization of these antimicrobial agents. This review article is a comprehensive description of natural plants like Coriandrum sativum L., Carica papaya L., Cinnamomum tamala, etc., containing those extracts used as antimicrobial agents listed, and numerous in vitro antimicrobial susceptibility testing methods are reported. These identified plant species and antimicrobial screening techniques hold the potential for formulating these plants into antimicrobial drugs, warranting further study and exploration in the field of medicine.

The revival of fosfomycin

There is a need for natural preservatives in cosmetic, food and pharmaceutcal industries especially at a time when the safety and acceptability of currently used synthetic antimicrobial agents and antioxidants are being re-visited. The aim of this study was to evaluate the antimicrobial and antioxidative activities of the essential oils of Tagetes minuta, Lippia javanica and Foeniculum vulgare wildly growing or grown in the Eastern Cape, South Africa. The antibacterial and antifungal activities of the oils were determined using the agar diffusion and dilution assays while the antioxidative activity was evaluated using the ß-carotene diffusion assay. All the essential oils tested exhibited strong antimicrobial activities against the common test organisms. However, the antimicrobial activity differed and depended on the type and concentration of the oil. Foeniculum vulgare and T. minuta oils also displayed remarkable antioxidative activities in addition to their antimicrobial activity. The latter oils could be considered as potential sources of raw materials for the development of natural antimicrobial and antioxidative agents.

Bacteria and fungi are among the most ancient creatures found on Earth. Since the advent of medicine, humankind has always sought new means and ways to combat these microorganisms. In modern scientific society, the tendency to seek new antimicrobial and antifungal agents is only increasing. 1,2,4-triazole derivatives, among which effective drugs and new molecules have already been found, make quite an interesting platform for the creation of new antifungal and antimicrobial agents. A promising direction for the search for antimicrobial and antifungal agents are 2-((5-(2-bromophenyl)-4-substituted-4 H -1,2,4-triazol-3-yl)thio)acetates. The aim of work was the investigation of antimicrobial and antifungal activity among new 2-((5-(2-bromophenyl)-4-substituted-4 H -1,2,4-triazol-3-yl)thio)acetates. Materials and methods. The substances were synthesized at the Department of Natural Sciences for International Students and Toxicological Chemistry. The antimicrobial and antifungal activity of the newly synthesized 2-((5-(2-bromophenyl)-4-substituted-4 H -1,2,4-triazol-3-yl)thio)acetates was studied with the method of serial dilutions. Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, and Candida albicans ATCC 885–653 were used as a set of standard test strains. Results. As a result, the antimicrobial and antifungal activity of 14 new compounds was investigated. The most active compounds with antifungal effect are IIf–IIh. Substances IIf–IIh and IIj, IIk have moderate antimicrobial effect to P. aeruginosae. Conclusions. Some results are obtained regarding “structure – antimicrobial and antifungal effect” dependence: the replacement of the ethyl radical with phenyl or methyl in the fourth position of the 1,2,4-triazole cycle in the 2-((5-(2-bromophenyl)-4-R-4 H -1,2,4-triazol-3-yl)thio)acetate acid molecule results in reduction of the antimicrobial effect; сonversion to 2-((5-(2-bromophenyl)-4-substituted-4 H -1,2,4-triazol-3-yl)thio)acetate salts and the choice of dimethylammonium as cation leads to an increase in the antimicrobial and antifungal effect.

Presently, the effective antimicrobial agents have been limited by the emergence of microbial strains with multidrug resistance and biofilm formation potential. In the present study, we report remarkable antimicrobial activity of silver nanoparticles (AgNPs) synthesized from Streptomyces calidiresistens IF11 and IF17 strains, including inhibition of biofilm formation and synergistic effect of AgNPs and antibiotics against selected bacteria and yeasts. Cytotoxic effect of AgNPs on mammalian cell lines was also evaluated. Analysis of biosynthesized AgNPs by Fourier Transform Infrared Spectroscopy and transmission electron microscopy revealed their spherical shape, small size in the range of 5-50 and 5-20nm, respectively, as well as the presence of capping agents. Study of antimicrobial activity of AgNPs against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Candida albicans and Malassezia furfur evaluated by minimum inhibitory concentration (MIC) and minimum biocidal concentration (MBC) assays revealed that MICs of AgNPs from IF11 and IF17 strains against bacteria and yeasts were found to be in the range of 16-128 and 8-256μgml-1 , while MBCs were in the range of 48-192 and 32-256μgml-1 respectively. AgNPs inhibited biofilm formation of microbial strains, which was tested by using crystal violet stain. The highest synergistic effect determined by fractional inhibitory index of AgNPs with antibiotic (kanamycin or tetracycline) was found against Staph. aureus; while in case of yeasts, M. furfur showed highest sensitivity to AgNPs-ketoconazole combination (FIC=0·12). The cytotoxic activity of AgNPs towards HeLa and 3T3 cell lines was studied by MTT assay. The IC50 of AgNPs estimated against mouse fibroblasts was found to be 8·3 and 28·3μgml-1 and, against HeLa cell line, 28·5 and 53·8μgml-1 respectively. It can be concluded that AgNPs synthesized from S. calidiresistens IF11 and IF17 strains have potential as an effective antimicrobial and cytotoxic agent, especially when used in combination with antibiotics/antifungal agents. This study indicates potential application of biogenic silver nanoparticles as an antimicrobial agent in nanomedicine.

Water hyacinth (Eichhornia crassipes) is an oceanic macrophyte and one of the worst sea-going weeds in the world and Egypt. The grouping of substantial metals in water hyacinth was higher in roots than in shoots. Nutrient content e.g. ammonia, nitrate, nitrite and phosphate was higher in the shoots of water hyacinth. Water hyacinth is a wellspring of phytochemicals which have a medicinal function. In this study, the high degree of peroxidation and nonenzymatic antioxidant systems were detected in leaf extracts. The chemical analysis of the leaf extracts was performed using acetone, ethanol, methanol and n-Butyl alcohol. The fractionated extracts showed the presence of some medicine compounds that include phenols, protein, alkaloids, amino acids, carbohydrate, flavonoids, glycosides, tannins, and terpenoids. The fractionated extracts were screened for antimicrobial and antifungal activities. The leaf extracts of E. crassipes exhibited antimicrobial activities against some bacteria: Escherichia coli, Bacillus subtilis, Bacillus cereus, Lactobacillus casei and Pseudomonas aeruginosa, and antifungal activity against six pathogenic fungi i.e. Aspergillus flavus, A. niger, Alternaria alternata, Colletotrichum gloeosporioides, Candida albicans, and Fusarium solani by using the well diffusion method. Streptomycin and Fluconazole were used as a standard as antimicrobial and antifungal agents, respectively. B. subtilis, F. solani, C. gloeosporioides and A. alternata were the most susceptible to plant extracts. The most effective antifungal and antimicrobial agent against some plant pathogens was the n-Butyl alcohol extract. Also, it reduced the production of mycotoxin e.g. Aflatoxin B1 and Ochratoxin A. The application of the formulation of the plant extract of E. crassipes protected wheat plants against net blotch diseases. The results indicated that the leaf extractsof E. crassipes have potential as biofungicides and can be used in the industry.

The global rise in bacterial resistance poses a significant challenge, exacerbated by the overuse of antibacterial agents. Schiff base-metal complexes have gained attention for their potential antimicrobial properties, attributed to their unique three-dimensional structures and modes of action, such as cell wall inhibition and membrane disruption. The aim of this study was to synthesize and characterize a novel Schiff base (L) derived from sulfamethazine and benzoin, and to develop metal complexes of it with Cu2+, Co2+, Ni2+, and Cd2+. This research sought to evaluate the antibacterial and antifungal activities of the synthesized Schiff base and its metal complexes to explore their potential as effective antimicrobial agents. The compound L and its metal complexes were synthesized. The compounds were characterized with 1H NMR, 13C NMR, mass spectrometry, UV-vis spectroscopy, and FTIR. Antimicrobial activities were tested on E. coli ATCC 8739, S. aureus ATCC 6538, P. aeruginosa ATCC 9027, and C. albicans ATCC 10231. To investigate the molecular properties of compound L, density functional theory calculations were also performed. The synthesized compounds (L, 1, 2, 3, 4) were tested for antibacterial and antifungal activities. Copper-based compound 1 showed the best overall antifungal activity, while zinc-based compound 3 demonstrated notable antibacterial efficacy against P. aeruginosa. Although all compounds outperformed L in antifungal tests, none surpassed ciprofloxacin, the reference drug, in antibacterial assays. These results highlight the potential of Schiff base-metal complexes as promising antimicrobial agents. This study highlights the potential of Schiff base-metal complexes as effective antimicrobial agents in response to the growing challenge of bacterial resistance. The synthesized Schiff base (L) and metal complexes of it with Cu2+, Co2+, Ni2+, and Cd2+ exhibited promising antibacterial and antifungal activities, with copper-based compound 1 showing the most potent antifungal action and zinc-based compound 3 demonstrating significant antibacterial efficacy against P. aeruginosa. Although none of the compounds outperformed ciprofloxacin in antibacterial assays, all were more effective than the parent Schiff base in antifungal tests. These findings suggest that Schiff base-metal complexes, particularly copper and zinc derivatives, hold considerable promise for developing new antimicrobial agents. Further optimization and testing could enhance their clinical application in combating resistant infections.

IFST Winning Articles

Background/Objectives: The emergence of antimicrobial resistance necessitates the development of new and effective antimicrobial agents. In this study, three different series of imidazole-based chalcones (IBC1-25) were designed and synthesised using a sustainable approach, with the aim of identifying compounds with enhanced antimicrobial activity. Methods: A series of monoalkoxy, dialkoxy, and trialkoxy imidazole-based chalcones (IBC1-25) were synthesised and evaluated for their antimicrobial and antifungal activities against a range of microbial strains. Structure-activity relationships were analysed, and molecular docking studies were performed to investigate potential binding interactions with biofilm-associated regulatory proteins. In addition, ADME properties were predicted to assess drug-likeness. Results: Among the monoalkoxy derivatives (IBC1-14), IBC5 exhibited the broadest spectrum of activity, particularly against S. epidermidis. Several dialkoxy analogues (IBC17-21) demonstrated improved potency, with IBC20 showing notably high activity. While IBC22 and IBC25 were largely ineffective, IBC23 and IBC24 displayed significant antibacterial and antifungal activities. Overall, dialkoxy and trialkoxy derivatives exhibited enhanced efficacy, whereas monoalkoxy compounds with bulky or long-chain substituents were generally less active. The presence of multiple alkoxy substituents, such as methoxy and ethoxy groups, on the phenyl ring significantly improved activity, particularly against fungi and Gram-positive bacteria. Molecular docking studies revealed that IBC20 and IBC23 showed favourable binding to the biofilm-associated regulator TcaR, suggesting a potential allosteric inhibition mechanism, while weak interactions were observed with TagF. ADME predictions indicated good oral absorption and compliance with key drug-likeness criteria. Conclusions: The results demonstrate that both the number and type of alkoxy substituents play a critical role in antimicrobial activity. In particular, IBC20 and IBC23 emerge as promising candidates for further development as antimicrobial agents targeting biofilm-associated pathways.

Natural oils, particularly essential and fixed oils derived from plants, have garnered increasing interest as effective antimicrobial agents and natural preservatives across the food, pharmaceutical, and cosmetic industries. This review explores the chemical composition, antimicrobial mechanisms, and practical applications of key natural oils such as tea tree, clove, oregano, lemongrass, neem, and coconut oil. These oils exhibit broad-spectrum activity against bacteria, fungi, and viruses due to their bioactive constituents like terpenoids, phenolics, and aldehydes. Their modes of action include disruption of microbial membranes, interference with enzymatic functions, inhibition of biofilm formation, and induction of oxidative stress. Natural oils serve as promising alternatives to synthetic preservatives, addressing consumer concerns over chemical toxicity and environmental impact. In food preservation, they effectively extend shelf life and inhibit spoilage microorganisms. In pharmaceuticals, they are utilized for their antibacterial, anti-inflammatory, and wound-healing properties. Despite their advantages, challenges such as variability in composition, potential toxicity, and regulatory compliance must be addressed to ensure safe and standardized use. This review emphasizes the need for continued research and regulatory oversight to harness the full potential of natural oils as sustainable and effective antimicrobial and preservative agents.

Antimicrobial resistance (AMR) is a significant concern to society as it threatens the effectiveness of antibiotics and leads to increased morbidity and mortality rates. Innovative approaches are urgently required to address this challenge. Among promising solutions, two dimensional (2-D) nanomaterials with layered crystal structures have emerged as potent antimicrobial agents owing to their unique physicochemical properties. This antimicrobial activity is largely attributed to their high surface area, which allows for efficient interaction with microbial cell membranes, leading to physical disruption or oxidative stress through the generation of reactive oxygen species (ROS). The latter mechanism is particularly noteworthy as it involves the degradation of these nanomaterials under specific conditions, releasing ROS that can effectively kill bacteria and other pathogens without harming human cells. This study explores the antimicrobial properties of a novel biodegradable nanomaterial based on 2-D transition metal trichalcogenides, FePS3, as a potential solution to drug-resistant microbes. Our findings indicate that FePS3 is an exceptionally effective antimicrobial agent with over 99.9% elimination of various bacterial strains. Crucially, it exhibits no cytotoxic effects on mammalian cells, underscoring the potential for safe biomedical application. The primary mechanism driving the antimicrobial efficacy of FePS3 is the release of ROS during biodegradation. ROS has a crucial role in neutralizing bacterial cells, conferring significant antipathogenic properties to this compound. The unique combination of high antimicrobial activity, biocompatibility, and biodegradability makes FePS3 a promising candidate for developing new antimicrobial strategies. This research contributes to the increasing body of evidence supporting the use of 2-D nanomaterials in addressing the global challenge of AMR, offering a potential pathway for the development of advanced, effective, and safe antimicrobial agents.

1. Cynthia B. Schofield, MPH, MT (CAMT)[⇑][1] 1. VA San Diego Healthcare System (retired), San Diego, CA 1. Address for Correspondence: Cynthia B. Schofield, MPH, MT (CAMT), VA San Diego Healthcare System (retired), 7050 Weller St., San Diego, CA, (858) 450-9651, cschofield{at}san.rr.com 1. Explain the physician's choice of antibiotic agents according to CLSI guidelines. 2. Define and discuss MIC breakpoints. 3. Compare qualitative versus quantitative antimicrobial susceptibility testing (AST) methods. 4. Contrast the various quantitative AST methods. 5. Discuss the broth dilution reference method. 6. Discuss genotypic methods and their purposes. The importance of antimicrobial susceptibility testing (AST) as a clinical laboratory function escalates as organism resistance to the available antimicrobial agents increases. As patient outcome is based on antimicrobial therapy, standardization of the AST methods determining therapy is essential. The Clinical and Laboratory Standards Institute (CLSI) in the US and the European Committee on Antimicrobial Testing (EUCAST) in Europe determine these standards that are updated annually. Of concern to physicians are questions regarding the establishment of breakpoints, which are the interpretive cut-off values of minimal inhibitory concentration (MIC) in quantitative testing and inhibition zones for antimicrobial agents in qualitative testing based on geographical surveys of clinical isolates. The CLSI performance standards in the US and the EUCAST in Europe, the groups responsible for developing the breakpoints, may differ in perception and usage.1,2 Interpretation of qualitative results as susceptible (S), intermediate (I) or resistant (R) and quantitative measures (MICs) of antimicrobial activity are standardized by CLSI in the U.S. with breakpoint guidelines updated annually for each antibiotic tested.1,2,4 Antimicrobial Agents The clinical laboratory's list of antimicrobial agents is chosen by the infectious disease specialists and pharmacologists at the individual institution according to the institution's unique guidelines. A formulary of agents that physicians regularly prescribe is derived from the susceptibility testing of organisms typically isolated at each clinical laboratory and is guided by a monthly updated antibiogram. The number of agents tested is designated by the AST method used, e.g. 12 disks per 150 mm Mueller-Hinton agar plate or similar number per… ABBREVIATIONS: AST-Antimicrobial Susceptibility Testing; CLSI-Clinical and Laboratory Standards Institute; EUCAST-European Committee on Antimicrobial Susceptibility Testing; FDA-Food and Drug Administration; MIC-minimal inhibitory concentration; MH-Mueller-Hinton; CAMHB-cation-adjusted Mueller Hinton broth; ATCC-American Type Culture Collection; MRSA-methicillin-resistant Staphylococcus aureus ; VRE-vancomycin-resistant Enterococcus ; S, I, R-sensitive, intermediate, resistant; PCR-polymerase chain reaction; DNA-deoxyribonucleic acid; RT-reverse transcriptase; CAI-community-associated infection; HAI-hospital-associated infection; MSSA-methicillin-sensitive Staphylococcus sp.; PBP-penicillin-binding proteins; ESBL-extended-spectrum beta lactamase; KPC- Klebsiella pneumoniae , carbapenem-resistant; CFU-colony forming units; TSB-trypticase soy broth; CSF-cerebrospinal fluid; ESBL-extended-spectrum beta lactamase 1. Explain the physician's choice of antibiotic agents according to CLSI guidelines. 2. Define and discuss MIC breakpoints. 3. Compare qualitative versus quantitative antimicrobial susceptibility testing (AST) methods. 4. Contrast the various quantitative AST methods. 5. Discuss the broth dilution reference method. 6. Discuss genotypic methods and their purposes. [1]: #corresp-1

Extracts of Cardiospermum gradiflorum and Cardiospermum halicacabumwere screened for antimicrobial activities against Candida albicans, Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Streptococcus viridans using the agar diffusion method. The two plants produced antimicrobial activities against all the test organisms. C. grandiflorum exhibited higher antimicrobial activity than C. Halicacabum. S.aureus was the most resistant to the extract of C. Gradiflorum among the organisms assayed while E. coli was the most resistant to the leaf extract of C. halicaca. The mimumum inhibitory concentration of the extracts ranged between 35mg/ml and 50mg/ml. Cardiospermum Gradiflorum could be a more effective antimicrobial agent than Cardiospermum halicaca.. Keywords: Extracts, antimicrobial activities, agar diffusion, resistant Nigerian Journal of Health and Biomedical Science Vol. 6 (1) 2007: pp.31-24

Evolutionary and in silico guided development of novel peptide analogues for antibacterial activity against ESKAPE pathogens

The growing incidences of drug-resistant pathogens have increased the attention on several medicinal plants and their metabolites for antimicrobial properties. These pathogens are the main cause of nosocomial infections which led to an increasing mortality among hospitalized patients. Taking into consideration those factors, this paper reviews the state-of-the-art of the research on antibacterial agents from native Brazilian plant species related to nosocomial infections as well as the current methods used in the investigations of the antimicrobial activity and points out the differences in techniques employed by the authors. The antimicrobial assays most frequently used were broth microdilution, agar diffusion, agar dilution and bioautography. The broth microdilution method should be the method of choice for testing new antimicrobial agents from plant extracts or isolated compounds due to its advantages. At the moment, only a small part of the rich Brazilian flora has been investigated for antimicrobial activity, mostly with unfractionated extracts presenting a weak or moderate antibacterial activity. The combination of crude extract with conventional antibiotics represents a largely unexploited new form of chemotherapy with novel and multiple mechanisms of action that can overcome microbial resistance that needs to be further investigated. The antibacterial activity of essential oil vapours might also be an interesting alternative treatment of hospital environment due to their ability in preventing biofilm formation. However, in both alternatives more studies should be done on their mode of action and toxicological effects in order to optimize their use.