Search for new antimicrobials against extended spectrum β-lactamase-positive Escherichia coli: Potential of fungi from the phylum Ascomycota

Abstract

Pathogenic strains of Escherichia coli can cause gastrointestinal infections, urinary tract infections (UTIs), bacteremia, and other severe infections. Some isolates of this species are capable of producing extended-spectrum β-lactamase (ESBL) enzymes, which mediate resistance against penicillin derivates and cephalosporins. Fungi of the Ascomycota phylum are known to produce antibiotics from different classes with activity against various bacterial agents. Among them, the genera Penicillium, Cephalosporium, Acremonium and Fusidium are known for the production of antimicrobial substances such as penicillin derivates, cephalosporins and fusidic acid. Currently, the search for new antimicrobials produced by species of the Ascomycota phylum includes the assessment of less explored habitats including aquatic environments, extreme environments, and the interior of plants/animals. The genus Penicillium remains promising for the discovery of new antimicrobial substances against resistant bacteria. In addition, those fungi have also been investigated regarding their usefulness for the biosynthesis of nanoparticles with antimicrobial activity. This narrative review introduces clinically relevant Escherichia coli pathovars, the historical contributions of the phylum Ascomycota to the production of antimicrobials, aspects of bioprocesses in the production of antimicrobial metabolites and different approaches of research targeting new antimicrobials such as screenings for fungi in environments not yet studied and the green synthesis mediated by fungi with antimicrobial activity.

Keywords:
Escherichia coli; Resistance; Antimicrobial drug; Ascomycota; Drug development

INTRODUCTION

β-lactam resistance in Gram-negative bacteria is most frequently caused by β-lactamase enzymes. β-lactamases are bacterial enzymes that inactivate β-lactam antibiotics by hydrolysis breaking the β-lactam ring present in these antibiotics. This action causes resistance to β-lactam antibiotics like cephalosporins, penicillin derivates and monobactams, resulting in medically ineffective compounds (Di Lodovico et al., 2022). Since the first records in 1983, members of the Enterobacterales such as Escherichia coli have globally been shown to express potent new β-lactamases enzymes called “extended-spectrum β-lactamases” or “ESBL”. Globally, the impact of antimicrobial-resistant (AMR) bacterial infections is substantial, both in terms of health and economic costs. In the United States of America, there was a 50% increase in infections caused by ESBL-positive bacteria between 2012 and 2017, negatively affecting death rates and health care costs (CDC, 2019). Similarly, in South America, the prevalence of ESBL-producing E. coli infections has markedly risen, posing significant challenges to public health systems (Bastidas-Caldes et al., 2022). The CDC’s 2019 Antimicrobial Resistance Threats Report (CDC, 2019) indicated that dedicated prevention and infection control efforts had reduced deaths from antimicrobial-resistant infections by 18% overall and by nearly 30% in hospitals. However, the 2022 special report highlighted that much of this progress was lost due to the effects of the COVID-19 pandemic, emphasizing the need for more action to protect people from AMR. The 2022 Global Antimicrobial Resistance and Use Surveillance System (GLASS) 2022 report highlights alarming resistance rates among prevalent bacterial pathogens. Data from 76 countries showed median resistance rates of 42% for E. coli resistant to third-generation cephalosporins and 35% for Staphylococcus aureus resistant to methicillin, both of which pose significant public health concerns.

Increasing rates of bacterial resistance stresses the importance of the development and production of antimicrobials in order to control mortality. Antimicrobials can be synthesized through various methods, including chemical synthesis (synthetic), modification of existing compounds (semi-synthetic), and extraction from microorganisms (Hutchings, Truman, Wilkinson, 2019). The Ascomycota phylum, widely known for its medicinal properties, is a significant source of medicinal compounds, providing a variety of biologically active substances used in pharmaceuticals to combat various infectious diseases. Research on genera including Penicillium, Acremonium, Cephalosporium, and Fusidium has significantly contributed to the development of antimicrobials like penicillin derivates, cephalosporins, and fusidic acid. Fungus-borne substances can be effective against various bacteria, including many E. coli strains, both susceptible and ESBL-producing variants.

Recent studies have expanded the exploration of the Ascomycota phylum’s antimicrobial properties beyond soils and plants to include diverse environments like glaciers, wastewater, and lakes (Grossart et al., 2019). In aquatic environments, Ascomycota have been identified as promising sources for new molecules with bioactive properties (Samuel et al., 2018). Abiotic factors such as temperature, salinity and nutrient cycling can result in the selection of microorganisms that produce secondary metabolites with antimicrobial activity (Youssef et al., 2019). Further, Ascomycota have also shown capability of producing antimicrobial nanoparticles, emphasizing their role in facilitating eco-friendly nanoparticle synthesis for medical uses (Mayegowda et al. 2023). This review has been focused on the role of Ascomycota fungi in developing new antimicrobial agents, also addressing the increasingly important issue of antibiotic resistance, especially in Gram-negative bacteria like E. coli. It addresses the historical importance and current potential of Ascomycota in antibiotic discovery and innovative methods like green synthesis. This Aims at informing on their role in combating resistance, on their potential as a novel source of antibiotics, and at contributing to a better understanding of the fungi from the phylum Ascomycota. The methodology involved a systematic literature search across the databases PubMed, Scopus, and Web of Science. This search utilized a combination of keywords pertinent to our study’s focus: ‘Ascomycota,’ ‘antimicrobial resistance,’ and ‘novel antibiotics.’ Our search included articles published from 2010 to 2023, with a primary focus on recent research, significant developments, and relevant case studies in the field. Additionally, manual searches through reference lists of pertinent articles were conducted to ensure a comprehensive coverage of the topic. Keywords were meticulously chosen to capture the essence of Ascomycota’s potential in antibiotic discovery and the challenges in addressing antimicrobial resistance. While the review aimed to be inclusive, potential limitations included the exclusion of contents from non-English publications and “grey literature”, a decision which may have narrowed the breadth of our findings.

Antimicrobial Resistance: The Emergency of ESBL-Positive E. coli

Since the discovery and introduction of first antibiotics, evolutionary adaptation of both Gram-positive and Gram-negative bacteria has led to resistance patterns that render many commercially available antibiotics partially ineffective (Chen, Kumar, Wu,2023). Over-use of antibiotics in humans, animals, and agriculture has been identified as a key factor contributing to a significant increase in bacterial resistance.

Antibiotic resistance in pathogenic or opportunistically pathogenic bacteria is often associated with high rates of morbidity and mortality among infected individuals. Annually, about 700,000 deaths worldwide are attributed to antimicrobial resistance (AMR) in case of infections, with projections suggesting a rise to ten million deaths by 2050 unless significant global control measures are implemented World Health Organization (WHO). In 2019, the Global Research on Antimicrobial Resistance (GRAM) Project reported that 1.27 million deaths were directly caused by drug-resistant infections, contributing to a total of 4.95 million AMR-associated deaths. The Center for Disease Control and Prevention (CDC) indicated over 2.8 million annual infections and at least 35,000 deaths in the U.S. due to resistant bacteria. Similarly, the European Center for Disease Prevention and Control (ECDC) reported in 2018 on 33,000 annual deaths in Europe associated with antibiotic-resistant bacteria.

Worldwide, clonal lineages of E. coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa have consistently been associated with typical resistance genes. The global spread of resistant lineages of these species is primarily attributed to the dissemination of genes located on mobile genetic elements such as plasmids, integrons, and transposons (Singh et al., 2022). Combination of genes on mobile genetic elements, along with chromosomally encoded resistance genes, occasionally results in bacteria resistant to all major classes of available antimicrobials. For Gram-negative bacteria, resistance often involves the expression of extended-spectrum β-lactamases (ESBLs) in Enterobacterales, particularly in E. coli and Klebsiella species.

E. coli is globally among the most common Gram-negative bacterial pathogens or commensals isolated from human samples. While it typically occurs as a harmless colonizer of the gastrointestinal tract in humans and warm-blooded animals, E. coli can cause various clinical infections. It spreads easily through direct contact, fecal-oral routes, and contaminated water or food. E. coli’s phenotypic and genetic characteristics allow it to evolutionary adapt and survive in diverse environments, including soil and water (Geurtsen et al., 2022). The number of sequenced E. coli genomes available in public databases has exceeded 140,000 (Horesh et al., 2021). This extensive collection reflects the significant scientific efforts to understand the genetic diversity of this bacterial species, which is crucial for both medical research and public health monitoring. The most well-known pathotypes of E. coli are categorized into two major groups: gastroenteric and extraintestinal, each associated with specific types of infections (Figure 1).

FIGURE 1
Overview on E. coli groups, corresponding pathotypes, and associated diseases and symptoms caused by pathogenic E. coli strains. *Image created using BioRender.com.

After introducing ampicillin in 1960, resistance to β-lactam antibiotics emerged. The first β-lactamase, TEM-1, (Temoniera patient - Greece) was identified in E. coli, leading to widespread dissemination across various bacteria via genetic transfer (Datta, Kontomichalou, 1965). Similarly, SHV-1-like enzymes (Sulfhydryl Reagent Variable) evolved and spread. In the 1980s and 1990s, TEM-1 and SHV-1 were prevalent in hospitals (Pitout, 2012). The introduction of third-generation cephalosporins and heavy antibiotic use in healthcare led to the emergence of TEM-2 and SHV-2 variants, especially in Klebsiella spp. and E. coli, marking the rise of ESBL-producing isolates (Kliebe, 1985). These enzymes, presently including more than 150 TEM and 50 SHV types, can hydrolyze cephalosporins and monobactams, while other ESBL-type beta-lactamase variants like OXA, PER, VEB, and GES have also emerged (Bradford, 2001).

From the 2000s on, CTX-M-type β-lactamases, first identified in Munich, Germany, (“CTX” for “ceftriaxone”, “M” for “Munich”) have shown strong hydrolytic activity against 3rd generation cephalosporins, notably associated with E. coli ST131 strains (Bradford, 2001). These resistant strains significantly complicate the treatment of infections like bloodstream and urinary tract infections, especially in case of combined resistance against both cephalosporins and fluoroquinolones (Fong, 2023). Between 2000 and 2010, CTX-M variants like CTX-M-14 and CTX-M-15, discovered in the early 2000s, have achieved global prevalence (Bevan, Jones, Hawkey, 2017). CTX-M-type ESBLs, capable of inactivating multiple cephalosporin generations, pose a public health threat, especially in regions with limited access to newer drugs. TEM, SHV, and CTX-M ESBLs quantitatively dominated globally in recent years (Rana et al., 2022).

Currently, the SMART program (Study for Monitoring Antimicrobial Resistance Trends), which is active since 2022 at 192 sites in 54 countries, monitors ESBL-producing Enterobacterales, especially focusing on urinary and intra-abdominal infections. This global study primarily evaluates the in vitro susceptibility of clinical Gram-negative bacteria, with a keen interest in uropathogenic E. coli (UPEC). According to WHO (2019), hospital infections caused by ESBL-expressing bacteria significantly impact patient mortality (26.7%), especially in intensive care units (42.6%).

Over the recent years, the increase in ESBL-producing E. coli, especially in hospital environments, has been significant. These strains have demonstrated substantial resistance to first-choice antibiotics such as carbenicillin, ampicillin, azithromycin, piperacillin, cefuroxime, ceftriaxone, trimethoprim/sulfamethoxazole, ciprofloxacin, and ofloxacin. Accordingly, resistant E. coli strains, particularly those of the ST131 clone, pose substantial public health risks, complicating the treatment of infections such as urinary tract and bloodstream infections (Castanheira, Simner, Bradford, 2021). Recently, resistance has also emerged against nalidixic acid and other quinolones in UPEC strains (Bhat et al., 2022). Key virulence factors, including adhesion molecules, significantly contribute to the severity of associated infections by facilitating bacterial adherence and proliferation within host tissues (Di Lodovico et al., 2022).

Recent advancements in laboratory detection of ESBL-positive E. coli are crucial for tailoring patient treatment. Current methods, as recommended by Clinical and Laboratory Standards Institute (CLSI) interpretation guidelines, employ disk diffusion assays using cephalosporins with inhibitors, and automated systems like VITEK and MICROSCAN for rapid, standardized detection. E-TEST strips can aid phenotypic identification of ESBLs by assessing changes in antibiotic minimum inhibitory concentration (MIC), while microbroth dilution assays are less common in routine diagnostics. These preliminary tests lead to confirmatory testing with advanced susceptibility assays to ensure accurate diagnosis and treatment of resistant strains (Dash, Sahu, Paty, 2022).

The increasing resistance of E. coli strains has emerged as a significant public health concern. The evolution and spread of resistance mechanisms, particularly CTX-M-type ESBL enzymes, are also on the rise in healthcare environments. Understanding the intricacies of ESBL production and resistance in E. coli is critical for developing innovative therapeutic approaches and containment strategies for ESBL-producing enterobacteria. Consequently, this situation demands control and surveillance policies for resistant pathogens and a concerted effort to discover novel treatments and strategies. This approach is imperative to address the growing challenge of antimicrobial resistance in E. coli and to ensure effective management of infections caused by resistant isolates.

Search for antibiotics

The history of combating infectious diseases with natural substances dates back about 3,000 years, with ancient civilizations using molds, plants, and salts. The development of medicinal drugs, particularly since the 16th century through alchemy, marks a significant era in the history of antimicrobials, including antiseptics, disinfectants, anesthetics, and early antimicrobials. However, it was not until the 20th century that the first antibiotics with strong therapeutic action were developed for the treatment of infectious diseases caused by specific agents (Smith, 2014).

The discovery of penicillin by Alexander Fleming in 1928 revolutionized the treatment of bacterial infections. Fleming observed that Penicillium chrysogenum cultures inhibited Staphylococcus spp. growth. The compound’s purification began in 1941 (Chain and Florey, 1939) and was extensively used in World War II. From the 1940s onwards, natural and semi-synthetic antibiotics became clinical mainstays, including β-lactams like cephalosporins, monobactams, and carbapenems. In addition, β-lactams combined with β-lactamase inhibitors target penicillin-binding proteins (PBP’s), disrupting cell wall synthesis (Hutchings, Truman, Wilkinson, 2019). Some important examples of substances obtained from microorganisms can be found in Table I.

TABLE I
Chronological overview of significant therapeutic advances achieved through microbial production of antimicrobial substances over the years

Microorganism	Antimicrobial product	Spectrum of action as intended by the developers	Year
Penicillium Notatum/P. chrysogenum*	Penicillins	Bacterial infeccions	1928
Penicillium griseofulvum*	Griseofulvin	Skin	1939
Streptomyces griseus	Streptomycin	Tuberculosis/Brucellosis	1944
Bacillus subtilis/B. licheniformis	Bacitracin	Skin/mucosa	1945
Bacillus polymyxa	Polymyxin	UTI, meningitis	1947
Acremonium chrysogenum*	Cephalosporin	Bacterial infeccions	1948
Streptomyces fradiae	Neomycin	Skin/mucosa	1949
Streptomyces rimosus	Oxytetracycline	Eye infections	1950
Streptomyces noursei	Nystatin	Candidiasis	1950
Pleurotus mutilus	Pleuromutilin	Respiratory infections	1951
Streptomyces erythraeus	Erythromycin	Acute/chronic inflammation	1952
Streptomyces ambofaciens	Spiramycin	Respiratory infections	1954
Streptomyces nodosus	Amphotericin B	Meningitis	1955
Streptomyces aureofaciens	Chlortetracycline/Tetracycline	Bacterial infeccions	1955
Streptomyces lincolnensis	Lincomycin	Bacterial infeccions	1955
Streptomyces orientalis	Vancomycin	Bacterial infeccions	1956
Streptomyces kanamyceticus	Kanamycin	Ears/eyes	1957
Streptomyces mediterranei	Rifamycin	Skin/mucosa	1959
Cephalosporium acremonium*	Cephalosporin	Bacterial infeccions	1960
Fusidium coccineum*	Fusidic acid	Skin	1960
Micromonospora purpurea	Gentamicin	UTI, meningitis	1963
Chomobacterium violaceum	Aztreonam	Cystitis/bacteremias	1986

For decades, β-lactam antibiotics, distinguished by the chemical structure of their β-lactam ring, have been pivotal in the treatment of bacterial infections due to their high efficacy and low toxicity. These antibiotics specifically disrupt the synthesis of peptidoglycan in bacterial cell walls. A significant portion of natural β-lactam antimicrobials, about 64%, is produced by actinomycetes, predominantly by Streptomyces species, about 10-15% by bacteria and about 20% by fungi. Among those fungi, the genera Penicillium, Acremonium, and Cephalosporium are considered as mainly responsible for producing various β-lactam antibiotics, including different penicillin derivates and cephalosporins (Silber et al., 2016). The most important antibiotics produced by fungi from the phylum Ascomycota are represented in Figure 2.

FIGURE 2
Schematic representation of the action mechanisms of various antibiotics produced by genera within the phylum Ascomycota, highlighting their impact on ribosomal sites and the inhibition of peptidoglycan proteins in bacterial cell walls. *Image created using BioRender.com.

Penicillin derivates and cephalosporins contain a β-lactam ring, crucial for their antimicrobial efficacy, fused with either a five-membered (thiazolidine) or six-membered (dihydrothiazine) ring. The β-lactam ring’s stability, often compromised by nucleophilic attacks from molecules like zinc ions, water, or hydroxyl groups, is vital to maintain the antimicrobial activity. To enhance stability and counteract the ring’s inherent susceptibility to hydrolysis, modifications have been made, including the addition of electron-withdrawing groups on the side chain carbons. These modifications to 6-aminopenicillanic acid (6-APA) in penicillin derivates and 7-aminocephalosporanic acid (7-ACA) in cephalosporins have led to the development of various antibiotic classes. New radicals on the side chains introduced unique pharmacological properties, spurring efforts to further refine the chemical structure (Kim et al., 2023). The goal has been to increase solubility, expand the spectrum of action and routes of administration, and optimize pharmacokinetic properties, focusing on absorption, distribution, and elimination of the drugs and their metabolites.

As the challenge of treating infections aggravated by antimicrobial resistance intensifies, the pharmaceutical industry is redoubling its efforts in researching, isolating, and discovering new compounds with antimicrobial properties. Applying different tools such as genomics and compound collection screening, the industry is broadening its focus to encompass novel antibiotics beyond conventional methods. This shift has partially overshadowed the screening of natural microbial products, particularly those produced by fungi of the phylum Ascomycota, despite their potential in yielding effective antimicrobial agents.

Producing Antimicrobials with Fungi of the Phylum Ascomycota

Ascomycota, a diverse group of fungi comprising about 57,000 species across 6,100 genera, including molds and yeasts, are saprobes, plant parasites, or lichen-formers with a cosmopolitan distribution (Wijayawardene, 2017). They are known for their sexual reproductive structures, ascospores, produced within asci, and for septate hyphae. Asexual reproduction predominantly occurs through conidia, a general term for asexual spores (Kirk et al., 2008). Ascomycota biosynthesize secondary metabolites through heterotrophy, involving absorption or exchange. These fungi are prevalent in soil but are also found in aquatic environments and on plants, where they are often linked with phytopathogens (Vera et al., 2017). Ascomycota, the most extensively studied fungal group for antimicrobial substance production, generates a variety of secondary metabolites like alkaloids, terpenoids, polyketides, and more, thus offering significant pharmaceutical potential (Blackwell, 2011).

The genera Penicillium, Cephalosporium, Acremonium, and Fusidium are known to produce a range of secondary metabolites with antimicrobial properties, including various types of penicillin derivates, cephalosporins, and fusidic acid. These antimicrobials hold significant economic value (Adrio, Demain, 2003).

Up to 1995, approximately 22% of known antibiotics were derived from Ascomycota fungi. In 2009 alone, cephalosporins generated sales of $11.9 billion, followed by penicillins at $7.9 billion, accounting for over 40% of the total drug market (Verma et al., 2022). Their economic significance is considerable, as they are used for the production of fermented foods, antibiotic drugs, and various chemicals.

The process of discovering new antibiotics has evolved significantly, transitioning from simple observation of natural phenomena to complex, structured projects. These projects typically span 10 to 15 years from discovery to market availability. They comprise the selection of an appropriate microorganism; the identification of a chemical molecule with antimicrobial properties; modifications of chemical compounds for better antibacterial activity; the evaluation of the activity and toxicity in vitro and testing of the substance in clinical trials. Such processes demand time lines and high financial cost (Fair, Tor, 2014).

Industrial scale production of antibiotics frequently employs fermentative bioprocesses, both liquid and solid. Submerged liquid fermentation offers the advantage of managing large volumes, optimizing nutrient absorption and metabolite excretion by the chosen microorganism, thereby reducing cultivation time and enhancing productivity (Pereira, Bon, Ferrara, 2008). On the other hand, solid-state fermentation replicates the natural environment of microorganisms by using solids with little to no free water, which can increase microbial activity and potentially yield higher amounts of products (Takahashi, Lucas, 2008).

For the production of fungal-derived secondary metabolites with antibiotic properties, certain critical aspects ensure the success of the bioprocess, irrespective of the fermentation medium used. Key factors include maintaining purity of the fungal isolate, monitoring biosynthetic stability, and regulating critical parameters like oxygen, biomass concentration, culture volume, pH, temperature, aeration, and agitation. Additionally, incorporating synthetic substrates such as polymers, carbon sources (commonly glucose), and nitrogen sources (including minerals and amino acids) into the culture medium is crucial (Figure 3).

FIGURE 3
General scheme illustrating the production process of antimicrobial substances. *Image created using BioRender.com.

After the discovery of penicillin, only a small fraction of the antibiotics approved over the past 40 years represents new compound classes, while the majority of substances was derived from already known chemical structures. The most recent new class of commonly used antibiotics was discovered during the 1980s (Shore, Coukell, 2016). Currently, only approximately 30 to 40 new antibacterial compounds are in the clinical trial stages of development, and significantly, those targeting pathogens prioritized by the WHO are derivatives of existing antibiotic classes. In fact, less than 25% of drugs presently undergoing clinical trials belong to novel classes or employ new mechanisms of action. Importantly, none of these drugs demonstrate efficacy against Gram-negative pathogens or those categorized as critical threats by WHO (Miethke et al., 2021). Innovative strategies have been developed to explore natural products of Ascomycota fungi. These include studying these fungi in unique habitats, using genomics for bioprospecting, applying advanced techniques like mass spectrometry and Nuclear Magnetic Resonance (NMR) spectroscopy, and utilizing synthetic biology to modify biosynthesis pathways (Miethke et al., 2021). The diversity of Ascomycota, along with environmental influences, provides key insights into their metabolites’ antimicrobial potential against various pathogens.

Table II presents recent research on Ascomycota known for producing substances effective against ESBL-positive E. coli. Contemporary studies have highlighted several key findings: a) the significance of marine environments in isolating Ascomycota; b) the association of genera such as Penicillium, Aspergillus, Stachybotrys, and Trichoderma with the production of bioactive compounds, especially highlighting Penicillium for its extensive research and variety of metabolites; and c) the identification of these metabolites as belonging to diverse classes like alkaloids, terpenes, cyclopentanes, polyketides, xanthones, and naphthopyrones.

TABLE II
Summary of bioprospecting research focusing on Ascomycota fungi and their metabolites with antimicrobial potential against ESBL- producing E. coli isolates

Fungi	Target microorganisms	Associated environment	Reference
Penicillium erubescens KUFA 0220	Escherichia coli (ESBL)	Marine sponge (Neopetrosia sp.)	(Kumla et al., 2019)
Aspergillus terreus	Escherichia coli (ESBL)	Marine-coral	(Liu et al., 2019)
Stachybotrys chartarum TJ403-SS6	Escherichia coli (ESBL) ATCC 35218	Marine-coral (Sarcophyton subviride)	(Yang et al., 2019)
Neosartorya fischeri	Escherichia coli (ESBL) ATCC 35218	Arthropod (Cryptotympana atrata)	(Lin et al., 2020)
Trichoderma reesei PDSP 5.7	Escherichia coli (ESBL)	Marine sponge (Cinachyrella sp. PPD.SP.05)	(Sibero et al., 2020)
Penicillium chrysogenum TJ403-CA4	Escherichia coli (ESBL) ATCC 35218	Arthropod (Cryptotympana atrata)	(Nweze et al., 2020)
Penicillium cremeogriseum W1-1	Escherichia coli (ESBL)	Soil	(Sang et al., 2021)
Aspergillus elegans KUFA0015	Escherichia coli (ESBL)	Marine sponge (Monanchora unguiculata)	(Kumla et al., 2021)
Penicillium steckii P2648	Escherichia coli (ESBL) ATCC 35218	Marine fungus (marine coral)	(Yao et al., 2021)

An emerging strategy in the quest for new antimicrobials is the “green synthesis” of antimicrobial nanoparticles, where fungi play a crucial role. This environmentally friendly approach seeks to streamline production processes and also aims to elucidate the mechanisms of action of potentially promising antibiotic agents. Fungi are instrumental in this process, particularly in the reduction of silver ions to form silver nanoparticles when exposed to substances like silver nitrate. These nanoparticles possess significant antimicrobial properties, making them medically interesting (Durán et al., 2011; Guilger-Casagrande, Lima, 2019).

Several studies have demonstrated the efficacy of biological synthesizing of antimicrobial agents (Table III). The optimization of variables such as culture medium, pH, fungal biomass, temperature and AgNO₃ is critical for enhancing antimicrobial potential. Research has shown that Sclerotinia sclerotiorum can successfully inhibit E. coli growth in case of increasing AgNO₃ concentrations and temperature (Saxena et al., 2016). Penicillium aculeatum Su1 was shown to effectively inhibit the growth of several Gram-negative bacteria when reaction conditions were optimized (Liang et al., 2017). Similarly, silver nanoparticles (AgNPs) synthesized by Botrytis cinerea (14G), Penicillium expansum (14S), and Fusarium graminearum (31084) have shown inhibitory effects on E. coli growth, resulting in cellular membrane damage and the inhibition of bacterial protein synthesis (Sanguiñedo et al., 2018). Optimization of the synthesis process, affecting nanoparticle size, shape, distribution, and yield, is a key factor in augmenting antimicrobial efficacy.

TABLE III
Compilation of studies from the last decade assessing the antimicrobial efficacy of silver nanoparticles synthesized by filamentous fungi against pathogenic Gram-positive and Gram-negative bacteria, including both susceptible and resistant strains of E. coli

Producer organism of silver nanoparticles	Bacterial target pathogens	Reference
Aspergillus flavus	Multi-drug resistant gram negative bactéria	(Naqvi et al., 2013)
Macrophomina faseolina	Escherichia coli (ESBL)	(Chowdhury et al., 2014)
Aspergillus terreus	Escherichia coli (MDREC 1 Multidrug- resistant strains)	(Rani et al., 2017)
Tritirachium oryzae W5H	ESBL producers	(Qaralleh et al., 2019)
Botryosphaeria rhodina	Escherichia coli (ESBL)	(Akther et al., 2021)
	Escherichia coli ATCC 25922

The green synthesis of silver nanoparticles (AgNPs) using fungi has emerged as a promising, eco-friendly strategy for developing new antimicrobial agents. This biosynthesis process is straightforward, cost-effective, and easily scalable, making it an appealing option for large-scale production. Research has already demonstrated the efficacy of AgNPs synthesized by various fungi regarding the inhibition of the growth of E. coli and other Gram-negative bacteria, highlighting their potential in therapeutically addressing antibiotic-resistant pathogens (Guilger-Casagrande, Lima, 2019). Continued exploration and development of this innovative approach may lead to the discovery of more potent antimicrobial agents and provide new approaches for dealing with the critical issue of antibiotic resistance.

CONCLUSIONS

The growing resistance of bacteria like E. coli against conventional antibiotics, especially β-lactam antibiotics, highlights the urgency for new treatment strategies. Ascomycota fungi have historically been pivotal in antibiotic discovery, producing a range of vital antimicrobial agents and secondary metabolites. Their diversity and adaptability make them a promising resource in the search for innovative antimicrobials. Recent advancements in green synthesis, particularly the fungal production of silver nanoparticles (AgNPs), provide a sustainable approach in order to therapeutically address antibiotic-resistant bacteria. This method’s proven effectiveness regarding pathogens like E. coli underlines its potential for antimicrobial drug development. Future research focusing on Ascomycota fungi, utilizing advanced biotechnological tools, is crucial for exploring their biosynthetic capabilities and harnessing their potential for providing new antimicrobial agents. Ascomycota fungi represent a largely untapped reservoir of biochemical diversity, potentially holding the key to next-generation antimicrobials. The synergy of conventional knowledge and the use of modern scientific techniques can unlock new frontiers for antibiotic discovery, offering options to address the challenge of antimicrobial resistance.

ACKNOWLEDGMENTS

We would like to thank Fundação de Amparo à Pesquisa do Estado do Amazonas (FAPEAM) for the funding of the research by J.V.B. Souza under the Universal Call FAPEAM-010/2021 and for the POSGRAD 2020 grant. All the authors also thank the other funding agencies involved: CNPq and CAPES. The bursary of W.O.P.F. Segundo study was financed by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

REFERENCES

• Adrio JL, Demain AL. Fungal biotechnology, International Microbiology, 6, Springer, 2003, 191-9.
• Akther T, Ranjani S, Hemalatha S. Nanoparticles engineered from endophytic fungi (Botryosphaeria rhodina) against ESBL-producing pathogenic multidrug-resistant E coli. Environ Sci Eur. 2021; 33: 1-8. doi.org/10.1186/s12302-021-00524-9.
  » https://doi.org/10.1186/s12302-021-00524-9
• Bastidas-Caldes C, Romero-Alvarez D, Valdez-Vélez V, Morales RD, Montalvo-Hernández A, Gomes-Dias C. Extended-spectrum beta-lactamases producing Escherichia coli in South America: a systematic review with a One Health perspective. Infection and drug resistance, 2022; 5759-5779. doi: 10.2147/IDR.S371845.
  » https://doi.org/10.2147/IDR.S371845
• Bhat BA, Mir WR, Sheikh BA, Rather MA, Mir MA. In vitro and in silico evaluation of antimicrobial properties of Delphinium cashmerianum L., a medicinal herb growing in Kashmir, India. J Ethnopharmacol. 2022; 291: 115046.
• Bevan ER, Jones AM, Hawkey PM. Global epidemiology of CTX-M b-lactamases: temporal and geographical shifts in genotype. J Antimicrob Chemother. 2017; 72: 2145-55. doi.org/10.1093/jac/dkx146.
  » https://doi.org/10.1093/jac/dkx146
• Blackwell M. The fungi: 1, 2, 3 5.1 million species? Am J Bot. 2011; 98: 426-38.
• Bradford PA. Extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev. 2001; 14: 933-51. doi: 10.1128/CMR.14.4.933-951.2001.
  » https://doi.org/10.1128/CMR.14.4.933-951.2001
• Centers for Disease Control and Prevention. CDC. US Dep Heal Hum Serv Centres Dis Control Prev; 2019: 150. Avaiable from: https://www.cdc.gov/drugresistance/pdf/threats-report/2019-ar-threats-report-508.pdf
  » https://www.cdc.gov/drugresistance/pdf/threats-report/2019-ar-threats-report-508.pdf
• Castanheira M, Simner PJ, Bradford PA. Extended-spectrum β-lactamases: an update on their characteristics, epidemiology and detection. JAC-antimicrobial resistance, 2021; 3(3), dlab092. doi: 10.1093/jacamr/dlab092.
  » https://doi.org/10.1093/jacamr/dlab092
• Chen L, Kumar S, Wu H. A review of current antibiotic resistance and promising antibiotics with novel modes of action to combat antibiotic resistance. Archives of Microbiology, 2023; 205(11): 356.
• Chowdhury S, Basu A, Kundu S. Green synthesis of protein capped silver nanoparticles from phytopathogenic fungus Macrophomina paseolina. Nanoscale Res Lett. 2014; 9(1): 365. doi 10.1186/1556-276X-9-365.
  » https://doi.org/10.1186/1556-276X-9-365
• Dash S, Sahu SK, Paty BP. Phenotypic detection of ESBL-producing Enterobacteriaceae using combined disk diffusion, ESBL HiCrome agar, and E-test: A comparative study. Journal of Dr. NTR University of Health Sciences. 2022; 11(3): 200-207. doi: 10.4103/jdrntruhs.jdrntruhs_105_21.
  » https://doi.org/10.4103/jdrntruhs.jdrntruhs_105_21
• Datta N, Kontomichalou P. Penicillinase synthesis controlled by infectious R factors in Enterobacteriaceae. Nature. 1965; 208: 239-241. doi: 10.1038/208239a0.
  » https://doi.org/10.1038/208239a0
• Di Lodovico S, Fasciana T, Di Giulio M, Cellini L, Giammanco A, Rossolini GM. Spread of Multidrug-Resistant Microorganisms. Antibiotics. 2022; 11(7): 832. doi 10.3390/antibiotics11070832.
  » https://doi.org/10.3390/antibiotics11070832
• Durán N, Marcato PD, Durán M, Yadav A, Gade A, Rui M. Mechanistic aspects in the biogenic synthesis of extracellular metal nanoparticles by peptides, bacteria, fungi, and plants. Appl Microbiol Biotechnol. 2011; 90: 1609-24. doi.org/10.1007/s00253-011-3249-8. doi 10.1007/s00253-011-3249-8.
  » https://doi.org/10.1007/s00253-011-3249-8
• European Center for Disease Prevention and Control. ECDC; 2018. Avaiable from: https://www.ecdc.europa.eu/en/news-events/33000-people-die-every-year-due-infections-antibiotic-resistant-bacteria
  » https://www.ecdc.europa.eu/en/news-events/33000-people-die-every-year-due-infections-antibiotic-resistant-bacteria
• Fair RJ, Tor Y. Perspectives in Medicinal Chemistry Antibiotics and Bacterial Resistance in the 21st Century. Perspect Medicin Chem. 2014; 6: 25-64. doi.org/10.4137/PMC.S14459.
  » https://doi.org/10.4137/PMC.S14459
• Fong IW. Antimicrobial Resistance: A Crisis in the Making. New Antimicrobials: For the Present and the Future. 2023; 1-21.
• Geurtsen J, de Been M, Weerdenburg E, Zomer A, McNally A, Poolman J. Genomics and pathotypes of the many faces of Escherichia coli. FEMS microbiology reviews, 2022; 46(6): p. fuac031. doi.org/10.1093/femsre/fuac031.
  » https://doi.org/10.1093/femsre/fuac031
• Global Research on Antimicrobial Resistance. GRAM. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis; 2019. Avaiable from https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(21)02724-0/fulltext
  » https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(21)02724-0/fulltext
• Grossart H-P, Wyngaert S Van den, Kagami M, Wurzbacher C, Cunliffe M, Rojas-Jimenez K. Fungi in aquatic ecosystems. Nat Rev Microbiol. 2019; 17(6): 339-54.
• Guilger-Casagrande M, Lima. R de. Synthesis of silver nanoparticles mediated by fungi: a review. Front Bioeng Biotechnol. 2019; 7: 287. doi.org/10.3389/fbioe.2019.00287.
  » https://doi.org/10.3389/fbioe.2019.00287
• Horesh G, Blackwell GA, Tonkin-Hill G, Corander J, Heinz E, Thomson NR. A comprehensive and high-quality collection of Escherichia coli genomes and their genes. Microbial genomics. 2021; 7(2). doi.org/10.1099/mgen.0.000499.
  » https://doi.org/10.1099/mgen.0.000499
• Hutchings MI, Truman AW, Wilkinson B. Antibiotics: past, present and future. Curr Opin Microbiol. 2019; 51: 72-80. doi.org/10.1016/j.mib.2019.10.008.
  » https://doi.org/10.1016/j.mib.2019.10.008
• Kirk P, Cannon P, Minter D, Stalpers J. Ainsworth & Bisby’s Dictionary of the Fungi. 10th ed. 2008.
• Kim D, Kim S, Kwon Y, Kim Y, Park H, Kwak K. Structural Insights for β-Lactam Antibiotics. Biomolecules & Therapeutics. 2023; 31(2): 141. doi: 10.4062/biomolther.2023.008
  » https://doi.org/10.4062/biomolther.2023.008
• Kliebe C. Evolution of plasmid-coded resistance to broad-spectrum cephalosporins. Antimicrob Agents Chemother. 1985; 28: 302-7. doi.org/10.1128/aac.28.2.302.
  » https://doi.org/10.1128/aac.28.2.302
• Kumla D, Dethoup T, Pereira JA, Freitas-silva J, Costa PM, Silva AMS, et al. A Xanthonopyrone SPF-3059-26 from the Culture of Penicillium erubescens KUFA 0220 and Antibacterial Activity Evaluation of Some of Its Constituents. Molecules. 2019; 24: 1-10. doi.org/10.3390/molecules24010208.
  » https://doi.org/10.3390/molecules24010208
• Kumla D, Sousa E, Marengo A, Dethoup T, Gales L, Freitas-Silva J, et al. Phytochemistry 1,3-Dioxepine and spiropyran derivatives of viomellein and other dimeric naphthopyranones from cultures of Aspergillus elegans KUFA0015 and their antibacterial activity. Phytochemistry. 2021; 181. doi.org/10.1016/j.phytochem.2020.112575.
  » https://doi.org/10.1016/j.phytochem.2020.112575
• Liang M, Wei S, Jian-Xin L, Xiao-Xi Z, Zhi H, Wen L. Optimization for extracellular biosynthesis of silver nanoparticles by Penicillium aculeatum Su1 and their antimicrobial activity and cytotoxic effect compared with silver ions. Mater Sci Eng. 2017; 77: 963-71. doi.org/10.1016/j.msec.2017.03.294.
  » https://doi.org/10.1016/j.msec.2017.03.294
• Lin S, Yu H, Yang B, Li F, Chen X, Li H, et al. Reisolation and Configurational Reinvestigation of Cottoquinazolines E−G from an Arthropod-Derived Strain of the Fungus Neosartorya fischeri. Nat Prod. 2020; 83: 169-73. doi.org/10.1021/acs.jnatprod.9b01000.
  » https://doi.org/10.1021/acs.jnatprod.9b01000
• Liu M, He Y, Shen L, Anbari WHA, Li H, Wang J, et al. Asperteramide A, an Unusual N-Phenyl-Carbamic Acid Methyl Ester Trimer Isolated from the Coral-Derived Fungus Aspergillus Terreus. European J Org Chem. 2019; 18: 2928-32.
• Mayegowda SB, Roy A, Manjula NG, Pandit S, Alghamdi S, Almehmadi M. Eco-friendly synthesized nanoparticles as antimicrobial agents: an updated review. Frontiers in Cellular and Infection Microbiology, 2023; 13. doi 10.3389/fcimb.2023.1224778.
  » https://doi.org/10.3389/fcimb.2023.1224778
• Miethke M, Pieroni M, Weber T, Brönstrup M, Hammann P. Towards the sustainable discovery and development of new antibiotics. Nature Reviews Chemistry. 2021; 10: 726-749.
• Naqvi SZH, Kiran U, Ali MI, Jamal A, Hameed A, Ahmed S. Combined efficacy of biologically synthesized silver nanoparticles and different antibiotics against multidrug-resistant bacteria. Int J Nanomed. 2013; 8: 3187-95. doi.org/10.2147/IJN.S49284.
  » https://doi.org/10.2147/IJN.S49284
• Nweze JA, Mbaoji FN, Huang G, Li Y, Yang L, Zhang Y, et al. Antibiotics Development and the Potentials of Marine-Derived Compounds to Stem the Tide of and Protozoa. Mar Drugs. 2020; 18: 145. doi.org/10.3390/md18030145.
  » https://doi.org/10.3390/md18030145
• Pereira JN, Bon EP da S, Ferrara MA. Tecnologia de Bioprocessos. Séries Biotecnol. Rio de Janeiro: 2008.
• Pitout JD. Extraintestinal pathogenic Escherichia coli: an update on antimicrobial resistance, laboratory diagnosis and treatment. Expert Rev Anti Infect Ther. 2012; 10: 1165-76.
• Qaralleh H, Khleifat KM, Al-limoun MO, Alzedaneen FY, Al-tawarah N. Antibacterial and synergistic effect of biosynthesized silver nanoparticles using the fungi Tritirachium oryzae W5H with essential oil of Centaurea damascena to enhance conventional antibiotics activity. Adv Nat Sci Nanosci Nanotechnol. 2019; 10: 11.
• Rana C, Rajput S, Behera M, Gautam D, Vikas V, Vats A, et al. Comparative Immunology, Microbiology and Infectious Diseases Global epidemiology of CTX-M-type β -lactam resistance in human and animal. Comp Immunol Microbiol Infect Dis. 2022; 86: 101-815. doi.org/10.1016/j.cimid.2022.101815.
  » https://doi.org/10.1016/j.cimid.2022.101815
• Rani R, Sharma D, Chaturvedi M, Yadav JP. Green synthesis, characterization and antibacterial activity of silver nanoparticles of endophytic fungi Aspergillus terreus. J Nanomed Nanotechnol. 2017; 8.
• Samuel P, Maheswari M, Vijayakumar J, Selvarathinam T, Amirtharaj K, Deenathayalan R. Bio-prospecting of marine-derived fungi with special reference to production of keratinase enzyme-A need-based optimization study. J App Biol Biotechnol. 2018; 6(3): 35-41. doi.org/10.7324/JABB.2018.60306.
  » https://doi.org/10.7324/JABB.2018.60306
• Sang XY, Wang ZJ, Yang Y, Wang YL, Xiang ML, Zhu DY, et al. Antimicrobial Natural Products Produced by Soil-Derived Fungus Penicillium cremeogriseum W1-1. Indian J Microbiol. 2021; 61: 519-23. doi.org/10.1007/s12088-021-00957-z.
  » https://doi.org/10.1007/s12088-021-00957-z
• Sanguiñedo P, Fratila RM, Estevez MB, Fuente JM, Grazú V, Alborés S. Extracellular biosynthesis of silver nanoparticles using fungi and their antibacterial activity. Nano Biomed Eng. 2018; 10: 165-73.
• Saxena J, Sharma PK, Sharma MM, Singh A. Process optimization for green synthesis of silver nanoparticles by Sclerotinia sclerotiorum MTCC 8785 and evaluation of its antibacterial properties. Springerplus. 2016; 5.
• Shore CK, Coukell A. Roadmap for antibiotic discovery. Nature microbiology. 2016; 1(6): 1-2.
• Sibero MT, Diponegoro U, Zhou T. Antibacterial activity of semi purified extract of marine-derived Trichoderma reesei PDSP 5.7 using bioguided fractionation method antibacterial activity of semi purified extract of marine-derived Trichoderma reesei PDSP. Bul Oseanografi Mar. 2020; 9: 45-54. doi.org/10.14710/buloma.v9i1.29192.
  » https://doi.org/10.14710/buloma.v9i1.29192
• Silber J, Kramer A, Labes A, Tasdemir D. From Discovery to Production: Biotechnology of Marine Fungi for the Production of New Antibiotics. Mar Drugs. 2016; 14: 137.
• Smith HA. Production of antimicrobials and antioxidants from filamentous fungi. Doctoral dissertation, National University of Ireland Maynooth; 2014.
• Singh A, Shahid M, Singh GP, Khan HM. Mobile Genetic Elements. In Beta-Lactam Resistance in Gram-Negative Bacteria: Threats and Challenges. Singapore: Springer Nature Singapore. 2022; 141-152.
• Takahashi JA, Lucas EMF. Ocorrência e diversidade estrutural de metabólitos fúngicos com atividade antibiótica. Quim Nova. 2008; 31: 1807-13.
• Vera J, Gutierrez MH, Palfner G, Pantoja S. Diversity of culturable filamentous Ascomycetes in the eastern South Pacific Ocean off Chile. World J Microbiol Biotechnol. 2017; 33: 1-13.
• Verma T, Aggarwal A, Singh S, Sharma S, Sarma SJ. Current challenges and advancements towards discovery and resistance of antibiotics. J Mol Struct. 2022; 1248: 131-380.
• World Health Organization. WHO. New report calls for urgent action to avert antimicrobial resistance crisis; 2019. Avaiable from: https://www.who.int/news/item/29-04-2019-new-report-calls-for-urgent-action-to-avert-antimicrobial-resistance-crisis
  » https://www.who.int/news/item/29-04-2019-new-report-calls-for-urgent-action-to-avert-antimicrobial-resistance-crisis
• Wijayawardene N. Notes for genera: Ascomycota. Fungal Divers. 2017; 86: 1-594.
• Yang B, He Y, Lin S, Zhang J, Li H, Wang J, et al. Antimicrobial Dolabellanes and Atranones from a Marine-Derived Strain of the Toxigenic Fungus Stachybotrys chartarum. J Nat Prod. 2019; 82: 1923-1929.
• Yao G, Chen X, Zheng H, Liao D, Yu Z, Wang Z, et al. Genomic and Chemical Investigation of Bioactive Secondary Metabolites From a Marine-Derived Fungus Penicillium steckii P2648. Front Microbiol. 2021; 12. doi.org/10.3389/fmicb.2021.600991.
  » https://doi.org/10.3389/fmicb.2021.600991
• Youssef FS, Ashour ML, Singab ANB, Wink M. A comprehensive review of bioactive peptides from marine fungi and their biological significance. Mar Drugs. 2019; 17: 559.

Edited by

• Associated Editor:
  Silvya Stuchi Maria-Engler.

Publication Dates

• Publication in this collection
  20 Jan 2025
• Date of issue
  2025

History

• Received
  10 Aug 2023
• Accepted
  21 Mar 2024