Antifungal Compounds Discovery: Natural and Synthetic Approaches

Antifungal Compounds Discovery

Natural and Synthetic Approaches

First Edition

Marina Soković

Konstantinos Liaras

Table of Contents

Cover image

Title page

Copyright

Dedications

Acknowledgments

Chapter 1: Introduction

Abstract

1.1: Toward novel antifungal agents

Chapter 2: Fungal infections—Background to specific fungal species

Abstract

2.1: Superficial mycoses

2.2: Cutaneous and subcutaneous mycoses

2.3: Mucosal infections

2.4: Opportunistic mycoses

2.5: Community-acquired and nosocomial mycotic infections

2.6: Systemic mycoses

2.7: Specific fungal species

2.8: Dermatomycetes

2.9: Dematiaceous fungi

Chapter 3: Antifungal therapy

Abstract

3.1: Antifungal therapy in the past

3.2: Antifungal therapy nowadays

3.3: Antifungal resistance

Chapter 4: Natural products as antifungals

Abstract

4.1: Fungal metabolites

4.2: Plant extracts and metabolites

4.3: Natural compounds from sponges

4.4: Other tested compounds

4.5: Morphophysiological changes in fungi due to inhibition activity by natural compounds

Chapter 5: Synthetic antifungal compounds

Abstract

5.1: Allylamines

5.2: Azoles

5.3: Thiazolidinones

5.4: Echinocandins

5.5: Other synthetic antifungals

Chapter 6: Future trends

Abstract

6.1: Connecting traditional medicines with modern technologies

6.2: Novel approaches to overcome antimicrobial resistance to existing drugs

Chapter 7: Concluding remarks

Abstract

Index

Copyright

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Dedications

I dedicate the book to the person who taught me to love books, to my father, and to my children to maintain and keep this habit.

Dr. Marina Soković

I dedicate this book to my mother for her unconditional love and continuous support in everything I do.

Dr. Konstantinos Liaras

Acknowledgments

Dr. Marina Soković

I am very thankful to my husband Miroslav and my children Jana and Luka for their love, understanding, patience, and continuous support to complete this book. I am grateful to them for the time they spent without my attention, when I was dedicated to writing this book.

I am extremely grateful to my parents for their love, caring, and sacrifices for educating and preparing me for my future and to complete all my ideas successfully, including this. I am extending my thanks to my brother for supporting me all the time.

Also, I express my thanks to my mother and sister in law for their support and valuable help with children.

I would like to say thanks to my friends and research colleagues, Dr. Jasmina Glamočlija, Dr. Ana Ćirić, Dr. Jovana Petrović, Dr. Marija Ivanov, Dejan Stojković, Marina Kostić for their constant help and for understanding me all the time. Their work and results are also presented in this book.

Dr. Konstantinos Liaras

I am very grateful to my family for their love and patience during this long period that I was focused in writing this book.

I would also like to thank my colleagues in the pharmacy for their understanding and extra work when I was concentrated in completing this book.

I would also like to express my gratitude to all my lab colleagues from Aristotle University of Thessaloniki for our fruitful collaboration in our common research projects and papers, some of which were also reviewed in this book.

Common acknowledgements of the authors

We would both like to thank Prof. Athina Geronikaki who introduced us years ago and worked with us in many research projects the results of which are included in this book.

We are also grateful to the architect and artist Sophia Spirliadou for her substantial help in designing the artistic figures of the book.

Chapter 1: Introduction

Marina Sokovića; Konstantinos Liarasb    a Mycological Laboratory, Institute for Biological Research Siniša Stanković, University of Belgrade, National Institute of Republic of Serbia, Belgrade, Serbia

b Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki, Greece

Abstract

This chapter introduces the reasons why fungal infections still remain a therapeutic problem with emphasis on the issue of antimicrobial resistance and the causes of its emergence. Facts and ideas, which could be taken into account in future approaches and research projects in the field of antifungals, are mentioned. Terms, which became very important recently concerning fungal infections such as biofilm formation, quorum sensing, and autoinducers, are explained and indicated as potential targets for innovative drugs. A recent update on studies is presented regarding natural and synthetic antifungals. The aims and general content of this book are explained in order to provide some aspects of the following chapters.

Keywords

Fungi; Fungal infections; Antifungal activity; Antimicrobial resistance; Quorum sensing; Biofilm; Antifungals

Chapter outline

1.1.Toward novel antifungal agents

References

Infectious diseases in humans have markedly increased during the past 10 years, especially in immunocompromised patients. Consequently, as high as 10% of hospital-acquired systemic infections are caused by fungi (Soković et al., 2006). Fungal infections could be provoked by primary and opportunistic pathogens. Primary pathogens are capable to provoke infection in the healthy population, while opportunistic ones could be responsible for infection in immunosuppressed organisms.

The research efforts concerning antimicrobials were focused mainly to bacterial infections and antibacterial resistance, delaying that way the progress in testing fungal diseases and resistance to antifungal agents. First of all, fungal diseases were for many years neglected and fungi as pathogens were not recognized and accepted as important (Wey et al., 1988). A large increase in fungal infections was noticed in late 1960s, when antibiotic treatments were developed in high extent and fungi started to represent a global health problem and threat (Vandeputte et al., 2012). The increase of fungal diseases is associated with changes in medical practice such as uncontrolled usage of antibiotics (especially broad-spectrum ones), frequent use of therapies that suppress immune system, utilization of indwelling intravenous devices, and spread of chronic immunosuppressive viral infections such as AIDS (Ghannoum and Rice, 1999).

Fungal infections still remain a therapeutic problem despite the availability of a number of treatments and can become an important issue in the care of patients with compromised immune system (Nychas, 1995). This could be a consequence of infectious diseases, chemotherapy, transplantations, and treatment of autoimmune diseases such as rheumatoid arthritis, AIDS, Crohn’s disease, etc., or might be a result of aging, but also as consequence of uncontrolled use of antimycotics. Increased numbers of immunocompromised persons lead to a rise in the number of people that have succumbed to fungal diseases. The frequency of fungal diseases is higher in low socioeconomic status communities: bad living conditions lead more often to skin-to-skin contact and close proximity to animals, while hygiene may be suboptimal (Havlickova et al., 2008).

Diseases provoked by fungi are often more difficult to treat than bacterial infections. Either topical or oral treatments are long term and with limited success in controlling the fungus, or completely ineffective. The majority of infections are chronic, tending to recurrence of the disease. One of the difficulties in antifungal therapy is that existing antifungal drugs usually cause harmful side effects due to similar cellular structure and functions between eukaryotic human and fungal cells.

Fungi are in general being controlled by drugs and the use of antimycotics is limited due to their undesirable effects, including teratogenicity, carcinogenicity, acute but also chronic toxicity and extended degradation period with consequences affecting environmental pollution (Shin and Lim, 2004).

It is obvious that some barriers are present in the effective reduction of mycoses: difficult elimination of fungi due to the fact that they are widespread in the nature; difficult diagnosis because fungi have various clinical manifestations that are host dependent; difficult to establish proper therapy due to restricted access to a number of available drugs; successful treatments are available only for a limited number of fungi (Marković et al., 2016).

Two main populations are in risk to acquire invasive infections by fungi. In the first group are included individuals who are susceptible to infection because of their geographic location and their infections can be referred to as endemic mycoses. In the second group are included individuals who possess increased host susceptibility, developing in that way opportunistic infections (immunocompromised, severely ill, malnourished patients, etc.) (Fridkin and Jarvis, 1996).

There is also some discordance and limitation in the interpretation of results obtained from in vivo and in vitro data; infections provoked by susceptible strains respond to appropriate therapy in ∼  90% of cases, whereas infections due to resistant strains respond in ∼  60% of cases (Rex and Pfaller, 2002).

Determination of minimal inhibitory concentration (MIC) in vitro that could inhibit fungal growth is also limited, because MIC levels do not always represent the most optimal measurement of resistance. Knowing that for the majority of fungal species, or at least for those which are important in medicine, there is a determined MIC for a specific studied drug, it is necessary to estimate its average MIC. Consequently, one of the common ways to define a strain as resistant is by observing a MIC higher than the average for the corresponding drug. There are suggestions and considerations that determination of minimum effective concentration of some drug should be done at hyphal cells, not only at spores and that might be a more appropriate end point than MIC obtained by testing the susceptibility of filamentous fungi to echinocandins (Kurtz et al., 1994). In any case, it is widely accepted that testing antifungal activity means determination of MIC and minimal fungicidal concentration (MFC) against conidia rather than sensitivity of clinically more important hyphal structures (Kanafani and Perfect, 2008).

The interpretation and comparison of the results obtained from different research groups are often very difficult due to differences in methodology, fungal strains, control drugs, etc. This has to be adapted at first for in vitro investigation and afterwards for testing antifungal activity in vivo and in clinical trials. Once reproducible methodologies and standards are established, it will be much easier to transfer knowledge from labs to patients.

One of the acknowledged ways to define clinical resistance is by evaluating the persistence of progression of a microbial infection despite administering the appropriate therapy. Especially for fungal infections, characterizing clinical response to therapy as successful, depends not only on the susceptibility of the pathogenic fungus, but also on the immune system condition of the host, patient compliance, drug pharmacokinetics, and absence of a persistent or protected focus of infection (f.i. abscess or catheter).

One of the greatest scientific achievements of 20th century is the development of new, effective, and mass-produced antimicrobial agents. Antibiotics and antimycotics comprised western medicine’s primary defense against infectious diseases for more than 60 years. After a long antibiotics era, when millions of lives were saved, our chemical shield has become increasingly leaky. World Health Organization published that the world is on the brink of losing these miracle cures (antibiotics) and that in the absence of urgent corrective and protective actions, the world is heading towards a post-antibiotic era, in which many common infections will no longer have a cure and, once again, kill unabated (Liljeqvist et al., 2012).

Antimycotic agents are in most of the cases the choice of preference in order to treat this kind of infections, being alongside with antibiotics the most widely used therapeutics around the globe. Despite the successful application of conventional antifungals for many years, their uncontrolled and injudicious use, as mentioned, led to the emergence of numerous resistant fungal species.

It should also be noted that in many countries, the administration of topical antifungal drugs is relatively uncontrolled. The use of dermatological products that contain antifungal drugs, alone or in combination with corticosteroids, has become very familiar to patients, since they consider them as totally safe, giving extreme dimensions to the problem.

Another factor that should be taken into account is the economic status of patients. There are many cases in which due to financial problems, combined with difficult access to public health institutions, patients turn to the dangerous solution of self-treatment. This phenomenon has the result of overuse of certain Over-The-Counter (OTC) antifungals with dosage/duration treatment schemes, which are self-defined from the personal experience of each patient. False self-diagnoses and wrong treatment schemes can prove to be even worse than no treatment at all.

We should also take into account some kinds of human behavior that can also potentially promote the emergence of resistance. An excellent example is the case of noncompliance of patients with the appropriate treatments. Noncompliance takes place when patients forget to take their medication, prematurely forfeit treatment when they start to feel better, or are not able to cover the whole cost of their therapeutic scheme.

In a number of countries, the issues of noncompliance and self-medication become even greater due to the fact that a series of the available antimicrobial drugs are not produced under the proper standards, they are counterfeit, or have expired in terms of efficacy (Knobler et al., 2003).

Another key factor that contributes to the resistance issue is the administration of series of antimicrobials in animals that are raised for food (poultry, pigs, cows, etc.). The only thing that has not been clarified yet is the contribution of that kind of agricultural in the emergence and spread of resistance pathogens that are dangerous for humans. While in some studies it was stated that this problem is under control by different programs, in others it was proposed that the level of risk is much higher than we suppose. It was mentioned that by using antimicrobial compounds in animal care, either for growth promotion or for therapeutic reasons, there is a possibility to induce the emergence of drug-resistant strains that can ultimately be transmitted to humans via food products (Knobler et al., 2003).

It is clear that the benefits that derive from the use of antimicrobials in animals reflect on more factors than just the animal health, taking into account the economic benefits for the producers and healthier and safer food products for the public. Despite the previously mentioned benefits, many scientists suggest the reduction of current use of antimicrobials due to serious risks, connected to resistance, for both animal and human health. Others propose that such restrictions in the use of antimicrobials in animals could turn out to be economically unfavorable for producers and ultimately, customers. Nevertheless, the vast majority of scientists agree that the injudicious use of antibiotics and/or antifungals should be taken into serious consideration and potentially be restricted when alternative solutions are available and recommended, or when conventional drugs are found to be undoubtedly ineffective.

Having all these facts in mind, human and veterinary doctors are entitled to promote the judicious and safe use of antimicrobial drugs. In addition, through health systems and higher education we should be in position to ensure the sufficient training of human and animal health professionals concerning the rational and considerate use of antimicrobials.

Despite the already-known facts about the way hospitals could minimize the spread of infectious diseases, intensive studies are still in need to give us better perspective on the matter. In addition, the development of fast and effective diagnostic methods for the identification of an infection, the specific microorganism and its susceptibility to marketed drugs remains a constant priority.

Precision in diagnostic tests is crucial in our efforts to alternate the current strategy of extensive empiric administration of antimicrobial agents in infected patients. Moreover, development of materials that can be used with medical devices (e.g., catheters) and are designed to be resistant to colonization by pathogenic strains is still drawing the attention of scientists and companies around the globe, while the ongoing production of innovative antimicrobials also remains a high priority (Knobler et al., 2003).

The progress in techniques and methods in the field of molecular biology led to the deduction that genes, which are responsible for resistance, can be also horizontally transferred among species in higher extent than it was previously thought by scientists. Consequently, this fact leads to a pessimistic scenario for future generations, when most of the known antimicrobial agents could possibly prove to be ineffective, bringing us back to a preantimicrobials era where common bacteria and fungi will again become life threatening.

Regarding the transfer of resistance to pathogens that can infect humans, the number of pathogenic species that can serve as recipients is of great importance. Having that in mind, it is implied that the human microbiome might act in this process, as intermediary reservoir for the emergence of resistance (Sommer et al., 2010).

However, this role of human microbiome has not been studied extensively by scientists so far. Moreover, environmental strains could possibly carry resistance genes into the human microbiome, where they could be subsequently transferred to human-associated species, including pathogens. Nevertheless, for the majority of this kind of microorganisms, the interaction periods with microorganisms of human microbiome can be short. It should be mentioned that animals can also act as intermediates for resistant species and offer an ideal ground for transferring resistance to human pathogens (Allen et al., 2010).

All mentioned facts could potentially lead to antimicrobial resistance (AMR) of fungal species that makes the problem of fungal infections even worse and more difficult to control and treat.

Antimicrobial resistance occurs when microorganisms (such as bacteria, fungi, viruses, and parasites) change at their exposure to antimicrobial drugs (such as antibiotics, antifungals, antivirals, antimalarials, and anthelmintics). Microorganisms that develop antimicrobial resistance are sometimes referred to as superbugs (WHO, 2017).

There are two main types of resistance that can be observed in vitro: primary (intrinsic) and secondary (acquired) resistance. When a fungus is resistant to a particular drug before exposure, then it is described as acquiring primary resistance. On the other hand, secondary resistance is obtained when there is previous exposure to an antifungal compound. Additionally, the main factors that could be responsible for the presence of a resistant fungal strain are: intrinsic resistance of endogenous strains, replacement with more resistant species or strains, genetic modifications that lead to endogenous resistant strains, temporary expression of certain genes that have as a result the production of an endogenous strain that is resistant for a certain period of time, cell type alterations, overcrowding and, in general, modifications in the size and variability of population, etc. (White et al., 1998).

This undesirable situation became a constant threat for public health, while the dramatic scale of the issue has alerted health care organizations, governments and pharmaceutical companies.

Economic factors and high costs of antimicrobial agents production make their development unprofitable (Nathan and Goldberg, 2005), while facts that uncontrolled use of antibiotics and antimycotics influence AMR turn us to search for new approaches, which will help us to fight the pathogens that are capable to kill. This idea is not futuristic or even theoretical: we can tap into an ancient arms race between bacteria and fungi and novel natural and synthetic antimicrobials to combat drug-resistant bacteria and fungi. Natural therapy, or the use of new semisynthetic or synthetic compounds based on combined scientific results from different branches (biology, molecular biology, proteomics, chemistry, pharmacy, etc.), represents potentially significant weapons in our ongoing battle against fungal diseases.

Vandeputte et al. (2012) reported that fungi possess mechanisms to counteract the fungistatic or fungicidal effects of different antifungal drugs. There are three basic mechanisms: possibility of fungi to decrease the affinity of the drug for its target, to reduce the accumulation of the drug in fungal cell, and potential to modify metabolism to counterbalance the drug effect.

According to mentioned facts, the need for novel approaches, methodologies, and studying of all of three basic mechanisms of resistance is now greater and more necessary than ever.

Although resistance to antimicrobials is a natural biological phenomenon, the extensive introduction of antibacterial and antifungal agents into clinical practice was followed by the laboratory detection of resistant strains, able to multiply in drug concentrations that are higher than the therapeutic ones. This kind of resistance may either characterize the entire species or be acquired by strains of a normally susceptible species via mutation or transfer of genes. Various mechanisms that promote resistance to particular antimicrobials are encoded by corresponding genes of microorganisms. These mechanisms can also promote resistance to antimicrobials of the same or even in some cases of different classes.

Alongside with the extensive use of antimicrobials worldwide was observed an increase of resistance prevalence to each new drug. Although the prevalence of resistance varies in diverse geographical areas, sooner or later applies also on every antimicrobial agent (WHO, 2001). Consequently, resistance has been observed to be acquired for every antimicrobial agent some time after their introduction to clinical practice. A fine example is the acquisition of resistance by Staphylococcus aureus to penicillin in hospitals, only a few years (mid-1940s) after its introduction to therapeutic use. Subsequently, the initial emergence of antimicrobial resistance in hospitals soon became widespread also in community-acquired infections.

To support these facts, it would be interesting to take a glimpse into some information regarding infections that are caused by Aspergillus spp. This type of infections is still in the spotlight of numerous medicinal research fields mainly due to the rapid development of diagnostic and therapeutic techniques and methods, having always as ultimate goal the reduction of morbidity and mortality rates. Especially invasive pulmonary aspergillosis (IPA) is a serious disease with increasing incidence and fatality rates. The first described case of IPA is from 1953. The incidence of this disease has increased in the past 10–20 years; in the years between 1978 and 1992 IPA incidence increased from 0.4% to 3.1%, while nowadays from 17% to 60%. Moreover, the mortality of IPA increased till 50% in patients with neutropenia and 90% in recipients of hematopoietic stem-cell transplantation (HSCT) (Kousha et al., 2011). Conventional treatment of aspergillosis is not effective in preventing death in up to two-thirds of the patients. The condition is even more severe for the immunosuppressed patients in the form of invasive and life-threatening aspergillosis. Moreover, aspergillosis is responsible for the presence of a series of risk factors, such as neutropenia and myeloablative-immunosuppressive treatments that are connected to different kinds of transplantations (hematopoietic stem cells, whole-organ, primarily lung and heart transplantations). It should be pointed out that the mortality rates for this illness range between 30% and 90%, proving that conventional treatments don’t exhibit the desirable results for the patients (Steele et al., 2005).

An important variable in such cases and conditions is the immunity that a host develops after exposure to the fungus or on the other hand the pathogenesis that can occur after transition from saprophytic form of some fungi (e.g., Candida albicans).

A side effect of antibiotic therapy is the increased growth of some fungal species, e.g., Candida sp., Aspergillus sp., and others. Candida albicans is a commensal microorganism and is usually present in human microbiota (mouth, urogenital, and gastrointestinal tract). Hormones, stress, weak immune system could affect the levels of C. albicans. Antibiotic treatment could dramatically increase the number of Candida colonies, while organisms treated with high doses of antibiotics and for long duration are more susceptible to C. albicans colonization and infection (Noverr et al., 2004). Rational use of antibiotics is very important for controlling C. albicans and other fungal species.

To summarize, in that kind of cases both innate and acquired immunities play a crucial role in confronting pathogenic fungal strains (Bellocchio et al., 2004).

There are numerous factors, which play important role in the emergence of resistance, including overuse of drugs, infections of inmmunosuppressed patients, and increased use of internal medical devices that provide an ideal environment for pathogens.

In order to avoid this threat it is necessary to work not only on simple biological screening of new antimicrobials, but also to find new approaches and new methodologies taking into account common experience and efforts of scientists from different branches (mycologists, bacteriologists, microbiologists, molecular biologists, chemists, pharmacists, medical doctors).

1.1: Toward novel antifungal agents

In addition, under particular stress conditions, pathogenic microorganisms have the ability to evolve via epigenetic mechanisms and genetic variations. Manifold approaches are in need to control and combat effectively resistance to antimicrobials. Such strategies contain rational use of existing antimicrobials, combination of different therapeutic approaches, and development of innovative antimicrobial agents and methods of treatment. The use of microbial genomics gives incredible opportunities for introduction of molecular diagnostic tools in order to overcome resistance.

Incorporating innovative technologies from diverse fields like chemistry, computer-aided studies, bacterial bioengineering, microbiology, and pharmacology could be the answer to the ominous problem of resistance to antimicrobial agents, lowering the cost, speed of development, and potency of drugs.

Despite the discovery of numerous antimicrobial molecules to date, design of new and efficient drug candidates is an issue that has not yet been fully addressed. To overcome the existing problem, combining skills and funds from academia, pharmaceutical companies, and public health systems would prove to be essential. This synthesis of skills could lead to synergy between innovative technological developments, academic sector, and industry, bringing promising results concerning the hunt for novel antimicrobials.

In the recent years, it has come to recognition that traditional therapeutic targets of antimicrobials, such as protein synthesis and cell wall peptidoglycan biosynthesis, can be subjects to rapid emergence of resistance. This led scientists to begin a search for alternative targets for antimicrobial agents like quorum sensing, protein (enzyme) folding, and formation of biofilms.

Biofilm is described as a strongly adherent assemblage of differentiated microbial cells enclosed in a matrix of polysaccharides (Stoodley et al., 2002). Another way to describe biofilms can be an assemblage of microbial cells that is irreversibly associated (not removed by gentle rinsing) with a surface and enclosed in a matrix of primarily polysaccharide material (Donlan, 2002). In the form of biofilms, microorganisms stick together and on a surface, embedded in a polymeric matrix containing polysaccharides, proteins, lipids, and DNA, produced by themselves, which is generally named extracellular polymeric substance (EPS) (Flemming and Wingender, 2002). This matrix is highly hydrated and transport of nutrients to the cells within the biofilm is provided through the water channels. Bacteria and fungi are capable to detach and migrate through the biofilms and hence to colonize other surfaces. They can also remodel biofilm matrix by using specific enzymes.

There are many kinds of surfaces and in general places where pathogens can form biofilms. These can be organic or inorganic and diverse, such as lungs, teeth, intestines, lenses, and water tubes. Microorganisms involved in biofilms cause serious infections to humans and animals (endocarditis, cystic fibrosis, periodontitis, rhinosinusitis, and osteomyelitis) and can be often seen in urinary catheters and medical implants. This is also a big problem in the industry, in every procedure where a water-based process is involved (paper manufacturing, nautical shipping, drinking water facilities, cooling systems, health care, medical devices, and food