Ayhan Filazi-Eng-Alternative Antimicrobial Approaches ... · “Virusesthat infect bacterial cells by transferring their own geneticmaterial (DNA or RNA)” 4.04.2019 KANATLI SEKTÖRÜNÜN

ALTERNATIVE ANTIMICROBIAL APPROACHES DEVELOPED TOWARDS TARGETMICROORGANISMSPROF. DR. AYHAN FİLAZİ

ANKARA UNIVERSITY FACULTY OF VETERINARY MEDICINE

DEPARTMENT OF PHARMACOLOGY AND TOXICOLOGY

4.04.2019 KANATLI SEKTÖRÜNÜN GÜNCEL SORUNLARI-VETERİNER TAVUKÇULUK DERNEĞİ 1

Why use antimicrobials?- To treat or prevent infections caused by microorganisms

- Increase performance and efficiency in animals

- Extending the shelf life of foods

Therefore, the newly developed substances must also have these properties.

KANATLI SEKTÖRÜNÜN GÜNCEL SORUNLARI-VETERİNER TAVUKÇULUK DERNEĞİ 24.04.2019

Why were new antimicrobials needed?- Residues in food

- Changes in the beneficial/pathogenic microorganism ratio in the gastrointestinal system

- Suppression of the immune system

- Host organ toxicity

- The emergence of environmental problems

- Resistance development (WHO says «Currently, every year, 700,000 patients die globally due to antimicrobial resistance. It has been estimated that this death toll will increase to 10 million by 2050.)


In this presentation- Since this topic is highly detailed, alternative approaches targeting bacterial infections will be discussed.

- The differences between the classical antimicrobials and the alternatives that are described here are more likely to target specific microorganisms and not to target non-target organisms. (narrow spectrum).

For simplicity, strategies have been arranged in three categories:

(i) naturally occurring alternatives,

(ii) synthetically designed strategies,

(iii) biotechnology-based strategies.


Naturally occurring alternatives- Probiotics, prebiotics, enzymes, organic acids

- Bacteriophages,

- Bacteriocins,

- Antimicrobial peptides,

- Predatory bacteria,

- Fecal Transplantation Therapy


Bacteriophages or phages (bacterial viruses)“Viruses that can be found in any environment that do not infect mammalian or plant cells and only infect bacterial cells all living beings are exposed to at high ratios through water or food”

or

“Viruses that infect bacterial cells by transferring their own genetic material (DNA or RNA)”


Bacteriophages - Not a new concept (1896-Why does not Vibrio cholera live on the Gange River? starts )

- First clinical applications: they were introduced to treat dysentery in humans and poultry typhus caused by Salmonella gallinarum, in 1915 and 1917.


Bacteriophages Interest in bacteriophages decreased with the introduction of conventional antibiotics (Excluding former Soviet countries - like Georgia-Poland). They came up with the resistance again.

To date, there are very few clinical studies performed in humans and accepted by health authorities such as the FDA and EMA.


Complex pyobacteriophage

Table 1. Bacteriophages That Are Approved for Human UseProduct Company Condition Phase (status)

ListShield Intralytix (USA) Food industry (Listeria monocytogenes) Approved

EcoShield Intralytix (USA) Food industry (Escherishia coli O157:H7) Approved

SalmoFresh Intralytix (USA) Food industry (Salmonella spp.) Approved

Septaphage (tablet) Biochimpharm

(Georgia)

Treatment and prophylaxis of bacterial purulent–inflammatory infections

(multiple microorganisms)

Approved

Phagyo (liquid) Biochimpharm

(Georgia)

Treatment and prophylaxis of bacterial purulent–inflammatory infections

(multiple microorganims)

Approved

Phagedys Biochimpharm

(Georgia)

Treatment and prophylaxis of dysentery (Shigella) Approved

Phagetyph and

Phagesal

Biochimpharm

(Georgia)

Treatment and prophylaxis of enteric fever and salmonellosis

(Salmonella)

Approved

Pyo-Phage Eliava Institute

(Georgia)

Urogenital infections; pyo-inflammatory gynecologic diseases;

enteric infections; dysbacteriosis, surgical infections

Approved

Intesti-Phage Micro-gen

(Russia)

Bacterial dysentery; salmonellosis; dyspepsia; dysbacteriosis;

enterocolitis, colitis

Approved


Bacteriophages - Intravenous or intraperitoneal phage applications?

The intravenous treatment route was not found to be ideal since it is impossible to ensure that the bacteriophage solution is completely free of pyrogens. However, intraperitoneal route is possible.

- Is it possible intranasally route?

The delivery of the PAK-P1 phage and the SS-phage intranasally in that order in mice to treat pulmonary infections caused by Pseudomonas aeruginosa and Klebsiella pneumoniae isrelatively less effective than intraperitoneal delivery.

- Therefore, choosing the delivery route is also an important aspect of phage therapy


Bacteriophages - In contrast to antimicrobial treatment, phage therapy targets specific bacteria, which minimizes their effects on untargeted bacteria.

- Empirical studies claim that phages could also be of the narrow or broad spectrum and that this range effect is mostly related to the phage titer, and makes it important to measure the sideeffects of phage therapy on commensal bacteria.

- Another important factor is the potential of the microorganism to become resistant to these specific phages.

However, this condition causes a temporary problem for the treatment. The development of resistance can be reduced by using a cocktail containing more than one phage.


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https://www.ampliphibio.com/science/how-bacteriophage-work

How Bacteriophages attack and kill bacteria?

Bakteriyofajlar- Combination: Some researchers argue that treatment will be more successful and resistance will be reduced if phages are used in combination with antibiotics instead of being used separately.

For example, phages combined with antibiotics may eradicate biofilm-forming Klebsiellapneumoniae strains and prevent resistant variants from emerging in vitro. This activity is known as phage-antibiotic synergy (PAS).

Some in vitro combinations

- The combination of T4 phage +cefotaxime against E.coli biofilms,

- The combination of phage MR-5 and tetracycline, linezolid or ketolide (telitromycin) antibioticsagainst MRSA,

- The combination of ceftriaxone and three phages against P. aeruginosa

- The bactericidal effects of penicillin and gentamicin against Streptococcus pneumoniae increase in the presence of phage lysin Clp-1.


when compared to conventional antibiotic treatments phage treatments have the following advantages

- They are widespread in the environment, Phage particles are specific to certain types or strains of bacteria depending on the receptor type that recognizes them. No adverse effect on eukaryotic cells.

- They penetrate into the skin well, hence they can reach chronically infected wounds that are located deeper in the skin. Phage particles can easily pass the blood-brain barrier, and they can be used to treat bacterial infections of the central nervous system,

- They exponentially grow inside the bacteria and therefore, accumulate in high concentrations as long as the bacteria are in the infected area,

- There is no need for continuous phage administration because the phage particle in the target bacteria will continue to replicate until the bacterial load drops to levels where they are no longer dangerous for the organism in question,

- They can penetrate through bacterial biofilms well,

- Procedures to isolate new bacteriophages are simpler and more economical than developing new antibiotics,

- They offer a safe therapeutic option for patients with allergies to antibiotics.


when compared to conventional antibiotic treatments phage treatments have the following disadvantages

- It is necessary to identify the infectious agent causing the infection and its specific phage needs to be isolated from the environment,

- When phages are administered systematically, they induce the immune response that leads to antibody production, and this response reduces the effectiveness of antimicrobial treatment,

- Its ideal route of administration, optimal dose, frequency of administration and average treatment duration have to be determined beforehand,

- The new genes of some bacteriophages need to be identified in an organized way,

- Bacteria can become resistant to phages by means of different mechanisms,

- Inadequate purification during phage preparation may manifest as inadequate stability and low viability rates,

- The heterogeneity and species-specific mechanisms of phages have not been explained entirely by scientific authorities yet.


Use of phages is limited? - 1. To perform phage therapy, the bacterial pathogen must be identified correctly after which its safety must be determined by assessing its susceptibility to current phages in vitro.

-2. Although phages have a promising future as alternatives to antimicrobials, there are a limited number of controlled studies towards their safety and effectiveness

- 3. Perhaps, the most important obstacles are the lack of a specific regulatory framework to evaluate individual therapies and the difficulty in registering patents.

It is believed that these uncertainties might cause the drug industry to hesitate from investing inphage development research efforts.


Antimicrobial Peptides (AMPs)

- AMPs are low molecular weight proteins that are secreted continuously or in response to induction from epithelial cells, liver, lymphoid tissue, phagocytes, gills and skin of mammals, plants and insects, they usually contain less than 100 amino acids, and their molecular weight varies between 1 to 5 kDa.

- They kill microorganisms directly, and

- are also regarded as protective components of the host defense system and the natural immune response. In other words, these peptides stimulate the immune system to act against invasive pathogens.



Antimicrobial peptides isolated in different groups of organisms by using proteomics and peptidomics. On the left side all classes were described. A MALDI ToF is represented in the centre of figure. Reference: Franco OL (2014). J Proteomics

Bioinform S8: 001. doi:10.4172/jpb.S8-001

Antimicrobial Peptides (AMPs)AMPs carry out the following functions;

- They stimulate the accumulation of immune cells (neutrophils, macrophages, and lymphocytes) in the infected area,

- They neutralize lipopolysaccharide endotoxins produced by gram negative bacteria,

- They stimulate angiogenesis,

- They act as immunomodulators and control the immune system response to certain microorganisms,

- They have anti-inflammatory characteristics.



- It has been reported that AMPs have inhibitory effects on pathogenic organisms, and a broadspectrum of activity including some yeasts and filamentous fungi (Broad spectrum).

- They might have antibacterial, antifungal (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Pasteurella haemolytica, Rhodococcus equi, Listeria spp., Salmonellaspp. and Bacillus spp. ), and in fish antiparasitic (against the protozoan ectoparasiteAmyloodinium ocellatum) effects even at micromolar doses and that they prevent fooddecomposition

- Today it is seen that they are widely used to prevent infections caused by some microorganisms and to strengthen the immune system mostly in livestock and fish.



- Despite the many advantages of AMPs until today only a few AMPs have been approved by the FDA and the EMEA and only for topical use in humans.

Because high concentrations of AMPs are required in the infected area to obtain therapeutic effects in in vivo clinical studies. However, such high concentrations are close to toxic doses.

AMPs can be filtered through the kidneys easily because they are small molecules and therefore their half-lives are rather short

- Although AMPs are drawing increasing attention as alternatives to traditional antimicrobials, some studies have reported that they are not good candidates for treatment because they are toxic to mammalian cells and that similar bacteriocins have been developed due to this reason.


Table 2. Table 2. Table 2. Table 2. Primary advantages and disadvantages of antimicrobial peptides

Some Antimicrobial

peptids

Advantages Disadvantages

Buforin II

Pleurocidin

Dermaseptin

Sekropin P1

Defensin

Pyrrocorocin

Bactenecins

Papiliocin

-Immunomodulatory

-Anti-inflammatory

-Bactericide

-Production and

purification are expensive

-Storage conditions are

difficult.

-Toxic on eukaryotic cells


Bacteriocins- These compounds are defined as peptides generally secreted by bacteria and synthesized by small ribosomes.

- Bacteriocins act by inserting themselves into the plasma membranes of target bacteria, forming pores and causing lysis.

- Bacteria can produce bacteriocin in nearly every generation, and some estimates suggest that at least 99% of all bacteria produce at least one bacteriocin.

- It is supposed that most of the commensal bacteria produce endogenous bacteriocin and that their use as alternatives to antimicrobials could be of potential use.



Bacteriocins - It is now widely used in food preservation

The most widely used bacteriocins as biopreservatives in the food industry: Bacteriocins fromLactic acid bacteria or “lantibiotics”. These bacteria occur in cheese, yogurt, and other fermented milk products and are generally accepted to be safe.

- For example: Nisin A has bactericidal effects and it is used as a food preservative in more than fifty countries.

- Some LAB bacteriocins can be used to treat infections caused by Helicobacter pylori, E.coli and Salmonella in the gastrointestinal canal

- Enterocin RM6: isolated from Enterococcus faecalis found in raw milk. It has been shown that this bacteriocin prevents the proliferation of Listeria monocytogenes, Bacillus cereus and MRSA strains.


Why are they widely used as biopreservatives in food?

- Antimicrobial activity,

- Since they are colorless, tasteless, odorless and heat resistant, they can be used in meat and milk products which are processed at high temperatures,

- They can be added to foodstuffs alone or in combination (such as nisin and pediocin),

- They do not cause adverse effects when they are used with a variety of substances with defined synergistic effectiveness (such as AMPs, prebiotics, proteins, salt, nitrite, organic acid, chelating agents, essential fats) may be counted among the reasons

- Therefore, their characteristics are more suitable for them to replace chemical preservatives in the preservation of normal and fermented foods (biopreservatives) and to prolong their preservation than they are for them to be used in the prevention of infectious diseases.


Bacteriocins in infections- Some lantibiotics such as nisin A and F, mersacidin, mutacin 1140, lactacin 3147 and pediocinAcH/PA-1 that are commonly used in the food industry as biopreservatives can also be effective against MRSA and vancomycin-resistant enterococcus strains and they could potentially be used in the treatment of multiple drug resistant and other bacterial infections

For example, nisin F were found to inhibit the bacteria when it is injected into mice infected with Staphylococcus aureus within at least 15 minutes

- And also that nisin A could be used as an alternative to antibiotics in women with mastitis caused by Staphylococcus aureus

- Mersacidin that is synthesized from Bacillus spp. is effective against some strains of MRSA and that it eliminates these bacteria from the nasal mucosa in mice.



Bacteriocins- Resistance development is difficult.

For example in routine use, it has been observed that nearly no resistance develops even with the widespread use of nisin A.

This is due to the narrow spectrum of effect.

- Thuricin CD kills C.difficile selectively, and it does not effect non-pathogenic microbiota .

- Resistance to bacteriocins could only develop in in vitro conditions. In one study, resistance to bacteriocins were found to develop in E.coli and Listeria monocytogenes strains when they were exposed to bacteriocins at increasing concentrations over long periods

- Therefore, in order to limit the development of resistance, careful and controlled practices should be implemented and when needed bacteriocin cocktails should be used just as they are in phage therapies.


Why do they have no clinical use?- Drug industry is unwilling to allocate resources to clinical research and the production of bacteriocin containing preparations.

Main reasons;

- the low efficiency of the fermentation procedure,

- the production of unstable products,

- costy and time-consuming purification procedure, and

- unclarified legislations about these products


Combination with other substances?Example 1: Colistin (polymixin E) with Nisin A and Pediosin PA: strong synergy against E.coliO157:H7 and decrease effects of colistin’s neurotoxic and nephrotoxic

Example 2: Lactic acid bacteria bacteriocins pediocin-PA, sackacin P and curvacin A cannot inhibit the growth of E.coli alone, but they increase the inhibitor potency of the antimicrobial peptide pleurocidin by four times.

Example 3: When the bacteriocin nisin A, the antimicrobial peptide and polymyxin B are used in combination they produce a synergy against both gram-positive and gram-negative bacteria (L. innocua HPB1 or E. coli RR1, respectively)

Finally: The combinations of various bacteriocins, antimicrobial peptides, and conventional antibiotics have the potential to increase the variety of treatment options and the efficiency against potential pathogens


Table Table Table Table 3333. . . . Primary advantages and disadvantages of bacteriocins.

Some Bacteriocins Advantages Disadvantages

Nisin A

Mersacidin

Pediocin PA-1

Plantaricin S

Thuricin CD

Enterocin RM6

-Synergistic when used

together with antibiotics

-Low potential for

resistance development

-Production and

purification are expensive

-Legislation is uncertain


Predatory Bacteria- Studies that researched Bdellovibrio and similar organisms (BALOs) as potential alternatives to antimicrobials appear rather promising.

- BALOs are motile deltaproteobacteria that strictly consume gram-negative bacteria to fulfill their need for energy and nutrients

- The genome of many BALOs codes hydrolase enzymes that help digestion and also affect bacterial biofilms. Because biofilm coated bacteria are nearly 1000 times less susceptible to antimicrobials than those found in planktonic cells, biofilms cause challenges during the treatment of infections in both humans and animals. Because BALOs affect bacterial biofilms, they have a therapeutic advantage as an alternative to antimicrobials.

- They enter the periplasm of prey bacteria by forming a localized pore. Upon entry into the prey, they form a hybrid called a bdelloplast. These then often deform into spherical cells due to degradation and remodeling of the peptidoglycan.



Predatory bacteria- BALOs are particularly useful in controlling diseases caused by complex microbial structures that are difficult to be reached by antimicrobials such as polymicrobial infections in patients with cystic fibrosis or periodental infections.

- Clinical research has shown that they can be effective against multiple drug resistant pathogens such as Acinetobacter baumannii, E. coli, K. pneumonia, P. aeruginosa and P. putida, and other persistent infections

- In chicken, it was shown that the oral administration of BALOs reduced the ceacal S.enteriticapopulation and also inflammation in an experimental infection induced by Salmonella enteritica.

- The activity against a wide range of bacteria, low immunogenicity, low toxicity, and negligible propensity to induce resistance make this bacterium a promising candidate for the treatment ofinfections.


Fecal BacteriotherapyFecal bacteriotherapy, or with its current name fecal transplantation therapy (FTT)

- is the procedure of applying feces obtained from healthy donors to patients.

- They can particularly be used in humans to protect the microflora during colonic diseases, in irritable bowel diseases and in the treatment of diarrhea caused by Clostridium difficile.

- FTT is actually an old procedure; it has been used in China to treat intestinal diseases since the fourth century. In the western world, it has been used to treat rumen acidosis in cattle at the beginning of the seventeenth century.

- Although its mechanics are not understood completely, it is accepted that FTT usually helps patients regain the bacterial diversity that is changed by antibacterial treatment, infections or other factors that cause dysbiosis.


Fecal Bacteriotherapy- FDA has approved the use of FTT for the treatment of patients suffering from CDI that is unresponsive to standard therapy.

- It has also shown promise in clearing colonization with multidrug-resistant Enterobacteriaceaesuch as E. coli, Salmonella, Klebsiella pneumoniae, Acinetobacter baumannii, methicillin-resistant S. aureus, and vancomycin-resistant enterococci (VRE). The investigation of the efficacy of FTT against these pathogens in humans is limited.

- However, this treatment method requires donor standardization and measures such as protection against unknown pathogens to succeed.


Antibodies- To fight the invasion of pathogens, the immune system produces antibodies – proteins thatrecognize specific components of the pathogen and neutralize them. Antibodies are thus usefulalternatives for the treatment of intractable bacterial infections.

They could be used to treat bacterial infections either by directly targeting the bacterial surface or indirectly by neutralizing the bacterial toxins and the virulence factors that are responsible for infection.

- Numerous antibodies against staphylococci, P. aeruginosa, Bacillus anthracis, and C. difficile are in various stages of clinical development and, in fact, some of them have already been approvedby the FDA.

- A major drawback of using antibodies for antibacterial therapy is the cost of the production and poor shelf life.


TableTableTableTable 4444.... Antibodies Approved or in Clinical Phases for the Treatment of Bacterial Infections

Antibodies Company Pathogen Target Condition Clinical studies

Bezlotuxumab Merck Clostridium difficile Toxin B C. difficile

associated diarrhea

CDAD

FDA approved

Raxibacumab GlaxoSmith Kline Bacillus anthracis Protective antigen Anthrax FDA approved

Anthim Elusys therapaeutics B. anthracis Protective antigen Anthrax FDA approved

Pagibaximab Biosynexus Staphylococci Lipoteichoic acid Staphylococcal

sepsis

Phase III

Shigamabs Taro

Pharmaceuticals

Shiga-toksin üreten

E. coli

Shiga toxin (tx1 ve

tx2)

Shiga-toksin üreten

bakteriyel

enfeksiyon

Phase II


Synthetically Designed Strategies- Synthetic Mimics of Antimicrobial Peptides (SMAMPs)

- Innate Defence Regulatory Peptides

- Nanoparticles


Synthetic Mimics of Antimicrobial Peptides (SMAMPs)

- Most of these strategies try to overcome the problems of protease lability, toxicity, and the high cost of manufacture of AMPs.

- SMAMPs exhibit strong antimicrobial activity and are resistant to degradation.

- They are able to re-sensitize resistant bacteria against conventional antibiotics.

- They are still under development.

- Brilacidin (Innovation Pharmaceuticals) for oral mucositis and skin infections (Phase II)

- AMC-109 (Amicoat AS) for impetigo and nasal decolonization as antistaphylococ (Phase II)


Innate defence regulatory’ (IDR) peptides- AMPs are also known to modulate the immune system for defense against invading pathogens.

- In an interesting concept, peptides with no antibacterial activity but with antiendotoxin andimmunomodulatory activities were designed.

- Called ‘innate defence regulatory’ (IDR) peptides, they were shown to protect mice from succumbing to severe bacterial and malarial infections, without having any direct antimicrobial activity.

- IDR peptides are a promising alternative to conventional antibiotics, and one of them has completed Phase I clinical trials for the treatment of bacterial infection (SGX 942)


Nanoparticles (Nanoparticles (Nanoparticles (Nanoparticles (NPsNPsNPsNPs))))- The use of nanotechnology in modern medicine has been defined as the greatest engineering innovation of recent times.

- The fact that nanoparticles increase durability, performance, strengthening and flexibility and their unique physicochemical characteristics have particularly increased the demand for their derivative products by modern medicine ever since they were discovered by the health industry

- Meanwhile, the fact that human are continuously exposed to NPs in their work environments may lead to unpredictable health problems. The air pollution they lead to may also disrupt the ecosystem and negatively impact other biological species in the environment.


Nanoparticles (Nanoparticles (Nanoparticles (Nanoparticles (NPsNPsNPsNPs))))

- The use of NPs is an important alternative strategy to antimicrobials.

- NPs usually have a size or 0.2-100 nanometers, and they have a high surface-to-volume ratio. This feature increases their interactions with microorganisms and subsequently their antimicrobial effects.

- The physicochemical and biological characteristics of NPs can be manipulated depending on the procedure selected.

- They may be of organic or inorganic structure. It has also been shown that they are more resistant to negative reaction conditions of inorganic NPs.


Nanoparticles (Nanoparticles (Nanoparticles (Nanoparticles (NPsNPsNPsNPs))))- They have found a field of use in many areas and especially in the development of new diagnostic methods (imaging), targeted vaccines and therapeutic agents.

- Currently, there are commercial NP products suitable for species-specific use in humans and animals.

However, the use of these products has been rather limited due to their undesirable side effects and low bioavailability.

- To eliminate such negative effects and to improve their therapeutic indexes and safety profiles different NP medicine transportation systems (such as polymeric-, solid fat-, inorganic-, ceramic-, carbon-, metalic-, nanoparticles, mycelles, nanoemulsions and liposomes).

-Currently, it has been reported that among more than 200 products designed for targeted treatments in humans, only nearly 30 nanoparticles with anti-neoplastic, antibiotic, analgesic, and anti-inflammatory effects have been developed that have been approved for use in clinical trials.


Nanoparticles (Nanoparticles (Nanoparticles (Nanoparticles (NPsNPsNPsNPs))))- Scientific studies have shown that nanoparticles are effective

- in some tumoral diseases in cats and dogs (breast adenocarcinoma, oral melanoma, hemangiosarcoma, osteosarcoma or soft tissue sarcoma),

- in painful conditions, leishmaniasis and anemia in dogs

- in E.coli infections and Foot-and-Mouth Diseases in pigs,

- in brucellosis and leukemia in cattle,

- in babesiosis and imaging methods in horses,

- in Staphylococcus mastitis and Fasciola hepatica infections in sheep,

- in colibacillosis and Salmonella infections in poultry and bursal infections caused by viruses.

- It can be noted that concerns about the expensive cost, the variability of the sensitivity of NP formulations in different cells, tissues or liquids, and the fact that their species-specific pharmacokinetic and toxicokinetic differences have not been entirely established yet has significantly limited their use in veterinary practice

--4.04.2019 KANATLI SEKTÖRÜNÜN GÜNCEL SORUNLARI-VETERİNER TAVUKÇULUK DERNEĞİ 46

Biotechnology-Based Approaches- Genetically Modified Bacteriophages

- CRISPR-Cas 9


Genetically Modified Bacteriophages

- The advancement in the field of genetically modified phages has been very well reviewed recently.

- For example, bacteriophages were engineered to overexpress lexA3 that represses the bacterial SOS DNA repair system.

E. coli treated with bacteriophages, engineered to suppress the SOS network, became more susceptible to bactericidal antibiotics such as ofloxacin.


Genetically Modified Bacteriophages- Re-sensitizing bacteria.

For example: Temperate bacteriophages were used to deliver genes rpsL and gyrA that rendered E. coli susceptible to two antibiotics, streptomycin and nalidixic acid.

- Silencing the resistance genes in bacteria.

A M13 bacteriophage was developed to silence the two genes (chloramphenicolacetyltransferase and kanamycin phosphotransferase) which gave E. coli resistance.

Numerous bacteriophages are currently used clinically or under clinical trials, Like most otherfields, phage therapy also has several problems to address but the incorporation of newertechnologies might be a way forward.


CRISPR-Cas 9 - Ever since its discovery, the CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 (CRISP-associated protein 9) system has taken the world by storm.

- CRISPR-Cas9 is a genome editing tool that creates excitement in the scientific world. It is faster, cheaper and more accurate than previous techniques and has a wide range of potential applications.

- CRISPR-Cas9 is a unique technology that allows geneticists and medical researchers to modify, add or subtract from various parts of the genome. As it is the simplest, versatile and sensitive method of genetic manipulation that is currently available, it attracts great attention in the world of science.

How was it developed? Some bacteria have a gene regulation system similar to the CRISPR-Cas9 system. Bacteria use this system in response to invading pathogens, such as viruses; like an immune system.

Using CRISPR, bacteria remove parts from the virus DNA and, if they attack again, hide some to help them get to know the virus and to defend it. It is aimed to develop new antimicrobials by utilizing this mechanism.


CRISPR-Cas 9Some authors used bacteriophages or bacterial plasmids to deliver novel RNA-guided nucleases (RGNs) to DNA sequences encoding virulence and antibiotic resistance in carbapenem- resistant Enterobacteriacae and enterohemorrhagic E. coli.

The introduction of RNA-guided nucleases also silenced antibiotic resistant bacteria in a complex bacterial population, which potentially allows for programing or remodeling of the microbiota.

This technology was also applied in resistant S.aureus strains and successful results were obtained. Overall, it can be argued that this technology will be a very good alternative to antimicrobials. However, it is still in its early stages.


TableTableTableTable 5.5.5.5. Major Alternatives to Antibiotics and Their Advantages and DisadvantagesStrategy Advantages over conventional antibiotics Possible disadvantages

Phage therapy - Selective towards specific strains of bacteria- Amenable to genetic engineering

-Immunogenicity- Pharmacokinetics- Release of bacterial endotoxins- Inadequate preparations – failure to remove endotoxins and pyrogenic substances- Resistance development

CRISPR/Cas9 - Can be tuned for a variety of antimicrobial applications- Reversal of antibiotic usage- Specificity towards pathogenic strains

- Expensive large-scale production-Toxicity

Antimicrobialpeptides

- Not prone to resistance development- Broad-spectrum activity is an advantage, depending upon application

- Expensive large-scale production- Susceptible to proteolysis- Toxicity

Bacteriocins - Specificity towards pathogenic strains of bacteria- Resistance to heat and UV

- Expensive large-scale production- Susceptible to proteolysis

SMAMPs - Ease of synthesis- Not prone to resistance development- Broad-spectrum activity is an advantage, depending upon application

- Toxicity- Route of administration

IDR peptides - Work by modulating the immune system- No resistance development as no direct antimicrobial activity

- Expensive large-scale production- Susceptible to proteolysis

Antibodies - Selective towards specific strains of bacteria- Do not damage the microflora

- High cost of production- Poor shelf life



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Ayhan Filazi-Eng-Alternative Antimicrobial Approaches ... · “Virusesthat infect bacterial cells by transferring their own geneticmaterial (DNA or RNA)” 4.04.2019 KANATLI SEKTÖRÜNÜN