Overview bacteria [309162]

This thesis is a take on current antimicrobial resistance by taking a step back into medieval medicine and recreating the ancientbiotic Bald´s eyesalve. This ancient remedy is being described in Bald´s Leechbook from the 10th century. Harrison et al provides a [anonimizat] S. aureus. Fuchs et al took it a step further and proved activity against S. aureus and P. aeruginosa. With our work we hoped to widen the spectrum of antibiotic activity of Bald´s eyesalve by testing it also on E.coli and E. faecalis. To monitor our progress we additionally tested on S. aureus and P. aeruginosa to be able to compare to Harrison et al and Fuchs et al. The preparation was performed in a student’s kitchen using medieval equipment. Today we know about the antimicrobial activity and most of their mechanism of the ingredients of Bald´s eyesalve. What remains unknown is the whole spectrum of its action as well as how these ingredients are functioning in combination

Overview bacteria

Staphylococcus aureus

S. aureus belongs to the genus Staphylococcus together with 44 other species. [anonimizat] 1 μm [anonimizat]. S. [anonimizat]. They cannot move nor produce spores. [anonimizat], glistening and smooth colonies on solid growth media that can range in colour from grey to golden. Unlike the Streptococci the Staphylococci produce catalase which allows them to process carbohydrates into lactic acid and not into gas. [anonimizat] (10% sodium chloride). S. aureus is part of the micro biota in around 20-50% of people and can also colonize the skin. In health care workers these numbers increase up to 80%. [anonimizat], towels, clothes and foods. Especially susceptible to an infection with S. [anonimizat], [anonimizat]. Furthermore it may occur as a secondary infection on top of a viral one. S. aureus causes superficial infections that are characterized by massive suppuration leading to abscesses filled with pus and local necrosis. [anonimizat], furuncles, carbuncles and abscesses. [anonimizat], endocarditis, sepsis and deep abscesses. [anonimizat] (without fever) and toxic shock syndrome that may be fatal.

The virulence factors of S. [anonimizat].

Enzymes:

S. aureus possesses two forms of coagulase that convert fibrinogen into fibrin. A free one that is responsible for plasma clotting and a linked one that gives it the possibility to cover its surface with fibrin deposits. This again alters their potential to be phagocytised and they are the most significant marker of pathogenicity.

It contains four different types of hemolysins. α-hemolysin is capable of acting on eukaryotic cell membranes. β-hemolysin destroys sphingomyelin and is dangerous for red blood cells. δ-hemolysin is indicated in diarrheal diseases produced by S. aureus and γ-hemolysin can lyse leukocytes by forming pores.

Panton–Valentine Leukocidin is an enzyme which is encoded on a mobile phage, has similar actions as γ-hemolysin and destroys white blood cells.

Staphylokinase is responsible for breaking down fibrin networks that normally keep an infection localised.

Leukotoxins destroy the membrane of eukaryotic cells.

Hyaluronidase is produced to increase the mobility of S. aureus. It dissolves the intercellular cementing substance allowing it to more easily infiltrate the body.

Deoxyribonuclease is used for degrading DNA.

Phospholipase helps S. aureus to colonize sebaceous areas such as the face and scalp by transforming lipids into fatty acids. These fatty acids are then utilized in the production of energy.

Penicillinase is an enzyme of antibiotic resistance towards β-lactam antibiotics and are easily transferable between strains thus giving a rapid spread of resistance.

Exotoxins

Pyrogenic exotoxins are bacterial super antigens that lead to a massive immune system activation with secretion of inflammatory cytokines, ultimately resulting in hypotension, shock and then organ failure.

16 types of enterotoxins (A-P) can be produced by S. aureus. They are all heat stable proteins and cannot be destroyed by digestive enzymes. They lead to food poisoning with vomiting and diarrhoea. Being heat stable they survive long cooking even in 100°C water.

Exfoliative toxins also called epidermolytic toxins break up the desmosomal cadherins located in the stratum corneum of the skin producing a disease called staphylococcal scaled skin syndrome (Ritter´s disease). There are two types of exfoliative toxins. Staphylococcal exfoliative toxin A is heat stable and splits the epidermis through the stratum granulosum, having an increased chance of fluid loss with higher risks of secondary bacterial infection. Staphylococcal exfoliative toxin B is heat labile, acts on desmoglein 1, causing splitting in between the stratum spinosum and stratum granulosum.

Alpha toxin also known as alpha-haemolysin forms small transmembrane pores with a diameter of 1 to 2 nm in lipid bilayers. This can cause septic shock if it is released systemically.

Toxic shock syndrome toxin 1 is very similar to enterotoxin F and causes toxic shock syndrome. Symptoms include fever, respiratory failure, multi organ failure and death can occur in up to 30% of patients.

Surface proteins

A polysaccharide capsule that is possessed by multiple strains of S. aureus allows them to bypass polymorph nuclear leukocytes since they cannot phagocytise them. This capsule can only be detected by electron microscopy thus being termed a microcapsule. If this capsule is expressed, these bacteria cannot colonize damaged heart valves probably by preventing adhesion.

Protein A is found in over 90% of S. aureus and is an immunoglobulin-binding protein. It prevents phagocytosis by binding to Fc portion of antibodies (IgG). This is achieved through wrong orientation binding of the antibody on the bacterial surface. Furthermore protein A helps S. aureus to bind to von Willebrand factor making it more infectious at the site of skin entry. It also has a role in staphylococcal pneumonia where it binds to tumour necrosis factor 1 receptors.

Today around 90% of S. aureus possess the ability to produce β-lactamase thus being resistant to penicillin G. Around 65% of it express the mecC or mecA genes making them not only resilient for nafcillin, methicillin and oxacillin but also to penicillins, cephalosporins and carbapenems. Despite this ceftaroline which is a new cephalosporin was developed and has action against methicillin-resistant S. aureus. Its nasal colonization is being tackled by using mupirocin. In the case of nafcillin-resistant staphylococci vacomycin is used. For the treatment of MRSA endocarditis or sepsis linezolid and daptomycin are being deployed. Thestrains that do not possess the lactamase can be treated with penicillin G.

Figure 1: Gram-stain of S. aureus

Enterococcus faecalis

E. faecalis is a Gram-positive bacteria that belongs to the family of Enterococcus and the genus Enterococcaceae. They possess the streptococcal group D Lancefield antigen (a teichoic acid) which formerly classified them as group D Streptococcus. E.faecalis is found as a normal constituent of human intestinal flora. Surviving in this harsh environment is only possible for it by production of lactic acid but also the possibility to get energy out of carbohydrates, citrate, glycerol, malate and lactate. Furthermore E.faecalis is very resistant. It is able to replicate at 10 to 45°C, at decreased and increased pHs, bile salts and in the presence of 6.5% NaCl. In plus it is not destroyed by ethanol or detergents. On a Gram-stain they are usually diplococci or form short chains. E.faecalis is an opportunistic bacterium and causes up to 90% of enterococcal infections. It plays a big role in nosocomial infections especially in intensive care units. If antibiotic treatment for another bacterial infection with cephalosporins, vancomycin or aminoglycosides is given it can selectively promote E.faecalis which can be resistant to them. The consequences are urinary tract infections, soft tissue infections and sepsis. Endocarditis and meningitis are also a possible outcomes. If abdominal trauma is present it may lead to intra-abdominal infections. Treatment of E.faecalis infections is challenging due to their resistance to multiple antibiotic agents. These include most β-lactams, aminoglycosides and even vancomycin. Agent of choice is ampicillin for UTIs. Severe infections such as endocarditis combinations of aminoglycosides and penicillin are used. If vancomycin resistant E.faecalis is the causing agent, linezolid is the antibiotic of choice .

Escherichia coli

E. coli is a Gram-negative, rod shaped, facultative anaerobic bacteria belonging to the genus Escherichia and the family of Enterobacteriaceae. In 1885 this bacteria was discovered by Theodor Escherich and has a size of around 1–3 µm × 0.4–0.7 µm. It shares its family with, Salmonella, Shigella, Klebsiella, Enterobacter, Proteus and Serratia and some others. Enterobacteriaceae have 3 three major antigens being represented by the K antigen (from the polysaccharide capsule), the H antigen (from flagella in Salmonella species) and the O antigen (somatic antigen). E. coli is part of the normal human microbiome but may also cause diseases. Within a few hours after birth the gastrointestinal tract of almost all warm-blooded animals is colonized by it. Its transmission is by food, water or directly from other humans. E. coli has its optimal growth at 37°C and it is not able to form spores. On nutrient agar medium it forms circular, smooth, convex, colonies with sharp borders and a greyish colour. On Eosin Methylene Blue Agar E. coli will give a green metallic colour while maintaining the circular, smooth, convex, colonies with sharp border aspects. If inoculated on a Blood Agar medium some strains can show β-haemolysis. Furthermore it possesses the enzymes tryptophanase positive indole test), lysine decarboxylase and is capable of mannitol fermentation. Additionally is has the ability to process glucose into gas. Almost all strains of E. coli have the enzyme β-glucuronidase which is necessary for breaking down complex carbohydrates. The cell wall of E. coli has an outer membrane that contains lipopolysaccharides underneath which lies the periplasmic space. This space has a peptidoglycan layer and is screened from the cytoplasm by the inner cytoplasmic membrane. On the external cell surface adhesive fimbriae are present. For some stains it is possible to exchange plasmids amidst each other thus increasing their chances of survival. Although being a part of the normal human flora it is capable of inducing disease. Human colonization is facilitated through a gangliozidic receptor that binds the brush border of enteric cells. Furthermore it can attach to the epithelia of the lower urinary tract by using a fimbriae that bind to mannose. In young women E. coli is responsible for almost all first urinary tract infections leading to urinary frequency, pyuria, haematuria, dysuria and flank pain if it ascends into the upper urinary tract. It may also cause diarrheal diseases. These types of E. coli are to be discussed in the chapter of diarrheal disease: EPEC (Enteropathogenic Escherichia coli), ETEC (Enterotoxigenic Escherichia coli), STEC (Shiga toxin-producing Escherichia coli also known as Enterohaemorrhagic Escherichia coli), EIEC (Enteroinvasive Escherichia coli) and EAEC (Enteroaggregative Escherichia coli). EPEC is a major cause of diarrhoea in infants giving them usually self-limited watery diarrhoea and fever. Traveller´s diarrhoea is caused by ETEC. Some strains produce a heat-labile enterotoxin that is similar to the cholera toxin leading to excessive secretion of chloride and water and a concomitant block of sodium reabsorption. Other strains produce a heat-stable enterotoxin that increases fluid production by stimulating guanylyl cyclase in the enterocytes. STEC produce two cytotoxins that are similar to the shiga toxin. The first shiga toxin is identical to the toxin produced by Shigella dysenteriae and the second one share many properties with it. They lead to light non-bloody diarrhoea, a more severe diarrhoea in the form of haemorrhagic colitis and haemolytic uremic syndrome. EIEC produces mostly diarrhoea in children of developing countries and peoples visiting them. This diarrhoea is closely resembling the diarrhoea produced by S. dysenteriae. EAEC is responsible for diarrhoea in industrialized countries, traveller´s diarrhoea and chronic diarrhoea in aids patients. Especially in immunocompromised patients E. coli can lead to much more serious diseases including sepsis and meningitis. In infants it is one of the major causes of meningitis . For treatment ampicillin, sulfonamides, fluoroquinolones, cephalosporins and aminoglycosides are all good choices, but high rates of resistance are appearing. In vitro it was possible to inactivate E. coli using bismuth subsalicylate. Because prevention is better than treatment, caution is advised in concerning the choice of water and food if there are questions about the hygienic and sanitation situation.

Pseudomonas aeruginosa

P. aeruginosa is a rod shaped Gram-negative bacteria that is motile due to a single flagellum and has a size of around 0.6 × 2 μm. It is obligate aerobe and can be singular, in pairs but also in short chains. It belongs to the family Pseudomonadaceae and the genus Pseudomonas. It is oxidase-positive (it can utilise oxygen to produce energy), catalase positive and non-lactose fermenting. P. aeruginosa can have a capsule that allows it to form micro colonies in the lung which are hard to be eliminated by phagocytes. It is ubiquitous in water and soil. When letting it grow on media it easily grows on many different culture media while on MacConkey agar it forms clear colonies due to being non-lactose fermenting. It can give a characteristic smell of grape like odour. Furthermore P. aeruginosa is capable of producing some pigments depending on its strain. These pigments include pyocyanin which gives a blue-greenish colour, pyoverdine which gives a yellow-greenish colour, fluorescent), and pyorubin which gives red-brownish colour. P. aeruginosa strains can produce exotoxin A that inhibits protein synthesis similar in action as the diphtheria toxin. This toxin acts mainly on the liver. Patients who have recovered from a severe pseudomonas infection may develop an antitoxin. Risk factors for an opportunistic infection include damaged skin like in burn patients, patients with neutropenia, the usage of urinary or intravenous catheters and cystic fibrosis. Wound infection caused by P. aeruginosa leads the formation of bluish-greenish pus. Transmission of P. aeruginosa is by hands, foods and flowers as well as respiratory equipment and intravenous fluids. Especially if transmission occurs through respiratory equipment necrotizing pneumonia can be a serious consequence. Ecthyma gangrenosum (haemorrhagic pustule that develops into a necrotic ulcer) is often seen in pseudomonas sepsis. It is rarely seen in patients with sepsis caused by other bacteria. The treatment of P. aeruginosa is quite difficult and should never done with just one antibiotic agent. It is important to test antibiotic susceptibility in every case. Current recommendations include piperacillin together with an aminoglycoside. Furthermore aztreonam, imipenem, meropenem can be chosen. Neutropenic patients profit from the combination of ceftazidime with an aminoglycoside. Because P. aeruginosa is ubiquitous in water special attention should be brought to disinfection and pasteurisation of it, especially if it is to be used in equipment such as respiratory apparatuses. Catheters should be changed frequently and flowers or raw vegetables are prohibited in burn units.

Overview Antibiotics

„Antibiotics are drugs that kill (bactericidal) or slow down (bacteriostatic) the growth of bacteria“. They are ubiquitous in doctors’ offices and are used on a daily basis. Treatment of bacterial diseases would be exceedingly difficult if we wouldn’t have them. The first part of this thesis contains a short synthesis of the current classes of antibiotics.

β-Lactam

Penicillin was the first one antibiotic to be discovered by Sir Alexander Fleming in 1928 when he used the mold Penicillium notatum. The β-lactam nucleus is the characteristic sign which all of this group need to have in their molecular structure. Today this group includes penicillins and derivatives, cephalosporins, carbapenems, monobactams and β-lactam inhibitors.Their mode of action is by binding to penicillin binding proteins inside of the bacteria. These binding proteins are not present within the human cells. Due to its structural similarity with D-Alanyl-D-Alanin it can bind to serine in the active centre of D-Alanin-transpeptidase, which is essential for cell wall synthesis in bacteria. The end result is that the bacterial wall is weaker than before, allowing water to enter into the bacteria. This water is being attracted by the high osmotic concentration inside of the cytoplasm and thus leads to osmotic death. They are therefore considered to be bactericidal. The transpeptidases are more lightly accessible in Gram-positive bacteria since they are located in the periplasmic space. Gram-negative bacteria are protected by their outer membrane, which β-lactams can cross by porin channels. The representatives of penicillins are penicillin G and penicillin V. Penicillin G is acid-labile thus there is no oral administration of it. It can be destroyed by β-lactamase and has no effect on intracellular bacteria. Its main range of action are Gram-positive bacteria and Gram-negative cocci. Nevertheless around 80% of Staphylococci are resistant to it due to the formation of penicillinase. Penicillin V in comparison is acid-resistant and therefore can be given orally. It has the same range of action as penicillin G. A specific Staphylococcal penicillin is flucloxacillin. It is resistant to penicillinase and can be given orally. However it has a bad tissue penetration and is thus only recommended for light infections with staphylococci. The cephalosporins can be classified according to generations. The first generation is against Gram-positive bacteria with only little effect on Gram-negative bacteria. They include cefazolin, cephalothin and cephalexin as examples. The second generation have a lower spectrum on Gram-positive bacteria than the first generation, but they are more active against Gram-negative bacteria. They include cefuroxime, cefoxitin and cefaclor as examples. The third generation are considered to be broad spectrum. They are active on Gram-positive and Gram-negative bacteria although they are best used on Gram-negative bacteria. Ceftriaxone, ceftazidime and cefixime are examples of them. The fourth generation are extended spectrum antibiotics since they have a resistance to beta lactamases. They include cefipime and ceftolozan. The fifth generation are also extended spectrum antibiotics and consist of ceftobiprole and ceftaroline. Carbapenemes have the ability to easily diffuse into the bacteria and are considered to be broad spectrum β-lactam antibiotic. This group includes imipenem and meropenem.Some bacteria have learned to produce a β-lactamase which can inhibit most β-Lactams. Against them β-lactamase inhibitors have an action. Although they alone show almost no antimicrobial activity, in combination with a β-lactam they can overcome this way of resistance. Clavulanic acid is an example of a β-lactamase inhibitor. Another group belonging to β-lactams are the aminopenicillins. These include ampicillin and amoxicillin. They are both acid-stable. It is preferred to give ampicillin on parental route since its enteral resorption is limited. Amoxicillin on the other hand has a better enteral resorption with less gastrointestinal adverse effects. Both of them are sensible to penicillinase. Furthermore aminopenicillins have a strong action against Gram-negative bacteria without acting on Pseudomonas. Their spectrum on Gram-positive bacteria are enterococci and listeria. The group of ureidopenicillins are active against Pseudomonas. They include piperacillin and mezlocillin. Ureidopenicillins are acid-labile and non-resistant to β-lactamase. Their spectrum of action is on Gram-negative bacteria.

Side effects of β-lactam include allergies which are highest for penicillin. Due to the cross-allergy we cannot substitute with other β-lactams. These allergies can be immediate or appear after 8-14 days. Other adverse effects include gastrointestinal afflictions (mostly on oral dose) and coagulopathies. These include a dysfunction of platelets and problems with vitamin K dependent clotting factors. This adverse effect is given by the high biliary elimination of cephalosporins, which kill Bacteroides fragilis and E. coli the main vitamin K producing enteric flora. If aminopenicillins are given to patients with infectious mononucleosis on the tenth day of therapy a maculous exanthema will appear. ß-lactams show interactions with oral contraceptive, anticoagulants, inhibitor of platelet aggregation, NSAIDs, probenecid, bacteriostatic antibiotics. Older cephalosporins have interactions with aminoglycosides, polymyxin B, colistin and high dose loop diuretics. They increase the nephrotoxicity of these drugs.

Macrolides

In 1952 the first macrolide, erythromycin A was isolated from Saccaropolyspora erythrea by J. M. McGuire. Its main purpose was to treat patients with penicillin-resistant S. aureus. Together with oleandomycin and spiramycin it belongs to the first wave of macrolides. They were followed by the second wave which included roxithromycin, clarithromycin, azithromycin and dirithromycin. These were produced to improve the negative features of the first wave, such as being poorly absorbed, bitter taste and chemical instability. Furthermore they were to be used against atypical bacteria. The third wave consists of ketolides. They are semisynthetic derivatives of erythromycin A and are used to treat resistance to erythromycin A. Characteristic for macrolides is a 14-, 15-, or 16- membered macrocyclic lactose rings with unusual deoxy sugars L-cladinose and D-desosamine attached.Compared to penicillins, their antibiotic spectrum is wider and they are frequently used in patients allergic to penicillin. When a new protein is formed by the ribosome, it exits it at the nascent peptide exit tunnel into the cytoplasm. This tunnel is plugged by the macrolides. Now recent studies found macrolides also are highly selective modulators of protein synthesis who can have action on the catalytic centre of the ribosome. They thus have a bacteriostatic effect. Macrolides are mainly metabolised in the liver and eliminated through the bile. So no reduction in dose is necessary when treating a patient with renal insufficiency. Bacteria can rapidly develop a resistance to this macrolides and there is a partial cross-resistance with clindamycin. It is preferred to use clarithromycin, azithromycin and roxithromycin compared to erythromycin because they have a stronger antibacterial effect and a better absorption. When being compared with penicillins, their antibiotic spectrum is wider and they are frequently used in patients allergic to penicillin. It incorporates Gram-positive Streptococcus pyogenes, Streptococcus pneumonia, Streptococcus fecalis, Listeria, Clostridium and Gram-negative Neisseria, Bordetella pertussis, Legionella, Haemophilus and Helicobacter pylori. Furthermore it includes Mycoplasma, Chlamydia and Ureaplasma. They are used acute respiratory tract infections, skin infections, genito-urinary tract infections and are frequently used in paediatrics due to their tolerability. Clarithromycin is part of the combination therapy for the eradication of H. pylori and spiramycin is in usage for toxoplasmosis during pregnancy. Adverse effects include gastrointestinal troubles such as nausea, vomiting, diarrhoea and smell/taste dysfunction. Furthermore they can be hepatotoxic (especially erythromycin), QT-interval prolongation or lead to allergic reactions. Contraindications are allergies to macrolides (cross-allergy) and QT-interval prolonging medication or risk factors for QT-interval prolongation such as bradycardia, hypokalaemia and hypomagnesemia. A lot of interactions are due to the inhibition of cytochrome P450 especially with substrates for CYP 3A4. Together with statins they increase their rhabdomyolysis risk. Tacrolimus has a higher nephrotoxicity and combined with digoxin it raises it´s risk for bradycardia. All substances with QT-interval such as class 1A and class 3 antiarrhythmic, but also some neuroleptic drugs and medication that can lead to hypokalaemia can lead to a higher risk of ventricular arrhythmias. The combination of macrolides with clindamycin or lincomycin is useless because they have a similar mechanism of action and decrease each other’s effect.

Tetracycline

In 1948 Benjamine Duggar, retired professor of plant physiology and economic botany from the University of Wisconsin, published his work with Streptomyces aureofaciens in the Annals of the New York Academy of Sciences. He had extracted aureomycin from the Streptomyces strain. With this a new broad band antibiotic was born. In 1950 Alexander Finlay who worked with his colleagues for Charles Pfizer Co isolated terramycin from the bacterium. Doxycycline was the first from the second-generation semisynthetic tetracyclines that was approved in 1967. In 2006 tigecycline was accredited by the FDA and formed the first of the third-generation. It was custom built to tackle tetracycline-resistant mechanisms.Today most often used are tetracycline, doxycycline and minocycline. Tetracyclines have a bacteriostatic effect. They bind to the 30 s subunit of the prokaryotic ribosome which during translation is responsible for connecting and reading mRNA. This blocks the synthesis of the polypeptide chain. Because they have a low affinity for eukaryotic ribosomes this gives the tetracycline a pharmacological advantage. With the exception of doxycycline and minocycline which have a renal and hepatic elimination, tetracyclines are mostly eliminated through the kidney. Being a broad-spectrum antibiotic tetracyclines are active against Gram-positive and Gram-negative bacteria, but also on Borrelia burgdorferi and intracellular bacteria such as Mycoplasma, Chlamydia and Rickettsia. Because of their frequent usage, nowadays there are high rates of resistance (especially Gram-negative rods) to them. Side effects of tetracycline include gastrointestinal troubles and mucosal damage up till rare pseudomembranous enterocolitis. Due to a high affinity for calcium, they can be incorporated into bone and teeth. This leads to general growth retardation and yellow coloration of the teeth. If a patient using it is exposed to sun it can lead to photo dermatitis. They are furthermore hepato/nephrotoxic. Contraindications for tetracyclines are pregnancy, breast feeding, children until 8 years and heavy liver or renal insufficiency. Interactions consist with antacids, iron, magnesium and calcium. They lead to the formation of complexes or an increase in ph. This also means that tetracyclines are not allowed to consume together with these substances, milk or food. Substrates for cytochrome P450 such as oral antidiabetic, oral anticoagulants, digoxin or cyclosporine A are in competition with tetracyclines thus having an increased effect. If combined with oral contraception it can lead to an increased risk of pregnancy.

Aminoglycosides

In 1944 streptomycin, the first aminoglycoside was isolated from Streptomyces griseus. It was soon followed by neomycin in 1949, gentamicin in 1963 and recently (1972) amikacin was produced as a semisynthetic derivative. All have two or more amino sugars that are linked with an aminocyclitol core. They are highly polar molecules which cross the outer membrane of Gram-negative bacteria by disrupting Mg2+ bridges between adjacent lipopolysaccharide molecules and not by porin channels due to their large size. They are moved by an energy dependent electron transport through the inner membrane. This is a rate limiting step and can be negatively influenced by hyperosmolarity, low pH, divalent cations and anaerobiosis. They now bind to the 30S subunit of prokaryotic ribosomes. Here they disrupt the elongation, not the formation of a polypeptide chain. The abnormal protein may be inserted into the cell membrane. This alters the permeability of it and increases the uptake of more aminoglycosides. Thus they are considered to be bactericidal for proliferative and resting bacteria. At oral route they have minimal resorption leading to sterilization of the enteric flora. If given intravenously they distribute good in the extracellular space and can cross the placenta, but have a bad intracellular, CNS and bone penetration. After minimal metabolisation they exit through the kidney and are reabsorbed there. This provides them with a high concentration in the urine. Aminoglycosides are broad band antibiotics thus acting on Gram-negative and Gram-positive bacteria. But they are non-efficient on Streptococcus, Haemophilus and anaerobes. Side effects include nephrotoxicity which is almost always reversible. It can go from progressive renal insufficiency up to acute renal failure. The next adverse effect is the accumulation of aminoglycosides in the endo- and perilymph, damaging the sensory cells of the auditory system. Initially these changes are reversible but severe damage is irreversible. The symptoms range from hearing defect to deafness and even to balance disorders. Furthermore aminoglycosides can be neurotoxic leading to neuromuscular blocks and diaphragmatic paralysis. Contraindications contain heavy renal insufficiency, internal ear damage, pregnancy and breast feeding. Caution is advised for patients with myasthenia gravis. Interactions are with cephalosporins which increase the nephrotoxicity and other medication that can me ototoxic or/and nephrotoxic (amphotericin B, cisplatin, vancomycin).

Combinations with halothane or non-depolarizing muscle relaxants increase the neuromuscular blocks.

Quinolones

In 1962 George Lesher isolated the first quinolone, nalidixic acid . Since then more members were discovered and in 1998 the Paul-Ehrlich-Gesellschaft divided them into four groups. The first group is made by norfloxacin. The second group is composed by enoxacin, ofloxacin and ciprofloxacin. The third group contains levofloxacin. The fourth group is includes moxifloxacin . Regarding their structure quinolones are heterocyclic containing a bicyclic core. A major role in their activity plays the position 3 that has a carboxylic acid group and position 4 with a carbonyl group. Quinolones convert topoisomerase IV and gyrase into cellular toxins that destroy the bacterial chromosome. DNS gyrase is only found in bacteria. It is needed for the formation of negative supercoils into DNA which is necessary for chromosome condensation. Topoisomerase IV is needed to unlink newly replicated daughter chromosomes otherwise cell division can´t be completed. Furthermore it has the same function as gyrase and relax positive supercoils. In conclusion quinolones block DNA replication thus disabling cell division. Today for Pseudomonas and other multi-resistant Gram-negative bacteria they are the only oral antibiotic applicable. They have a good tissue penetration and also enter into the CSF (except norfloxacin). There is a cross-resistance among quinolones but not with other antibiotics. Adverse effects include gastrointestinal troubles, neurotoxicity, hepatotoxicity (increase in transaminases and alkaline phosphatase). They can lead to leukopenia and tendinitis even till tendon rupture. Quinolones are contraindicated during pregnancy, breast feeding, in children and in teenagers. You can´t use them if you have a tendon disease after quinolone usage. Especially for moxifloxacin QT-prolongation and its risk factors is another contraindication. Quinolones have interactions with antacids, calcium, magnesium, iron and zinc. They all decrease their absorption. If combined with theophylline, coffin, cyclosporine A or oral anticoagulants they block the metabolism of these substance thus potentiating their effect. Group 1 is active on Gram-negative rods and Pseudomonas. Thus they are used for urinary-tract infections. Group 2 has a good activity on Enterobacteriaceae and Haemophilus but weaker action on Staphylococcus, pneumococcus, and Enterococcus. Furthermore they act on atypical bacteria such as Chlamydia, Legionella and Mycoplasma. They have different activity against Pseudomonas where ciprofloxacin has the strongest effect. The 2nd group is used for respiratory tract infections with Gram-negative pathogens, urinary-tract infections, ENT infections, skin and soft infections, gastrointestinal infections and pulmonary anthrax. Group 3 has a better activity on Gram-positive pathogens and atypical bacteria. They have good effects on mostly all pathogens involved in upper and lower respiratory tract infections. Furthermore they are used for skin and soft tissue infections, complicated urinary-tract infection and pulmonary anthrax. Group 4 has similar activity as group 3 but even better actions on Gram-positive bacteria and atypical bacteria. Additionally they act on anaerobes. Considering this they are used for respiratory tract infections and infection of the female genital tract including salpingitis an endometritis.

Glycopeptides

Vancomycin which is produced by Amycolatopsis orientalis was the first glycopeptide to be used by hospitals in 1958. It was soon (1978) followed by teicoplanin. Their structure consists of a core heptapeptide scaffold that contains aromatic amino acids. They bind to the d-Ala-d-Ala dipeptide end of peptidoglycan precursors that are needed for proper synthesis of the bacterial cell wall. The bacterial death occurs through osmotic damage. The peptidoglycan precursors are exposed on the extrinsic side of the cytoplasmic membrane of Gram-positive bacteria. On the other hand Gram-negative bacteria have an external lipopolysaccharide membrane that is impervious for big biomolecules. Thus we can conclude that glycopeptides are selective for Gram-negative bacteria. If they are given orally there is no absorption. They have a bad tissue penetration, do not reach the CSF and are eliminated by the kidneys. Adverse effects include ototoxicity, nephrotoxicity, reversible neutropenia, hypersensitive reactions and even anaphylactic reactions. Contraindications are acute kidney insufficiency, deafness and attention when giving them in combination with aminoglycosides to a patient with decreased kidney function. Interactions with aminoglycosides increase both the nephrotoxicity as well as the ototoxicity. In combination with muscle relaxants they potentiate the muscle relaxation. Attention should be paid to patient with inflammatory gastrointestinal diseases and infections. After oral dosage of glycopeptides they can develop a clinical significant serum concentration. Glycopeptides are used for heavy staphylococcal infections with oxacillin resistance or penicillin allergy. Furthermore they have activity against Streptococcus, Clostridium difficile and Corynebacterium.

Oxazolidinones

In 1978 linezolid was the first member of the oxazolidinone antibiotics. It was introduced for control of plant diseases. It took until the year 2000 when the US Food and Drug Administration approved it as antibiotic. Another member is represented by tedizolid that was approved in 2015. They act by binding to rRNA on its 30S and 50S ribosomal subunits. This blocks the formation of an initiation complex, thus diminishing the length of growing peptide chains and minimizing the rate of protein synthesis. But in contrast to other protein synthesis inhibitors this inhibition ensues earlier. Due to its unparalleled site of blockage there was no demonstration of cross-resistance to other protein synthesis inhibitors until now. They act bactericidal on Staphylococcus and bacteriostatic on Streptococcus and Enterococcus (also Vancomycin-resistant strains). Linezolid is indicated for pneumonia and complicated skin-/soft tissue infections. Tedizolid is only approved for skin-/soft tissue infections. Adverse effects include gastrointestinal troubles, headache, increases transaminases, changes in haemogram and bone marrow suppression. Contraindications are combination with MAO-A or MAO-B blockers, combination with SSRI, tricyclic antidepressants, severe liver-/kidney insufficiency and children under 18 years.

Lincosaminde

This group contains clindamycin. It is used as a reserve antibiotic for therapy resistant Staphylococci and anaerobes. It´s mode of action is similar to macrolides by blocking bacterial protein synthesis. This is why there can be a partial cross-resistance with macrolides. It has a good penetration into soft tissue, bone, placenta and breast milk. But it doesn´t pass into the CSF. It has a very good impact on abscesses because it is concentrated in macrophages and granulocytes. The main metabolism happens in the liver and elimination is done by the kidney and bile. Gastrointestinal troubles appear prevalently as adverse effects but it rarely goes till pseudomembranous enterocolitis. Other side effects include allergic reactions, hepatotoxicity and seldom neuromuscular block. Contraindications are allergies for lincosamine and new-borns. Caution is advised for severe liver insufficiency, impaired neuromuscular transmission, chronic inflammatory gastrointestinal disease and penicillin allergy. Interactions occur with macrolides, oral contraceptives, muscle relaxants and gases used for anaesthesia. Clindamycin is indicated for anaerobe infections such as severe intraabdominal or gynaecological infections but also abscesses. Furthermore it comes into use for staphylococcal bone infections in patients with penicillin allergy or intolerance. Other uses are actinomycosis and CNS-toxoplasmosis in HIV-patients.

Fosfomycin

Fosfomycin intravenously is a reserve broad band antibiotic. Orally it is the first choice for women with uncomplicated cystitis. It acts by blocking pyruvyltransferase which is necessary for cell wall synthesis. It thus acts bactericidal on proliferating pathogens. Fosfomycin has a good soft tissue penetration, enters the CSF and also crosses the placenta. Because it has no chemical relationship with other antibiotics there is no cross-resistance or cross-allergy. Adverse effects are gastrointestinal trouble, Changes in haemogram, increase in transaminases and increase in sodium (1g of fosfomycin contains 4.5 mmol of sodium). The only contraindication is pregnancy. It acts on Gram-negative bacteria such as Neisseria, Enteroobacteriacea, Haemophilus and some anaerobes (not bacteroides). On Gram-positive bacteria it acts on Staphylococcus (including penicillinase producing) and some anaerobes. Therefore it can be used for sensible pathogens involved in sepsis, meningitis, osteomyelitis and abscesses, but only as intravenous reserve antibiotic.

Fusidic acid

It is a reserve antibiotic for severe staphylococcal infections and allergy or therapeutic failure of other common antibiotics that was discovered in the 1960s. The bacteriostatic fusidic acid inhibits protein synthesis by blocking peptide translocation and the disassembly of ribosomes. Being metabolised in the liver it is soon eliminated by the kidneys. It easily enters soft tissue, bone, pus and synovial fluid. We can give it orally, local or intravenous. If given intravenously there is the risk of haemolysis, that’s why it should be given as infusion. Because of local necrosis it can´t be given intramuscularly. Adverse effects include gastrointestinal trouble and blockage of leucocyte migration. It acts on Gram-negative bacteria such as Meningococcus, Gonococcus, Bacteroides but not on Gram-negative rods. Its spectrum of Gram-positive bacteria include Staphylococcus (including penicillinase producing), Streptococcus, Clostridium and Corynebacterium. Fusidic acid is used as reserve antibiotic for severe staphylococcal infection in patients with penicillin allergy or oxacillin resistance. Other indications are osteomyelitis, sepsis, pneumonia and skin-/soft tissue infections. It is usually used in combination therapy.

Nitroimidazole

The main nitroimidazole antibiotic that is in use is metronidazole. It blocks the synthesis of nucleic acids in anaerobe bacteria and protozoa. This leads to the breakdown of DNA strands. It is therefore a bactericidal antibiotic. It has a good tissue penetration and can be given intravenous, orally, rectally or intravaginal. Following a hepatic metabolism it is eliminated by the kidneys. Adverse effects include gastrointestinal troubles such as stomatitis, glossitis or a metallic taste. Others are alcohol intolerance, neurotoxicity and changes in liver function (increased transaminases and bilirubin). Furthermore it demonstrated carcinogenic potential in animal trials. According to this, the maximum therapy length has to be less than 10 days. Contraindications are hypersensitivity for nitroimidazoles, therapy longer than 10 days, pregnancy and caution is advised for heavy liver insufficiency or disturbed haematopoiesis. There are interactions with alcohol, barbiturates, oral anticoagulants, lithium and cimetidine. Metronidazole works on all obligatory anaerobe bacteria with the exception of Actinomyces and Propionibacterium. Here it is used for intra-abdominal and gynaecological infections as well as abscesses. Prophylaxis for colon and gynaecological infections is another usage as well as antibiotic associated enterocolitis. Metronidazole also functions on Gardnerella vaginalis that is responsible for vaginitis. In addition to this it acts on protozoa such as Entamoeba histolytika, Trichomonas vaginalis and Giardia lablia. Therefore it is used in clinics for amoebiasis, Trichomonas infection and giardiasis.

Sulphonamides

In 1939 Gerhard Domagk was awarded with the Nobel Prize for his discovery of prontosil, the first sulphonamide that was used in clinical practice. Today sulfadiazine and suflamethoxazol are in usage. They have bacteriostatic action by blocking the bacterial synthesis of folic acid. Sulphonamides enter very easily into the CSF plus the have a good entry into tissue. They are mostly eliminated by the kidneys. Because there is a rapid progression for resistance, combination therapy is the way to go. Adverse effects include gastrointestinal troubles, allergic exanthemas, nephrotoxicity and for premature babies there is the danger of developing hyperbilirubinaemia. Contraindications are allergies against them, pregnancy, breast feeding, new-borns, severe skin reactions and severe kidney of liver insufficiency. They have interactions with oral anticoagulants, oral ant diabetics, methotrexate, thiopental, Indomethacin and salicylates. Sulphonamides are used together with trimethoprim for Neisseria, Enterobacteriaceae, Streptococcus and Staphylococcus for urinary-tract infections, Typhus, Paratyphus and are first line therapy for Pneumocystis jirovecii induced pneumonia. In combination with pyrimethamine they are used for toxoplasmosis.

Diaminopyrimidine

This group is represented by trimethoprim and pyrimethamin. They are important in the combination with sulphonamide. Their function is by blocking bacterial dihydrofolate reductase which is necessary for the synthesis of folic acid and ultimately purines and thymidine (major components of DNA). So they act bacteriostatic. Adverse effects are similar with sulphonamides, including gastrointestinal troubles, nephrotoxicity and the risk for hyperbilirubinemia. But they show less skin reactions. Contraindications are severe kidney insufficiency (GFR under 10 ml/min), disturbed haematosis and new-borns. Caution is advised in folic acid deficiency, liver insufficiency and mild kidney insufficiency. Interactions are with oral anticoagulants, cyclosporine A, phenytoin and oral contraceptives. They are rarely used for monotherapy because of rapid development of resistance. Diaminopyrimidines are used together with sulphonamides for the bacteria discussed in the chapter sulphonamides.

Cotrimoxazole is a combination of trimethoprim and sulfamethoxazole with a broad band spectrum. There is a symbiotic effect when combining these two bacteriostatic. It increases the spectrum and delays resistance. The optimal combination to increase efficiency is 1(trimethoprim): 5(sulfamethoxazole). Adverse effects include gastrointestinal troubles, nephrotoxicity, risk for hyperbilirubinemia and changes in haemogram. Contraindications are allergies for sulphonamide or trimethoprim and their contraindication. Interactions are with oral anticoagulants, oral antidiabetic, NSAIDs, antacids, barbiturates, diuretics, digoxin and rifampicin. It has a broad spectrum of action including Gram-positive and Gram-negative cocci and Gram-negative rods. These are Neisseria, Enterobacteriacea, Streptococcus and Staphylococcus. It has no effect on Pseudomonas, Bacteroides, Clostridium and Spirochaetes. It is indicated for acute and chronic urinary-tract infection, bronchitis, sinusitis, typhus and paratyphus and drug of choice for pneumocystis pneumonia.

Streptogramine

This is a new group of cyclic peptide-antibiotics, who block bacterial protein synthesis in the same manner as macrolides and lincosamide. Together with them they are combined in the MLS-group. The representatives of streptogramine are quinupristin and dalfopristin. Alone they are only bacteriostatic but combined with each other (called Synercid®), they are bactericidal, especially on Gram-positive cocci. They have to be given over a central venous catheter because they strongly irritate veins. Synercid® is a solely reserve antibiotic and can therefore only be used for vancomycin-resistant Enterococcus facium, nosocomial pneumonia and complicated skin-/soft tissue infection if no other antibiotic shows effect.

Daptomycin

Daptomycin is a member of the cyclic lipopeptides a new antibiotic class. They bind to the membrane of Gram-positive bacteria and form pores through which ions (mostly potassium) exit. This leads to the depolarization of the cell membrane and in the end to cell death. Adverse effects include headache, gastrointestinal troubles and myopathy. They can lead to an increase in creatine kinase and rhabdomyolysis. Regarding this it is advised to avoid other medication that has the risk of myopathies such as cyclosporine A, fibrates and statins. Daptomycin is indicated for complicated skin-/soft tissue infections and infectious endocarditis with or without staphylococcal bacteraemia.

Tigecycline

Tigecycline belongs to the new antibiotic class of glycylcycline that are derived from tetracycline. They bind to the 30S-subunit of ribosomes thus blocking protein synthesis. It is effective against numerous Gram-positive and Gram-negative bacteria including multi-resistant pathogens like methicillin-resistant Staphylococcus or vancomycin resistant Enterococcus. It is not effective against Pseudomonas. Adverse effects are similar to tetracycline and are mostly made up of gastrointestinal troubles. Tigecycline is indicated for complicated skin-/soft tissue infections and complicated intra-abdominal infections.

Antimicrobial Substances:

Allium species

While the recipe clearly states to use garlic (Allium sativum), it is unsure which other allium species should be added. Following the material and methods of Harrison et al we used leek (Allium ampeloprasum) and yellow onion (Allium cepa). Garlic itself has antimicrobial properties, can decrease LDL-cholesterol as well as total-cholesterol without touching HDL-cholesterol, it can reduce blood pressure, inhibit platelet aggregation and is even indicated in the prevention of cancer. . The study by Fuchs Al identified allicin to be the essential antibacterial compound in Bald´s eyesalve. Cavallito proved that allicin is the main antibacterial substance from garlic . Allicin is an organosulfur compounds produced from its precursor alliin with the help of the enzyme alliinase. This process happens when the clove of garlic is cut and crushed. The product is dehydroalanine and allyl sulfenic acid who spontaneously combine and form allicin. Its mechanism of action is still unclear today. It is hypothesized that allicin oxidizes thiol groups that can be present in glutathione or cystein residues. If the thiol of a protein is oxidized this can result in a structural change for instance in a disulfid bond formation. The result is the gain or loss of function of this protein. This affects proteins such as alcohol dehydrogenase, triosephosphate dehydrogenase, hexokinase and succinic dehydrogenase who are involved in primary metabolism. If a thiol containing compounds such as glutathione and cysteine was added to allicin its inhibitory function was negated. Glutathione has a high concentration in mammalian cells in comparison to a low concentration in microbial cells. This explains why allicin only has a low toxic effect on mammalian cells. Furthermore it was shown that allicin has partial inhibitory effect on protein and DNA synthesis. In other studies it was shown that 40 minutes after the addition of allicin there was a 90% inhibition of RNA synthesis. In addition to that it was suggested that mRNA degradation was blocked.

Ajoene were also found in Allium sativum. They showed antifungal, antiviral as well as antibacterial effects. Their antimicrobial actions included E. coli, Klebsiella pneumoniae, P. aeruginosa and S. aureus . Ajoene has two isomers the E- and Z-forms. They are not found in the bulb of garlic but are formed when the garlic pulp is polar solvents. In vegetable oil Z-ajoene is more prevalent, while in alcohol or acetone E-aloene is in higher concentrations. Z-ajoene has a stronger bioactivity while E-ajoene is more stable. It is presumed that ajoene have the same mechanism of antimicrobial activity as allicin.

Thiosulfinate derivatives are direct decomposition products of allicin. They differentiate from each other by the alkyl groups connected to the thiosulfinate group in length of the chain as well as in its branching. They are compounds such as diallyl monosulphide (DAS), diallyl disulphide (DADS), diallyl trisulphide (DATS) and diallyl tetrasulphide (DATTS) were identified to have antimicrobial properties. DAS present in Allium sativum showed activity against fungi and bacteria such as K. pneumoniae and S. aureus. DADS is present in Allium sativum and Allium cepa has antifungal and antibacterial properties including H. pylori, K. pneumonia and S. aureus. DATS present in Allium sativum and Aliium cepa showed antifungal and antibacterial properties including H. pylori and S. aureus. Furthermore it acts on parasites such as Entamoeba histolytica and Giardia lamblia. DATTS is present in Allium sativum and Allium cepa. It has antifungal and antibacterial properties including H. pylori and S. aureus.

Cammue et al depicted in 1995 that Allium cepa has antimicrobial activity on Bacillus megaterium and Sarcina lutea both Gram-positive bacteria but no effect on Gram-negative bacteria. It furthermore displayed antifungal actions. This action was implicated to be due to Ace-AMP1. Other studies showed that kaempferol and quercetin who are major flavonoids produced by Allium cepa have activity against E. coli, B. cereus, S. aureus and P. aeruginosa. They hypothesised that quercetin was inhibiting DNA gyrase while kaempferol blocks the NorA pump in S. aureus which is a multidrug efflux pump.

In the roots and bulb of leek as well as in garlic high concentrations of N-feruloyltyramine and N-feruloytyrosine were found. Both of them were found to have activity against Fusarium culmorum a fungal plant pathogen.

Copper

Copper was already mentioned in one of the oldest books of humankind, the Smith Papyrus. In this ancient egyptian text, that originated at around 2600 B.C. copper was used for sterilization of chest wounds and to sterilize potable water. In 2008 copper was registered as antimicrobial surface by the Environmental Protection Agency. Copper is capable of killing E. coli in around 65-75 minutes, P. aeruginosa in 180 minutes and even strains of methicillin-resistant S. aureus in 180 minutes. This killing was even accelerated when a higher copper concentration, a higher humidity and an increased temperature were present. On the other hand a heavy copper oxide layer and inhibitors of corrosion decreased its effectiveness. Even bacteria that form endospores such as Clostidium difficile which can resist to radiation, heat, denaturing chemicals and aridification were destroyed by copper. In 3 h 99.8% of endospores were killed by solid copper and in 24-48 h complete Inactivation was achieved. Parra et al showed that copper surfaces were capable of rapidly dealing with food borne pathogens such as Listeria monocytogenes and Salmonella enterica and even destroyed them when the copper surface was covered by organic matrix from poultry carcasses. The clear mechanism by which copper destroys bacteria is still unknown. P. aeruginosa that lacked the cinR gene responsible for a copper-responsive regulator as well as well as a strain that lacked the cinA genre responsible or an azurin-like protein that is indicated in copper resistance were both more easily destroyed than any wild types. For E. coli the lack of three systems was involved in being more rapidly destroyed. The first system was cueO that is necessary for a periplasmic copper oxidase. The second system cues encodes a periplasmic copper efflux pump and the third was copA that is responsible for a cytoplasmic copper extrusion pump. It is speculated that copper kills the cells in four stages. In the first stage the copper dissolves from its surface and starts to damage the cellular membrane. In the second stage there is a loss of cellular membrane potential leading to ruptures and influx of copper ions. In the third stage there is formation of reactive oxygen species by the copper ions leading to even more cellular damage. In the fourth stage there is DNA fragmentation followed by cell death. The Asklepios Hospital,Hamburg Germany started a test run to decrease the bacterial load in the hospital and replaced door knobs and touching surfaces with brass (copper and zinc mix). They reported a 63% reduction in bacterial load on copper surfaces compared to their aluminium control.

Bile

Bile is a watery solution composed of inorganic and organic compounds. It has a yellow to greenish colour and contains phospholipids, cholesterol, bile acids and the pigment biliverdin. 50% of the organic compounds is represented by bile acids. Cholic acid and chenodeoxycholic acid are primary bile acids produced by the liver from cholesterol. Lithocholic acid and deoxycholic acid are secondary bile acids that resulted from modification of primary bile acids by bacteria. Bile is secreted by the liver by the pericentral hepatocytes. They release it into the bile canaliculi from where it flows into the collecting bile ducts and then into the hepatic ducts. The right and left hepatic duct join up to form the common hepatic duct. At the junction with the cystic duct that comes from the gall bladder they form the common bile duct. The common bile duct unites with the main pancreatic duct forming the hepatopancreatic ampulla. This opens into the duodenum by the major duodenal papilla. The bile is continuously secreted by the hepatocytes and is stored in the gall bladder. Furthermore the gall bladder concentrates the bile around 5-10 times. It removes electrolytes and water plus acidifies it by usage of the Na+/H+ exchange. Bile plays an essential role in digestion of fats and the excretion of substances that can´t be eliminated through the kidneys. But it also has a role in controlling the gut bacteria. Hofman et al show that in patients with liver cirrhosis due to a decreased bile secretion, bacterial overgrowth can occur in humans as well as in animals. They further stated that bacterial overgrowth was observed in animals after ligation of the bile duct. When conjugated bile acids were fed to these animals the bacterial overgrowth was remitted as well as a lower level of bacterial translocation into lymph was observed. Experiments with erythrocytes showed that the main mechanism of destruction of bile is by acting on the membrane. This can be concluded because erythrocytes have no cell organelles and no mechanism for the uptake of bile but still there was haemolysis after exposure to bile. Under the electron microscope the erythrocytes were shrunken and void. Bile salts act on the membrane lipids and rapidly dissolve them. The membrane proteins are also destroyed. At physiological pH the conjugated bile acids are fully ionized thus being lipophobic and can´t cross the cellular bilayer without active transport system. Unconjugated bile acid are lipophilic and easily cross the membrane. This passive transport is dependent on the hydroxy groups. Dihydroxy bile acids found in porcine bile cross more rapidly than trihydroxy bile acids from bovine bile. Furthermore bile acids were indicated in damaging DNA and changing the structure of RNA in bacterial cells. In E. coli it was shown, that bile salts stimulated the genes micF and osmY, which are involved in the response to oxidative stress which can be induced by the production of free oxygen radicals by bile. Iron and calcium are also targets of bile. It links to them forming chelates thus decreasing their intracellular concentration and consequently leads to a delay in bacterial growth.

Wine

It is widely known that the consumption of moderate amounts of wine can have beneficial effects on health. These may include reduction in the risk of coronary heart disease and even in some forms of cancers. This is given by the antiradical and antioxidant properties of wine mostly constituted by a high polyphenol amount. In 1995 Weisse et al. showed that red and white wines were able to reduce E. coli, Shigella sonnei and Salmonella enteritidis within 20 minutes to 0 Colony forming units. Dilutions of white wine was superior to red wine dilutions although weaker than the undiluted wine. They also found that the antibacterial effect was not due to the ethanol in the wine. They speculated that its antimicrobial action was because of polyphenol that was released by fermentation and acts on bacteria at a lower pH. Sugita-Konishi et al. disproved this hypothesis by showing antibacterial effect of wine on S. enteritidis, E. coli and Vibrio parahaemolyticus after removal of the polyphenol. They concluded that the antimicrobial effect of wine is due to acetic acid since the effect was lost after evaporation of acetic acid. Daglia et al. tested the wine on several oral Streptococci and found that the antibacterial effect was not only due to acetic acid but also due to lactic, citric, succinic, tartaric and malic acid. The antibacterial mechanism is not known though it can be speculated that they act on the cell wall. This conclusion can be drawn since peracetic acid that is formed by the combination of acetic acid and hydrogen peroxide can destroy proteins and enzymes as well as it makes the well wall more permeable by splitting sulphur and sulfhydryl.

Mechanism of bacterial resistance

Figure 2: Spreadsheet CDC Mechanisms of bacterial resistance

In a review on antimicrobial resistance from 2016 it was estimated that around 700,000 people die every year from drug resistant strains of common bacterial infections . According to WHO there was an incidence of 558 000 new patient in 2017 with resistance to the most efficient first line therapy which is rifampicin. 82% of these cases were with multidrug-resistant tuberculosis. They furthermore state that approximately 55% of multidrug-resistant tuberculosis were successfully treated. In India, antibiotic-resistant neonatal infections cause the deaths of nearly 60,000 new-borns each year . It is estimated that in the year 2050, 10 million live per year are at stake due to drug resistant infections if there is no change in the current increase of resistance. With the spreadsheet from the CDC we can try to understand the five mechanism of antibiotic resistance that result in these drastic numbers.

Firstly is by inactivation of the antibiotic. Two distinct mechanisms have to be discussed here:

Obliteration of the antibiotic molecule is done by hydrolysis. This mechanism is performed by β- lactamases. They target the β-lactam ring at its amide bond and remove it, thus making the active site ineffective. Today over 1,000 various types of β-lactamases are known with rising tendency. They are grouped by two different systems, the Ambler classification which is based on molecular structure and the function based Bush-Jacoby classification. The Ambler classification has four groups. Group A are extended spectrum β-lactamases. These organisms are resistant to penicillins, 1st, 2nd and 3rd generation cephalosporins as well as monobactams. These organisms are still sensitive to cephamycins, carbapenems and the combination of a β-lactam combined with a β-lactamase inhibitor such as clavulanic acid or sulbactam. Group B are metallo beta-lactamases. They are resistant to the same antibiotics as group C furthermore including carbapenems. Group B are not sensitive to the combination of β-lactam with a β-lactamase inhibitor. Group C are resistant to to all penicillins and cephalosporins including cephamycins but are still sensitive to carbapenems. Similar to Group B they are not sensitive to the combination of β-lactam with a β-lactamase inhibitor. Group D are oxacillinases. They have the same antibiotic spectrum as group C but also include carbapenems.

Chemical alterations of the antibiotic is the second possible mechanism. Here the antibiotic avidity for its target site is decreased thus increasing the bacterial MIC. This can happen by three mechanisms. Firstly acetylation for aminoglycosides, streptogramin and chloramphenicol. Secondly phosphorylation for chloramphenicol and aminoglycosides. Thirdly by adenylation for aminoglycosides and lincosamides.

The second mechanism is by changing the antibiotics target. This is done either by target protection or my modification of the target site. Target protection is seen in the resistance to tetracycline, fluoroquinolones and fusidic acid. Best described is it for tetracycline. The bacteria produce tetracycline resistance determinants in two forms M and O. They displaces tetracycline from its ribosomal binding site and furthermore change the structure of the target site so tetracycline can´t rebind to it. This allows protein synthesis to continue working and the bacterium survives. Modification of the target site can happen by point mutation in the encoding genes or by enzymatic changes in the binding site itself. The resistance to rifampin is an example of point mutation. Rifampin acts on the β subunit of RNA polymerase blocking transcription. This β subunit is encoded by rpoB gene. Modification of the target site can happen by a point mutation, by enzymatic alteration of the target site or by complete replacement of the target. If a point mutation that leads to amino acid substitution in this gene happens it decreased the affinity of rifampin for its target. This is only a slight change that permits the transcriptase to continue its work but confers antibiotic resistance. The point mutation mechanism is also seen in the resistance to fluoroquinolones and oxazolidinones. The enzymatic alteration of the target site is best represented by the resistance to macrolides. The bacteria have the erythromycin ribosomal methylation gene (erm gene). This leads to methylation of a specific adenosine residue of the 23rRNA of the 50S ribosomal subunit. This blocks the binding capability of macrolides, streptogramin B and lincosamides which all have the same binding site, thus the bacteria are cross-resistant.

Complete replacement of the target is seen in methicillin resistance of S.aureus. The resistant strain possesses the mecA gene which it most likely acquired from Staphylococcus sciuri. This gene encodes Penicillin-binding-protein 2a that replaces the transpeptidase function of PBP but not the transglycosylase, for this native PBP is required. PBP 2a has a low binding capacity for β-lactams, with exception of the 5th generation on cephalosporins. Another replacement resistance is seen in vancomycin resistance. Glycopeptides act on D-alanine-D-alanine that blocks PBP linkage and ultimately cell wall synthesis. The presence of the van gene allows the bacterium to replace the last D-Ala with either D-lactate or D-serine. Furthermore the normal D-Ala-D-Ala precursors are removed to prevent binding of vancomycin. Vancomycin resistance is seen especially in enterococci. The transfer of this van gene from enterococcus to S. aureus has already been described in 2002.

The third mechanism is by changing its own metabolic pathways so that it doesn’t have to use the antibiotics target. Enterococci for example have the possibility to use exogenous tetrahydrofolic acid. This increased the MIC of trimethoprim/sulfamethoxazole by 25-fold. Co-trimoxazole acts by inhibiting folate synthesis inside of bacteria .

The fourth mechanism is to decrease the permeability of the cell wall for the antibiotic. This mechanism involves porins that are barrel proteins crossing cell membranes. This is seen in the outer membrane of Gram-negative bacteria and in Mycobacteria. They act as pores and allow the passive diffusion of hydrophilic molecules. There is a natural resistance of Gram-negative bacteria for vancomycin which is too big in size to cross these porins. Reduction in permeability can be achieved by three mechanisms. It has been described a change in the type of porin, a decrease in the number of porin that are expressed in the cell membrane and a decrement in the function of the porins. P. aeruginosa uses the porin OprD that functions for the absorption of imipenem. Mutation in the gene encoding OprD was shown to lead to resistance. This mechanism can stand alone or be combined with an efflux pump or a carbapenem-hydrolyzing enzyme. K. pneumoniae uses OmpK35 and can exhibit an alteration to OmpK36, which is a channel of smaller size. The result was a 4 – 8 fold reduction of susceptibility for β-lactams.

The fifth mechanism is the usage of membrane efflux pumps that are able to expel the antibiotic molecule thus decreasing their intra bacterial concentration. These efflux pumps can be specific for one antibiotic (Tet for tetracycline or Mef in Pneumococcus for macrolides) or they can be broad spectrum as seen in multi-drug-resistant bacteria. They are fused into five families based on their structure and energy usage

major facilitator superfamily (MFS)

small multidrug resistance family (SMR)

resistance-nodulation-cell-division family (RND)

ATP-binding cassette family (ABC)

multidrug and toxic compound extrusion family (MATE)

The Tet efflux pump (part of MFS family) is responsible for resistance to tetracyclines. Currently 20 different Tet genes are described with the majority being present in Gram-negative bacteria. They expel tetracycline and doxycycline using a proton exchange system without having an effect on tigecycline or minocycline. Furthermore multi-drug-resistant efflux pumps such as AcrAB-TolC in Enterobacteriaceae are able to expel tetracyclines. Resistance to macrolides is given by the Mef pumps. These are found in Gram-positive bacteria foremost S. pyogenes and S. pneumoniae .

Introduction

This licence thesis focuses on the reproduction of a medieval antibiotic substance that was described in Bald´s leechbook written in the tenth century. It consists of Allium sativum (garlic) mixed with another Allium species which is either Allium cepa (yellow onion) or Allium ampeloprasum (leek). This is combined with copper ox-gall and white wine and then rests for nine days. The book describes that it is to be used on a wen which is a staphylococcal infection of the eyelash follicle today known as a sty. There are only a few studies that focus on this topic. Notable are Harrison et al. with “A 1000 year Old Antimicrobial Remedy with Antistaphylococcal Activity” and Fuchs et al. with “Characterization of the antibacterial activity of Bald´s eyesalve against drug resistant Staphylococcus aureus and Pseudomonas aeruginosa”. We used this study as template since the original text is written in old english and they give a translation of it. Our goal was to see if a medical student was able to recreate such a functional antibacterial substance. If we succeed with this, and in act prove that Bald`s eyesalve is bactericidal, its usage could remerge in the world where not everybody has access to antibiotics, but has its basic ingredients. Furthermore these studies tested it even with success on multiple antibiotic resistant bacteria that have become a rampant problem in today’s world. Because we didn´t have access to multiresistant bacteria we extended the spectrum of Gram-positive bacteria to also include Gram-negative bacteria. Although Fuchs et al already gained positive results on P.aeruginosa we were interested to see if we could replicate these results and wanted to see if other Gram-negative bacteria were also susceptible.

Materials and method

Materials for Bald´s eyesalve

We bought the garlic, yellow onion and leek freshly from a local shop located at the intersection of Strada Louis Pasteur and Strada Zorilor in Cluj-Napoca. For the garlic concentrate we used 100% Knoblauchextrakt from AQUA LIGHT and for the onion concentrate Naturreiner Pflanzensaft Zwiebel from Schoenberger. The white wine was Schwaben Wein Feteasca Regala Vin Alb Demisec (5942063000021) from Recas, 307340, Jud Timis, Romania. We received the fresh pork bile from Metzgerei Der Ludwig a german butcher from Schlüchtern (36381). The dried ox gall powder (Ochsengale Fel Tauri sicc 25g) was bought from Apotheke am Theater 79098 Freiburg, Germany and can be identified by the number PZN- 13359412. The copper plates were Thermal Pad 20x20x1 mm bought from Richter-R over Amazon. The linen cloth was 100% pure linen bought from Axel Suijker Textilagentur Quakenbrücker Landstraße 24 Menslage (49637) Germany. The tests were performed in the microbiology department of UMF Iuliu Hațieganu located on Str. Pasteur, Nr. 6.

Fresh garlic (from local grocery shop)

Fresh yellow onion (from local grocery shop)

Fresh leek (from local grocery shop)

Garlic concentrate

Onion concentrate

White wine (Schwaben Wein from Recaṣ,România)

Bile (pork bile from local butcher)

Dried ox gall powder

Copper plates

Linen cloth

Sterile 125ml container

Manual mortar and pestle

Juice extractor from HOMEVER model GS-353

Batch 1:

This batch contains Bald`s eyesalve prepared with fresh garlic and yellow onion. They were hulled and then chopped into coarse pieces. They were then crushed using a mortar and pestle. We now weighted them to get 25 ml of each compound. According to a study (Fuchs et al, 2018) this equals 14.2 g of garlic and 22.2 g of onion. These were placed in a sterile container and 25 ml white wine, 25 ml bile and 2 copper plates were added to it. It was then placed in a fridge at 4°C for 9 days. We proceeded by pouring it through a linen cloth into a sterile container, to remove any coarse material.

Batch 2:

This batch contains Bald´s eyesalve prepared with fresh garlic and leek. The garlic was hulled and then chopped into coarse pieces. From the leek we only used the green end which was also cut into chunky pieces. They were then crushed using a mortar and pestle. We now took 25 ml of each substance which were 14.2 g garlic and 8.8 g leek according to (Fuchs et al, 2018). These were placed in a sterile container and 25 ml white wine, 25 ml bile and 2 copper plates were added to it. Continued we placed it in a fridge at 4°C for 9 days. We proceeded by pouring it through a linen cloth into a sterile container, to remove any coarse material.

Batch 3:

This batch contains Bald`s eyesalve prepared with fresh leek and garlic concentrate. From the leek only the green end was used. We cut it into small pieces and crushed them with a mortar and pestle. According to Fuchs et al (2018) we weighted 8.8 g and combined them with 25 ml of garlic concentrate in a sterile container. We then added 25 ml white wine, 25ml bile and 2 copper plates. It was then placed it in a fridge at 4°C for 9 days. We proceeded by pouring it through a linen cloth into a sterile container, to remove any coarse material.

Batch 4:

This batch contains Bald`s eyesalve prepared with fresh yellow onion and garlic concentrate. The onion was dehulled and chopped into small pieces and crushed them with a mortar and pestle. We took 22.2 g (Fuchs et al, 2018) and combined them with 25 ml of garlic concentrate in a sterile container. We then added 25 ml white wine, 25 ml bile and 2 copper plates. It was then placed it in a fridge at 4°C for 9 days. We proceeded by pouring it through a linen cloth into a sterile container, to remove any coarse material.

Batch 5:

This batch contains Bald´s eyesalve preparation with garlic and Onion concentrate. We combined 25 ml of each in a sterile container. 25 ml white wine, 25 ml bile and 2 copper plates were added to it. It was stored at 4°C in a fridge for 9 days. We then poured it through a linen cloth into sterile container to remove any coarse material.

Batch 6:

This batch contains Bals´s eyesalve prepared with fresh garlic and yellow onion. They were peeled and crushed with a mortar and pestle so that we achieved 25 ml of each. 2.175 g of dried ox gall was dissolved in 25 ml of water to achieve a concentration of 87 mg/ml. The bile, garlic and onion were placed in a sterile 125 ml plastic container and combined with 25 ml white wine as well as 4 Copper plates. After closing the lid we wrapped it in aluminium foil and stored it at 4° in a fridge for 9 days. We then poured it through a linen cloth into sterile container to remove any coarse material.

Batch 7:

This batch contains Bals´s eyesalve prepared with fresh garlic and leek. They were peeled and crushed with a mortar and pestle so that we achieved 25 ml of each. From the leek we used only the green leafs. 2.175 g of dried ox gall was dissolved in 25 ml of water to achieve a concentration of 87 mg/ml. The bile, garlic and onion were placed in a sterile 125 ml plastic container and combined with 25 ml white wine as well as 4 Copper plates. After closing the lid we wrapped it in aluminium foil and stored it at 4° in a fridge for 9 days. We then poured it through a linen cloth into sterile container to remove any coarse material.

Batch 8:

This Batch contains Bald´s eyesalve prepared with garlic and yellow onion juice that were gained with a juice extractor. Both of them were peeled and chopped into small pieces before being processed through it. 2.175 g of dried ox gall was dissolved in 25 ml of water to achieve a concentration of 87 mg/ml. 25 ml of bile, garlic and onion were placed in a sterile 125 ml plastic container and combined with 25 ml white wine as well as 4 Copper plates. After closing the lid we wrapped it in aluminium foil and stored it at 4° in a fridge for 9 days. We then poured it through a linen cloth into sterile container to remove any coarse material.

Batch 9:

This Batch contains Bald´s eyesalve prepared with garlic and leek juice that were gained with a juice extractor. Both of them were peeled and chopped into small pieces before being processed through it. From the leek only the green leafs were used. 2.175 g of dried ox gall was dissolved in 25 ml of water to achieve a concentration of 87 mg/ml. 25 ml of bile, garlic and onion were placed in a sterile 125 ml plastic container and combined with 25 ml white wine as well as 4 Copper plates. After closing the lid we wrapped it in aluminium foil and stored it at 4° in a fridge for 9 days. We then poured it through a linen cloth into sterile container to remove any coarse material.

Figure 3: Materials used in the third test. From front to back one can see the copper plates, garlic yellow onion, leek, linen cloth, mortar and pestle, dry ox bile powder and the sterile water for its preparation, white wine, 125 ml cups and the food processor.

First Test: 25.6.2019

Pseudomonas aeruginosa ATCC 254992 on Müller-Hinton medium

Escherichia coli ATCC 00201502 on Müller-Hinton medium

Staphylococcus aureus ATCC 254995 on Müller-Hinton medium

We took the P. aeruginosa from its blood agar with a sterile wire and combined it in a sterile test tube with saline solution. Before doing that we checked with a McFarland device that the saline has 0.0 MF. We now achieved a suspension of 0.5 MF. With a cotton swab we went into this suspension and removed extra fluid on the wall of the tube. Proceeding, we moved in zigzag lines over a Müller-Hinton medium. We did this 3 times while turning the plate with 120° each turn to ensure that a wide spread was achieved. Now we waited for 5 minutes to let it dry onto the media. On the Müller-Hinton medium we had marked with 5, 10, 15 and 20 how many microliter we were going to place there. After placing a sterile 6 mm disk at each marking we used a pipet to place the according amount of test substance onto each disk. We did this 5 times for each prepared batch of ancient biotic. They were then placed in an incubator for 18-24 hours. In the same manner we repeated the process for E. coli and S. aureus.

Second Test: 27.6.2019

Pseudomonas aeruginosa ATCC 254992 on Müller-Hinton medium

Escherichia coli ATCC 00201502 on Müller-Hinton medium

Staphylococcus aureus ATCC 254995 on Müller-Hinton medium

We took the P. aeruginosa from its blood agar with a sterile wire and combined it in a sterile test tube with saline solution. Before doing that we checked with a McFarland device that the saline has 0.0 MF. We now achieved a suspension of 0.5 MF. With a cotton swab we went into this suspension and removed extra fluid on the wall of the tube. Proceeding, we moved in zigzag lines over a Müller-Hinton medium. We did this 3 times while turning the plate with 120° each turn to ensure that a wide spread was achieved. We waited 5 minutes to let it dry onto the media. Using a glass pipette we punched 6 mm holes into our media and marked them with 30, 40, 50 and 60. Each hole was filled with the according amount of microliter of test substance. In this second test we only used batches 1 and 2 as well as the wine. They were then placed in an incubator for 18-24 hours. We then repeated the process for E.coli and S. aureus with exactly the same execution.

Third Test: ceased due to COVID-19:

Pseudomonas aeruginosa ATCC 254992 on Müller-Hinton medium

Escherichia coli ATCC 00201502 on Müller-Hinton medium

Staphylococcus aureus ATCC 254995 on Müller-Hinton medium

Enterococcus faecalis on Müller-Hinton medium

We took the P. aeruginosa from its blood agar with a sterile wire and combined it in a sterile test tube with saline solution. Before doing that we checked with a McFarland device that the saline has 0.0 MF. We now achieved a suspension of 0.5 MF. With a cotton swab we went into this suspension and removed extra fluid on the wall of the tube. Proceeding, we moved in zigzag lines over a Müller-Hinton medium. We did this 3 times while turning the plate with 120° each turn to ensure that a wide spread was achieved. We waited 5 minutes to let it dry onto the media. Using a glass pipette we punched 6 mm holes into our media and marked them with 30, 40, 50 and 60. Each hole was filled with the according amount of microliter of test substance. In this second test we only used batches 1 and 2 as well as the wine. They were then placed in an incubator for 18-24 hours. We then repeated the process for E.coli, S. aureus and E.faecalis with exactly the same execution.

Figure 4: McFarland densiometer with suspension of S. aureus at 0.5 MF-units

Figure 5: Batches 1, 2 and 3 after preparation and before filtration. There is visible coarse garlic in batches 1 and 2 while in batch 3 bought garlic concentrate was used.

Figure 6: Batches 6 to 9 before refrigeration. There is visible coarse onion, garlic and leek in batches 6 and 7 compared to the finely ground substances in batches 8 and 9. All four batches contain the same quantity of tested substances.

Figure 7: Batches 8 and 9 after refrigeration and before filtration. Visible is the oxidation of copper in the lower 1 cm. The darkish brown colour in the upper 1 cm is given by accumulation of bile. Both colours were also present in batches 6 and 7 but were not as distinct as in batches 8 and 9.

Results

After 24 h in the incubator we read the results using a ruler. Batch 1 and Batch 2 showed the most effect in test 1 and 2 on P.aeruginosa ranging from 6 to 21 mm of inhibition zones. They furthermore showed some effect on E. coli in test 2 with zones of 7 to 8 mm. In table 1 and 2 are no zones of inhibition written down for S. aureus, this is because when reading the results we realized that in every sample there were colonies growing till the centre. We therefore couldn´t measure their zone of inhibition. Nevertheless it is worth to mention that there were fewer colonies growing close to the batches compared to the rest of the plate as seen in figure 8 as well as clear spaces in between these colonies.

Table 1.

Table 1 shows us the zones of inhibition (in mm) from the first test on the 25.6.2019 of small quantities of batches 1 to 5 (5 to 20 µl) on S. aureus, E. coli and P.aeruginosa.

Figure 8: Batch 3 on S.aureus.

Figure 8 shows a zone of inhibition in all quantities. In all these zones there exist colonies of S.aureus thus no real inhibition is present. This phenomenon was present in all batches tested on S.aureus.

Figure 9: Batch 1 on E.coli.

Figure 9 shows a round zone of inhibition that increases in size with increasing quantity of tested substance. Nevertheless there are visible colonies inside of this ring thus only leaving us with the result of no real inhibition.

Figure 10: Batch 2 on E.coli.

Figure 10 reveals similar results as batch 1 on E.coli. Again no real inhibition is observed, but there are similar ring sizes.

Figure 11: Batch 2 on P.aeruginosa.

Figure 11 shows that there are zones of inhibition present at all 4 quantities but only at 20 µl are no colonies present inside of this ring.

Figure 12: Juxtaposition of the zone of inhibition of batches 1 and 2 with the zone of inhibition of 30 µg Amikacin on P.aeruginosa. Only batch 1 is able to reach 15 mm of inhibition thus showing same efficacy as Amikacin. The cutoff value of Amikacin was determined from data of EUCAST .

Figure 13: Juxtaposition of the zone of inhibition of batches 1 and 2 with the zone of inhibition of 10 µg Ceftazidime-avibactam on P.aeruginosa. Neither batch 1 nor batch 2 was able to reach the 17 mm of zone of inhibition of Ceftazidime-avibactam. Thus they are less effective than it. The cutoff value of Ceftazidime-avibactam was determined from data of EUCAST .

Table 2.

Table 2 shows us the zones of inhibition (in mm) from the second test on the 27.6.2019 of larger quantities of batches 1 to 5 (30 to 60 µl) and of pure wine on S. aureus, E. coli and P.aeruginosa.

Figure 14: Only wine on S.aureus and P.aeruginosa.

Figure 14 shows that no zones of inhibition were observed.

Figure 15: Batch 1 on S.aureus.

Figure 15 shows zones of inhibition that increase in size with corresponding increase in quantity. Inside of these rings are colonies of S.aureus thus no real inhibition is present. Similar sizes were obtained in batch 2. In batches 3, 4 and 5 much smaller zones of inhibition were observed.

Figure 16: Batch 1 on E.coli.

Figure 16 shows that there are visible zones of inhibition present at all quantities. But only at 50 µl and 60 µl the zones are clear and without colonies reaching the centre.

Figure 17: Batches 1 and 2 on P.aeruginosa.

Figure 17 shows that with the exception of 30 µl and 40 µl of batch 2 there are zones of inhibition present without colonies present inside, thus having real inhibition.

Figure 18: Juxtaposition of the zone of inhibition of batches 1 and 2 with the zone of inhibition of 20 µg Eravacycline on E.coli. Neither batch 1 nor batch 2 was able to reach the 17 mm of zone of inhibition of Eravacycline. Thus they are less effective than it. The cutoff value of Eravacycline was determined from data of EUCAST .

Figure 19: Juxtaposition of the zone of inhibition of batches 1 and 2 with the zone of inhibition of 100 µg Nitrofurantoin on E.coli. Neither batch 1 nor batch 2 was able to reach the 11 mm of zone of inhibition of Nitrofurantoin. Thus they are less effective than it. The cutoff value of Nitrofurantoin was determined from data of EUCAST .

Figure 20: Juxtaposition of the zone of inhibition of batches 1 and 2 with the zone of inhibition of 30 µg Amikacin on P.aeruginosa. 50µl and 60 µl of Batch 1 were able to reach the 15 mm of zone of inhibition of Amikacin and even surpass it with 5 and 6 mm. Thus they show the same, if not even stronger efficacy than it. The cutoff value of Amikacin was determined from data of EUCAST.

Figure 21: Juxtaposition of the zone of inhibition of batches 1 and 2 with the zone of inhibition of 10 µg Ceftazidime-avibactam on P.aeruginosa. 50µl and 60 µl of Batch 1 were able to reach the 17 mm of zone of inhibition of Ceftazidime-avibactam and even surpass it with 3 and 4 mm. Thus they show the same, if not even stronger efficacy than it. The cutoff value of Ceftazidime-avibactam was determined from data of EUCAST.

Discussion

In the first test from the 25.6.2019 we started with small quantities of tested substance, to be more specific we used 5, 10, 15 and 20 µl. We obtained no results for S. aureus and E.coli. Only batches 1 and 2 showed effect on P.aeruginosa. If we look at figure 12 and compare the power of inhibition with a standard antibiotic used on P.aeruginosa, we find that only the highest dose of batch 1 was able to reach the same zone of inhibition on P. aeruginosa as the antibiotic amikacin. If we follow that up and compare it with another antibiotic as in figure 13, we see that no quantity of batches 1 and 2 was able to reach the cut off zone of inhibition of ceftazidime-avibactam on P.aeruginosa.

In the second test from the 27.6.2019 we used larger quantities, respectively 30, 40, 50 and 60µl. There was no effect recorded on S. aureus, but inhibition was observed for E.coli and P.aeruginosa. We wanted to see if the effect of the alcohol in the wine itself was responsible for some of the inhibition but figure 14 shows that absolutely no zone of inhibition was observed with it. Now we wanted to see how the results from test 2 compared to the zones of inhibition of some antibiotics. Figure 18 shows us that no quantity of batches 1 and 2 was able to reach the cut off zone of inhibition of eravacycline on E.coli. Similar results were seen in figure 19 where no quantity of batches 1 and 2 was able to reach the cut off zone of inhibition of nitrofurantoin on E.coli. Following this we compared the effect of batch 1 to amikacin on P.aeruginosa in figure 20 and found out that in quantities of 50 µl and 60 µl it was able to reach the cutoff value. Batch 2 is 1 mm short of reaching having the same effect. In figure 21 we compared it to ceftazidime-avibactam on P.aeruginosa. Here batch 1 crosses the cutoff zone of inhibition of in quantities of 50 µl and 60 µl. Batch 2 is 3 mm short of reaching the cutoff value.

From this we conclude that only batches containing self-crushed garlic, onion and leek possess antibiotic properties with only batch 1 mimicking the strength of amikacin and ceftazidime-avibactam. Furthermore they showed strong effect on P.aeruginosa and only weak effect on E.coli. What came as a surprise was that no real effect was observed on S.aureus. This is because Harrison F et al showed that Bald´s eyesalve was even strong enough to kill methicillin-resistant S.aureus that was inoculated in a mouse. They compared it with vancomycin and showed that Bald´s eyesalve was more effective than it. For them both preparations with onion and leek showed antibacterial effects, with onion being a bit more powerful. They emphasized that it was important to follow the recipe to the detail. They removed the leek, onion or oxgall only to obtain weaker bactericidal activity. When they eliminated the wine or garlic no bactericidal activity was seen anymore. It was surprising that when they removed the brass no change in bactericidal activity, compared to the full recipe was noticed. They speculate that the concentration of copper might have been too low or that it wasn’t an essential ingredient and only allowed the medieval physician to start with a clean copper pot that was not contaminated.

Similar results were obtained by Fuchs Al et al who showed that Bald´s eyesalve inhibited the growth of S.auerus but also of P.aeruginosa. The anti-staphylococcal effect was stronger than the anti-pseudomonal effect. Their most potent formula lacked onion and leek. They explained it by stating that they didn´t test on a synthetic wound model, but on planktonic S.aureus. Furthermore they identified allicin derived from garlic to be the main antibacterial agent to be present in Bald´s eyesalve. This was shown by the addition of free cysteine, which consumes allicin, after that no antimicrobial action was observed. Against S.aureus allicin was antagonised by other components of Bald´s eyesalve while against P.aeruginosa they were acting in synergism.

In our experiment we only saw partial anti-staphylococcal activity of our tested substances. At S.aureus we observed zones of inhibition with similar sizes as seen as in P.aeruginosa, but they were incomplete. These rings always had colonies growing inside of them. One explanation for this might be the theory of heteroresistance. El-Halfawy et al give the definition of “Heteroresistance as a population-wide variation of antibiotic resistance, where different subpopulations within an isolate exhibit various susceptibilities to a particular antimicrobial agent.”. With the elimination of the susceptible bacteria, the resistant bacteria have the possibility to grow in the zone of inhibition, which is non-lethal for them, without any competition. These resistant bacteria in the tested S.aureus population might have this resistance due to a mutation in resistance genes. This was observed in heteroresistant strains of H. pylori for metronidazole where a mutation in the rdxA gene lead to more resistant populations . Furthermore non-genetic mechanisms have been described for S.aureus and include heteroresistance to methicillin in the presence of glycine .

We observed no significant results for batches 3, 4 and 5. In them we used the bought garlic concentrate. Allicin being the main antibacterial agent present in Bald´s eyesalve is a highly instable compound. Its maximum half-life time was observed in water being around 11 days. Because we don´t know when the garlic concentrate was produced, we assume that at the time of the experiment it was already deprived of all allicin.

If we compare the effectivity against S.aureus and E.coli we recognize that Bald´s eyesalve was more active against S.aureus even if it was an incomplete activity. This might be explained by the higher lipid concentration in the cell membrane of E.coli that traps the active allicin. Thus preventing it from reaching the internal of the bacterium where it would exert its antibacterial function.

With our third experiment we wanted to see is there was any difference in the effectivity of Bald´s eyesalve if we used the dried oxgall powder, as in Harrison et al and Fuchs et al , in comparison to the fresh pork bile as in the first two tests. We used the pork bile because we didn´t have access to the dried oxgall powder at the first two test. Furthermore we wanted to evaluate if there is a difference in using mortar grounded substances or a food processor. In a future experiment we might be able to perform this third test. We expect from the third test to be more effective on S.aureus since its preparation is much more identical with Harrison et al than our previous two tests.

Conclusion

In this study we found out that a student using medieval methods and materials is able to recreate the ancientbiotic Bald´s eyesalve. Furthermore this recreation was able to have similar antibacterial effect on P.aeruginosa as commonly used antibiotics. This effectivity was exclusively seen in the preparation that contained yellow onion as second allium species and garlic that was crushed by a mortar. Batch 2 that used leek showed also antibacterial properties but not to the same extend. Only slight antibiotic activity was observed for E.coli with batches 1 and 2. In the case of S.aureus we found incomplete antibiotic activity for all 5 batches that might be explained with heterogenous resistance. For S.aureus batches 1 and 2 were also more effective than the others.