“Mining signs of resistance. It is the development of

“Mining Soil Microbes for Novel Antimicrobials Against MRSA.”Name: Lisa BrennanStudent Numbers: 114321711/R00120933Supervisor: Dr. Máire BegleyIntroductionAntibiotic resistance is a global epidemic, and is threatening the end of the Golden Age of Antibiotic Therapy, (Spellberg et al., 2008). Antimicrobial resistance has a profoundly negative impact on all areas of healthcare, and poses difficulties to the success of empirical therapy. Staphylococcus aureus displays an unusual ability to resist new antibiotics rapidly, (Schaaff, Reipert and Bierbaum, 2002). This started with penicillin, until the most recent, linezolid and daptomycin. Methicillin resistant S. aureus (MRSA) has become endemic today in hospitals worldwide.  If antibiotic resistance continues at such an alarming rate, without the development of more antimicrobials, humanity could return to the dangers that were a pre- antibiotic era.Methicillin Resistant Staphylococcus Aureus (MRSA) is a gram-positive bacterium in the genus Staphylococcus, (Rice, 2006). It is characterized by its resistance to the antibiotic methicillin and to related semisynthetic penicillin’s, (Schneewind, 2016). Vancomycin was an antibiotic of choice to treat MRSA infections, however it is now showing signs of resistance. It is the development of resistance to many antimicrobials that makes MRSA infections difficult to treat, and creates the need for new antimicrobials against it.Review of LiteratureMethicillin Resistant Staphylococcus AureusS. aureus is a facultative anaerobic, gram-positive coccal bacterium also known as “golden staph” and “oro staphira”, (Masalha et al., 2001). S. aureus is non-motile and does not form spores. It is a member of the normal flora of the body, often present in the nose, respiratory tract, and skin. It appears as staphylococci under the microscope. On blood agar, round golden colonies may be observed, frequently with haemolysis. The bacterium reproduces asexually by binary fission. Autolysin ensures full separation of the daughter cells, in autolysin inhibition clusters are observed, (Zhou et al., 2015). S. aureus is catalase positive. Catalase is an enzyme that catalyses the conversion of hydrogen peroxide to water and oxygen. Catalase-activity tests can be performed to differentiate staphylococci from enterococci and streptococci, (Vitko and Richardson, 2013).Natural genetic transformation is a reproductive process involving DNA transfer from one bacterium to another through the intervening medium, and the integration of the donor sequence into the recipient genome by homologous recombination, (Lorenz and Wackernagel, 1994). S. aureus was found to be capable of natural genetic transformation, but only at low frequency under the experimental conditions employed, (Haaber, Penadés and Ingmer, 2017). Further studies suggested that the development of competence for natural genetic transformation may be substantially higher under appropriate conditions, yet to be discovered. Although S. aureus is not always pathogenic (and is regularly found existing as a commensal), it is a common cause of skin infections including abscesses, respiratory infections such as sinusitis, and food poisoning. Pathogenic strains often promote infections by producing virulence factors such as potent protein toxins, and the expression of a cell-surface protein that binds and inactivates antibodies. The emergence of antibiotic-resistant strains of S. aureus such as Methicillin Resistant Staphylococcus aureus (MRSA) is a worldwide problem in clinical medicine. MRSA is a strain of S. aureus and was first isolated in the early 1960s, shortly after methicillin came into use as an antibiotic, (NIAID, 2016).  Although methicillin is no longer used, MRSA has become widespread. Studies show that about one in three (33%) people carry MRSA in their nose, usually asymptomatic. Two in 100 people carry MRSA. There is no published data illustrating the total number of people who get MRSA skin infections in the community, (CDC, 2017). Two main types of MRSA have been identified. These are community-associated MRSA (CA-MRSA) and health care-associated MRSA (HA-MRSA). CA-MRSA was once rare but is becoming more common. HA-MRSA accounts for well over half of the total number of staph infections, (David and Daum, 2010).Methicillin is a ?-lactam antibiotic which acts by inhibiting the activity of penicillin-binding proteins (PBPs) that are involved in the synthesis of peptidoglycan, a very important polymer that surrounds the cell. S. aureus can become resistant to methicillin and other ?-lactam antibiotics by expressing foreign PBP, PBP2a, that is resistant to the action of methicillin but can still perform the essential functions of the host PBPs. Methicillin-resistant S. aureus isolates are often resistant to other classes of antibiotics (through different mechanisms) making treatment options limited, and making it a cutting-edge area of research, (Stapleton, 2002).  Although Staphylococcus aureus is a commensal of humans, it is also a common cause of human infections. These may be serious infections if caused by antimicrobial resistant strains, (Monecke et al., 2011). MRSA globally spread quickly after the introduction of methicillin to clinical medicine, in 1960, (Chambers and DeLeo, 2009). This sparked an increase in interest into the use of clindamycin for treatment of S. Aureus infections. However, there is an increase in numbers of strains of S. aureus that are acquiring resistance toward clindamycin,(Prabhu, Rao and Rao, 2011). Vancomycin was the traditional drug of choice for treatment of MRSA infections. Vancomycin Resistant Staphylococcus Aureus and Vancomycin Intermediate Staphylococcus Aureus have recently been reported, along with reports of treatment failure of the infections caused by MRSA having MIC of vancomycin just below cut off value. Drug resistance to many antibiotics may be indicated by the observation of high vancomycin MIC for MRSA which are susceptible to vancomycin, (Kshetry et al., 2016).MRSA is resistant to entire classes of ?-lactams and has a high risk of developing resistance to quinolones, aminoglycosides, and macrolides, (Baddour, Abuelkheir and Fatani, 2006). Methicillin resistance in S. aureus is due to an alteration in low-affinity penicillin binding protein (PBP2a). PBP2a is encoded by mecA gene. This gene is located in a chromosomal mobile genetic element called Staphylococcal cassette chromosome mec, (Grundmann et al., 2006). due to the possibility of a link between MRSA and multiple antibiotic resistance, and a relatively challenging and more expensive treatment, the precise, accurate and quick identification of MRSA is vital in medicine for timely management of the infections caused by this microbe, (Johnson, 2011). MRSA associated medical complicationsIt is commonly found on the skin, in the nose, or in the blood or urine. Staphylococcus aureus is a commensal bacterium that colonizes the axillae, nares, vagina, damaged skin, and/or pharynx. Infections can occur when there is a breach of the skin or mucosa, which can then grant the organism access to adjoining tissues or the bloodstream. If invasion of the bloodstream occurs, bacteraemia is pronounced, which can be fatal, (Loewen, 2017). Risk for infection is increased by the presence of foreign materials, eg. intravenous catheters, (David and Daum, 2010). Biofilm formation is a feature of S. aureus, making it more difficult to treat, (Oyama et al., 2016). This is a deadly cocktail of circumstance for someone with an illness that requires a lot of IV therapy, eg. malignancies. Biofilm formation can be observed in IV lines, catheters, pacemakers and port-a-caths. This biofilm formation, along with the immunocompromised state, can be fatal. S. aureus is unique in its ability to invade and cause disease in previously normal tissue at virtually all sites. Common manifestations of S. aureus infection include skin and soft-tissue, respiratory, bone, joint, and endovascular infections.MDR-MRSA strains have serious clinical implications. MRSA strains are labelled as a “superbug” in the heath sector, with a special concern for “YOPIs”- the young, old, pregnant and immunocompromised, in which inoculation may be fatal, (Mayo Clinic, 2017).Why is an antimicrobial needed against MRSAIncreasing rates of antibiotic resistance in Gram-positive pathogens has become a major issue worldwide as there are less, or occasionally no, effective antimicrobial agents available for infections caused by these bacteria. This pressing issue is even more dangerous when one considers the very few new antimicrobials that are in development, (Ventola, 2015). Given the speed at which new antimicrobials are released, Staphylococci have developed efficient mechanisms to neutralize them; which reduces the amount of bactericidal antibiotics to treat these often fatal infections.A variety of activities lead to the increasing speed of antibiotic resistance: • Extreme over prescription: Doctors prescribing antibiotics for illnesses such as viral infections, to which they will not aid the recovery, (Llor and Bjerrum, 2014).• Misuse by the food industry: The addition of antibiotics to animal feed for non-curative reasons, such as prophylaxis, metaphylaxis, and growth promotion, (Landers et al., 2012). • Human independent resistance.Antibiotic resistance is a very real problem in human health and welfare. 30% of all deaths were from bacterial infections in pre- antibiotic USA. This shocking percentage could be within reach again if resistance occurs without new antimicrobials being developed. Approximately 20% of the US population carry S. aureus as a commensal, and 19000 deaths occur annually in the US due to MRSA and the associated complications. This costs the healthcare sector $3 billion per year. These statistics will rise instead of fall if new antibiotics are not found, (Fair and Tor, 2014). Multidrug-resistant (MDR) Staphylococci are a serious issue in mammalian health, (Onanuga, 2011). The increase of drug-resistant virulent strains of Staphylococcus aureus, especially MRSA, is a serious concern in the treatment and control of Staphylococcal infections. Methicillin-resistant Staphylococci (MRS) can cause infections that are difficult to treat. Some strains of MRSA have been described with concomitant resistance to many first line antimicrobials, with groups including aminoglycosides, fluoroquinolones, macrolides, tetracycline and chloramphenicol.Antimicrobial resistance in S. aureus may be defined using a special rule specifically created for this purpose. Once a S. aureus isolate is determined to be an MRSA, it is automatically classified as an MDR, because when a microbe is resistant to both cefoxitin and oxacillin, one may infer nonsusceptibility to all categories of penicillins, cephalosporins, ?-lactamase inhibitors, and carbapenems. Thus, MDR-MRSA paradigmatic pathogen that is continuously evolving, (Kaur and Chate, 2015).Drug resistant infections are already on the rise with numbers suggesting that up to 50,000 lives are lost each year to antibiotic-resistant infections in Europe and the US alone, (O’Neill, 2014).First reported in 1960, the growing problem in the Indian scenario is that MRSA prevalence has increased from 12% in 1992 to 80.89% in 1999. MRSA has become endemic today in hospitals covertly worldwide, and 30% of S. aureus isolates are MDR, 2 decades ago, as conjectured from surveillance in the US, (Kaur and Chate, 2015).More than 95% MRSA worldwide do not respond to the first-line antibiotics, which is a strong reason to search for new antibiotics. This concretes the law that duplicate genes accumulate mutations quicker than single ones. The following antimicrobials have become available during the last 5 years: Quinupristin/dalfopristin, linezolid, daptomycin, and tigecycline. Novel lipoglycopeptide agents liketelavancin, novel cephalosporins (ceftaroline) with enhanced activity against MRSA are in the pipeline.Several studies have reported the resistance to the newer antimicrobial agents like linezolid, vancomycin, teicoplanin, and daptomycin. The fear of pandrug-resistance (resistance to all antibiotics and drugs in present use), as warned in Gram-negative pathogens, may also be an unpleasant reality in S. aureus, which would cause a return to a pre-antibiotic era- making many routine procedures and injuries far more dangerous, eg. Childbirth, superficial wounds, surgical incisions etc. This frightening possibility makes research into new antimicrobials crucial for public health. Bacteriocins are proteinaceous or peptidic toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strain(s), (Okuda et al., 2013). Applications of bacteriocins are being tested to assess their clinical application as alternatives to antibiotics, with some success to date, (Al Atya et al., 2016).Databases are available to research current bacteriocins, eg. BAGEL, (van Heel et al., 2013).  The classification of bacteriocins is shown in fig.1.Figure 1: Classification of Bacteriocins. Table adapted from Heng and Tagg, 2006 and Yang et al., 2014.Class I Bacteriocins Small peptide inhibitors and include nisin and other lantibiotics.Class II Bacteriocins • IIa cause cell-leakage by permeabilizing the target cell wall, and are pediocin-like bacteriocins.• IIb are two-peptide bacteriocins, eg. lactococcin G, which permeabilizes cell membranes for monovalent sodium and potassium cations.• IIc encompasses cyclic peptides, in which the N-terminal and C-terminal regions are covalentely linked.• IId are single-peptide bacteriocins, which are not post-translationally modified and do not show the pediocin-like signature.Class III Bacteriocins Large, heat-labile (>10 kDa) protein bacteriocins. 2 sub classes, IIIa (bacteriolysins) and IIIb (disrupt membrane potential).Class IV Bacteriocins Complex bacteriocins containing lipid or carbohydrate moieties.Fatalities are higher in MRSA than Methicillin Susceptible Staphylococcus Aureus (MSSA), due to the wider availability of drugs to treat MSSA. Therefore, it may be postulated that if more antibiotics were found for MRSA, the fatality rate would drop, and equal that of MSSA, (Yao et al., 2015).Why are we looking at soil?Two thirds of all conventional antibiotics have been isolated from soil, (Fair and Tor, 2014). Therefore, it is the logical place to search for more. The Golden Era of Antibiotic Discovery occurred between the 1940s and 1960s, with some key antibiotics being discovered in that time, as shown in fig.2.Figure 2: Some antibiotics discovered from soil isolates during The Golden Era of Antibiotic Discovery, obtained from Wiest, Cochran and Tecklenburg, 2012; Davies, 2006; Nelson and Levy, 2011; Levine, 2006.Chloramphenicol 1947 Neomycin  1949 Vancomycin 1952Tetracyclines 1953 Selman Waksman and his students, at Rutgers University, created the first screening procedures to detect antimicrobial agents produced by microbes. This deliberate search for chemotherapeutic agents was inspired by the discovery of penicillin, which came through a chance observation by Alexander Fleming, who discovered that petri dish contaminated with mould had inhibited the growth of a bacterial pathogen. During the 1940s, Waksman and his students isolated over fifteen antibiotics, the most famous of which was streptomycin, the first effective treatment for tuberculosis, which was isolated from soil. In 1952 he was awarded the Nobel Prize in Physiology or Medicine due to his discoveries from soil microbes. He also coined the term “Antibiotic”, (Woodruff, 2013).The biosphere is rich in microbes, however the culturable fraction is low, less than 1%, (Pham and Kim, 2012). This massive gap is referred to as “The Great Plate Count Anomaly”, (Connon and Giovannoni, 2002). If more bacteria could be cultured, it is likely that new antimicrobials could be produced. The Isolation chip (or ichip) was developed by NovoBiotic Pharmaceuticals. This was founded by Kim Lewis and Slava Epstein. It is a new method of culturing bacteria. Under standard conditions, 99% of bacterial species cannot be cultured as they do not grow in conditions made in a laboratory, ie. “Great Plate Count Anomaly”. The ichip is different. It cultures bacterial species within the soil environment, so they have access to the nutritional and environmental requirements that they need to successfully grow. The soil is diluted in molten agar and nutrients such that only a single cell, on average, grows in the ichip’s small wells. The chip is then enclosed in a semipermeable plastic membrane and returned to the soil to allow the microbes to grow in nutrients not available in a laboratory environment, (Hunter, 2015).With this culturing method, fifty to sixty percent of bacterial species can be cultured, instead of the previous one percent. The bacterial species Eleftheria terrae, which produces the antibiotic Teixobactin that has shown promise against MRSA, was discovered using the ichip in 2015. Teixobactin works differently than other antibiotics used in human medicine. Teixobactin inhibits peptidoglycan synthesis in S. aureus by binding to a motif of lipid II (a peptidoglycan precursor) and lipid III (teichoic acid precursor). No teixobactin-resistant S. aureus or M. Tuberculosis were isolated at four times the MIC. No resistant S. aureus were noted after serial passage in subinhibitory concentrations of Teixobactin. This led to the conclusion that it could be difficult for MRSA to become resistant to teixobactin, (Piddock, 2015).It is the availability of the iChip along with the history rich in antimicrobial discovery that allowed for the decision to be made to choose soil as the material to screen for new antimicrobials against MRSA, (Nichols et al., 2010).A soil horizon is a layer of soil, parallel to the surface, with different physical characteristics from the layers above and beneath. Each soil type usually has three or four horizons. Horizons are defined in most cases by obvious physical features, usually colour and texture.Horizon A is recommended for soil sampling for screening for antimicrobials, because it is the richest in microorganisms. This soil is topsoil, located immediately under the O horizon, and is typically dark in colour. Samples will be taken in the region of 7 cm beneath the surface layer. Soil from Cork City centre will be used. The decision to use urban soil is due to the rich findings in previous studies in New York City, (Charlop-Powers et al., 2016).  Cities are areas of higher activity than coastal and rural areas, with a broader array of individuals, foods and initiatives taking place. Theoretically, the soil could therefore have a broader spectrum of microbes, and the chance of finding an antimicrobial against MRSA would be higher.From this, a hypothesis may be formed: novel antimicrobials against MRSA may be found from soil bacteria