Microbe-Host Analysis
Microbe-Host Analysis
Introduction
It is unknown how long Staphylococcus Aureus (S. Aureus or staph) has been on this planet. We have been too busy trying to conquer all of successful attempts that it has made to evade our weapons against to establish an intricate epidemiologic profile of it. We do know when it was discovered to be a harmful bacteria, though. One of the primary toxins of staph, PVL, was first discovered by an assistant in Belgium by the name of H. van de Velde in 1894. He was taking care of some rabbits in a laboratory and noticed a yellow pus exuding from a couple nodules in one of the animals. A look through the microscope and a couple tests later, this assistant noticed that the pus was actually fragmented leukocytes, most animals first line of immune defense to foreign organisms in their body. It was termed leukocidin until 1932, when Panton and Valentine first associated the leukocidin with staph.
Years later, we have seen several antibiotics be released specifically for the treatment of Staph. Every time, we have seen these antibiotics fail. From Methicillin resistance in 1953, a year after the drug was released, in the United Kingdom, to 1975, when it was first described in the United States. Vancomycin came and went, and now we are down to our very last antibiotic options, Zyvox, Torezolid (pending approval), Telavancin, and occasionally Daptomycin for super-resistant substrains of the US300 community associated strain. The problem seems to be getting worse, as CA-MRSA and HA-MRSA, combined, have been killing as many or more people than AIDS on a consistent annual basis in developed countries.
Research Problem
The research problem I am addressing is MRSA, and how we can prevent and treat it in the midst of the end of the antibiotic era. MRSA has been around from a year after Methicillin was developed, but was limited by its lack of virulence factors, and was only seen in the hospital community for many years. At some point, PVL and MRSA were introduced to each other genetically, forming a new gateway for pathogenesis, which led to an abundance of virulence factors that today makes it one of the most easily spread lethal bacteria we have. As our antibiotic options cease, new novel ways of attacking the bacteria are coming into light.
Human-Microbe Relationship
One-third of the the United States is now colonized with staph. This is not usually a problem. In fact, many symbiotic relationships are formed when innocuous staph is introduced to a human body — we provide them with a nice environment to live and reproduce, and they provide extra protection towards bacteria and viruses that could be dangerous for us. This staph is PVL negative and has no TNF-α genes associated with it. Unfortunately, that’s not the case every time. One to two percent of all Americans are colonized with MRSA, a dangerous form of staph (Shrestha, Singh, et al. 2014). There are six prevalent strains of staph in the US, with two accounting for about 85 percent of all cases: US100, the hospital strain; and US300, the community strain (Chambers and DeLeo, 2009).
The US300 strain is of major concern because it can infect anybody, whereas US100 is much more opportunistic, and usually stays around health care facilities. Though US100 can deal the final heavy blow to a person weakened from poor health, it is usually concomitant with a variety of other diseases that patient was enduring: cancers, AIDS, amputations, untreated diabetes, the list of tragic illnesses goes on. A kid who is perfectly healthy with a little scratch on their thumb can go to school one day, catch the US300 strain through contact with a door handle, a desk, a poorly sanitized lunch tray or table, or someone sneezing without covering their mouth. In less than 72 hours, that child can be on death’s door. It could just stay on the thumb, but most children have an aptitude for fidgeting with their face. Exposure from the thumb to the nose can lead to direct contact with the child’s lungs, a favorite part of the human body for staph to cause necrosis, or cell death.
Clinical Study of MRSA
It should go without saying that the average high school microscope, though powerful, is not really enough to make specific observations of bacteria. At 1000X, all that can really be observed are little specs that look like they are freezing cold because of how much they appear to be shaking. The most practical microscopic method we have for most bacteria, and especially MRSA, is Immunofluorescence Microscopy. It makes all main strains of staph easily identifiable because of how antigen-specific they all are. First, tagged groups of antibodies are introduced to the sample. If they hit a target or targets in the MRSA cells, we can determine a lot about the microbiology of the substrain we have (Robinson, Sturgis, and Kumar, 2009).The process is extremely simple, though can be time-consuming if one comes across a rare phenotype.
Microscopy might be easy and san be a great tool for the observation of phenotypes, but the trade-off is it is not how microbiologists obtain the most elusive answers to the superbugs ability to change, usually at the genotype level. We have to go further into the trenches to get more answers. Things get a little more complicated, but generally, in schools, labs and hospitals that cannot afford the newest microarray technology, the process starts with cultivation, which accounts for about 90 percent of all MRSA testing done, and there specifically two different types of cultivation in this method (Rapidmicrobiology, 2016).
The first part is PCR amplification. The approach to amplification is different with each PhD, but usually several different samples, which have been gene clipped by and purchased from a biotech company, are clipped again with a tagging primer and fluids, then put through several heating a cooling, denaturation and annealing steps, which altogether takes from an hour to 24 hours, again depending on the PhD. This process increases the population of each sample by thousands of times over. After that, samples are determined to be either mecA positive (MRSA) or negative (oxacillin susceptible).
The second part of Cultivation is plating. There are several ways of plating, and Oxacillin screening is by far the most popular, probably having to do with it being the cheapest (CDC, 2010). Whatever way is chosen, the process includes a plate of agar gel, the samples, and whatever drug one wants to examine for resistance. If the bacteria continues to grow after the antibiotic or other property is introduced, it is resistant. There is still no one single cultivation method that can test against all substrains of MRSA.
Unfortunately, with more strains than ever before, this simply will not do. Today, there is a strain still fairly foreign to the US in the United Kingdom known as EMRSA-15. The E stands for epidemic. We know that it takes roughly 22-46 years for strains of MRSA to penetrate the US from overseas (1953-1975, UK to NYC; 1953-1999 unknown to San Francisco), (Marshall Medical Center, 2016). EMRSA was first identified in 1991 as a pure strain from HA-MRSA, meaning it crossed the HA-CA barrier without help. As of 2013, the strain only accounts for .22 percent of Americans with MRSA, but has overcome traditional CA-MRSA in the UK, now representing 60 percent of the county’s cases (Holden, Hsu, 2013).
Microchips and microarray analysis, a non-cultivation method, is conclusive in its results nearly all of the time. A chip is made with a bath of the pathogen over the chip. Genotype tags for ATCGU are brushed on by a lab tech, and the chip is analyzed, giving a full genetic profile of the bacteria. As the genetic profiles of multiple strains have been catalogued in GenBank (Kegg Genome, 2005). Anomalies are found against the standard Staph strain, and are uploaded to a computer for analysis. If the anomalies match a profile in the system, a diagnosis can be made. If not, the anomalies are ran through to see if protein matches can be determined to find the resistance genes that substrain has.
Virulence Factors, Patient Outcomes
There are six main virulence factors when determining the pathogenesis of a microbe. Adhesins such as pili attach themselves to the the host cell. Invasion factors are genes that encode surface components that allow the bacteria to attack. These are thought to be mediated by microRNA. Capsules are extra layers that some bacteria have that add an extra layer of protection from immune cells. Endotoxins, associated with gram negative bacteria, usually promote inflammation already being caused by the immune system. Exotoxins include PVL, and other cytotoxins, neurotoxins and enterotoxins. Finally siderophores steal iron from competing cells for their own use. A lot of recent research has been focused on this last category (Peterson, 1996).
Patient outcomes are extremely unpredictable, largely because we usually don’t know the substrain we are dealing with. It could be a strain that is easily treated with Bactrim and mupirocin. In that case the prognosis is usually great. An extremely virulent strain can lead to death within 48 hours if the pathogen is not identified in time, or if the patient happens to be in a hospital that does not carry the latest drugs. For instance, if a person comes down with a strain that has neurotoxins, and they develop meningitis or encephalitis, there is a small window in which the person can be flooded with steroids, given high doses of Telavancin and Daptomycin, possibly both. If they are able to to inhale enough Thermazene or Altabax, that would probably be helpful too. Still, it will be a fight. Obviously, antibiotics are becoming an unsustainable source of treatment.
Prevention and New Treatments
The three most important keys to prevention have never changed: wash hands with soap that contains absolutely no Triclosan, keep places that are touched frequently sanitized, and antibiotics need to stop being abused by the patients, and more importantly, not given out by doctors when they are unnecessary. Hands-free soap is the best option, or at least soaps that are dispensed through a tube, rather than a bar. There are multiple products that are great but expensive. All that is needed is rubbing alcohol or bleach if the irritation can be tolerated. Doctors who fall prey to patient pressure or time allocation per patient pressure need to be put on license probation, and learn more about the harm they are doing by not performing proper tests or by not standing their ground with an aggressive patient.
The part that all good microbiologists are excited for are the advances being made through all the hard work they do, whether their name appears first or last on an publication (both have about the same distinction), or if they were one of the lab techs that incubated broths for the last ten years. In the case of MRSA, very strident research and advancement has taken place, and hopefully we can come closer to reaching the end of this growing epidemic. The most promising research is in bacteriophages. These are viruses whose rods (drills used to perforate a living cell) cannot penetrate any animal cells, but are able to drill right through the cell walls of both gram positive and gram negative bacteria.
The success rate is higher than any antibiotic ever researched, and is extremely close to 100 percent effective across all cases in which the correct bacteriophage was matched to the correct substrain. The next phase of this is to learn whether or not we can control and manipulate the DNA or RNA of these various phage (Pincus, Reckhow, et al., 2015) . Hinted at earlier was Thermazene. This is a topical bacteriocide (usually) that uses nanosilver. Silver is now used almost across the board for any soft tissue wound. Finally, Manuka Honey, which seems to have very fundamental bactericides, seems to do something else to kill bacteria aside from the chemical processes we know of — one of which leads to the development of hydrogen peroxide. Research on this is at warp speed because, unfortunately, Manuka bees are becoming endangered.
Conclusion
After the antibiotic era has passed and is well behind us, probably one of the greatest debates in medicine, and certainly the single greatest debate in microbiology about the era will be: “Would humanity have been better off without the discovery and use of antibiotics?” What if Alexander Fleming had listened to his contemporaries as they predicted that antibiotics would confer resistance? What if that “fungus” had never grown on one of his cultures? Sulphonamides were actually used before penicillin, but they never became quite as popular, they were never termed “the miracle drug.”
Many people’s lives have been saved by antibiotics. The tragedy is, though, today, it is more likely that they were saved from something that was caused by antibiotics to begin with. There really is no way to fairly quantify each person’s life, however, we can say that antibiotics of the past are now doing more harm than good, and as some doctors incessantly continue to over-prescribe these medications, and patients continue to misuse them, the problem will only get worse. Zyvox treatment runs about $3,000 a month. It’s been out on the market for fifteen years. Bacteriophages will no doubt be infinitely more expensive when they are approved. It is a question that most of us cannot definitively answer right now, because the problem continues to this day.
References
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