Figure … Bisulfite conversion of unmethylated cytosine followed by PCR. (methyaltion ffpe—11.8.9 –bs con)
Bisulfite-conversion of unmethylated cytocine to uracil is a three step process. First step is the reversible addition of HSO3– to cytosine. Second step is the liberation of NH3 by hydrolysis, and third step is the release of HSO3– to form uracil. The [C-SO3–] formed in first step rapidly regenerates in to [C] because it is unstable in neutral solutions. On the other hand, [C-SO3–] is stable in acid. The equilibrium between [C] and [C-SO3–] is reached rapidly at pH 7 when bisulfite concentration is 0.5M or higher. Second step is the key process of the overall procedure. It is the rate-limiting step for the conversion of [C] to [U-SO3–]. [U-SO3–] is stable in neutral conditions, but can be easily converted to [U] by alkali (Step 3). With respect to pH, the [C] to [U-SO3–] conversion is optimal at pH 5–6. (Hayathsu et al 2008) (Coffee et al 2009)
Figure …. Bisulfite conversion of cytosine to uralic
5- methyl cytosine can be deaminated by bisulfite, but the rate is much more slower than cytosine. The rate of deamination is approximately two orders of magnitude lower than that for cytosine. T he deaminated products, thymine-bisulfite adducts are a mixture of two diastereomers. The structures of these isomers are shown in Fig. …..
Figure … Two diastereomeric thymine -bisulfite adducts formed in the deamination of 5- methylcytosine.
They are produced in approximately 1 to 1 ratio. The treatment of trans isomer with alkali rapidly generates thymine. In contras the cis adduct, is quite stable. It can be converted to thymine only with a harsh alkaline treatment because cis elimination is difficult. This situation creates an issue in bisulfite modification of methylated cytosine. (Hayathsu et al 2008)
DNA degradation is the major side reaction occurs during this conversion. If the incubation temperature and time of the deamination step is increased DNA degradation will be increased. Therefore bisulfite modification is carried out in 55 C temperature for 3 hours. A total of 500 ng genomic DNA is enough for this modification. (BS 1— BS CONVERSION)
1.12.6. Melting curve analysis of combined direct 5’ and 3’ triplet primed PCRs
PCR is performed in the presence of intercalating dyes such as SYBR green 1, eva green and BRYT green. The intercalating dye binds to dsDNA producing fluorescence. When the DNA is heated, the strands begin to separate and the dye is released and fluorescence is reduced. The temperature at which of the 50% DNA is in single stranded is the melting temperature (Tm). Tm is influenced by GC content and length, therefore Tm is an indicative of the identity of a PCR product.
Individuals with expanded alleles (PM and FM) from both males and females are produced melt peak profiles that are distinct from those individuals with the NL alleles. Individuals with high GC content have higher Tm than individuals with low GC content.
This approach consists of 2 complementary FMR1 triplet-primed PCR (TP-PCR) assays that use unmodified DNA as template. Both assays are based on the same principal feature use of a primer that anneals within the triplet repeats. The combined analysis of both 5’ and 3’ TP-PCRs avoid false negatives caused by flanking-sequence deletion. This assay works as a rapid first-line screen for FMR1 expansions, thereby reducing the number of samples that require more labor-intensive approaches such as Southern analysis for exact repeat sizing and methylation analysis.
Previously described methods such as conventional PCR and MS PCR are two step procedures, initially PCR and then product analysis. dTP-PCR followed by MCA is a single step assay with high sensitivity and specificity for rapid screening of FXS.
The closed-tube format of this screening strategy eliminates post-PCR transfers, thereby reducing the possibility of human error and sample mix-ups or contamination, an important consideration in the multi-sample screening setting, which is currently minimized via automated sample handling. By eliminating the electrophoresis step, we also reduced the total hands-on and turnaround time.
1.12. 7. Triplet primed PCR and capillary electrophoresis
The CE is the most successful analytical tool for high-throughput, cost-effective, and reliable DNA analysis. CE separates ions based on their electrophoretic mobility with the use of an applied voltage. CE is predominantly used in nucleic acid fragment analysis and DNA sequencing. Triplet-primed repeat PCR combined with CE simplifies detection of FX expanded alleles.
In triplet primed PCR, forward and reverse primer flanks the CGG repeat region. (CGG)5 primer anneal at multiple targets within the repeat region generating multiple amplicons with a length difference of 3 bases. When electrophoresed in agarose, the multiple products appear as a smear. If PCR products subjected to the CE, the smear becomes a characteristic stutter pattern. The number of peaks corresponded to the number of repeat units (tp pcr 2—tppcr 2010). PMs and FMs can be discriminated as expanded from NLs in males and females by evaluating the peak pattern. Then individuals with expanded alleles can be subjected to Southern blot analysis to determine precise allele sizing and methylation pattern. ( Nahhas et al 2012 ). This largely overcome the need for Southern analysis, (Lyon, et al 2010). However CE provides resolution up to approximately 200 repeats. Above 200 all FM alleles migrated late as an aggregate peak independent of length, located at approximately 850 to 1100 bp range () (TPPCR 1—TP PCR 2010)
Figure …. Schematic representation of triplet primed PCR
1.12.8. Methyaltion specific triplet primed PCR and capillary elecrophoresis
Another simplified strategy based on fluorescent methylation-specific PCR (ms-PCR) and CE analysis for molecular diagnosis of FXS is available. Sodium bisulfite modification is performed to selectively modify genomic DNA. Then msPCR is perforemed using fluorescently labeled three primer sets with different fluoropores to amplify modified methylated or unmethylated DNA and amplicons are resolved by CE. This assay accurately size the NL and PM in both male and females according to the fluorescent peak sizes and patterns on the electropherogram. (Zhou et al 2006)
1.12. 9. Methyaltion specific triplet primed PCR and Melting curve analysis (msTP-PCR MCA)
In this assay MCA is performed for the amplicons generated from MS PCR. The melting temperature of an amplicon is dependent on its length and base composition. Amplicons generated from methylated templates have 5-methylcytosine in the positions corresponding to cytosine and they have higher melting temperatures than amplicons from unmethylated templates, which contain thymines. When methylation-independent primers are used, this allows the amplification of both methylated and unmethylated sequences which are seen as distinct melting peaks. (Candiloro et al 2011)
Previously described dTP-MCA assay does not discriminate between PMs and FMs. However the msTP-PCR MCA can differentiate PMs from FMs. Therefore only FMs cab be subjected to sizing by Southern analysis. The msTP-PCR MCA assay is rapid and less labor-intensive than Southern blotting or even the newer PCR and capillary electrophoresis– based tests. This assay also ideal for population-based newborn or early childhood screening of FXS. (bs-mca——–ref nus 2013)
1.12.10. Matrix-assisted laser desorption/ionization-time of ï¬‚ight mass spectrometry (MALDI-TOF MS)
Southern blot analysis provides the number of the CGG repeats and methylation status for all types of FMR1 alleles. But one of its limitations is that it targets methylation of only a few CpG sites located on the CpG island in 5? of the repeat region. Recently developed test for FXS targets biomarkers located in the CpG sites.
Two novel epigenetic markers called fragile X–related epigenetic elements 1 and 2 (FREE1 and FREE2) were identified and they are related to FMRP expression (inversely correlated with FMRP production) and cognitive impairment in FM individuals. These elements are located on either side of the repeat region and are different from the sites routinely analyzed using methylation-sensitive SB.
FREE1 is located on the 5‘ region of CGG repeats. FREE2 is located on the exon 1/intron 1 boundary of the FMR1 gene. FREE2 allows identifying FM males and females with 100% specificity and sensitivity but could not distinguish between PM carriers and healthy controls or between PM carriers and high functioning males with un methylated FM alleles (Godler et al 2010, Godler et al 2011, Godler et al 2012, Godler et al 2013, ) .
1.13 Ethical aspects
Genetic testing is a complex ethical and social concern. The use of patient sample requires informed consent from individual. If the study population is children or mentally impaired consent should be obtained from parent or guardian. Explanation of the research and its potential outcome is must. Each individual is entitled to privacy and confidentiality in terms of the protection of their personal data and research findings.
Current guidelines state that genetic testing of children is recommended only if a clear benefit can be offered. Fragile X testing for children less than 18 years of age must be approached carefully, with medical and emotional benefits to the child weighed against potential harms.
Are We Entering the Post Antibiotic Era?
Introduction Antibiotics have been the main course of treatment for microbial infections throughout the years since Sir Alexander Fleming first discovered that Staphylococcus aureus colonies could be destroyed by Penicillium chrysogenum (Houbraken et al., 2011), leading to the production of Penicillin G Procaine in 1942 by Howard Florey and Ernst Chain (Ligon, 2004). The following years saw improvements being made in the treatment of infectious diseases due to the fact that symptoms were recognized earlier in patients, thus prompting a faster response of treatment. This in turn managed to reduce the mortality rate of the patients that had these illnesses (Tishler, 2005). The reason as to this would be due to the rapidly expanding understanding of the molecular biology of infectious diseases, the mechanisms involved in it and studies done on understanding the pathophysiology of an infectious disease. Further down the road, antimicrobial treatments have evolved rapidly by increasing its safety and efficacy. Antibiotics were able to target the foreign source of infection and is able to eliminate it, thus eliminating the infection. Despite this, antibiotics such as prontosil, the first sulfa drug, penicillin G and streptomycin (Finkelstein and Birkeland, 1938; Houbraken et al., 2011; Bruton and Horner, W.H. 1966) were extremely expensive and were initialy produced to be used for the military in World War I (Tishler, 2005). The cascade of antibiotic discovery continued, causing an increase in the manufacturing of antibiotics, research into the area of antibiotic development was increasing in order to keep up with the types of infections that were appearing in the world. Antibiotics became the only treatment anyone had in mind when a patient described symptoms that could be of bacterial origin and they were being widely prescribed throughout the world at an alarming rate. Penicillin was one such antibiotic whereby resistance was seen in just 3 years after it was available to the public. A serious number of strains of Staphylococcus aureus were becoming resistant to penicillin G (Levy and Marshall, 2004). The problem has escalated to a point where bacteria are mutating and multiplying faster than scientists are able to refine and modify drugs. Such examples of resistant bacteria are Clostridium difficile, Pseudomonas aeruginosa, methicillin resistant S. aureus and several other bacteria that initially only caused hospital acquired infection that increased the mortality rate of hospital confined patients, begun spreading to the community, causing infections in what should seem as health individuals (Barrozo, 2003).
Antibiotic Resistance Just like any other living organism, bacteria also constantly work towards adapting to the environmental conditions that are changing and evolving such as weather and the availability of oxygen in the ‘survival of the fittest’ pattern (Darwin et al., 1958). Due to bacteria having a remarkable capability to endure harsh environments and evolve alongside it, they have been able to develop various methods of resistances to several antimicrobials that in turn make these drugs ineffective as first choices for treatments caused by these pathogens. As mentioned earlier, certain bacteria do cause hospital acquired infections but they are also able to stay viable in the clinical environment. The chronology of the development of antibiotic resistant strains of bacteria is impressive seeing as it has been occurring since the 1930s where S. aureus begun showing resistance to penicillin and sulphonamides such as sulfamethoxazole (Suvorov et al., 2007). Neisseria gonorrhoea and Haemophilus influenza were the next to follow suit by becoming resistant to penicillin (Lind, 1990). S. aureus also became resistant to methicillin in the late 1970s. That was also the same period where Mycobacterium tuberculosis became resistant to several drugs (Soini and Musser, 2001). The next group to have resistance was Group A Streptococci that build resistance towards macrolides like erythromycin (Martin et al., 2002). Several other antibiotics have experience resistance during treatment regime such as vancomycin whereby enterococci have become resistant to it (Donskey et al., 2000).
Mechanisms of resistance There are various classes of antibiotics that have been produced and marketed worldwide, to name a few, quinolones, macrolides, penicillins and cephalosporins of several generations. Despite this, bacteria have developed at least one way of resisting each of those classes of antibiotics. Mycobacterium tuberculosis has even developed resistance to both rifampicin and isoniazid which are the first line treatment for tuberculosis (Weinstein and Hooper, 2005). This increases the difficulty in choosing a suitable regime for treatment, it also increases the cost and morbidity in the community. The world has seen the emergence of acquired immunodeficiency syndrome (AIDS), severe acute respiratory syndrome (SARS) and quite recently in the past year, Middle East respiratory syndrome (MERS) (de Groot et al., 2013) despite the existence of antimicrobials. The trend that is noticed here is that bacteria, even common bacteria are developing resistance to multiple drugs and the types and frequency of infectious diseases in the population are also increasing, bringing about the problem of treatment seeing as the number of effective antimicrobials are decreasing. In order to understand and develop effective targeted treatment, scientists must first understand the mechanisms in which a bacteria becomes resistant (Sefton, 2002). In most instances of resistance, it occurs in the genetic level whereby the genetic makeup of the bacteria evolves thus modifying the biological action of the bacteria that in turn determines the resistance type in the bacteria.
In the development of antibiotic resistance, a specific antibiotic’s presence is required as well as the targeted colony of bacteria, that has the genetic capability to develop resistance (Sefton, 2002). Once treatment is administered, susceptible colonies die while resistant strains survive and catalogue the resistance magnitude that is to be depicted in the cell. These genes are usually located in the transposons that make genetic information transfer between plasmids easier. Multidrug resistance usually occurs when the resistant strains of bacteria contain an integron in the transposons in their DNA that facilitates the integration of multiple antibiotic resistant genes. Both gram-positive and gram-negative bacteria have been identified to have integrons in their genetic makeup. Once a change in DNA has occurred, the genetic material is transferred amongst bacteria. The first method of transfer would be conjugation whereby a pilus forms between close lying bacteria that links them for a short while to transfer the DNA fragments (Sefton, 2002; Leclercq, 2002). A second form of transmission is known as transformation where naked DNA from dead bacteria travel to a nearby bacteria and the DNA is subsequently taken up into the cytoplasm to be incorporated into the living bacteria. Transduction is also a popular methods of genetic methods of resistance whereby a vector is used to transfer the genetic material from one bacteria to another by infecting the bacterial cell. The vectors are usually bacteriophages (Patterson, 2001). The biological action that follows the transfer of genetic material determines the ability of the bacteria to express the transferred gene. One of the mechanisms is by the destruction of the antibiotic which occurs when enzymes produced by the bacteria chemically modify the drug rendering it inactive. An example of this mechanism is seen in the production of β-lactamases in S. aureus, P.aeruginosa, Klebsiella pneumoniae and several other microorganisms that work against β-lactam drugs such as penicillins and cephalosporins. Another mechanism that makes bacteria resistant specifically towards tetracyclines and fluoroquinolones is the antibiotic active efflux where it occurs when the bacteria develops a mechanism of active transport in order to remove the antibacterial component inside the bacterial cell until it the concentration decreases to a point where bactericidal activity stops (Moise et al, 2008).
Risk factors associated with antibiotic resistance One of the main reasons as to why resistance is an issue to begin with would be that antibiotics are being prescribed to patiently both hospitalized and outpatients in an excessive and alarming manner, not just for treatment but also for prophylactic use. After being prescribed a fairly broad spectrum antibiotic, as the practice is nowadays, most colonies of bacteria in the patients, including commensals, leaving resistant strains in the body to grow and multiply and eventually use the host as a reservoir (Gonzales et al., 2001). This is one reason as to why the prescription of antibiotics should be controlled and only given to those who absolutely need it. The increase in invasive procedures that utilise prosthetics such as valve replacements, pacemaker implants and plastic surgery, whereby foreign bodies may cause infections.
New drugs? As scientists understand the pathology of diseases and the molecular biology of it, the knowledge of new drug targets and different substances that have the potential of eradicating these diseases have come to light. Also, pharmacology companies have moved their focus from infections to designing drugs for other chronic diseases that affect the daily lifestyle of a patient such as osteoporosis and because of this diversion, the interest in developing new antibiotics that work against resistant strains has waned. The development of antiretroviral drugs have also caused divisions of infectious diseases research groups to have limited funding in their studies as more importance is placed on obtaining a cure for AIDS. New technological approaches that have emerged such as high throughput screening and genetic engineering has failed to actually give us a new class of antibiotics that could be utilised (Barett, 2005). The creation of novel classes of antibiotics would exhaust resources, would be extremely expensive and would be time consuming when compared to tweaking drugs that are already in the market, due to the waning interest in novel antibiotics, it is important that different platforms of society, i.e government, academics and pharmaceutical companies come together to collaborate on these projects. Alongside this, it is important to instil basic hygiene practices to control infections in the hospital and healthcare setting. Practices such as washing hands, proper disposal of clinical waste amongst other would ensure that the spread of infection in a healthcare setting is curbed (Shlaes and Moellering, 2002). Despite these measures being practical, the development of antibiotics is still deemed to be unprofitable due to the growing investments in other areas of scientific research that is deemed more important. If scrutinized properly, one does notice that investment in discovering new antibiotics and their subsequent production have been solely carried out by private pharmaceutical companies (DiMasi et al., 2003). Pharmaceutical companies currently do spend a huge amount of money in discovering and developing suitable new drugs. The growing costs of carrying out scientific experiments and sourcing the materials required, not to mention the overall time frame of drug discovery and development of around 10 to 20 years do cause pharmaceutical companies to rethink the need to focus on antimicrobials. Unless attractive incentives are offered to pharmaceutical companies, it is unlikely that these companies are going to show an interest in the development of antibiotics in the near future due to the reasons explained above. As of now, several tertiary research group have taken the initiative to involve the government and administrative force to collaborate with them and to include incentives towards pharmaceutical companies that do indulge in the development of potential antibiotics (Talbot et al., 2006).
Conclusion I would say that creating newer antibiotics will not solve the overall problem of resistance towards antibiotics seeing as microorganism will continue mutating and evolving in accordance to their need to survive, thus continuing to build resistance towards antimicrobials. Despite this, antibiotics will still be prescribed until a different form of treatment can be solidified and distributed to the public. It is now up to the scientists, clinicians, the pharmaceutical industry and governments worldwide to collaborate in preventing the occurrence of infection, controlling an outbreak of infection that is aided by the development of diagnostic tools as well as identifying patients that truly need antibiotics to begin with. Even in this case, patients should always be given a narrow spectrum medication unless multiple infection is detected to prevent the occurrence of multidrug resistance. More vaccines should also be created to prevent infections to begin with.
We are not living in the post antibiotic world yet, but it doesn’t seem far off unless collaborations are done to develop new antibiotics. Not only would it be harder to treat infections, but many other aspects of medicine would suffer as well, such as cancer therapy, organ transplantation that heavily rely on immune system suppression. We will also not be able to treat trauma patients who have been in a car crash for instance as antimicrobials would be needed as well. Childbirth would also be a problem as there are occurrences of postpartum infections. We are heading to a post antibiotic era unless measures are taken to develop new antibiotics or alternative form of treatment that is efficient.