Microarray in Determining Anitimicrobial Drugs
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Microarray in determining antimicrobial drug resistanceClaire D’melloRoll no.: 04UID No.: 138605MSc Part II Microorganisms include bacteria, viruses, fungi and parasites, most of which cause infections in humans, animals and plants. Antimicrobial drugs, namely, antibiotics, antivirals etc. have been successfully used to treat patients with bacterial or viral infection respectively. However, over the years, many infectious organisms have become resistant to the drugs designed to kill them, rendering the products useless. Antimicrobial resistance (AMR) occurs via many different mechanisms. However, two main genetic events generate majority of antimicrobial resistance currently observed: when microorganisms evolve by replicating erroneously leading to mutations of native genes to resistant alleles; and when resistant traits are exchanged between them. This enables microorganisms to withstand attack by antimicrobial drugs, making standard treatments ineffective, leading to persistence of infections and increase in the risk of spread to others. The use and misuse of antimicrobial drugs accelerates the emergence of drug resistant strains. Poor infection control practices, inadequate sanitary conditions and inappropriate food handling encourages the further spread of AMR. Resistant bacterial phenotypes are characterized by growth in the presence of antimicrobials using test methodology like broth dilution, disk diffusion and Etest. Initially, identification methods for the genes that cause resistance was limited to PCR and southern blotting, which is cumbersome and can only detect one or a few genes at a time. Therefore, identifying the genes responsible for resistance required laborious screening for hundreds of possible AMR genes. However, over the past decade, an enormous amount of progress has taken place in developing genotypic drug resistance assays that are more accurate and rapid. Genotypic drug resistance assays are used more in clinical microbiology laboratories, especially for detection of antimicrobial resistance in microorganisms that grow slowly, such as multidrug-resistant M. tuberculosis, or for rapid detection of difficult-to-detect resistance mechanisms such as those found in MRSA and VRE. It is now possible to detect thousands of genes simultaneously using DNA microarrays and determine if specific antimicrobial resistance genes are present in a particular microbe. Microarray, commonly known as gene chips have been used for many years to detect a multitude of signals simultaneously and may be used to detect genetic (DNA) differences or differences on expression (mRNA). They are useful in determining which genes are expressed and which are silenced in response to different stimuli. The microarray consists of thousands of hybridization sites, each of which provides information regarding the nucleotide composition of the amplicon. The signal generated from the microarray may be fluorescent or electrical. There are many types of microarray namely DNA microarray, protein microarray, peptide microarray, tissue microarray, cellular microarray, antibody microarray etc. DNA microarray, also known as DNA chips, are made up of glass and are divided into small square fields. Each field contains copies of a specific synthetic DNA probe and about 20 nucleotides in length attached to the glass. Along a row of fields, the sequence of the probe differs by one nucleotide from field to field. Thus, a set of four fields is needed in order to test the nucleotide content of a given position in a DNA molecule. Modified microscope glass slides have become the most widespread format for custom microarrays. Microarrays can be used for several purposes like microbial identification, virulence factor, antimicrobial resistance and food safety analysis. Such microarray analysis methods have potential use in research, public health, clinical, industrial, agricultural and ecological settings.
For genetic testing, DNA is extracted from cells and cut with restriction enzymes. The resulting fragments are tagged with a fluorescent dye (often red or green), denatured into single strands and pumped into the microarray. Fragments with nucleotide sequence that exactly matched the probe sequence will bind, and those with a sequence that doesnt match are washed off. The microarray is then scanned by a laser. The fields where hybridization has occurred fluoresce (red or green). Software linked to the microarray analyses the pattern of hybridization and the data is then presented in several forms. DNA microarrays provide detailed information on the presence or absence of a large number of antimicrobial resistance genes in a single assay within several hours thus proving to be a time-saving and conventional tool. Microarrays have been successfully used to determine the antibiotic resistance in clinical samples. Now-a-days, this technique is also being used for environmental samples. However, it cannot detect the desired genes when the sample is in less quantity. This obstruction can be overcome by using PCR. The use of multiplex PCR for amplification of different target genes combined with detection on DNA microarray of capture probes represents one of the most powerful strategies and is being increasingly used. A prototype microarray developed in 2003 by Call et al demonstrates the feasibility of developing relatively high-throughput, planar microarrays for identification of resistance genes. The probe fragments were cloned into a common vector thus making the production process more efficient. Microarrays designed from PCR products require positive control constructs for the PCR template which proves to be a disadvantage. These long probes are less sensitive to minor point mutations, such as those required for protection against extended-spectrum beta-lactams. In 2013, a revised microarray developed by Card et al, proved to be a powerful alternative to PCR for the rapid, accurate and simultaneous detection of a large number of clinically important acquired genes in a diverse collection of gram-negative bacteria including E. coli and Salmonella, associated with human infections. As seen in PCR, only desired genes that are being amplified and present on microarray are detected even when not expressed. The rate of detection of these genes is highly accurate and specific. However, when resistance develops due to other genes or mechanisms which are not present on the microarray, they will not get detected. Thus, for the microarray, just like PCR, the absence of a signal doesnt indicate that genes or mechanisms leading to resistance are absent. The advantage of using microarray over multiplex PCR is that, in microarray, a chip is present at the bottom of each well of a 96-well plate, thus enabling a large number of genes and up to 96 samples, in this case, to be tested simultaneously, thus providing genotypic results within 5 hours post bacterial growth. This helps in reducing the risk of administration of antibiotics that may not be effective for treatment and prevents development of highly resistant strains.