Microarray in Determining Anitimicrobial Drugs

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Bacteria possess a broad genetic diversity and can have a number of mutations in diverse gene families associated with antimicrobial resistance (eg.bla, aac, van, gyr, mecA, etc.). For example, mutations in the beta-lactamase genes blaTEM-1 and blaTEM-37 lead to resistance of E. coli to ampicillin and to third- and fourth-generation cephalosporin. Single-Nucleotide Polymorphisms (SNPs) present in specific genes also leads to antibiotic resistance. For example, SNPs in the gyrase gene gyrA in E. coli leads to the resistance of the organism to nalidixic acid and fluoroquinolones. Presence of SNPs can be detected by microarrays using hybridization or enzymatic methods with either DNA polymerase or DNA ligase. A disposable microarray was developed for the detection of up to 90 Antibiotic resistance genes in gram-positive bacteria by hybridization. Multidrug-resistant strains of Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, a virulent strain of Bacillus anthracis harboring the broad-host-range resistance plasmid, Staphylococcus haemolyticus and Clostridium perfringens were detected using this technology thus indicating it's large potential in research, food safety and surveillance programs for antimicrobial resistance.

Mycobacterium tuberculosis is a slow growing pathogen and thus determination of its antibiotic resistance is challenging. To overcome this issue, a TB-Biochip oligonucleotide microarray was developed to detect mutations associated with rifampin resistance in Mycobacteria. This microarray developed proved to be rapid with a sensitivity of 80% and a specificity of 100% as compared to the conventional drug susceptibility testing results for rifampin resistance. Other microarrays are used for mapping of mutations of pyrazinamide-resistant Mycobacterium tuberculosis strains. Mutations in rpoB, katG, mabA, inhA, rpsLL, rrs and embB to antitubercular drugs have been detected using Combination microarrays. QIAplex-based suspension bead array developed by Gegia et al detects 24 mutations in Mycobacterium tuberculosis that converse resistance to isoniazid, rifampin, streptomycin and ethambutol.

Microarrays are also used in detecting and characterizing food-borne pathogens, strain discrimination within a particular patho-type and also identify genetic elements responsible for virulence and antimicrobial resistance. From an epidemiological point of view, microarray technology enables the screening of large numbers of bacterial isolates for the presence of a large number of antimicrobial resistance genes. Thus data about the prevalence and diffusion of resistance determined over time and geographic areas can be obtained. A key example of the potential or capability of DNA microarray technology for the detection of many resistant genes can be found in the microarray built through the use of the target gene sequences found in GenBank. This method is being used as a screening technique to study the prevalence, epidemiology and spread of resistance genes among bacteria namely, S. enterica, E. coli, MRSA, Camphylobacter spp., Listeria spp., Enterococcus spp. etc. Another microarray developed identifies common resistance genes in gram-positive and gram-negative bacteria. Azole resistance in Candida albicans is detected using a genome-wide expression microarray. Multiplex PCR followed by oligonucleotide array is being used to detect and identify polymorphisms associated with Plasmodium falciparum antimalarial-drug resistance. In the case of viruses like HIV and HBV, microarrays are used to detect mutations in viral genome that predict resistance to certain drugs and therefore direct appropriate therapeutic options. Oligonucleotide microarrays are used to detect polymorphisms in HBV that are important in regards to disease prognosis and antiviral resistance and also determine the relative proportion of wild-type HBV and lamivudine-resistant HBV in patient sera. However, in the case of HIV, development and maintenance of a microarray for its genotyping is difficult and challenging due to the constant and rapid changes taking place in its genome.

In addition, microarrays can also be used to determine antibiotic resistance mechanisms and pathways by observing the changes that takes place in gene expression in response to environmental changes. These results contribute to better understanding of drug resistance which enables development of new drugs. Resistance caused by enzymatic inactivation of antimicrobials can be detected using microarrays by studying the genes that code for these enzymes that modify aminoglycoside antibodies or degrade beta-lactam drugs, e.g. Penicillin and Cephalosporin. Microarrays enable study of permeability changes with respect to its role in resistance, e.g. Tetracycline resistance, and also study target alterations as in the case of resistance to macrolides and (fluoro) quinolone antimicrobials. In 2002, a specific serovar of S. enterica, which is resistant to multiple antibiotics, was investigated in a study by microarray technology, revealing genetic deletions in the glyoxylate pathways and in the second - phase flagellar gene. Microarrays are also used to obtain information about virulence-related genes. This, along with antimicrobial resistance, both detected by DNA microarrays, is now used to study the evolution and/or transfer of mobile antimicrobial resistance and virulence - associated genes.

The primary limitations of using microarrays are cost, data management and interpretation. Large amount of data generated from a single microarray may be overwhelming to the user and usually requires computers and advanced software packages for data analysis. Due to this, its routine use in clinical microbiology laboratories is highly unlikely. In the future, as costs decline and competition in this area increases, clinically useful microarray assays may become available for routine use in clinical microbiology laboratories. These technologies will compete directly with DNA sequencing for rapid identification of microorganisms and for the detection of genetic polymorphisms associated with antimicrobial drug resistance and human susceptibility to infection.

Bibliography:

- Call DR, Bakko MK, Krug MJ and Roberts MC. 2003. Identifying Antimicrobial Resistance Genes with DNA Microarrays. Antimicrob. Agents Chemother. 47(10):3290.

- Card R, Zhang J, Das P, Cook C, Woodford N and Anjum MF. 2013. Evaluation of an Expanded Microarray for Detecting Antibiotic Resistance Genes in a Broad Range of Gram-Negative Bacterial Pathogens. Antimicrob. Agents Chemother. 57(1):458.

- Frye JG, Jesse T, Long F, Rondeau G, Porwollik S, McClelland M, Jackson CR, Englen M, Fedorka-Cray PJ. 2005. DNA microarray detection of antimicrobial resistance genes in diverse bacteria. Int. J. Antimicrob.