Understanding microbial infections using microarray technology

Human microbial infections are symbiotic processes between pathogens and humans that often lead to human disease and death. Microbial infections involve the attachment, growth, and survival of microorganisms on human skin, inside the body, or inside specific cells. Microbial infections can be localized to one body region or migrate to secondary body locations utilizing various transport mechanisms. Understanding host-pathogen interactions related to the expression of essential genes during and after infection can lead to valuable information for biologists and clinicians. Microarray technologies allow researchers to perform genomic characterization experiments rapidly and efficiently. Microarray experiments support the resolution of underlying molecular events that play a role in normal and aberrant physiologic activities in living systems. Microarray technology, coupled with bioinformatics analysis, generates comprehensive insights into relevant genes, proteins, and protein-protein interactions. This review article explores recent microarray research studies from select protozoan and bacterial pathogens to illustrate how researchers utilize microarray technology to examine microbial infection aspects. Microarray studies of pathogen and host genomes at various stages of the infection process will generate a more precise understanding of pathogenic life cycles and pathogen survival strategies. Detailed knowledge of the genes involved in the microbial infection process will lead to discovering disease biomarkers and potent therapeutic solutions.


Introduction
Completing the human genome project paved the way for developing high throughput technologies in functional genomics such as DNA microarrays and next-generation RNA sequencing. DNA microarray protocols are widely practical methods for investigating genome-level transcription events in cells and tissues in a separate experiment [1]. DNA microarrays are solid surfaces that contain microscopic copies of complementary DNA or oligonucleotides of varying lengths (e.g., short or long) arranged in spots [2]. Common microarray platforms such as Affymetrix, Agilent, and Illumina are more ubiquitous in today's market and can even be customized to address specific research questions. In the past, the costs to conduct microarray experiments were not cost-effective for most life science laboratories. Today, however, costs associated with microarray experiments are somewhat manageable for laboratories with relatively small budgets.
Nucleic acid microarray experiments involve the selection of an appropriate biological process to examine. Next, mRNA is isolated and converted to complementary DNA (cDNA). The cDNA is then labeled with a fluorescent molecule and applied to a microarray surface containing oligonucleotides. Fluorescence intensity comparisons of experimental treatments and baseline samples, following hybridization of nucleic acid sequences, provide evidence regarding up-regulated genes, down-regulated genes, and non-active genes [1]. Comparing differential gene expression in normal cells vs. abnormal cells or healthy cells vs. diseased cells is a common strategy to elucidate the relevance of underlying genetic mechanisms that participate in biological processes. In addition to identifying particular activated genes and gene expression levels, analysis of microarray data sets can provide information about biological processes, transcription factors, signal transduction pathways, biomarkers, and diseases associated with gene expression clusters.
DNA microarrays are used to diagnose microbial pathogens in patients, food samples rapidly, and water samples [3][4][5][6]. Microarrays demonstrate the application in resolving antimicrobial resistance in clinically-relevant microbes [7][8] and biomarker elucidation [9][10]. Microbial-based microarrays are cost-effective compared to other microbe detection techniques and offer the added benefit of providing valid identification of test samples in less time. Clinical samples (e.g., including nasopharyngeal, respiratory, blood, buccal, fecal, urine, spinal fluid, and saliva) can be used to detect specific microbes known to cause disease and determine the difference between antibiotic-susceptible and antibioticresistant bacteria. Recognition of antibiotic resistance genes in clinical samples can impact therapeutic strategies and potentiate patient recovery and survival.

Microbial Infections
A microbial infection occurs when a microorganism (e.g., protozoa, bacteria, fungi) invades the human body through a specific entry portal. Humans can be exposed to microbes in various ways, including ingestion, inhalation, fecal contamination, physical contact with fomites, and body fluid transfer. Moreover, co-infections exist in which more than one type of microbe (e.g., bacterial and viral) participates in primary and secondary infections [11][12]. Figure 1 summarizes the stages of many microbial infections. Figure 1 does not account for all known microbial infections.
The first step involved in the infection process is the attachment phase. Microbes possess attachment factors that mediate attachment to the skin or mucous membranes. Specific receptor molecules on the surface of the skin and mucous membranes are complementary to microbial attachment factors. Some microbes remain attached to the skin or membranes; others penetrate these outer layers and become positioned in the internal environment. Many pathogenic microbes are ingested via contaminated food and water.

Figure 1 Basic microbial infection strategy
Some microbes also can invade specific cells, tissues, and organ systems using complex host-pathogen mechanisms in which microbial growth, differentiation, proliferation, and survival are the main biologic objectives. In terms of microbial infections, an examination of gene expression data can reveal how microbes activate human and microbial genes to complete the fundamental infection strategy shown in Figure 1. The next section of this review discusses research utilizing DNA microarrays to detect specific microbes and examine microbial gene expression profiles.

Protozoa and Microarray
Protozoa are unicellular eukaryotes that cause various human diseases such as Toxoplasmosis, Giardiasis, Malaria, and Trypanosomiasis. There is now a plethora of useful DNA microarray platforms to explore pathogenic protozoa [13][14][15]. Chen et al. [16] developed a DNA microarray system to concurrently detect 18 different human bloodborne protozoa species from 5 of the most common genera of protozoa found in mammalian blood (Plasmodium, Leishmania, Trypanosoma, Toxoplasma gondii, and Babesia). A diagnostic test with a relatively low detection limit detected waterborne pathogenic protozoa such as Cryptosporidium parvum [17]. The microarray was deemed adequate and clinically relevant based on comparative verification tests. The investigators utilized protozoan small ribosomal RNA (rRNA) nucleotide sequence probes on the microarray platform. Ribosomal RNA subunit gene sequences serve as excellent microbial probes for diagnostic microarrays for several reasons. First, both the small and large subunits of prokaryotic and eukaryotic ribosomes are highly conserved among species. Additionally, rRNA gene sequences undergo fewer nucleotide modifications when compared to their macromolecular counterparts.
Classification of gene profiles during the various stages of infection could create molecular countermeasure approaches that suppress microbial infection capacity and kill the microorganism or activate host immune mechanisms. Some protozoal parasites, such as amoeboid microbes, can transition into multiple biological and structural forms (e.g., trophozoite, cyst) depending on the type of host they inhabit. The trophozoite stage is the vegetative phase or feeding and amplification stage of the parasite. It is typically found in the human host, while the cyst stage is the microbe's protective form and is observed in the environment after being passed in feces. A cyst stage is a resistant form of the parasite that can survive harsh stimuli (e.g., climatic changes, chemicals). The cyst is converted back to the trophozoite stage inside the human host's intestines following human consumption of the cyst via contaminated water or food. Understanding the genetic changes that occur during trophozoite-cyst conversion is paramount to control efforts. Moon, Xuan, Chung, Hong, and Kong [18] performed a microarray study to map the essential genes responsible for Acanthamoeba castellanii encystation. Gene expression profiles of cysts were compared to Acanthamoeba trophozoites following microarray and bioinformatics procedures. There were 701 upregulated genes and 859 downregulated genes in the cyst stage compared to the trophozoite stage. Not surprisingly, a portion of the differentially expressed genes was associated with metabolic functions, according to KOG analysis. Since Acanthamoeba encystation within the host further exacerbates immunological eradication efforts, understanding the essential genes involved in the encystation process may be beneficial.
Recently, molecular parasitologists set out to identify biomarkers associated with miltefosine-resistance in visceral leishmaniasis. Following treatment with miltefosine, infected patients were evaluated. Comparing differential gene expression characteristics of parasites from relapsed and cured patients, Tiwary, Kumar, and Sundar [10] showed that a cysteine protease-like protein was highly upregulated from relapsed patients, suggesting that the cysteine proteaselike protein could serve as a biomarker to monitor patient relapse.

Bacteria and Microarray
Bacteria are unicellular prokaryotes that account for a large number of human microbial diseases. Some of the wellknown bacterial infections are leprosy, diphtheria, plague, tuberculosis, and cholera. Ranjbar, Behzadi, Najafi, and Roudi [19] recently designed a DNA microarray platform that contained distinct oligonucleotide sequences that were specific to medically relevant bacteria (Escherichia coli, Shigella boydii, Sh.dysenteriae, Sh.flexneri, Sh.sonnei, Salmonella typhi, Brucella, Legionella pneumophila, and Vibrio cholera). Following the experiment, the microarray successfully detected all of the bacteria at the same time. Moreover, researchers recently used microarray technology (FDA-ECID DNA Microarray) to identify and characterize virulence gene composition of non-O157 E. coli serovars [20]. Virulence gene identification in clinical isolates is equally as crucial as microbial identification and can provide a greater depth of understanding regarding the nature of infection and genetic factors influencing patient-related pathophysiological outcomes.
Nosocomial infections are contracted during a stay at a healthcare facility and were not present before the patient was admitted. Keum et al. [21] developed a DNA microarray-based detection system to identify nosocomial pathogenic Pseudomonas aeruginosa and Acinetobacter baumannii in clinical isolates. The microarray technology demonstrated a sensitivity of 84.6% and 96.2% for A. baumannii and P. aeruginosa. Both nosocomial pathogens displayed a positive predictive value of 100%. Purulent meningitis is characterized by acute inflammation of the membranes associated with the central nervous system and is particularly devastating in neonatal populations [22]. Bacterial and viral infections primarily cause purulent meningitis. Purulent meningitis leads to various unpleasant symptoms, which depend on the microbial agent and can lead to death if untreated. Hou et al. [3] designed a DNA-based microarray to enhance diagnostic efforts of purulent meningitis bacteria. A significant number of positive test results (87.5%) were generated using the microarray detection approach compared to 58.3% using the traditional cerebrospinal fluid culture detection method. The application of a rapid identification and detection procedure significantly reduces diagnostic deliberations.
Antibiotic-resistant bacteria are the scourge of healthcare facilities across the world. Antibiotic-resistant bacteria lead to unimaginable loss of life and account for millions in medical treatment costs. Antibiotic-resistant bacteria are a global health threat that could render today's powerful antimicrobial options essentially useless. Moreover, as many have pointed out, antibiotic resistance may be further complicated by severe acute respiratory syndrome coronavirus two infections and COVID-19 [23]. The use of rapid molecular recognition technology allows multiple bacteria to be tested for genetic signatures that confirm antibiotic-resistant genes are extraordinarily beneficial to physicians and patients. Carbapenemase and extended-spectrum β-lactamases (ESBLs) are particularly problematic enzymes produced by some bacteria because they confer antibiotic resistance to bacteria. Carbapenemase-and ESBL-producing bacteria have generated a significant number of hospital-acquired infections (HAI) worldwide. Uddin et al. [8] created a microarray platform designed to detect Acinetobacter baumannii carbapenemase and ESBL genes in patient specimens. Researchers demonstrated that their microarray-based method (CT 103XL Check-MDR) of antibiotic resistance genes detection is equally or more effective than other methods.

Conclusion
Less than 1% of the microbes on earth lead to human disease. However, the impact that microbial infections have on the economy, human health, and other societal factors elicit enormous medical and research communities' responses. Techniques such as DNA microarrays and now next-generation RNA sequencing or RNA Seq are becoming increasingly prevalent in life science laboratories because of their sensitivity, high throughput capacity, and application. Moreover, these techniques are more advantageous for microbial detection and identification than traditional sequencing and PCR arrays. Microbial detection microarrays are constructed by adding oligonucleotides (probes) from specific microbes to a solid matrix. From a microbial perspective, nucleic acid hybridization techniques are largely applied to clinical diagnostic assays. However, a growing segment of the literature points to a shift in DNA microarrays to study underlying genetic mechanisms of fundamental biological processes. The determination of the molecular constituency, biomolecular interactions, and canonical signaling pathways associated with microorganisms can provide a wealth of beneficial biological and clinical information.
A review of the DNA microarray investigations in this article highlights previous uses of this technology to examine microorganisms' biology. This review also focuses on using nucleic acid technology to accurately and rapidly detect unique microbial species from clinical, food, and water samples. In clinical and hospital environments, tests that can rapidly detect and identify microbes are paramount. A delay in diagnosing an infectious entity may provide the microbial agent more time to increase and potentially expand to other ectopic sites in the human body, thereby causing more damage to tissues and vital organs. Elaborate time-course studies could allow microbiologists an opportunity to map the global gene expression profiles of bacteria and protozoa at different phases of the infection process (e.g., attachment, penetration, proliferation, etc.). It is hypothesized that unique genes or gene families are involved in discrete stages of the microbial infection process. Further, microbial genes can be identified that likely play a role in host symptoms and clinical outcomes. With such detailed molecular characterizations, it would be possible not only to identify biomarkers but to link specific microbial gene changes with distinct phases of infection (i.e., pathogenesis markers). Moreover, correlating gene expression profiles with microbial responses to drugs and other treatments is also possible using DNA microarrays. Performing microarray procedures to analyze host gene expression profiles during a microbial infection can also have tremendous scientific and therapeutic value. New microarray platforms are needed further to assist microbiologists, clinicians, and other healthcare workers.
Future microbiological investigations will explore microarray technology to understand host responses following viral and bacterial infections at the molecular level. The advent of new quality control, normalization, and bioinformatics software are certain to have a constructive impact on microarray data usability and diversity of data visualizations. Findings generated from DNA microarray studies will open up new possibilities to treat and prevent microbial diseases.