Antimicrobial resistance (AMR) or drug resistance occurs when the microbes (fungi, viruses, bacteria, and others) areexposed to antimicrobial agents (antibiotics, antifungals, antivirals, and others); some microbes that develop resistance to the majority of antimicrobial agents are termed “superbugs.” This resistance is increasing worldwide. Thus, several actions may accelerate the emergence and spread of antibiotic-resistant bacteria, such as: misusing of antibiotics, poor infection prevention and control practices, working under unsanitary conditions and mishandling food that lead to spread illness, disability, and death¹. About 17 different classes of antibiotics have been produced to date
(Table 1)².

Table (1):
Major antibiotic classes by mechanism of action.

Modified from Levy and Marshall (2004)²

In the absence of antibiotics or antimicrobials for treatment and prevention of microbial diseases, other treatment procedures such as structure transplantation, cancer chemotherapy, diabetes therapy, and surgeries (e.g., caesarean servings or hip replacements) pose a high risk of infection.³ The total cost of healthcare may also increase depending upon drug resistant infections as well as longer stays in the hospitals and consequence more intensive care required.⁴

The discovery of antibiotics was a crucial moment in the history of mankind as it revolutionized scientific medicine and saved numerous lives. Unfortunately, the infectious strains are becoming resistant to antibiotics, and therefore, health professionals fear the return of the pre-antibiotic era. In this context, the bacterial genome has been examined as resistance strains, and was found that more than 20,000 possible examined bacterial genes have been successfully concluded as resistance strains⁵.

Antibiotic resistance against the antimicrobials was observed in the late 1950s and 1960s, among the intestinal bacteria, Salmonella sp, Shigella sp, and Escherichia coli.⁶ During this early period, these resistant strains caused enormous commercial losses and affected the clinical treatments worldwide; however, this phenomenon was presumed to be confined to the intestinal microbes. This misconception was later cleared in the late 1970s, after observing that Neisseria nebulae and Haemophilus influenzae were resistant to ampicillin and serotonin; moreover, few studies described their resistance to tetracycline and chloramphenicol. The increasing use of antimicrobial agents has led to several issues worldwide, specifically in developing countries, where these antibiotics are used without prescriptions. The hygiene settings prevented from transferring the confrontation and the trivial care boxes (T-box) that were manipulated for admission to new and competent antibiotics. T-boxes are structures that recognize when a cell is deficient in a specific amino acid, the building blocks of cells, and they allow bacteria to respond to this deficiency by initiating a process that generates more of that amino acid, including pathogens such as M. tuberculosis and B. anthracis, which causes the deadly anthrax disease^6,7.

Klebsiella pneumoniae, is a common intestinal bacteria causing several life-threatening diseases including pneumonia, and its worldwide spread is a major cause of hospital-acquired infections, as well as bloodstream infections, infections in neonates and intensive-care unit patients; this strain is usually resistant to carbapenem antibiotics⁸. In some countries, due resistance to this strain, the carbapenem antibiotic had no effect in more than half of people treated for pneumonia infection^8,9. Furthermore, another example of antibacterial resistance associated with urinary tract infection (UTIs), E. coli strain, a major bacterial species, and showed to be resistant to fluoroquinolone antibiotics (ciprofloxacin or norfloxacin), and Klebsiella pneumoniae is the second most important bacteria in this type of infection, the most common prescribed antibiotics to treat this infection UTIs are sulfamethoxazole, trimethoprim, fluoroquinolones, but on other countries showed that the bacterial resistance to sulfamethoxazole, trimethoprim and ciprofloxacin reached its critical^7,9.

In various health facilities and societies, certain microbes are resistant to the first-line drugs that treat infections caused by Staphylococcus aureus, a common cause of severe infections in these areas. In some cases, people infected with S. aureus resistant to methylation (methyl S. aureus) have a ≥64% mortality rate even with the nonresistant forms¹⁰.

In general, frequent of uses of antibiotics has several side effects; however, it has been observed that several patients use antibiotics to treat the wrong medical conditions. It is common to use antibiotics to treat respiratory infections caused by viruses such as the cold or flu (influenza), in this case uses of antibiotics had no affect on treat this infection¹⁰. The wrong uses of antibiotics lead to a new antibiotic-resistant strains. Based on data cited for to the centers for disease control (CDC), Overuses of antibiotic are a particular problem, and showed  to be higher in some regions of the world, as the Southeast. For example, carbapenems, a major class of beta lactam antibiotics, increased significantly from 2007 to 2010. carbapenems a group of broad-spectrum beta-lactam antibiotic agents are three parenteral preparations, used for treatment of severe or high-risk bacterial infections, similar to penicillins and cephalosporins¹¹.

Factors
Human factors
Human factors, caused by human behaviors regarding antibiotic use include: (i) self-medication, (ii) noncompliance of patients, (iii) antibiotic use for growth promotion in animal husbandry, (iv) ongoing transmission due to the lack of functional infection control programs,¹² and (v) accommodation of more than ten million expatriates, mainly hailing from endemic areas, for example, tuberculosis (TB) cases from endemic places, for work purposes as well as for religious rituals carried out in Mecca and Medina, Kingdom of Saudi Arabia.

Self-medication
Access to antibiotics is a main concern in several countries worldwide, including Saudi Arabia. The accessibility of antibiotics and community approaches toward the use and misuse of antibiotics have contributed tremendously to the increasing antibiotic resistance¹³. In several developing countries, including Saudi Arabia, antibiotics can be obtained as a commodity without a prescription from a qualified healthcare professional¹⁴. About 90% of upper respiratory infections are caused by viruses; unfortunately, physicians experience tremendous pressure from patients to prescribe antibiotics for these viral infections, as individuals erroneously think that antibiotics can treat such infections; hence, antibiotic prescription for viral infections is observed on a daily basis, despite that the rules and regulations forbid such act^15,16.

Noncompliance
Noncompliance arises due to various reasons including missing medication, early cessation of treatment as the patient starts to feel better, and inadequate access to appropriate antibiotics. Other potential reasons for noncompliance are dosage frequency, treatment duration, complexity of the treatment, and its side effects¹⁷. In addition, psychological distress can lead to noncompliance, as it has been found that some individuals do not comply because the medication changes the patient’s urine color¹⁸. Noncompliance undoubtedly promotes antibiotic resistance, as an unfinished course of antibiotics often wipes out most susceptible bacteria but allows the relatively resistant bacteria to survive and thrive^19,20. The infections caused by resistant bacteria resulting from patient noncompliance are difficult to treat, as patients remain sick for a longer period of time, which can necessitate long-term hospitalization and lead to the further spread of resistant bacteria²¹.

Antibiotics as a growth promoter in animal husbandry
Use of antimicrobial agents as growth promoters is an efficient technique for enhancing the productivity and health of livestock. Unfortunately, the existing use of antibiotics to treat animals and encourage animal growth has led to the emergence and spread of antibiotic resistance. For instance, avoparcin is a growth promoter that is used in the agricultural systems. Avoparcin significantly contributes to the emergence and spread of vancomycin-resistant enterococcal infections in the USA²². Another example is virginiamycin, which is used as an animal feed additive in the agriculture industry. The overuse of virginiamycin can lead to the acquisition of resistance to streptogramins. As a result of streptogramin resistance, the use of virginiamycin has been banned in Denmark and throughout Europe^23,24.

A study conducted in 2013 reported that Saudi Arabia has no published records on the use of antibiotics as a growth promoter, although several reports have described the isolation of various multidrug-resistant (MDR) bacteria from animal feed.²⁵ A survey was conducted by Al-Mustafa,²⁶ in which 23 randomly chosen poultry farms and all veterinary pharmacies in the eastern province of Saudi Arabia were identified and 29 antimicrobial agents were accessible for poultry use. Of the 29 agents, 22 antibiotics (75.9%) are vital for treating human infections. These antibiotics include ampicillin, neomycin, colistin, doxycycline, enrofloxacin, oxytetracycline, sulfamethoxazole, and erythromycin.²⁷ The prevalence of resistance to these antibiotics is unknown due to lack of data from both human and animal subjects²⁸.

Hospital-acquired infections (HAI)

These infections have been documented for more than a century as a serious problem disrupting excellence in healthcare²⁹. HAI are a major source of adverse healthcare outcomes. Hospitals, primarily intensive care units, are a significant breeding ground for the emergence and spread of antibiotic-resistant bacteria, as contact between the patients and hospital staff can generate risk for cross-infection^30,31. Despite various efforts considering the control of antibiotic-resistant nosocomial infections, only slight indication of better pathogen control within healthcare services has been observed in most countries, including Saudi Arabia³². To support patients with tuberculosis (TB), mainly multidrug-resistant tuberculosis (MDR-TB), patients are often hospitalized for prolonged time period³³. During long-term hospitalization, the patient may be cross-infected by a different strain of TB from another patient. Genotyping data have revealed that patients have indeed been cross-infected by different drug-resistant strains during the treatment period and hospitalizations³⁴. Additional genotyping data have revealed that Saudi Arabia is suffering from ongoing TB transmission, as indicated by high clustering rates of clades circulating in the community³⁵. Moreover, recent sequencing data have confirmed that Saudi Arabia faces tremendous challenges due to the ongoing transmission, which is mainly caused by MDR strains^36,37.

Influence of expatriates and pilgrims
Travel is recognized as a risk factor for the spread of various infectious diseases³⁸. Saudi Arabia faces certain challenges, as it accommodates more than 10 million expatriates that primarily descend from the endemic areas, carrying diseases such as tuberculosis (TB). In addition, as Saudi Arabia hosts two holy mosques, and also welcomes an additional 10 million individuals who visit annually for religious Islamic rituals³⁹. Those 10 million individuals stay at specific places at are in close vicinity to each other. Such conditions are ideal for the transmission and exchange of infectious diseases. Recent data have revealed that some travelers returning from the Hajj acquired New Delhi metallo-beta-lactamase-1-producing Escherichia coli and MDR Acinetobacter baumannii during the Hajj pilgrimage⁴⁰. In addition, data collected from two main hospitals in Mecca revealed that ceftazidime resistance was evident in 52.7% of Pseudomonas aeruginosa, 34.4% of K. pneumoniae, and 24.6% of E. coli isolates tested. Furthermore, another report indicated that the number of septicemia incidents in Mecca increases by 16.5% during the Hajj season due to the arrival of infected individuals from various countries^25,41.

Prophylaxis
Antibiotic prophylaxis can be used successfully to avoid infection in individuals with risk of bacterial infection due to other medical issues, such as recurrent cellulitis, meningococcal disease, spontaneous bacterial peritonitis associated with cirrhosis, infectious endocarditis, infections related to open fractures, prosthetic joint implantation, and wounds. Antibiotics may also be prescribed to prevent infection in individuals with destabilized immune systems, such as people receiving chemotherapy, those infected with HIV, and people who travel globally where they are likely to get an infection^42,43.

In all these circumstances, an authorized physician must decide whether antibiotics are necessary. Resistance resulting from the overuse of antibiotics, along with associated increases in healthcare costs and toxicity in patients, is eminent unless antibiotic use is restricted. In addition, drugs taken to prevent infection should only be used for a short time⁴².

Bacterial behaviors influencing drug resistance
One of the determinants for antimicrobial activity is the bacterial status. Under antibiotic exposure, bacterial phenotypes such as susceptibility, tolerance, resistance, and persistence differ.⁴⁴ These behaviors are enacted by pathogens as they attempt to protect themselves against antibiotics. Several bacteria exhibit natural (i.e., intrinsic) resistance to different types of antibiotics with various degrees⁴⁵. In addition, bacteria may develop resistance by two extrinsic mechanisms: acquisition of a resistance-encoding gene from another bacterium, which occurs via horizontal gene transfer (HGT) or by genetic mutation. Both of these extrinsic mechanisms enable the bacteria to expose a novel resistance determinant. The ability of bacteria to transfer resistance genes among themselves significantly contributes to the spread of antibiotic resistance⁴⁶. Bacterial factors include (i) horizontal gene transfer, (ii) physical or chemical mutations, (iii) epistasis, (iv) selective pressures, (v) biofilms, and (vi) outer membrane permeability by bacterial transport systems (Fig. 1).

Horizontal Gene Transfer (HGT)
HGT is known as the transfer of genetic material from one species to another species⁴⁷. Genetic transfer occurs by plasmids, bacteriophages, and transposons that can move from the chromosomal- and plasmid-associated resistance genes to disparate bacterial hosts. In addition, DNA released from dead bacteria can be obtained and recombined into the bacterial genome, thus emerging in new strains⁴⁸. Mobile genetic elements, such as plasmids, bacteriophages, genomic islands, and transposons, play a significant role in the progression of various bacteria and enable the spread of genes associated with drug resistance, pathogenicity, and fitness, causing the emergence of “hospital superbugs.”^{49, 50} Mechanisms of HGT vary widely among the bacteria, and it acts as a key mediator of antibiotic resistance and spread⁵¹.

Mutations
The mutation would cause a change in the genomic structure of the bacteria and produce new species and traits, including bacterial resistance to antibiotics. Mutations divided into three types spontaneous (natural), physical or chemical, and genetic manipulation by transferring genes from bacteria to other bacteria using different cloning methods that acquired the modified bacteria (mutant strain) new characteristic over than the wild type such as resistance to antibiotics, salt tolerance, and others. Some mutations allow bacteria to synthesize enzymes that inhibit antibiotics, whereas others may remove the antibiotic targets from the cell, thus inhibiting the antibiotic activity or restricting antibiotic entry into the cell⁵². When bacterial populations are treated with these specific antibiotics, resistant bacteria will be able to reproduce and can increase the number of bacteria, and the end result is the a group of different bacteria resistant mainly to the specific antibiotic. Mutations caused by antibiotic resistance can have a clinical significant impact on the effectiveness of specific antibiotic groups or specific bacterial pathogens. Mutations can also modify conditions in which the resistance genes are spread. In the long run, mutations may be crucial in the development and diversification of acquired resistance elements^52,53.

Epistasis is defined as the interaction between different genes, which is essential in both molecular and quantitative genetics⁵⁴. In recent years, Epistasis has become a hot topic in complex disease genetics, for complex traits such as diabetes, asthma, hypertension and multiple sclerosis. This is probably due to complicating factors such as an increased number of contributing loci and susceptibility alleles, incomplete penetrance, and contributing environmental effects⁵⁵. Epistasis is notable phenotypic differences among individuals with the same genotype at one locus depend on their genotypes at another locus⁵⁶. The progress of epistasis is mainly missing from classical population genetics research⁵⁷. In the past decade, however, this topic has received immense attention as the data related to the monogenetic systems have become accessible⁵⁸,functional⁵⁹, or physiological⁶⁰.

Epistasis is the effect of one gene (locus), which is reliant on the presence of one or extra “modifier genes,” that is, the genetic background⁶¹. On the other hand, this term indicates that the phenotypic effect of one gene is masked by a disparate gene (locus). Thus, epistatic mutations possess disparate effects in combination than individually. It was primarily a concept from genetics but is nowadays utilized in biochemistry, computational biology, and evolutionary biology, due to the gene interaction, managing the nonlinear effects. Epistasis has a colossal impact on the form of evolutionary landscapes, that leads to profound consequences for progress and phenotypic trait evaluability⁶². Epistasis can considerably affect antimicrobial resistance. Moreover, the combinations of resistance mutations may crucially affect the development of multidrug resistance⁶³.

At the genotypic level, epistasis occurs by the one locus gene masks, or prevents the phenotype of another locus gene for ascertaining fitness while not noticeable at phenotype level, as described by Lunzer⁶⁴. In other theoretical studies, on the progress of recombination, the form of the fitness used as a function of number of deleterious mutations shown to be critical⁶⁵. In contrast, negative epistasis, under certain conditions of the recombination (or sex), is favorable, with increasing mutation number and linear decrease in the log fitness⁶⁶. Contradictory to the positive epistasis, an approximate numerical treatment by Azevedo⁶⁷, revealed that the deleterious mutations can be recombined and should be targeted toward negative (synergistic) epistasis, and whether recombination can influence epistasis⁶⁸.

Selective pressure
The use of antibiotics in abundance is associated with the significant rise of multidrug resistance and the way antibiotics are used. Bacteria can also gain more resistance to antibiotics in response to environmental stresses; however, these antibiotics provide selective pressure that can lead to the emergence of several bacteria exposed to mutated antibiotics or that obtain slices of DNA to develop antibiotic resistance. DNA slices may encode these multidrug pumps and enable the flow of various antibacterial agents from the cell. The selection pressures resulting from the use and misuse of antibiotics significantly support the phenomenon of antibiotic resistance^69,71.

Biofilm formation
Biofilm is defined as a self-produced polymer matrix comprising polysaccharides, proteins, and the genetic material (DNA), which is the common mode of growth for most microorganisms in natural and medical systems and leads to protective bacterial growth that promotes the survival of bacteria in hostile environments⁷². Nevertheless, in biofilms, the prevalence of antibiotics is assumed to be extremely weak, due to nutrient constraints, slow growth, adaptive stress responses, and the formation of a series of coherent cells that form a multilayered defense system⁷³. Therefore, according to drug resistance, each gene and gene product can be used as a target for developing new chemotherapeutic agents; however, the presence of biofilm formation, that facilitate chronic infections and antibiotic resistance.⁷⁴ Thus, bacterial biofilm-specific resistance is considerable higher than any antibiotic resistance in planktonic bacteria. Consequently, biofilm-related infections are more problematic to treat and more prone to recurrence. The relation between biofilm and antibiotic resistance is essential for biomedical researchers⁷⁵.

The bacterial cells cannot be affected by the antibiotics used without genetic modifications; these types of cells are recognized as persister cells. In contrast, resistant cells have the ability to grow in the presence of antibiotics, and do not produce inhibitory cells in the presence of these antibiotics^76,77.

Outer membrane permeability and bacterial transport systems
The outer membrane of microorganisms crucially provides an additional layer of cellular defense to the organism without interfering with the exchange of essential substances necessary for cell survival. In addition, most of the antibiotics used have intracellular targets and cross the bacterial cell envelope to have an effective impact. Thus, the outer membrane of microorganisms is a difficult barrier that various antibiotics must cross in order to inhibit their growth. The lipid-mediated route is the main pathway that antibiotics can use to cross the bacterial outer membrane. The lipid and protein contents of the outer membranes have a strong potent effect on the bacterial sensitivity to various antibiotics, and resistance to these drugs relate to changes in these large molecules⁷⁸.

Bacterial transport systems comprising structured protein channels may also influence antibiotic resistance in microbes by assisting the bacteria in both the acquisition of essential nutrients and the expulsion of toxic molecules, such as host bactericidal molecules and antibiotics. Consequently, bacterial transport systems play a significant role in both intrinsic bacterial drug resistance and the failure of antibiotic treatment^78-80.