Escherichia coli (E. coli) Resistance against Last Resort Antibiotics and Novel Approaches to Combat Antibiotic Resistance

Abstract

An important feature complicating the treatment of infections caused by E. coli is the increase in resistance to different antibiotics, even to last resort antibiotics. When resistant bacteria spread to the community, resistance creates comprehensive infection control issues, increasing morbidity for non-hospitalized patients of all ages and sexes. New resistance mechanisms are constantly being described, and new genes and vectors of transmission are identified on a regular basis. This chapter reviews different mechanisms of E. coli resistance against different classes of last resort antibiotics such as fosfomycin, nitrofurantoin, and polymixins. In addition, E. coli vaccines, epidemiology, and novel approaches to combat antibiotic resistance will be discussed throughout the chapter. In the age of antibiotic resistance and precise microbial genome engineering, many new strategies are now being used to combat multidrug-resistant bacteria, hoping to be our end game weapon. These strategies include CRISPR-Cas antimicrobials, nanobiotics, phage therapy, and probiotics, which promise to have a substantial impact on the way we treat diseases in the future, as we will discuss in the chapter.

1. Introduction

Escherichia coli is the best-known member of the normal microbiota of the human intestine and a versatile gastrointestinal pathogen. E. coli infection is a major global problem in the clinical and community setting. The prevalence of E. coli among clinical specimens varies from country to country and even among two different institutions in the same country and continuously changes over time [1, 2, 3, 4]. More and more people die each year from hospital infections caused by multidrug-resistant E. coli [5].

According to the WHO, E. coli is considered a global critical pathogen that possesses the highest priority for research, discovery, and development of new antibiotics [6]. When antibiotics are consumed during bacterial infection treatment, the resistance of the commensal E. coli is developed after exposure [7]. Undeniably, commensal E. coli is one of the main reservoirs for antibiotic resistance transmission to other pathogenic bacteria through plasmid exchange, for example (Figure 1) [8, 9, 10]. Via contact with livestock or a contaminated natural environment, humans can be exposed to viable commensal antibiotic-resistant E. coli [11].

Figure 1.

Transfer of resistance between bacteria through plasmid exchange.

Indiscriminate use of antimicrobials and antibiotic overuse has led to the treacherous resistance rates in recent years, creating a very complicated therapeutic challenge that threatens to return clinicians and patients to a “pre-antibiotic era”. Furthermore, mobile genetic elements (plasmids, bacteriophages) carrying antibiotic resistance genes (ARGs) play a major role in transferring resistance to both human and nonhuman, contribute to the spread of antimicrobial-resistant organisms, and increases the risk factor of infections and diseases in both animals and humans [12].

This chapter will discuss various mechanisms, epidemiology, vaccines, and novel approaches to combat E. coli antibiotic resistance.

2. Antimicrobial resistance against last resort antibiotics in E. coli

Generally, there are five main mechanisms by which gram-negative organisms develop resistance: First, bacteria can carry genes coding for enzymes, such as beta-lactamases, hydrolyzing, and inactivating beta-lactam antibiotics. Second, mutations can occur in the genes for binding sites for antibiotics changing the specific target or its function. Third, alterations of the membrane porins result in reduced permeability. Fourth, bacteria can express efflux pumps to actively transport antibiotics out of the cell, and finally, fifth, alternate metabolic pathways can bypass paths inhibited by antibiotics [13, 14].

Resistance in gram-negative bacteria can be intrinsic, arise, or be acquired and is often composed of a combination of resistance mechanisms like beta-lactamases, porin deletions, and efflux pumps [15].

Acquired bacterial resistance may be due to mutations in chromosomal genes and by horizontal gene transfer. The intrinsic resistance appears due to inherent structural or functional characteristics (Figure 2).

Figure 2.

Proportion of anionic phospholipids in the cytoplasmic membrane is lower in gram-negative bacteria than gram-positive bacteria so the efficiency of the Ca²⁺-mediated insertion of daptomycin into the cytoplasmic membrane that is required for its antibacterial activity is reduced [16].

The intrinsic resistance of some gram-negative bacteria to many compounds is due to an inability of these agents to cross the outer membrane: For example, the glycopeptide antibiotic vancomycin inhibits peptidoglycan cross-linking by binding to target d-Ala-d-Ala peptides but is only normally effective in gram-positive bacteria as, in gram-negative organisms, it cannot cross the outer membrane and access these peptides in the periplasm [17].

2.1 Resistance to fosfomycin

Fosfomycin is receiving renewed worldwide attention as one of the most active agents for sparing carbapenems in extended-spectrum β-lactamase (ESBL)–producing isolates and for treatment of carbapeneme-resistant Enterobacteriaceae (CRE) in combination with colistin [18].

The mechanism of E. coli resistance to fosfomycin is through the production of fosA, a glutathione S-transferase that inactivates fosfomycin by the addition of a glutathione residue [19]. The mechanism of action of Fosfomycin is inhibition of the initial step in peptidoglycan synthesis by blocking MurA irreversibly in both gram-positive and -negative bacteria. It is imported through the inner membrane through the glycerol-3-phosphate (G3P) transporter GlpT and the glucose-6-phosphate (G6P) transporter UhpT. Reduced expression or mutations in glpT or uhpT genes are the most common causes leading to lowered susceptibility [20]. Another mechanism is the production of fosA, a glutathione S-transferase that inactivates fosfomycin by addition of a glutathione residue. This mechanism is particularly relevant because it is disseminative and frequently associated with ESBL-producing Escherichia coli. Plasmid-mediated fosA3 and, less frequently, fosA5 (formerly fosKp96), are mostly associated with CTX-M and co-harbored on a conjugative plasmid. The possible dissemination of this gene is worrisome because fosA3 is generally surrounded by the IS26 insertion sequence on a composite transposon borne by the IncFII conjugative plasmid, which is known to be a dissemination vector of resistance genes worldwide [21].

2.2 Resistance to nitrofurantoin

Nitrofurans are a group of compounds characterized by the presence of one or more nitro-groups on a nitroaromatic or nitroheterocyclic backbone. Examples of compounds belonging to this group include furazolidone, nitrofurazone, and nitrofurantoin: drugs that all display antimicrobial activity and are used clinically to treat different types of infections [22].

Nitrofurans need to be activated by E. coli nitroreductase reducing activity to show their antibiotic effect. E. coli nitroreductase activities may be insensitive to oxygen (type I) or inhibited by oxygen (type II). In type 1, reduction occurs via a sequence of toxic intermediates, including a nitroso and hydroxylamine state, to a biologically inactive end product where one of the intermediates is thought to be responsible for toxicity as it binds and disrupts bacterial DNA and protein. Increasing resistance is accompanied by a decrease in the activity of their reductive capacity [23].

Sequential increase in resistance was genetically shown to result from sequential inactivation of the diverse nitro-reducing activities present in E. coli. The mutations were genetically mapped and named nfsA and nfsB. The direct link between these genes, and the sequential loss of nitro-reducing activity, was established by mutant isolation and sequencing of nsfA and nsfB. Nitrofuran resistance has been mapped only to type I nitroreductase genes [23, 24].

2.3 Resistance to polymyxin (last defense line)

Colistin and the other polymyxins are cationic antimicrobial peptides. These agents interfere with the negatively charged outer membrane of gram-negative bacteria. When polymyxins bind to the outer bacterial membrane it will disrupt the membrane. Thereby promotes the killing of the bacteria [25].

The clinicians have reconsidered the value of colistin due to the rising number of hospital outbreaks with carbapenem-resistant gram-negative bacteria along with the deficiency of the development of new antimicrobial agents directed toward such MDR strains. Upon this, colistin systemic administration has been reintroduced as a final treatment option. In the light of this the WHO reclassified colistin in 2012 as a critically essential antibacterial agent for mankind’s remedy [26].

Worldwide, the increased use of colistin led to the appearance of colistin resistance. Colistin resistance rates have been noticed to increase more often [27].

2.3.1 Intrinsic resistance

Colistin resistance occurs normally via alterations n the two-component regulatory system phoPQ-PmrAB, both contain a sensor kinase (PhoQ and PmrB, correspondingly), these kinases sense the signals that originated from the surrounding environment [28].

2.3.2 Acquired resistance

Similarly, to the chromosomal mechanisms of colistin resistance, the acquired resistance to colistin is mainly involved with lowering the affinity of the colistin to bind to the LPS by decreasing its negative charge. MCR gens (mobile colistin resistance genes) are a member of the phosphor-ethanol-amino transferase enzyme family, with expression of this gene resulting in ethanolamine moiety addition to the lipid A [29].

This plasmid-mediated mechanism of resistance is of special due to the possibility of colistin resistance spreading among a wide range of enteric bacteria in mankind and animals. This type of resistance is resistance is associated with the low level of MIC (4–8 mg/L) [30].

2.4 Plasmid-mediated colistin resistance

On the 18th of November 2015, Liu et al. reported the first description of plasmid-mediated colistin resistance (mcr-1 gene) among samples from food-producing animals, food, and humans in China, the detection of the mcr-1 gene the horizontally transferred plasmid-mediated colistin resistance gene altered the previous idea about the colistin resistance in gram-negative bacteria which stats that Enterobacteriaceae only develop colistin resistance through chromosomal mutations or other adaptive mechanisms. In vitro studies on mcr-1 gene showed self-transfer of the gene from conjugative plasmids [31]. After the first detection of the mcr-1 gene in Enterobacteriaceae in China, within the 6 months after its first detection, the plasmids which carry the mcr-1 gene were found in isolates from animals, food, the environment, and humans worldwide [32]. On 3 March 2016, a literature review published in Euro surveillance showed that during 3 months of its discovery the mcr-1 gene had been spread to many parts of the world and found in isolates from different sources of food and environment and also from infected patients as well as asymptomatic human carriers [33]. It is worthy to mention that, the mcr-1 gene detected in a human was in the U.S on the 26th of May 2016 in an E. coli isolate [32].

Although the first detection of mcr gene was in 2015 it is believed that this gene was existed among Enterobacterial isolates for many years before, but it wasn’t identified and it was transmitted silently for years [29].

Surprisingly, a retrospective study by [34], on isolates from Chicken origin indicated that the emergence of mcr-1 gene among enterobacterial isolates was when the colistin was first used in food-producing animals in 1980, but it did not appear again in the isolates from the next 20 years. However, the mcr-1 gene was noticed again in random isolates belonged to the period from 2004 to 2006. In isolates from 2009, the outbreak of the presence of mcr-1 gene among isolates recovered from the chicken was noted [35].

Until now, there are 8 variants of the mcr gene (mcr-1, mcr-2, mcr-3, mcr-4, mcr-5, mcr-6, mcr-7 and mcr-8). mcr-2 gene is a variant of mcr gene which share about 76.7% nucleotide (81% amino acid) with mcr-1, this gen was first detected in Belgium [25]. The mcr-3 gene has nucleotide sequence 45.0 and 47.0% identity to mcr-1 and mcr-2 respectively [29].

3. Classification of E. coli according to their antimicrobial resistance pattern

The continuous emergence of resistance to antimicrobial agents among the prevalent pathogens is the most dangerous obstacle facing the treatment of infectious diseases. Many different definitions for multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR) bacteria are being used in the medical literature to characterize the different patterns of resistance found in healthcare-associated, antimicrobial-resistant bacteria [36].

Lists of antimicrobial categories proposed for antimicrobial susceptibility testing were created using documents and breakpoints from the Clinical Laboratory Standards Institute (CLSI), the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the United States Food and Drug Administration (FDA) [37].

1. MDR E. coli:

MDR was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories.

1. XDR E. coli:

XDR was defined as non-susceptibility to at least one agent in all but two or fewer antimicrobial categories (i.e., bacterial isolates remain susceptible to only one or two categories).

1. PDR E. coli:

PDR was defined as non-susceptibility to all agents in all antimicrobial categories. To ensure the correct application of these definitions, bacterial isolates should be tested against nearly all of the antimicrobial agents within the antimicrobial categories and selective reporting and suppression of results should be avoided [37].

4. Epidemiology of resistance in E. coli

The World Health Organization (WHO), through its Global Antimicrobial Surveillance System (GLASS) report, bared that there are treacherous levels of antibiotic resistance in both low- and high-income countries (LMIC) [38].

The European Centre for Disease Prevention and Control (ECDC) conveyed that 25,000 people died due to antibiotic-resistant bacterial infections in 2007, which is over half the number caused by road traffic accidents in the same countries [39]. In 2015, this number increased to about 33,000 deaths resulting from an estimated 671,689 infections of selected antibiotic-resistant bacteria leading to 874,541 total disability-adjusted life-years (DALYs) [40]. This indicates that the burden on the European Union and European Economic Area is on the rise. By 2050, the World Health Organization (WHO) predicted that, death because of antibiotic resistance would upsurge from 700,000 to 10 million per year globally [39]. As a result of antibiotic resistance, more than 2.8 million people are infected, and more than 35,000 die each year in the USA [41].

The estimated number of cases of uncomplicated cystitis per year, caused by E. coli alone, is 130–175 million globally and 2–300.000 in Denmark alone [42]. Consequently, infections caused by E. coli, susceptible and resistant, collectively result in considerable morbidity as well as direct and indirect financial costs seen as increased healthcare expenses, antibiotic treatment, and loss of productivity [43].

Furthermore, UTI patients experience morbidity and impaired quality of life with an estimated 20–40% of women having at least one UTI during their lifetime [43].

It is difficult to determine the precise incidence of UTI, but by using self-reported medical history the annual incidence in the USA was 13% among women and 3% among men [44]. Resistance in E. coli, besides β-lactam resistance, includes sulphonamides, trimethoprim, and ciprofloxacin [45].

In 2008, UPEC isolates from five countries, were commonly resistant to ampicillin 28%), sulfonamides (25%), trimethoprim (17%), and nalidixic acid (10%), with a significant increase in resistance to nalidixic acid and trimethoprim from 2000 to 2009. A total of 60%, only, of the UPEC isolates, were found to be fully susceptible [42].

Antibiotic resistance continued to increase throughout Europe, with 41% being fully susceptible in 2012, only. Especially, the current increase in resistance to extended-spectrum cephalosporins mean = 12% and aminoglycosides (mean = 10%) in combination with increased resistance to at least three antibiotic classes, is worrisome. The increased resistance is likewise worrying in Denmark. In 2012, the resistance in E. coli isolated from urine (primary health care) was 40% for ampicillin with 33% for sulphonamide and 10% were resistant to ciprofloxacin and 6% to mecillinam [42, 46, 47].

The continual increase in resistant E. coli has added to the enormous economic and human costs of infections with 400.000 infections caused by MDR bacteria in Europe in 2007 [46]. The economic costs associated with these infections, counted as extra hospital costs and productivity losses exceeds €1.5 billion in Europe and $20 billion per year in the United States [48, 49].

5. Novel technique for detection of antibiotic resistance

For appropriate treatment of antibiotic-resistant E. coli infected patients, it is crucial to recognize the pathogen species and drug-resistant gene accurately in a timely manner [50]. Traditionally, the conventional culture-based plating assay was commonly used for antibiotic-resistant bacteria diagnosis. However, this method is very time-consuming as it takes several days to confirm the growth of the targeted bacterial colony [51]. On the other hand, a molecular characterization via polymerase chain reaction (PCR) requires relatively less time than the culture-based plating assay, but still cannot fully avoid separation and bacterial pre-enrichment [52]. Therefore, using a novel rapid and accurate technique to detect resistance was an urgent goal. Matrix-assisted laser desorption ionization time-of-flight spectrometry has captured the attention for the rapid identification of resistant pathogens by profiling bacterial proteins from the whole cells [53]. Moreover, endogenous H2S evolution was recently developed for drug-resistant bacteria via in situ hybridization [54].

Furthermore, fluorescence in situ hybridization (FISH) is a technique for the identification and analysis of diverse organisms such as bacteria and animal cells, based on the hybridization of a fluorescently labeled oligonucleotide probe to complementary target sequences from organisms using epifluorescence or confocal laser scanning microscopy [55]. Unfortunately, weak and unstable fluorescent signals due to quenching caused by natural and artificial light remain the limitation for the detection of a single microbe using fluorescence microscopy.

In 2020, Lee et al., could develop a novel fluorescent nanoparticle-based probe (nanoprobe) for FISH technique and successfully applied the nanoprobe for the detection of antibiotic-resistant bacteria [56]. The stable nanoprobe was prepared by the modified sol–gel chemistry and consisted of fluorescent dye-loaded poly (d,l-lactide-co-glycolide) (PLGA) and silica nanoparticles (NPs) [57, 58]. For the identification of ampicillin-resistant E. coli, the nanoprobe was functionalized with two kinds of biotinylated single-stranded DNAs (ssDNAs) which can conjugate to E. coli-specific gene and ampicillin-resistance bla gene that encodes beta-lactamase conferring beta-lactams (e.g., ampicillin) degrading enzyme, respectively. Finally, ampicillin-resistant E. coli was successfully detected using a nanoprobe-ssDNA.

6. Development of MDR E. coli vaccines

Since 1969, many strategies were applied to develop an effective vaccine against E. coli infections but they all have failed [59, 60]. In the 1990s, traditional vaccine strategies were based on single-purified virulence factors like Hemolysin [61] or on the O-specific polysaccharide (OPS) chain of the lipopolysaccharide (named O-antigen), conjugated to r Pseudomonas aeruginosa endotoxin A (TA) or cholera toxin (CT) as carrier proteins [62].

Although the prevalence of K-antigen and O-antigen is different among the different pathotypes, there is an association between K (K1, K5, 30, and 92) and O (O1, 2, 4, 6, 7, 8, 16, 16/72, 18, 25, 50, and 75) antigenic groups and uropathogenic strains [62].

However, because of the high antigenic heterogeneity of the surface polysaccharides, the design of a polysaccharide vaccine able to prevent ExPEC infections has been extremely difficult [62]. An O18-polysaccharide conjugated to either cholera toxin or to P. aeruginosa exoprotein A (EPA) was shown to be safe and able to induce antibodies with opsonophagocytic killing activity (OPK) in human volunteers. IgG purified from immunized individuals was protective in mice in an E. coli 018 challenge sepsis model [2].

Vaccines based on whole or lysed fractions of inactivated E. coli were evaluated in human clinical trials and were so far the most effective in inducing a high degree of protection in subjects suffering from recurrent urinary tract infections [62].

Extraintestinal E. coli vaccines are either in the preclinical or clinical stage as follows:

New antigens in preclinical studies

1. Antigens involved in iron acquisition: FyuA, IutA, ChuA, Iha, IreA, Hma, IroN.

2. Highly conserved antigens: SsIE(YghJ) and FdeC (EaeH.

3. Fimbrial-based antigens: MrpH-FimH.

Vaccines in clinical studies

1. Uromune: Inactivated E. coli, Klebsiella pneumonae, Proteus vulgaris, and Enterococcus faecalis. Uromune showed clinical benefit in reduction of UTI recurrence in females [11].

2. Solco-Urovac: Inactivated six E. coli serotype, Proteus mirabilis, Morganella morganii, Klebsiella pneumonae and E. faecalis. Solco-Urovac showed minimal efficacy in Phase 1 and two Phase 2 trials in recurrent UTIs in females [62, 63].

3. OM89/Uro-vaxom: Lyophilized lysate of 18 E. coli strains and ExPEC-4 V: 4-valent O antigens conjugated to exotoxin A from P. aeruginosa.

7. Synthetic microbiomes and engineered vaccine probiotics

The World Health Organization (WHO) has defined probiotics as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host”. Dysbiosis of healthy gut microbiota plays a critical role in the dysregulation of microbial ecology that favors colonization of pathogenic bacterial strains and diseases [64, 65].

Interestingly, from healthy specific-pathogen-free chickens, competitive exclusion (CE) products could be designed and administered by crop gavage as feed supplements for broiler chickens [66, 67]. This type of bacteriotherapy effectively protects chickens from bacterial infections, notably salmonellosis [67]. Freeze-dried CE preparations are manufactured and commercialized in many countries [68].

Extensive basic and clinical studies with lactic acid bacteria strains such as Bifidobacteria spp., Bacteroides, and Akkermansia spp. have provided strong evidence of their health benefits. This is achieved through multiple mechanisms and effector molecules [69, 70]. In-feed supplementation with Bacillus, Bifidobacterium, Enterococcus, Lactobacillus, Streptococcus, Lactococcus spp. And Yeast Saccharomyces have been given as probiotics to inhibit infection of enteric pathogens such as E. coli and mitigate antibiotic-associated diarrhea [71, 72].

8. Novel engineered probiotics/postbiotics/synbiotics

Novel engineered probiotics based on yeast strains, mainly saccharomyces boulardii, have been constructed to secrete multispecific and single-domain antibodies directly targeting bacterial virulence factors, in particular, enterotoxins [73, 74].

In the last century, the uncontrolled use of antimicrobials has led to a massive increase in E. coli resistance representing a threat to public health [75] so the use of an alternative including probiotics and acidifiers was a must [76]. One of the most common substitutes is probiotics Lactobacillus spp. and Bifidobacterium spp. which are commonly used to combat E. coli infections like gastroenteritis [77], antibiotic-associated diarrhea [77], necrotizing enterocolitis [78], inflammatory bowel diseases, [79]allergic disorders and others [80].

Furthermore, bioactive molecules secreted by probiotics can effectively down-regulate virulence gene expression in enterohemorrhagic E. coli O157: H7 [81]. They can also reduce.

E. coli O157: H7 and E. coli O127: H6 adhesion to epithelial cells monolayers [82, 83]. Unfortunately, probiotics need to be further studied to evaluate their efficacy as anti-biofilm against pathogenic E. coli. One of the most common examples of probiotics is Protexin which is a commercially available multistrain potential probiotic [84]. The Protexin contained bacteria that showed antimicrobial activity against Salmonella Typhimurium LT2, E. coli NCFB 1989, Staphylococcus aureus NCTC 8532, E. faecalis NCTC 775, and Clostridium difficile ATCC 43,594 [85].

Probiotics are capable of controlling MDR bacterial agents by enhancing immune response and competitive exclusion [86]. They augment the activities of macrophages and natural killer cells, modulate cytokine and immunoglobulin secretion, promote intestinal epithelial barrier integrity [87, 88, 89, 90] and activate B lymphocytes [89, 91].

9. Novel approaches to control E. coli infections

Several innovative approaches have been developed to combat antibiotic resistance in MDR E. coli, including the use of peptide nucleic acid (PNA) as an ultra-narrow-spectrum antibiotic, phage therapy, zinc finger nucleases (ZFNs) [92], and clustered regularly interspaced short palindromic repeat – CRISPR-associated (CRISPR-Cas) systems, [93, 94] which are genomic engineering tools for gene knock-out and knock-in of sequence-specific DNA antibiotic targets. Fast development CRISPR-based synthetic biology may transfigure the way we treat disease in the upcoming years.

9.1 The clustered regularly interspaced short palindromic repeats – CRISPR-associated (CRISPR-Cas) system

The clustered regularly interspaced short palindromic repeats – CRISPR-associated (CRISPR-Cas) system is a bacterial adaptive immune system, which is used for controlling antibiotic-resistant strains. Moreover, the programmable Cas nuclease of this system can totally diminish or reduce the resistance of bacteria to antibiotics [93].

The Cas (CRISPR-associated) nucleases identify a specific sequence of DNA by establishing a complex with a CRISPR-RNA (crRNA) that has sequence homology to the target4,5. The crRNA-Cas complex binds to the target and leads to DNA damage [95].

Interestingly, the CRISPR-Cas system is precise and easily programmable, so CRISPR-based tools for genome editing are magnificently applied nowadays in eukaryotes and prokaryotes [96].

Eukaryotic cells can repair DNA breaks using the error-prone non-homologous end joining (NHEJ) mechanisms but most prokaryotes lack NHEJ mechanisms, wherefore continuous DNA damage leads to cell death if not repaired through homologous recombination. This phenomenon has been exploited for the development of CRISPR-Cas based antimicrobials [95]. The most important advantage for CRISPR-Cas antimicrobials is the discrimination and elimination of specific bacteria at the strain level such as E. coli [95, 96].

CRISPR-Cas provides acquired immunity against viruses and plasmids. In the treatment of E. coli, CRISPR-encoded immunity is provided by transcription of the repeat-spacer array, followed by transcript processing into small crRNAs (CRISPR RNAs), which are then used in combination with Cas proteins as guides to interfere with invasive DNA or RNA. In E. coli, few model systems have been established to study of CRISPR/Cas functionality [97].

The CRISPR2 and CRISPR4 systems present in the S. thermophilus DGCC7710 genome belong to the TypeIII (Mtube) and Type I (E. coli), respectively. Differences between types can be observed in terms of repeat, spacer, and Cas gene content and sequence. The multiplicity of CRISPR/Cas systems in S. thermophilus is explained by their susceptibility to horizontal gene transfer, and phage selective pressure [98].

9.2 Phage therapy

Bacteriophages are bacteria-specific viruses, which can specifically infect and lyse bacteria. Phage therapy has been used to treat MDR E. coli that are resistant to last resort antibiotics. It is considered one of the most effective weapons for combating MDR E. coli [99, 100].

An example of phages used to treat E. coli is VB_EcoS-Golestan which is a virulent phage that belongs to Kagunavirus genus of Guernseyvirinae subfamily, Siphoviridae family. VB_EcoS-Golestan has many advantages in the treatment of UPEC specifically such as broad host range specificity, a rapid adsorption time, large burst size, and high stability at a wide range of pH and temperatures, which makes it a promising agent against E. coli infections [101].

Since phage therapy is still an under-study therapeutic approach, further development of this method requires biological characterization of bacteriophages such as their host specificity, genome diversity, and adaption to their bacterial hosts.

9.3 Nanoparticles

Nowadays, nanoparticles are one of the safest, cost-effective, and most effective bactericidal materials, which can be efficiently used as carriers of therapeutic agents [102].

Unfortunately, one of the major obstacles facing us when using silver nanoparticles is their high toxicity toward mammalian cells but to a lesser extent than pathogenic bacterial cells [103]. On the other hand, silver is less dangerous to mammalian cells than other metals [104]. Silver nanoparticles (Ag NPs or nanosilver), a kind of nanosized silver particle, are widely used NPs and show strong broad-spectrum biocidal effects on pathogenic bacteria, including MDR E. coli [104, 105].

Furthermore, Gold NPs may become useful in the development of antibacterial strategies due to their polyvalent effects, versatility in surface modification, and nontoxicity [106, 107].

Cui et al. could develop a strategy to fight against MDR bacteria via presenting inactive small organic molecules, such as 4, 6-diamino-2-pyrimidinethiol on gold NPs (Au_DAPT NPs), which act on E. coli such as disorganizing cell membranes, binding to nucleic acids, and inhibiting protein synthesis [108].

The Gold NPs antibacterial mechanism of action is to change membrane potential and inhibit ATP synthase activities to decrease the ATP level, indicating a general decline in metabolism; and inhibition of the ribosome subunit for tRNA binding, resulting in a collapse of biological process [108, 109].

10. Conclusions

The treacherous E. coli resistance rates in recent years created a very complicated therapeutic challenge that threatens to return clinicians and patients to a “pre-antibiotic era”. Clinicians must be alert to the possibility of nitrofurantoin, fosfomycin, and colistin resistance among MDR and XDR bacteria. Finally, vaccines and probiotics, CRISPR-Cas systems, and phage therapy may be the means to combat AMR of E. coli.