**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:
