## 3 Antibiotic Resistance in Hospital and Municipal Wastewater  
### Hospital Wastewater  
Hospitals are regarded as one of the most important AMR reserves, due to the high and frequent usage of a wide range of antibiotics for the treatment of various diseases that create AMR through selection pressure and horizontal gene transfer between pathogens in hospital wastewater [45]. Similarly, the improperly treated hospital waste and other municipal waste disposed into the sewage also cause multiple drug resistance in microorganisms in municipal wastewater systems. Hospital wastewater is notorious for their unique ARG profile, due to the usage of a wide variety of antibiotics including Colistin, a last-resort antibiotic [46]. Hence, studies deciphering various patterns of the ARG profile have become a global requirement. Several studies on the antibiotic resistance of hospital and municipal wastewaters have been conducted [20; 47; 48; 49; 50; 51; 52; 53; 54; 55; 56]. The employment of molecular methods to detect AMR began as early as 2004 while real-time PCR (RT PCR) was used to detect AMR genes, namely _VanA_ of _Enterococci_, _ampC_ of _Enterobacter_ and mecA of _Staphylococci_ in various municipal and clinical wastewater samples by [PERSON] et al. [57]. This study also revealed that _mecA_ was found to be prevalent in clinical wastewater samples; however, in municipal water, _ampC_ (~80%) was the most dominant. [PERSON] et al. [47] reported the abundance of multidrug-resistant (MDR) bacterial isolates such as _Staphylococcus aureus_, _Klebsiella pneumonia_ and _Escherichia coli_ in hospital wastewater samples (~80%) when compared with non-hospital wastewater samples (~45%). A similar study was conducted to compare the AMR strains in hospital wastewater and water from the urban Clinton River for the presence of three groups of extended spectrum beta-lactamases (ESBLs) including _blaSHV_, _blatTEM_ and _blaCTX-M_ using the RTPCR technique [51]. The authors assessed the AMR of 156 isolates, out of which 34 isolates belonged to the ESBL category. It was found that the concentration of the _blatTEM_ gene was 100-fold higher in HWW than river water. In addition, the microbial isolates were reported to exhibit resistance against other antibiotics such as cefotaxime (40-65%), chloramphenicol (25-40%), tetracycline (30-40%), ciprofloxacin (25-65%) and gentamicin (30-50%). HWW analysis by quantitative PCR (qPCR) in a Romanian hospital revealed 14 different ARGs conferring resistance to aminoglycos, chloramphenicol, tetracycline, beta-lactam and sulfonamides; among those ARGs, _sull_ (sulfonamide resistance) was the most abundant ARG [21]. Yet another study using qPCR technique was employed for the analysis of ARG composition in a primary, secondary and tertiary hospital in East China [58] which showed quinolone-resistant ARGs (_qnrA_), and beta-lactam-resistant ARGs (_blaGES-1_, _blaGES-10_ and \(bla_{TEM}\)) to be the prevalent ARGs. [PERSON] et al. [59] assessed the antibiotic susceptibility of HWW samples from both rural and urban hospital settings through disk diffusion and E-strip tests. Genes resistant against ceftaxone, ceftazidine, gentamicin, amoxicillin/clavulanic acid, ciprofloxacin, fosfomycin and MDR were observed in both rural and urban hospital wastewater in different concentrations. For example, 49% of bacterial isolates were found to have ceftaxone resistance and 40% were resistant towards antibiotics such as ceftazidine, gentamicin and amoxicillin/clavulanic acid in rural HWW. However, the Co-trimoxazole and ceftaxone resistant genes were carried by 71% and 39% of isolates in urban HWWs, respectively. The study also revealed that the most common ARGs found were ESBL genes such as _blaCTX-M_ and _blaTEM_ and the most common quinolone resistance gene found was _qepA_. Shotgun metagenomics is the advanced high-throughput DNA sequencing tool that has been scarcely used to study antibiotic resistances from various environmental samples. [PERSON] et al.