key: cord-346308-9h2fk9qt
authors: Kaur, Rajwinder; Yadav, Bhoomika; Tyagi, R.D.
title: Microbiology of hospital wastewater
date: 2020-05-01
journal: Current Developments in Biotechnology and Bioengineering
DOI: 10.1016/b978-0-12-819722-6.00004-3
sha: 
doc_id: 346308
cord_uid: 9h2fk9qt

The study of hospital wastewater (HWW) microbiology is important to understand the pollution load, growth of particular pathogenic microbes, shift and drift in microbial community, development and spread of antibiotic resistance in microbes, and subsequent change in treatment efficiencies. This chapter investigates the potential microbes such as bacteria, viruses, fungi, and parasites present in HWW along with the diseases associated and methods of treatment used. Due to the indiscriminate release of antibiotics from hospitals, HWW serves as a hotspot for emergence of antibiotic-resistance genes (ARGs) and antibiotic-resistance bacteria. This chapter discusses the ARGs occurrence in HWW, their prevalence in the environment, the molecular tools used for identification, and different mechanisms of horizontal gene transfer. Thus better understanding of the microbiology of HWW could further help in development of advanced treatment technologies for effective removal of microbes and their bioproducts (toxins and infectious nucleic acid) from HWW and contaminated water.

Wastewater includes any type (e.g., from agriculture, domestic means, industries, human excretion, commercial sectors, pharmaceuticals, healthcare units) of water which quality has been deteriorated under anthropogenic influence [1] . Hospital wastewater (HWW) is quite different from the wastewater discharged from other sources and is hazardous and infectious. It consists of wide range of several micro-and macropollutants, discharged from operation (surgery) rooms, wards, laboratories, laundry, polyclinics, research units, radiology, and medicine and nutrient solutions used in microbiology laboratories [2] . The micro-and macropollutants include radioactive isotopes, pharmaceuticals, stock cultures, heavy metals, media, pathogens, drugs, cotton particles, disinfectants, and chemical compounds [3] . The contraceptive-rich pharmaceuticals present in HWW were reported to be associated with effects of endocrine disruption, for instance, exposure to pharmaceutical waste containing estrogen or androgen caused sex reversals in fishes and thus, reproductive impairment [4] . The hospital effluent discharged directly into the ponds has caused eutrophication, which is evident by oxygen depletion, dense algal blooms, and death of aquatic animals [1] . The discharge of wastewater depends upon the capacity of hospital and generally water varying from 400 to 1200 L/day/bed is consumed by the hospitals [5] .

The HWW with many microbes and emerging infectious particles such as prions, viroids, and toxins is hazardous for the environment, and ultimately human health. However, in many countries, HWW is directly discharged into sewage water without pretreatment, it then undergoes treatment along with the municipal wastewater but the treatment could not be sufficient to remove micropollutants from HWW [6] . Moreover, the pharmaceutical compounds present in HWW undergo biological transformation and form conjugate compounds, those toxicity could be even higher than the present metabolite [7] . Thus wastewater pretreatment methods designed for municipal wastewaters are not able to remove the pharmaceutical-related micropollutants or conjugate compounds, which are generally present in low concentration than other macropollutants (chemical oxygen demand, biological oxygen demand, phosphorus, and nitrogen) and thus, could not be removed using conventional wastewater treatment plant (WWTP) operations [8] . Moreover, the pathogens present in HWW could cause microbial population imbalance in existing and operating municipal WWTP and metals or heavy metals present in the HWW can affect the biological processes like nutrient removal. The heavy metals are in fact nonbiodegradable as compared with other organic pollutants and thus move to other pollutant sources [9] .

The HWW can act as an ideal growth medium for various pathogenic microbes including bacteria, viruses, fungi, and parasites. The wastewaters from hospitals also consist of several resistant bacteria and antibiotic residues as well, which could inhibit the growth of susceptible bacteria, thereby increasing the population of resistant bacteria in the receiving water. Resistant bacteria discharged into environment either act as vectors to carry a transmissible gene or as the reservoirs for antibiotic-resistance genes (ARGs) that could pose a threat to public health [10] . In addition, fungi which have capability to grow at faster rate and can spread their spores to external environment also pose a serious threat to environment and human health [11] . The absence of specific pretreatment technologies for HWW also increased the frequency of gastroenteric viruses in aquatic bodies [12] . The direct discharge of HWW into municipal wastewater containing disease-causing parasites has also increased the risk of skin infections and other harmful diseases in humans [13] . The specific and advanced technologies are required to be developed for HWW treatment to prevent the release of harmful contaminants into the environment. Therefore the microbial community analysis of HWW is utmost importance to assess the pollution load and to develop the specific treatment methods for protection of the environmental health. Thus authors explain about the various pathogens present in HWW, ARGs, and tools used to study the ARGs. Moreover, metagenomics, a recent approach used to study different microbial communities and gene-specific identification, are also explained in this chapter. In addition, horizontal gene transfer (HGT) approach, which can efficiently contribute to spreading of ARGs, is discussed briefly.

The HWW poses a serious threat to humans with respect to contagiousness and drastic spread of infective diseases in healthcare units, society, hospital employees, and environment [5] . Different solvents, pharmaceuticals, and radionuclides are used in hospitals for purpose of diagnostics, disinfection, and research. After the drug consumption, various drugs remain nonmetabolized or partially assimilate in the human body and thus excrete as such and end up into wastewater. The residual quantities of disinfectants used for treatment of skin microbial infection and to disinfect instruments and surfaces of hospitals, also end up in the HWW, resulting in an increase in the population of pathogenic microbes. The pathogenic microflora present in HWWs also come from medical devices, atmosphere, and water used in the hospital practice, and the pathogens are released mainly in the form of excreta of patients [14] . Therefore HWW consists of a mixture of pathogenic microbes including bacteria, fungi, yeasts, algae, viruses, protozoa, parasites, and bacteriophages. The effluent from hospitals is usually discharged and treated with domestic wastewaters without any prior treatment [15] . The pathogens in the receiving water, if untreated, survive for a long time in soil or water and enter into the food chain causing infectious diseases and health risks to human beings [16] .

Due to the economic reasons and lack of resources for analysis of actual pathogens, certain bacteria have been used as indicators for contamination of water since decades [17] (Box 4.1). However, it has to be considered that removal of coliforms, which is indicator of fecal contamination, could not be directly correlated with removal of viruses, pathogens, fungi, protozoa, or other bacteria from water (samples) [17] . In addition, the indicator bacteria such as Escherichia coli and Clostridium perfringens when compared with pathogenic bacteria, protozoa and viruses are more sensitive to inactivation through processes such as natural competition, wastewater treatment, and high temperature [18] . Traditional detection of pathogenic bacteria involves selective culture media and biochemical characterization methods. These techniques are inexpensive and simple; however, sampling error, time consumption (5À11 days), tedious, and monospecific detection (detection of only one type of pathogen) are the major limitations [17] . The enzyme-linked immuno sorbent assay (ELISA) is used for laboratory diagnostics of different pathogens. The polymerase chain reaction (PCR) has been adapted in many ways: nested PCR, multiplex PCR, real-time PCR, fluorescence, and digital PCR [19] (Box 4.2) for analysis of waterborne pathogens.

Criteria for selection:

• Should be present only in contaminated water, not in uncontaminated ones • Should not be able to grow and proliferate in water • Should be present in intestinal tract of warm blooded animals • Should have similar survival pattern as pathogens present outside the host • Should be easily detectable • Should be useful for all types of water • Should be relatively cheap to use Total coliforms are higher in number than any other pathogens. The subgroup fecal coliforms indicate fecal contamination of water and thus, indicate the presence of other pathogens as well, which are lower in number, hard to detect. However, lower number of pathogens are enough to cause morbidity and mortality to humans. They are detectable by inexpensive cultural methods and do not pose any health risk to laboratory workers. However, there are few limitations of fecal indicator bacteria, which have been discussed in the text. Biochemical tests, PCR, and sequencing [32] (Continued) bed-linens, or through infected patients [39] . The hospital-acquired infections resulted in increase of methicillin-resistant S. aureus (MRSA) isolates. The MRSA is associated with infections of skin and soft tissues and has been a global threat for human health. In the United States, 72,444 cases of MRSA infections were reported in 2014 [40] . E. coli and Pseudomonas aeruginosa are also commonly found in HWW along with other nosocomial pathogens including Candida albicans, Klebsiella Proteus, and Enterobacter species [37] . Most of the times, E. coli is harmless and is important part of human intestinal tract, but some of the strains are pathogenic to humans. The E. coli 0157:H7 is related to many outbreaks of water and food-borne illness and also responsible for 63,000 hemorrhagic colitis (bloody diarrhea) cases in the United States annually [41] . A study reported on HWW of Brazil also confirmed the presence of other bacterial species including Citrobacter freundii, Klebsiella ornithinolytica, Proteus mirabilis, Pantoea agglomerans, and Serratia rubidacea [28] . Many C. freundii infections have been reported in bloodstream such as septic shock, pneumonia, hypothermia, oliguria, and thrombocytopenia [42] . Another bacterium P. mirabilis, which is found in HWW, is responsible for urinary tract infections and is further accompanied by development of kidney stones [43] . P. agglomerans mostly affects plants but also causes opportunistic infections in immunocompromised individuals causing skin infections [44] .

An important prospect about fluctuations of pollution was discussed [44] , according to which, the microbial load present in wastewater was directly correlated to several hospital activities. The higher microbial load was present in HWW during the day period as compared with evening and early morning. Another study [44] was also conducted on HWW collected during early hours (EH) and late hours (LH) of the day in Abia state, Nigeria. The microbiological analysis revealed the presence of higher total bacterial count (7.9 3 10 4 CFU/ mL) at LH as compared with that (6.7 3 10 4 CFU/mL) at EH of the day. The similar results were also observed for total coliform count, which was also higher (2.2 3 10 3 CFU/mL) at LH as compared with that (1.8 3 10 3 CFU/mL) at EH due to the fact that hospital activities at night time were more restricted than day time. According to the recent study, the HWW samples were collected from three hospitals in different cities of Romania [35] . Three hospitals (H1, H2, and H3) were having different numbers of beds and inhabitants. The microbial community analysis results revealed that wastewater from H1 was dominant by 97.27% of the Proteobacteria phylum, while from H2 was dominant by 44.7% of Proteobacteria phyla followed by Firmicutes (33.68%), Bacteroidetes (16.4%), and Actinobacter (4.9%) and from H3 was dominant by 71.4% of Proteobacteria, Bacteroidetes (13.7%), and Firmicutes (13.1%) [35] . The similar taxonomic composition was also reported in various studies using wastewater collected from different hospital facilities, nonhospital medical facilities, and municipal treatment plant [26, 45, 46] . The dominant nature of Proteobacteria was correlated to their presence in human feces, long-term survival ability in wastewater, and exposure to several antibiotics present in HWW. Moreover, the dominant nature of Firmicutes could be correlated to their capacity to survive in extreme environmental conditions and high contaminant levels [47] . In 2018 Buelow et al. [48] detected abundant bacterial taxa to be different from urban and suburban WWTP influents and comprised of Camphylobacteraceae, Aeromonadaceae, and Carnobacteriaceae. HWW also consisted of several different members of human gut microbiota such as of genus Streptococcus and family Ruminococcaceae due to the sampling location in close proximity of the hospital sanitation areas as compared with WWTP influent. These microbes are not well suited to survive in such complex environment, which results in progressively decreased levels of these bacteria in urban WWTP influent [48] . Another study compared the relative abundances of the most abundant bacterial orders in WWTP for 7 years and showed that the WWTP environment was dominated by phyla Actinobacteria, Bacteriodetes, and Proteobacteria, and the wastewater community was highly stable and unique to its environment [49] .

Hospital sewage also contained high levels of anaerobic bacteria including genera such as Bifidobacteriales, Clostridales, Bacteroidales that were likely to originate from the human gut [1] . The release of antibiotics from hospitals results in creating a selection pressure on bacteria and as a result, effluents from hospitals contain high numbers of resistant bacteria as well as antibiotic residues. Prevalence and spread of antibiotic-resistance bacteria (ARB) in the environment are a major problem worldwide due to improper antibiotic usage and lack of effective HWW management systems. Therefore ARB and ARGs are particularly studied among the hospital contaminants. For example, one study reported E. coli from hospital effluent to be multiresistant toward ampicillin, streptomycin, sulfamethoxazole, cephalosporin, ciprofloxacin, and tetracycline [29] . The hospital water environment includes potable water, faucets, wastewater drainage systems, and effluents can be the reservoir of nosocomial pathogens (ARB such as MDR Enterobacteriaceae, Acinetobacter baumannii, and Pseudomonas species), thus, increasingly dominating the HWW microbiome [50] . Commonly found isolates such as P. aeruginosa, A. baumannii, and Enterobacteriaceace in hospital effluents have shown carbepenem resistance and have disseminated around the globe [50] .

Baricz et al. [51] reported antimicrobial effect of a crude bacterial extract of Janthinobacterium lividum against MDR bacteria of both clinical and environmental origin, for example, MRSA, methicillin susceptible S. aureus, Enterococci, and Enterobacteriaceae [51] . Such studies not only help to provide promising candidates for development of new antimicrobials but also to propose new or improved treatment technologies to reduce the burden of antimicrobial resistance (AMR) in environment.

The microbiological analysis of several HWWs showed the prevalence of fungal species along with bacterial and coliform species. The fungi have simpler nutritional requirements and have higher capability to grow at lower water activity as compared with bacteria [52] . Moreover, the fungal populations can easily spread their spores to external environment, hence affecting the human beings directly [11, 52] . This is the reason for its prevalence in hospital and clinical environment because if healthcare facilities are even considered free from fungus, but nature and environment factors such as temperature, moisture, and nutrients could provide easy and favorable conditions for the extensive growth of fungal species in storage containers holding clinical waste. The fungal infections could range from moderate to fatal depending upon the infection site as well as immune system of the affecting individual [53] . The prevalence of invasive fungal species has been reported to increase since three decades, due to increase in number of immunocompromised patients. The moderate fungal infections include athlete's foot and ring worm infections (cutaneous infections) in immunocompetent patients and life-threating infections include mucosal and systemic infections [53] . The fungal isolates, associated diseases, and treatment methods reported in various studies are presented in Table 4À2 .

The study reported by Neely and Orloff [54] stated the prevalence of Aspergillus species in the hospital waste collected from the United States and even worse than that, this species was capable enough to survive for more than a month on the hospital waste. The longer survival of Aspergillus can result in reoccurrence of fungi and has higher chance to cause disease. Moreover, many spores have capacity to remain dormant under adverse conditions and then again develop into fungi, when conditions become favorable. The other fungal species were also detected but the survival capacity of Mucar ( . 20 days), Paecilomyces (11 days), and Fusarium (10 days) species was lower than that of Aspergillus species. The survival efficiency of different fungal species is related to the presence of specific structure of spores. The spores of Aspergillus species are spiny and rough and thus capable enough to adhere to any type of waste and can survive longer time. However, spores of Fusarium species are smooth and thus have less capability to adhere and survive. The wastewater collected from Nigeria revealed the presence of total fungal count of 3.5 3 10 3 CFU/mL during the day time activities in the hospital [72] . The most prevalence fungal species in the HWW were Aspergillus (33.3%), followed by Candida (28.7%), Cryptococcus (14.9%), and Penicillium (14.2%) species. The fungal diversity (29 species) was reported to be higher for waste samples collected from hematology section of Malaysia hospital [72] . Moreover, gloves waste consisted of the highest number of fungal species (19 species) among all the different types of waste collected from hospital. The high fungal count of 7 3 10 5 CFU/mL was also reported in soil collected from hospital dumpsite, which was correlated with the presence of high organic material present in the hospital waste. The fungi are heterotrophic microorganism, which have capacity to consume organic compounds from the surrounding environment [72] . The common fungal species identified in hospital waste were Penicillium rubrum, Penicillium viricadum, Trichothecium roseum, Rhizopus nigricans, Aspergillus flavus, Aspergillus parasiticus, Microsporum canis, and Aspergillus niger. Among them, A. niger was found to be dominant fungal species with 34.5% due to the fact that it released variety of enzyme such as amylases, pectinases, and proteases and had capacity to utilize number of organic compounds as a substrate [73] . A. flavus and A. niger can cause disease named as aspergillosis and are considered as pathogens for both humans and animals. They can cause either bronchopulmonary (coughing and wheezing) or invasive aspergillosis (affects the lungs and could spread to throughout the body) [74] . More than 200,000 cases and 30%À95% mortality rate have been reported for aspergillosis infections worldwide. The nonpathogenic black bread mold called R. nigricans was also found in abundance with 27.5%. M. canis can cause infection in upper and dead layer of skin of domestic animals, including dogs and cats and later on the humans can also get affected by the infection [75] . Other fungal species including T. roseum, P. viricadum, and P. rubrum are nonpathogenic fungus, which can only affect the humans with weak immune system. The Curvularia species have also been reported to be present in hospital waste and can cause rare infections in the respiratory tract and cornea of human beings. Curvularia lunata is also responsible for eumycetoma disease caused in farmers of Chandigarh and Rajasthan. Most of the agricultural regions use azole fungicides (imidazole, metalaxyl, tebuconazole, and propiconazole). Therefore the farmers responded poorly to antifungal treatment and were referred for long-term therapies [76] . The few fungal species of Fusarium (1.5%) and Trichoderma (2.2%) have also been reported in hospital waste, but infection caused by these fungal species is difficult to cure [77] . Over 1.2 billion people worldwide are reported to be suffered from several fungal infections and 1.5À2 million people die due to fungal infections each year, which are far superior than malaria or tuberculosis death rate [76] . Antifungal drugs are mostly used for treatment of fungal infections as they target plasma membrane, sterol biosynthesis, DNA biosynthesis, and β-glucan biosynthesis of different fungi. However, since past decades, the increased use of antifungal drugs resulted in development of resistance, leading to increase in morbidity and mortality. The drug resistance could be due to several reasons such as alterations of the drug target site, increased efflux of drugs, and biofilm formation. Although several studies have reported, antifungal drugs resistance is not at the same level as resistance in bacteria against antibiotics. Other reasons of high mortality rate with fungal infections include availability of limited treatment options against invasive fungal diseases and less susceptibility of immunocompromised patients to antifungal drugs [78] . For instance, in the United States, until 2013, there were reports of 19,262 organ transplants, which resulted in increased susceptibility to fungal infections in immunocompromised patients [78] . In addition, adverse effects of antifungal drugs and lack of effective antifungal therapies are the other reasons, which necessities the development of novel treatment strategies and next-generations of antifungal drugs. The development of ultrahigh-throughput screening techniques could help in advent of novel antifungal drugs by providing rapid, effective, and economical drug screening.

Presently, the viral infections are in forefront as compared with pathogenic diseases caused by bacteria, fungi, and parasites (Table 4À3) . Moreover, the detection, analysis, characterization, and epidemiology of virus is entirely different from bacteria because the bacterial indicators used to represent water contamination cannot actually represent viral contamination.

It has been reported that each of the material present in the hospital waste can carry viruses, and thus, viruses are able to survive for 5À8 days [78] .

The viral hepatitis is very common and leading disease. Moreover, human immunodeficiency virus infections, Hepatitis B, and Hepatitis C are among the deadly infectious diseases, transmitted by direct contact of blood from infected to another. However, these are yet easy to prevent, once detected at early stages and obligatory precautions are followed by patients [5] . The samples collected from different WWTPs have been analyzed for detection and characterization, and even research has been focused on removal of viruses from wastewater [79] . However, in spite of all the health risks associated with viruses, very few research [5] has been conducted specifically for HWW, and thus, requires special attention due to the fact that, it is one of the main sources to spread the pathogenic and deadly viral diseases. The modern techniques of molecular biology including PCR assays offer several advantages such as specificity, sensitivity, and wider data analysis for easy and faster detection of viruses.

The occurrence of Rotavirus A, norovirus genogroup I and II, human adenovirus (HAdV), and hepatitis were detected in samples collected from two different hospital WWTPs. The load of rotavirus and HAdV present in hospital WWTP was 2 log cycle higher as compared with that present in urban sewage WWTP [80] .

The HAdV consists of double-stranded linear DNA (Fig. 4À1) and comes under category of nonenveloped viruses, which makes them resistant to heat, dry, and acidic conditions [78] . The adenovirus consists of outer capsid and inner core with several histone proteins. The elongated fiber proteins on the surface of the virus interact with receptor of host cell such as coxsackie and adenovirus receptor and MCP and start infection in the host cells. After interaction between virus fiber proteins and host cell receptors, uncoating of virus particles takes place resulting in dissolution of viral protein in the endosome. Ultimately, the translocation of adenovirus takes place with microtubules through cytoplasm toward the nucleus [81] . HAdV is mostly profound in patients with weak immune system and acute lower respiratory disease [82] . Seventy-nine types of HAdV have been reported [83] , and out of them, HAdV 40/41 is mostly prevalent in aquatic systems such as sea water, sewage, rivers, surface water, and drinking water [12] . The HAdV is placed among the contaminant candidate list for drinking water by U.S. Environment Protection Agency and is a real factor of concern for the human health [84] . It can cause several diseases in humans including ocular infections, conjunctivitis, genitourinary infections, pharyngoconjunctival fever, hemorrhagic cystitis, exanthema, encephalitis, and pneumonia [85] . The HAdV is more frequently found in wastewater than any other enteric virus. The HAdV is reported to be transmitted through fecal-oral transmission, aerosol droplets, and contaminated materials. It can survive for the extended period of 3À8 weeks in the environment without host. Due to its stable and persistence nature in aquatic systems, it could also be considered as an indicator of fecal contamination as suggested by various studies [86, 87] .

The prevalence of HAdV species varies according to the hospital environment. According to Prado et al. [80] , the HAdV 40/41 (species F) was among the most prevalence genotype found during molecular characterization of viruses, detected during analysis of samples collected from two different hospital WWTPs. The species F come from the hospitalized children reported with acute gastroenteritis disease. The other reported HAdV strains include species C and D, which are associated with conjunctivitis and respiratory tract infections [80] . The frequency of HAdV has also been tested for the samples (102 in number) collected throughout year from hospital WWTP located in Tunisia City, Tunisia and 64% samples were detected positive for HAdV and most prevalent species were species F (HAdV 40/41) [12] . In the similar way, the prevalence of HAdV was detected in samples collected from different regions of the world such as 92.1% in Poland, 76.9%À92.3% in Greece, 92% in Norway, and 100% in Brazil, while samples collected from Morocco (45.5%), Italy (21.6%), and Taiwan (27.3%) consisted of low prevalence of HAdV [78] . The high and low prevalence of HAdV reported in various countries could be collaborated by the fact that the circulation of virus varied according to the geographical regions and epidemiological community profile. However, whether it was high or low prevalence, still presence of HAdV questions the ability of WWTPs to remove the gastroenteric viruses and highlights the urgency for development of effective environmental control programs and innovative HWW treatment plants before discharging the HWW into the sewage system.

The detection of HAdV includes cell culture method, antigen detection, and PCR method. Serology is also used sometimes to detect adenovirus-specific antibodies. There is no effective treatment against HAdV, essential precautions should be taken to control infections, which include washing of hands, disinfection of instruments as well as application of infection control protocol in hospitals against adenovirus to prevent outbreaks [88] .

The rotavirus consists of 11 segmented double-stranded RNA and its surface is surrounded by three layers: the outer capsid, inner capsid, and central core. There are seven rotavirus groups that have been reported from A to G and studies have revealed that groups A, B, and C affect both humans and animals, while D to G are reported to cause infection mainly in animals. The dual (binary) classification system has been used for detailed classification of rotavirus on the basis of two outer capsid proteins; the glycoprotein VP7 and protein VP4 (Fig. 4À2) , where the glycoprotein VP7 defines for the stereotype G and protein VP4 defines for stereotype P [89] . The viral attachment to the host cell surface is by glycoprotein VP7 or surface protein VP8. After interaction with host cell, calcium-dependent endocytosis takes place and thus, uncoating of the particle occurs, resulting in release of double layer of the viral particle into cytoplasm. Further, transcription and translation takes place in the cytoplasm and viral proteins are synthesized by cellular ribosomes. After assembly, virus particle is released from the infected cell through cell lysis, and infection is further spread into the host [90] .

Rotavirus is associated with around 111 million cases of acute gastroenteritis in the newborn babies and children across the world, and 352,000À592,000 deaths have been reported for children with age of ,5 years globally. After infection and replication, rotavirus is discharged into surface water, groundwater, drinking water, and wastewater, able to transmit effectively through water due to its stable and resistant nature under adverse environmental conditions [91] . The rotavirus can survive at ambient temperature of 30 CÀ35 C and can exist on inanimate objects for 60 days without host. The rotavirus is mostly prevalent in humans and 11%À42% of positive samples are associated with group A rotavirus [92, 93] . The detection methods of rotavirus include electron microscopy, ELISA, passive particle agglutination tests, polyacrylamide gel electrophoresis, and RT-PCR. The vaccination with safety and efficacy profile for rotavirus A has been developed and licensed in 2006 after the withdrawn of first ever vaccination, which was associated to cause intestinal intussusceptions in children [94] .

The prevalence of rotavirus in different seasons (summer, autumn, and winter) was detected by collecting 60 different samples in the period of 9 months from HWW located in Shiraz, Iran. Enzyme immunoassays (EIAs) were performed and positive specimens were further investigated with nested-PCR by various primers. Moreover, the virus concentration method such as the pellet and two-phase is reported to enhance the concentration of virus by 50-to 100-fold before applying EIA. About 15 samples (25%) were found to be positive for rotavirus A and the highest prevalence was detected in autumn with frequency of 46.67% followed by 33.33% and 20% of prevalence in winter and spring, respectively [95] . Other studies also confirmed the prevalence of rotavirus A in cold weather as compared with other seasons [96] . The molecular characterization of rotavirus further revealed the predominance of G1 genotype during various clinical investigations followed by mixed genotype of G1G4, which is associated with frequent water contamination and further generation of novel strains by genetic reassortment. Moreover, various studies have reported the prevalence of rotavirus in treated wastewater as well, which is a real concern for public health [95] . The studies on rotavirus highlight the significance of environmental surveillance tools for detection of novel genotypes and to further analyze the distribution and treatment of the hazardous effects caused by rotavirus in human beings.

The norovirus belongs to the family Caliciviridae, and is reported to be nonenveloped and contains a positive sense, single-stranded RNA, which is organized into three open reading frames (ORFs). The three ORFs encode three different proteins; ORF1 is of size 5 kb and encodes nonstructural (NS) polyproteins, ORF2 is having 1.8 kb size and encodes structural capsid protein VP1, while ORF3 with size of 0.6 kb encodes a VP2 protein to maintain the stability of capsid protein [97] . The norovirus life cycle has been presented in Fig. 4À3 . The interaction of norovirus with the host cell surface takes place through histoblood group antigens. After release of the VPg-linked RNA genome of the virus into cytoplasm of host cell, the viral RNA translation takes place. Once the translation of the ORF1 polyprotein is done, further co-and posttranslation processing occurs by NS6 protease of virus, which results in the release of NS proteins for the formation of replication complex. Thereafter the viral replication takes place by RNA-dependent RNA polymerase using de novo and VPg-dependent mechanism. Furthermore, the replicated genomes are translated and further packed into the capsid for assembly of virion and exit [98] .

The norovirus is divided into seven genotypes, among them three genotypes GI, GII, and GIV are responsible for infection in humans, and GII with genotype 4 (GII.4) is a predominant infectious virus associated with 18% of the diarrheal diseases and gastroenteritis infections around the world. In addition to GII.4, various other non-GII.4 containing viral strains are also responsible for gastroenteritis infections, for instance, the gastroenteritis emerged in Southeast Asia in 2014À15 was associated with G11.17 viral strain. Norovirus is associated with 677 million gastroenteritis cases and 210,000 deaths annually around the world [99] . The emission of waterborne norovirus is caused by contamination of water systems including drinking water, surface water, mineral water, and groundwater [100] . As the infectious individuals shed the virus into the water system, the samples collected from HWW are considered useful for study of norovirus epidemiology at population level [99] . The transmission of norovirus is through fecalÀoral route from person to person or animal to person by contamination food or water intake. Depending on the surface temperature and conditions, the norovirus can survive for longer period outside the host. It has capacity to survive for weeks on the hard surface, until 12 days on contaminated fabrics and for years in the contaminated still water [101] . The norovirus infection in humans can be detected by real-time PCR, multiplex gastrointestinal platforms, EIAs, and genotyping as well. The development of real-time PCR for detection of norovirus is considered as significant advancement with various environmental applications. The prevalence of norovirus was studied by Ibrahim et al. [100] , where 102 samples were collected form 5 different basins of hospital WWTP located in Tunisia. The 65% of the samples were positive for norovirus GII followed by norovirus G1, which was detected in 1% of the samples collected from wastewater, while frequency of norovirus was lowered moving from one basin to another. However, there is requirement of tertiary wastewater treatment before recycling of water for bathing and other purposes. Different studies have revealed the prevalence of norovirus in different seasons, for instance, studies on HWW collected from Italy and Sweden have showed occurrence of norovirus GII in spring and summer seasons [102, 103] . However, the study from China has revealed the prevalence of norovirus in winter conditions, which could be explained by variability in temperature during different seasons, immunity level of the host, humidity as well as socioeconomic conditions of different countries [104] .

The emerging recombinant norovirus has been analyzed by illumine Miseq and Sanger sequencing technique by collecting different clinical and wastewater samples within Australia and New Zealand. The prevalence of pandemic variant (GII.Pe/GII.4) was found in Sydney during 2014 and 2015, while decline in pandemic virus was observed in 2016 with emergence of five new recombinant strains. These new viruses were held responsible for emergence of gastroenteritis outbreaks during November 2016 [99] . The use of new generation sequencing tools has advanced and reformed the genome sequencing of different pathogens present in wastewater.

The norovirus has been responsible for 210,000 deaths per year. In 2016 World Health Organization (WHO) identified norovirus as a priority disease for vaccine development. However, several vaccines are still in trails phase and preclinical stages and no licensed vaccine has been available for norovirus infection treatment. The cost effectiveness, target population, and public acceptance are the major considerations for vaccine development [105] . The public awareness about the norovirus and prevention measurement should be spread for protection of people from norovirus infection due to lack of treatment available.

Hepatitis A virus (HAV) is a nonenveloped virus, belongs to the Picornaviridae family, and has positive sense single-stranded RNA (Fig. 4À4) . The RNA consists of only one ORF, which encodes a large polyprotein [106] . There are six genotypes of the virus reported and genotype I, II, and III are infectious to human population and are further subgrouped into A and B, while IV, V, and VI affects other primates than human beings [107] . There is a genetic nucleotide variation of 7.5% in each of the subgenotypes, which has been evaluated with PCR by VP1/VP2A junction present in 168 nucleotide fragment [108] .

The interaction of the HAV with host cell involves endocytic pathway. The host cell receptor involved in interaction with virus is TIM-1 (HAVCR1) and it is reported to cycles between plasma membrane and lysosome of host cell with calthrin-mediated endocytosis. The replication, translation, and assembly of virus occurs inside the cytoplasm of host cell [108] . The genetic analysis of HAV could be correlated with the outbreaks of viral infections and transmission mode as well. The main transmission mode for the virus is fecalÀoral route, which could be directly transmitted through one human to another or indigestion of contaminated food as well [109] . The HAV has capacity to survive for months outside the host body, it can survive through freezing temperature but can be killed, when exposed to high temperature of .80 C. The geographical distribution of the virus has been mainly present in India, Africa, Middle East, Central America, and South America. The HAV is considered as one of the most important food-borne pathogens and is a major cause of hepatitis in humans with reported cases of around 1.5 million globally. It has been estimated that half of the hepatitis cases are related to HAV. The wastewater coming from the hospital could be emerging source of HAV due to the fact that human excretion contains enormous amount of virus; moreover, virus is highly resistant to harsh environmental conditions and is able to survive for longer periods [110] . The HAV is also detected in rivers, raw or treated water, and dam water as well [111] . These viruses are a real cause of concern for human health; moreover, there are no strict regulations in existence related to monitoring of these viruses in environment and water resources [112] . The consumption of contaminated food has also been directly linked with outbreak of HAV at several places. For instance, there were 397 reported cases of HAV in Michigan between August 2016 to October 2017 with 15 fatalities and 320 hospitalization. The outbreak was linked to consumption of raw scallops served in sushi chain. In another case at Hawaii, 292 cases suffering with hepatitis A infections were reported between June and October 2016, which were linked to contaminated frozen strawberries [113] . The prevalence of HAV was detected in different clinical samples collected over a period of 2 years from Patras and Alexandroupolis located in Greece. The nested real-time PCR revealed the presence of 100% genotype-1 and particularly subgenotype A [114] . The predominance of genotype-1 and subgroup A has also been reported in wastewater samples collected from Brazil [80] . The prevalence of virus was also reported to be higher during the lower number of cases reported for hepatitis A, which could be directly correlated to the shedding of virus into feces. The detection of IgM antibodies and anti-HAV in blood is related to hepatitis A infection. The presence of anti-HAV in blood indicates infection or past vaccination. The HAV vaccination within 2 weeks of infection is the prevention method for HAV infection and includes two-dose series for individuals above age of 12 months. The vaccination includes inactivated virus and is safe for immunocompromised persons [113] .

The HWW is heavily loaded with pathogens and discharged directly into aquatic bodies could be directly evident by skin infections and intestinal parasites. Around the globe, millions of people are affected by deadly parasites infections, for instance, 2.8 million suffer from Giardiasis, 50 million from Amebic dysentery, 740 million affected by hookworm infections, 795 million detected with Trichuriasis, and 1.2 billion are infected with Ascariasis [13] . These parasites are mostly transmitted through fecalÀoral route under poor hygienic conditions, contaminated water and food sources, and poor wastewater disposal practices. The parasites in their ineffective stages such as cysts, eggs, and oocysts survive under environmental adverse conditions and through many wastewater treatments processes due to the presence of protective outer layer. Therefore parasites have capability to survive in wastewater for extended time period in comparison to viruses and bacteria [115] . The samples collected from hospital sewage treatment plant located in Zaria, Nigeria contained several eggs, cysts, and oocytes of various parasites. About 1648 eggs, cysts, and oocysts were present per liter of wastewater. Ascaris lumbricoides was the common parasite found in HWW with 307 eggs per liter (18.67%) of wastewater followed by 287 eggs (17.42%) of Taenia spp., 253 eggs (15.35%) of Schistosoma spp., 176 eggs of Toxocara spp., 135 eggs (8.19%) of Ancylostoma spp., 130 eggs (7.89%) of Cryptosporidium parvum, and 58 eggs (3.52%) of Giardia lambila. Moreover, 92 eggs (5.58%) of Trichuris and Hymenolepis spp. were also found in HWW. The cysts of Entamoeba histolytica and A. lumbricoides were found in many studies and remained viable for longer period of time in pond effluents that was further used to irrigate raw vegetables, thus, entering the food chain and then directly to humans [13] . C. parvum causes the disease Cryptosporiadiasis and G. lambila causes Giardiasis, both could be life threatening if found in persons with weak immune system. The children diarrhea in many cases is caused by the Cryptosporidium species [116] . Taenia species can cause cysticerocosis in humans that can infect muscles, brain and can ultimately cause onset seizures in adults, while it can also cause bovine cysticerocosis in cattle after consumption of contaminated water. The main symptoms of parasitic infection include nausea, vomiting, malabsorption, diarrhea, and stomach cramps [117] . Therefore the wastewater should be treated before discharge into water bodies otherwise they could be a great risk to public health.

Antibiotics are one of the most successful and important drugs used in therapeutic applications and the indiscriminate use of these compounds has made their way into the environment. The overuse and misuse of antibiotics not only in human therapeutics but also in veterinary, agriculture, and aquaculture applications [118] has led to the emergence of ARGs and ARB compromising or decreasing the effect of antibiotic compounds as they are becoming resistant to multiple drugs thus, causing a major concern.

Some studies have shown that resistant bacteria can also be present where antibiotic concentrations are low, saying that subinhibitory concentrations can also promote resistance among bacteria as similar to concentrations found in some aquatic and soil environments [118] . Just as natural antibiotics have existed for billions of years, ARGs are also ancient [119] . There have been occurrences of ARGs in places where anthropogenic activities are minimal such as genes encoding for β-lactamase in a remote Alaskan soil suggesting the environment to be a reservoir of ARGs [120] . Antibiotic-resistant bacteria have been reported from Lechuguilla Cave, New Mexico which has been isolated for more than 4 million years including some strains which were resistant to over 14 different antibiotics [121] and in ancient permafrost samples where communities harbored resistance mechanisms minimum 5000 years ago [122] . Such studies improve our understanding of the prevalence of resistance genes in environments much prior to human use of antibiotics. Within past years, pieces of evidence have shown mobilization of these resistance genes from the environment into pathogenic bacteria causing health risks to humans and animals and also, demonstrating a link between environmental and clinical resistance [123] .

Moreover, due to the introduction of antibiotics from various human activities, the environment has turned into a reactor of ARB and ARGs contributing to the evolution and spreading of resistant genes. The phenomenon of emergence and spread of ARGs has increased so intensely that 70% of all hospital-acquired infections show resistance toward at least one family of antibiotics. HWW represents an important source of ARGs and ARB and such effluents are highly hazardous due to its infectious and toxic features [78] . Traditionally, resistance was viewed as a healthcare problem but now nonclinical environment has also been reported as an important factor in the dissemination of resistance genes [124] . A wide range of antibiotics and ARGs are being released from the hospitals and urban wastewaters which are received by WWTPs [125] . The WWTP also serves as a hotspot in the emergence and spreading of ARGs and ARB in the ecosystem [126] . Even after the treatment, some of the antibiotics and ARGs are still not completely removed and being released into the receiving water bodies making aquatic ecosystems an ideal place for acquisition and spread of such genes. Commensal bacteria of humans and animals also constitute a reservoir of such resistance genes which can enter the environment through sewer systems or use of animal manure [127, 128] . Other than WWTP effluents, ARGs can reach the soils, sediments, surface water and groundwater bodies including drinking water systems [129, 130] by surface runoff or infiltration of water that has been used for agricultural purpose [118] . It has been estimated by WHO that ARBs are responsible for 25,000, 23,000, and 38,000 deaths per year in European Union, the United States, and Thailand, respectively [131] . There has been increasing resistance in many human pathogens such as E. coli, A. baumannii, Enterococcus sp., Klebsiella pneumonia, S. aureus, P. aeruginosa, Serratia marcesens, Citrobacter sp., and other Enterobacter species, and there have been a great number of growing reports concerning the prevalence and dissemination of these pathogens into various environmental settings [132À134]. Knapp et al. [135] reported ARGs from different major classes of antibiotics tested from 1940 to 2008 had significantly increased since 1940, along with tetracycline ARGs around 15 times more abundant than in the 1970s [135] . The resistant gene can be specific to one antibiotic, for example, ciprofloxacin or a group of antibiotics such as β-lactums and as a result, hundreds of ARGs are being detected [136] . Genes resistant to antibiotics are commonly observed for aminoglycoside (aac, aad, aph), chloramphenicol (cat, cml), sulfonamide (sulI, sulII, sulIII, sulA), trimethoprim (dfrA, dfrB), quinolone (qnrA, B, S), tetracycline (tetA-E, G, H, J, Y, Z, etc.), vancomycin (vanA, vanB), macrolide (ermA-C, E, F, T, V, X), β-lactum, and penicillin (bla, mecA, penA) antibiotics in different environments [124, 136, 137] . Infections caused by resistant strains are difficult to deal and have led to increasing rates of infections, higher hospital costs, and high rates of morbidity and mortality, for example, strains producing extended-spectrum β-lactamase (ESBL) are responsible for higher rates of mortality [138] .

There is a widespread occurrence of ARGs originating from different settings that has contributed to a web of resistance among humans, animals, and the environment and, even after their treatment, they can be still present and can contribute to their spread in the environment. Furthermore, there still exists an unexplored pool of genes that may have the potential to be used as ARGs and may be passed to pathogenic bacteria. Various hypothesis have been given for the mechanisms of resistance genes to traverse WWTP and their influence by the treatment process [139] (Fig. 4À5) . Thus antibiotic resistance development includes a broader impact on the environment and human health rather than just a local health issue and has to be addressed. The current risk assessment is inadequate and requires advanced biological risk assessment evaluations to detect the proliferation of ARGs and antibiotics. This includes minimizing the resistance emergence and spread in the environment and their transmission to humans. To achieve this, it is necessary to achieve goals such as defining resistance in environmental samples and standardizing testing in those samples which will further require establishment of more comprehensible databases to combine both environmental and clinical metadata. It would help to understand relationships between resistomes of different settings and would improve the risk assessment of ARGs and ARB to further develop control strategies [140] .

The need for screening of ARGs present in bacteria is important for the optimal antimicrobial therapy to treat infections in patients and this need is increasing with increasing resistance among bacteria. Detection will also predict the spread of resistant organisms and genes throughout the environment. A diversity of detection methods is available for both phenotypic as well as genotypic determination of ARGs in isolates. Antibiotic resistance is a selectable phenotype and can be detected using growth inhibition assays using disc diffusion method, broth dilution, gradient strips, or other methods to determine the minimum inhibitory concentration (MIC) of antibiotics [141] . The MIC is calculated for each isolate and based on the results, isolate can either be susceptible or resistant to the antibiotic [142] . However, there remain certain problems associated with this method, such as gradation of resistance, time-consuming (can take several weeks for slow-growing bacteria such as Mycobacterium tuberculosis), and culture-dependent method, relate to only concentration of antibiotics (difficult to detect low-level resistance), and give no information about dissemination of resistance via mobile genetic elements (MGEs) [141, 143] . To overcome these disadvantages, genotypic or molecular characterization methods such as PCR, hybridization techniques including microarray, and whole genome sequence (WGS) are used extensively to determine specific resistance genes and providing results within hours. Genotypic characterization has led to the identification of ARGs in fastidious bacteria which expresses few phenotypic features, for example, Tropheryma whipplei, that causes whipple's disease showed mutations in gyrA and parC gene owning to fluoroquinolones resistance [144] .

The WWTP compartments like influent, activated sludge, effluent consist of contrasting conditions including different concentrations of metals, antibiotics, etc.

Changing stress concentrations may act as drivers for microbial community and resistomes thus, changing biomass per volume and persistence/enrichment of ARB This can result in a strong shift in the whole microbial community composition and antibiotic-resistance subset

These changes are expected to correlate with the changes of resistome. Contigs analysis allows further identification of marker genes for mobile genetic elements Finally, horizontal gene transfer may help in the transfer of resistance genes and may act on evolutionary timescales The use of a plethora of PCRs such as standard, real-time, multiplex PCR to detect the presence of genes encoding resistance to aminoglycoside, chloramphenicol, macrolide, β-lactum, penicillin, trimethoprim, and tetracycline in bacteria is quite common [136, 145, 146] . Isothermal amplifications using loop-mediated isothermal amplification [147] PCRs are very rapid, performed at one constant temperature and have been developed to detect ESBLs genes and carbapenemases genes in bacteria but they cannot be used in multiplex to detect several genes simultaneously. Very recently, a paper-based chip which is integrated with LAMP and a switch molecule for fluorescent detection of ARGs has been developed to allow more convenient and efficient detection especially in resource-limited conditions [148] . Many other detecting and genotyping techniques based on the PCR have been developed to identify ARGs such as restriction fragment length polymorphism-PCR, mismatch amplification mutation assay-PCR, or PCR-single strand conformation polymorphism that have been used to detect gyrA mutations in quinolone-resistant isolates [149] .

Schwartz et al. [150] used PCR technique to detect vanA (vancomycin-resistance gene), mecA (methicillin-resistant gene), and ampC (ampicillin-resistant gene) in wastewater, surface water, and drinking water biofilms [150] . Recently both PCR and RT-PCR have been used across Europe for detection of plasmid-mediated mcr-1 genes harboring resistance against colistin [151À154] (Table 4À4) . Multiplex RT-PCR has been used to identify Staphylococci from blood samples by targeting mecA and other species-specific genes [158] . Another example of using multiplex PCR is in the detection of prevalent ESBLs genes in E. coli that encoded mainly bla SHV , bla TEM , bla CTX-M , and bla OXA [156] . It is also possible to devise and use multiplex PCR to detect 35 genetically diverse resistance genes for [136] tetracycline thus, making it one of the most widely used techniques [159] . However, these methods have low-throughput, sometimes gives false results, mostly ignore potential reservoirs of resistance that are nonculturable bacteria, and depend on primers that leave less room for discovery of novel genes. Molecular hybridization is one of the oldest molecular techniques that have been used to detect the presence of specific ARGs and meanwhile several improvements have been done on probe designs and synthesis. Southern hybridization demonstrated tet and class-I integrons can be cotransferred from isolates present in soil to E. coli and/or Pseudomonas putida [160] . PCR-Southern blot assays were used frequently and reported tetracycline (tet39) and sulfonamide (sulII) resistance genes in Acinetobacter species from fish farms in Thailand [157] . Probes can be labeled with a variety of reporters including radioactive and nonradiolabeled systems. One nonradiolabeled method is fluorescence in situ hybridization that has been used for rapid detection of macrolide, clarithromycin, linezolidresistances [136] .

High-throughput DNA microarray is another technique that has been successfully used to detect ARGs in the test organism in comparison to a reference strain and works with high speed and high delicacy. In this technique, probes specific to the particular gene are spotted on a solid substrate (e.g., glass slide). DNA is labeled and hybridized, and the specific targetprobe duplexes are detected. Comparison of genomic diversity in a large number of test isolates can be done for which WGS is not available [145] . It enables detection of a large number of single genes, mutations, MGEs and also can characterize strain at the molecular level [161] . It has been used for antimicrobial genes detection in a diverse range of bacteria [162] . Glass-based microarray has been developed for the detection of 17 tetracyclineresistance genes and one β-lactamase gene in multiple bacteria that used microarray probes c. 550-base pair PCR products [134] . Perreten et al. [163] developed a rapid and efficient screening of gram-positive bacteria using microarray for the presence of 90 ARGs which was further improved by additional oligonucleotides for detecting 117 ARGs [163, 164] . Similarly, Alere microarrays have been developed capable of detecting 75 clinically relevant antibiotics and can be used in routine diagnostics laboratories [165] . Some of its disadvantages include, low detection limit, high cost (due to use of platforms with probes of short oligonucleotides or PCR products as well as fluorescent dyes) and problems in dealing with complex samples [166, 167] .

Just like PCR and microarray, WGS is another potential method to detect genes and mutations conferring antibiotic resistance. The main advantage is its ability to use and detect different targets simultaneously and to subtype-specific gene variants. One can possibly add new targets to the database for analysis and can perform rapid in silico reanalysis of sequenced isolates. The presence of ARGs within the WGS data has to be determined for which, comprehensive databases containing relevant target DNA is required along with the use of appropriate bioinformatics methods for obtaining information about the WGS data.

Some of the databases that have been used for detection of ARGs in curated WGS data are ResFinder, which is a web server and uses BLAST for identification of ARGs [168] , comprehensive antibiotic-resistance database that uses BLAST and resistance gene identifier as two analysis options [169] , antibiotic-resistance gene-annotation that gives user an opportunity to use local BLAST in Bio-Edit software and analysis can be done without web interface [170] , and antibiotic-resistance gene database that is a manually curated database unifying publicly available information on ARGs such as resistance profile, ontology, mechanism of action, and much more [171] . Other knowledge resources of AMR include antibioticresistance genes online, collection of antimicrobial peptides, database of antimicrobial activity and structure of peptides, antimicrobial peptide database, and BacMet which differ from each other on the basis of the knowledge on molecular level of ARGs they reflect and the scope of resistance mechanism they cover [172] .

However, one major drawback of using molecular techniques for ARG detection is that new emerging resistances against some antibiotics may be overlooked as observed for ndm-1 gene encoding resistance to carbapenem antibiotics which was first isolated using phenotypic methods and then characterized on the basis of genotype [141] . Thus, within the number of both phenotypic and genotypic methods, there are limitations involved with each one and one can perform a combination of these screening methods to monitor resistance. Thus the use of all these techniques helps in predicting the resistance genes, setting up control measures in hospital infections, adapt to a specific therapy and using them routine detection tools [173] .

The molecular analysis tools such as PCR, quantitative PCR, and 16s rRNA analysis have provided the in-depth knowledge about microbial communities present in wastewater since decades [174] . However, requirement of gene-specific primers, and species-specific approach used by these tools limit their activity for detection of certain targeted microbes providing incomplete information about microbial communities present in wastewater [175] . Recently, metagenomics analysis has been introduced, which overcomes the limitations related with conventional molecular analysis tools and is able to generate hundreds to thousands of sequences, providing complete profile of microbial communities present in unknown samples, thus high abundance of potential microbes are detected [176] . The metagenomics analysis consists of four steps; genetic material isolation followed by genetic material cloning, construction of library, and the analysis of genetic material from the metagenomics library.

Metagenomics along with Search Engine for AMR [177] can analyze the unknown samples collected from environment and can provide full-length ARG data. The HWW has been reported to have two overexpressed β-lactam-resistance genes (bla GES and bla OXA ) as compared with the water collected from other aquatic bodies, which could be correlated with antibiotic usage over the time in hospitals and discharge of the residues of antibiotics in the wastewater [176] . The HWW that is derived from clinical speciality ward is determined to be major spot for the AMR. The wastewater derived from different wards of the hospital and the final effluent of hospital was tested and compared for resistance genes. The wastewater has a high abundance of class A β-lactamase resistant genes (i.e., bla KPC , bla CTX-M , bla SHV , bla TEM ) and the wastewater from clinical ward also consists of high level of bla KPC-2 genes (142, 200 x/Gb), encoding for carbapenem resistance. Moreover, there was presence of assembled scaffolds of IncQ and IncF plasmids having quinolone-resistance genes (qnrS1, qnrS2) and the class A β-lactamase gene (bla TEM-1 ) in wastewater, which further helps in proliferation of AMR [178] .

Metagenomic functional selection has been integrated with deep metagenomic sequencing to trace validated ARGs in different environments. Studies have identified WWTP resistome consisting of novel ARGs thus, throwing the limelight on WWTP reservoir of uncharacterized resistance genes using these selections. Using such metagenomic methods relates the number of clinically relevant resistant genes to overall functional resistant genes and thus, helps in improving the risk classification in environments [49] .

Metagenomics strategy also revealed the association of isolated P. aeruginosa from HWW with AMR frequency. The isolated strain was selected as a bio-indicator, due to its ability to survive and colonize in harsh environments and it was used to assess its viability, antibiotic susceptibility, and diversity in HWW. The samples collected during different treatment steps of HWW revealed that each treatment step was able to decrease the bacterial population by four logarithm cycle; however, the antibiotic resistance profile did not decrease at each treatment level, while, on the contrary, there was increase in AMR microbes and genes as well. The microorganism was able to remain survived in the spore form during different treatment steps and again transform into vegetative form when released into surviving environment [179] . The healthcare clinics and hospitals, where antibiotic use is more common among the major contributor to disseminate the pathogenic microorganism into the environment. In addition, shortgun metagenomics analysis of HWW collected from Turkey revealed the presence of more than 100 antibiotic resistance determinants and most common genes belongs to aminoglycosides and β-lactameses. The prediction of high resistome diversity in HWW raises an alarm for health concern of human population.

An important step in coping with the serious problem of emergence and dissemination of ARGs is to understand the pathways for resistance gene spread. The ability of bacteria to respond to selective pressures and new environments can be explained by the acquisition of new genes by cells using horizontal transfer methods. Studies have shown around 75% of the genes in each genome have been transferred by HGT during evolution [180] . Many resistance genes are located on MGEs such as plasmids, transposons also known as jumping genes, and integrons that are capable of capturing and expressing gene cassettes [181] , which are responsible for antibiotic resistance that functions as vectors for their dissemination [182] . Horizontal acquisitions might be neutral or have deleterious effects in the chromosome or confer a selective advantage to the host. Other than mutations, HGT has been one of the methods which are most responsible for the dissemination of ARGs among different bacterial species. It has caused the spread of antibiotic resistance from commensal and environmental bacteria to pathogenic species. The ARGs transfer by HGT exists much back before the use of antibiotics by humans, for example, OXA β-lactamase genes that confer resistance against β-lactam antibiotics mobilized from chromosomes to plasmid millions of years ago [183] . But the rate of the resistance gene transfer has increased tremendously due to selective pressure caused by antibiotic use by humans in the last few decades.

Bacteria employ three mechanisms for HGT, that is, conjugation, transformation, and transduction (Fig. 4À6) . Conjugation is said to have the greatest influence over the spread of ARGs. Conjugation requires a physical contact between two cells via pili or adhesions to transfer the genetic material. This mechanism has been found in bacterial cells with an exception of Agrobacterium species that uses HGT in plants. The conjugative machinery encoded by the genes on plasmids or by integrative conjugative elements on chromosomes facilitates this process. ARGs are mostly associated with conjugative elements like plasmids and transposons, and the transfer of these elements is most likely to occur by conjugation because it provides a more efficient way to enter the recipient cell and gives better protection in the environment [119] . Conjugation can be of various types such as (1) transfer of a selftransmissible conjugative plasmid, for example, RP4 plasmid of E. coli. (2) Mobilization of nonself-transmissible plasmids by the action of conjugative plasmid, for example, IncQ plasmid RSF1010. (3) Cointegration of two different circular plasmids to fuse to become one. (4) Conjugated transposons which may facilitate mobilization of plasmids or cointegration. The MGEs conferring AGRs transfer have been found in bacteria ranging from soil, water ecosystems to food, and human pathogens [185] . The transfer of the conjugative elements between bacteria over distant phylogeny indicates the emergence of multiresistance between different reservoirs [186] . Bla CTX-M ESBL genes have been disseminated to various plasmids within Enterobacteriaceae and other pathogens and can be found in various geographical locations [187] . The conjugation of plasmids has caused dissemination of ARGs worldwide that encode resistance to β-lactamases, carbapenemases, quinolones, aminoglycosides, colistin, sulfonamide, tetracyclines, and other classes of drugs. Conjugation has been also reported between bacteria and eukaryotic cells and is observed in various environments such as soil, aquatic, marine sediment, wastewater, and activated sludge [124] .

The second mechanism is transformation, in which the cells take up the naked DNA from the environment. For transformation to take place, there are certain prerequisites like release and persistence of extracellular DNA in the environment, the recipient cells must be competent, and the DNA translocated must be stabilized by integration into the genome or by recircularizing into self-replicating plasmids [188] . The process takes place in two steps: first, the DNA substrate is transferred from surface to the cytoplasmic membrane which is mediated by type II secretion system, type IV secretion system, and type IV pili; and second, is the transport of DNA across the cytoplasmic membrane using cytoplasmic membrane channels [189] . With an exception of Neisseria gonorrhoeae that is a naturally transformable bacteria which responds to environmental conditions [188] , most species develop competence under autoinducers, peptides, or stressful conditions, for example, aminoglycoside and fluoroquinolone antibiotics sublethal concentrations induce competence in Streptococcus pneumoniae and Legionella pneumophila [190] . Mao et al. [191] developed a method to extract extracellular and intracellular DNA from water and sediments. They obtained a higher concentration of extracellular DNA than intracellular DNA in sediments from a river basin that serves as a major ARG reservoir that could be transferred to indigenous bacteria through natural transformation [191] . Also, it has been shown in pneumococcal genome that the conjugated transposon disseminates via transformation in addition to conjugation [192] .

Transduction is the process by which DNA is transferred with the help of bacteriophages. Bacteriophages can transfer DNA sequences like chromosomal DNA, MGEs such as plasmids, transposons and genomic islands that are advantageous to their bacterial hosts as well as serves in improving the survival of bacteriophages [193] . Transduction can be generalized where random host DNA is incorporated during cell lysis and specialized, where the prophage excises itself from the host genome and also incorporates flanking host DNAs [194] . ARGs transfer by transduction has also been reported in various bacteria, for example, β-lactamase gene transfers by P1-like bacteriophages in E. coli [195] , erythromycin resistance transfer by phage between Clostridium difficile strains [196] , tetracycline and gentamycin resistance between enterococci [197] , or transfer of resistance plasmids in MSRA [198] . Bacteriophages can have broad host range, that is, between different species or among different taxonomic groups [197] thus, indicating dissemination of ARGs via transduction in the environment, a common mechanism.

Other mechanisms such as gene transfer agents (GTAs) and cell fusion have also been described. GTAs are delivery systems that carry random pieces of host genome in capsids and get integrated into the host chromosome. The amount of DNA that GTA contain is insufficient to encode their proteins counterparts, unable to self-propagate thus, they do not necessarily carry GTA-encoding genes that is an important distinction from transduction mechanism [199] . The first GTA discovered was in Rhodopseudomonas capsulate called as RcGTA and they were able to transfer antibiotic resistance to bacteria by a mechanism similar to transduction which does not require cells contact and was resistant to DNases [200] . Apart from the evidence of GTA shown in various members of Rhodobacterales, for example, Ruegeria pomeroyi which contain a complete RcGTA-like cluster of these genes, Roseovarious nubinhibens and Ruegeria mobilis also showed rates of transfer of antibiotic resistance around 10 6 -fold higher than rates of transformation and transduction when added to microbial communities [199] .

McDaniel et al. [201] reported GTA frequencies to be much higher, around a million times higher the frequency of transformation and transduction measured previously in the marine environment [201] . There are several advantages of GTAs over other mechanisms such as protection of DNA from environmental factors, transfer not constrained to cellÀcell contact, and they contain random DNA from host rather than most of the bacteriophage DNA as observed in transduction. Another mechanism, cell fusion has been observed in Haloferax and Sulfolobus species [202, 203] . Symmetric and bidirectional cell fusion has been observed in Haloferax volcanii. Studies have shown interspecies gene exchange in halophilic archaea where cells fuse forming a diploid state containing two different chromosomes of parental cells thus, facilitating genetic exchange and recombination followed by separation of hybrid parental cells [202] . Although new mechanisms are continuously being developed by bacteria and identification, mapping of these resistance mechanisms provides us with knowledge of sources of resistance and helps in designing new antimicrobial drugs.

The prions are infectious protein particles present in brain, which are responsible for degenerative neurological disorders. In humans, the PrNP gene present on chromosome 20 produces the PrP protein and prions results in transformation of PrP protein into abnormal disease causing isoform [204] . These pathogenic prions cause the functional disruption of neural cells resulting in cell death and leading to memory problems, trouble with movement and personality changes as well. The prions can result in many infectious diseases such as Creutzfeldtjakob disease (CJD), variably protease-sensitive prionopathy, Gerstmann-Sträussler-Scheinker disease, scrapie, variant CJD, fatal insomnia, and kuru. These transmissible disease impacts number of mammalian species, including sheep and goats, cattle, deer, moose, elk, and humans. The scrapie and CJD are of particular concern because they can be transmitted horizontally and they remain infectious in the environment for very long time [205] . For instance, the scrapie infection was persistent even after burial in soil for 3 years. The CJD disease is most common and deadly human prion disease and can cause infection in three ways: sporadic, genetic, and acquired. Most of the times (85%À90%), CJD occurs sporadically and approximately 1À1.5 million people are affected annually, because there is no cure available for CJD and other prion diseases [206] . The recently developed methods for detection of prion infectivity include protein misfolding cycling amplification, quake-induced conversion, and quantitative tandem mass spectrometric techniques [207] . However, the study of prions is highly challenging due to the fact that the high-resolution structure of prion isotherm is still to be determined and change in structure directly affect infectivity and pathology of disease.

Prions are likely to enter through live, infected hosts, and can be shred through saliva, urine, feces, mucus, and blood; and they enter in wastewater through hospital effluents, research facilities, homes, slaughterhouses, and mortuaries [208] . The WWTPs are not able to treat prions, for instance, after entering into municipal WWTP, the prions bind to sewage sludge, survive through anaerobic digestion and are further present in treated biosolids. These biosolids are further used for land application, which results in their introduction into environment. Thereafter they can be swept by wind and contaminated water with prions could further migrate into lakes, rivers, and oceans and thus, directly affecting the humans, aquatic life, and animals as well [209] .

Viroids are small (B250À400 nucleotides), single stranded, circular RNA particles, which are distinguished from viruses by smaller size, lack of capsid and also they do not encode any protein. They have capacity to reproduce and are mostly known to cause infection in plants; however, hepatitis δ, which cause infections in humans, has many similar properties with viroids and could be related to viroids [210] . A total of 29 viroids species has been identified, which are grouped into Avsunviroidae and Pospiviroidae. The viroids are mainly infectious for plants such as tomatoes, cucumber, avocados, potatoes, apples, coconut palms, and chrysanthemums and are responsible for million dollars' loss in agriculture revenue each year. The most commonly reported viroid plant diseases caused by viroid include apple fruit crinkle, tomato chloric dwarf, and chrysanthemum chlorotic mottle [211] . The yellowish curled leaves of plants are due to pairing of viroid RNA with messenger RNA of plants resulting in interference of proper translation. Some of the viroids cause devastating effects, for instance, Coconut cadongÀcadong viroid has caused lethal effects in coconut palms in Philippines, resulting in loss of 40 million coconut palm [212] . Other reported effects of viroids include epinasty, rugosity, necrosis, chlorosis, stem shortening, color alteration of fruits, flowering, and ripening delays. The microarrays are identified as a significant tool for detection of many viroids simultaneously and even has ability to detect emerging or established viroid in new host.

Potent protein toxins such as tetanus and botulinum toxins, anthrax toxin, epsilon-toxin, and enterotoxin can cause some most significant diseases in humans and animals. These protein toxins are most common virulence factors encoded by plasmids in Clostridium and Bacillus species, for example, tetanus toxin plasmid and conjugative toxin plasmid of C. perfringens. Extracellular vesicles (EVs) are also infectious particles said to be produced by all types of microorganisms. These EVs can contain nucleic acids, toxins, lipoproteins, adhesins, nutrient scavenging factors, and enzymes that can play major role in pathogenicity in HWW. Such vesicles from Gram-positive bacteria, mycobacteria, and fungi contain virulence factors that can elicit host immune responses. For example, Crytococcus neoformans EVs have glucuronoxylomannan (capsular polysaccharide) which is a virulence factor [212] . EVs from Gramnegative bacteria originate from outer membrane and are called outer-membrane vesicles which have been associated with cytotoxicity, virulence, invasion of host cells, and antibiotic resistance proteins. In addition to these particles, certain factors and conditions may further increase the virulence of EVs, for example, a study indicated the role of iron-limiting conditions in the virulence of mycobacterium EVs. These emerging infectious particles are not easily removed by conventional treatment technologies. Thus there is a requirement of more novel methods and techniques to deal with such infectious particles. Therefore these particles have to be readily analyzed for their presence and have to be studied for their removal from HWW due to their potential negative effects on human and animal life.

The recent years have witnessed the emphasis toward management of HWW and various studies focused on the microbial communities present in wastewater have increased immensely. The pathogenic microbes present in HWW have affected the human health since decades and antibiotic resistance microbes are also increasing significantly with time. The development of resistance toward antibiotics has been observed worldwide and has challenged both public and animal health. The use and release of various antibiotic agents in different settings have not only led to the prevalence of ARGs in the environment but also spread and emergence of resistant bacteria. This has caused increased resistance in human pathogens and thus, making infections caused by them difficult to deal with, leading to higher mortality rates. However, the surveillance of its spread and prevalence in environment is limited and has to be expanded more due to the broad impact of antibiotic resistance on human health. Various culture-dependent and independent techniques have allowed characterization and exploration of ARGs and ARB, which have increased our understanding toward the evolutionary pathways for their dissemination within any community. Further, metagenomics tools have advanced the research toward analysis of complete microbial profile and increased the knowledge toward microbial abundance present in HWW. However, further planning and implementation of strategies, policies, and experimental approaches have to be done by collaboration of scientific community and public authorities to limit the use of antibiotics, detection of microbial communities (resistant and/or sensitive) from wastewaters, and mapping resistance mechanisms to clearly understand the role of MGEs and extracellular DNA in evolutionary process. 

The microbiome and resistome of hospital sewage during passage through the community sewer system

Evaluation of wastewater discharge from Al-Sadr teaching hospital and its impact on the Al-Khorah channel and Shatt Al-Arab River in Basra City-Iraq

Study of widely used treatment technologies for hospital wastewater and their comparative analysis

Microbiological and toxicological assessment of pharmaceutical wastewater from the Lagos Megacity

Contaminant properties of hospital clinical laboratory wastewater: a physiochemical and microbiological assessment

Occurrence of Pharmaceuticals in Finnish Sewage Treatment Plants, Surface Waters, and Their Elimination in Drinking Water Treatment Processes

Review on fate and mechanism of removal of pharmaceutical pollutants from wastewater using biological approach

Hospital effluents as a source of emerging pollutants: an overview of micropollutants and sustainable treatment options

Groundwater contamination by microbiological and chemical substances released from hospital wastewater: health risk assessment for drinking water consumers

Antibiotic resistant bacteria from treated and untreated hospital wastewater at Ayder Referral Hospital, Mekelle, North Ethiopia

Survival of opportunistic fungi in clinical wastes

Molecular detection and genotypic characterization of enteric adenoviruses in a hospital wastewater

Parasitological profile of raw wastewater and the efficacy of biosand filter in reduction of parasite ova and cysts

Disinfectant-resistant bacteria in Buenos Aires city hospital wastewater

Separate treatment of hospital and urban wastewaters: a real scale comparison of effluents and their effect on microbial communities

A global overview of treated wastewater guidelines and standerds for agricultural reuse

Determination of pathogenic bacteria in wastewater using conventional and PCR techniques

Pathogen detection methodologies for wastewater and reserviors

dPCR: a technology review

Lab-on-a-chip technology for environmental monitoring of microorganisms

The route of antimicrobial resistance from the hospital effluent to the environment: focus on the occurrence of KPCproducing Aeromonas spp. and Enterobacteriaceae in sewage

Hospital wastewater releases of carbapenem-resistance pathogens and genes in urban India

Assessment of antibiotic-and disinfectant-resistant bacteria in hospital wastewater, south Ethiopia: a cross-sectional study

Antibiotic susceptibilities of Enterococcus species isolated from hospital and domestic wastewater effluents in Alice, Eastern Cape Province of South Africa

Beta-lactamase-producing Enterobacteriaceae in hospital effluents

Insights into the relationship between antimicrobial residues and bacterial populations in a hospital-urban wastewater treatment plant system

Influence of hospital wastewater discharged from University of Benin Teaching Hospital (UBTH)

Multiresistance, beta-lactamaseencoding genes and bacterial diversity in hospital wastewater in Rio de Janeiro, Brazil

Enumeration and characterization of antimicrobial-resistant Escherichia coli bacteria in effluent from municipal, hospital, and secondary treatment facility sources

Antibiotic resistance and antibiotic resistance genes in Escherichia coli isolates from hospital wastewater in Vietnam

Antibiotic resistant bacteria in hospital wastewaters and sewage treatment plants

Dissemination of antibiotic resistance in methicillin-resistant Staphylococcus aureus and vancomycin-resistant S aureus strains isolated from hospital effluents

Detection of antimicrobial-resistant Gram-negative bacteria in hospital effluents and in the sewage treatment station of Goiânia Brazil

Antimicrobial resistance of 3 types of gram-negative bacteria isolated from hospital surfaces and the hands of health care workers

Abundance of antibiotics, antibiotic resistance genes and bacterial community composition in wastewater effluents from different Romanian hospitals

Vancomycin resistant enterococci: from the hospital effluent to the urban wastewater treatment plant

The microbiological effects of hospital wastes on the environment

Hospital effluent: a source of multiple drug-resistant bacteria

Hospital-associated microbiota and implications for nosocomial infections

Vancomycin resistance in Staphylococcus aureus

Escherichia coli (E. coli 0157 H7), StatPearls [Internet

Citrobacter freundii fitness during bloodstream infection

Pathogenesis of Proteus mirabilis infection

Pantoea agglomerans cutaneous infection

Influence of a non-hospital medical care facility on antimicrobial resistance in wastewater

Bacterial communities in full-scale wastewater treatment systems

Bacterial community characteristics under long-term antibiotic selection pressures

Limited influence of hospital wastewater on the microbiome and resistome of wastewater in a community sewerage system

Limited dissemination of the wastewater treatment plant core resistome

The hospital water environment as a reservoir for carbapenem-resistant organisms causing hospital-acquired infections-a systematic review of the literature

Investigating the potential use of an Antarctic variant of Janthinobacterium lividum for tackling antimicrobial resistance in a One Health approach

Inactivation of Aspergillus spores in clinical wastes by supercritical carbon dioxide, Arab

Overcoming antifungal resistance

Survival of some medically important fungi on hospital fabrics and plastics

Multidrug-resistant Candida: epidemiology, molecular mechanisms, and treatment

Treatment of infections due to Aspergillus terreus species complex

Candida and invasive mould diseases in non-neutropenic critically ill patients and patients with haematological cancer

The spectrum of fungi that infects humans, Cold Spring Harb

Emerging infectious diseases with cutaneous manifestations: fungal, helminthic, protozoan and ectoparasitic infections

Combination therapy for the treatment of pulmonary mold infections

Treatment of Paecilomyces variotii pneumonia with posaconazole: case report and literature review

First report of monomicrobial Candida parapsilosis necrotizing fasciitis

Genetic diversity and in vitro antifungal susceptibility of 200 clinical and environmental Aspergillus flavus isolates

Aspergillus niger infection in an immunosuppressed patient confined solely to the brain

Central lineÀassociated mucor velutinosus bloodstream infection in an immunocompetent pediatric patient

Microbiological and antimicrobial analysis of hospital wastewater discharged into the soil environment

Trichophyton rubrum infection characterized by Majocchi's granuloma and deeper dermatophytosis: case report and review of published literature

Darier disease complicated by terbinafine-resistant Trichophyton rubrum: a case report

Zygomycetes in human disease

Cryptococcosis, Diagnosis and Treatment of Human Mycoses

Management of candidemia and invasive candidiasis

Introduction to fungi, The Plant Health Instructor

How a fungus shapes biotechnology: 100 years of Aspergillus niger research

Fatal invasive aspergillosis caused by Aspergillus niger after bilateral lung transplantation

Fungal infections from human and animal contact

Upsurge in Curvularia infections and global emerging antifungal drug resistance

Fungal opportunist infection: common and emerging fungi in immunocompromised patients

Antifungal resistance: current trends and future strategies to combat

Longitudinal study on occurrence of adenoviruses and hepatitis A virus in raw domestic sewage in the city of Limeira

Quantification and molecular characterization of enteric viruses detected in effluents from two hospital wastewater treatment plants

The repertoire of adenovirus in human disease: the innocuous to the deadly

Acute lower respiratory tract infections in soldiers

First isolation of a new type of human adenovirus (genotype 79), species Human mastadenovirus B (B2) from sewage water in Japan

Quantitative real-time PCR assays for detection of human adenoviruses and identification of serotypes 40 and 41

Human Adenovirus Removal in Wastewater Treatment and Membrane Process

Evaluation of human adenovirus and human polyomavirus as indicators of human sewage contamination in the aquatic environment

Technical aspects of using human adenovirus as a viral water quality indicator

Human adenovirus surveillance-United States

Detection and molecular characterization of human rotaviruses isolated in Italy and Albania

Rotavirus Disease Mechanisms Diarrhea, Vomiting and Inflammation: How and Why

Occurrence of Common Pollutants and Pharmaceuticals in Hospital Effluents

Rotavirus detection in environmental water samples by tangential flow ultrafiltration and RT-nested PCR

First molecular detection of group A rotaviruses in drinking water sources in Beijing, China

First molecular detection of group A rotavirus in urban and hospital sewage systems by nested-RT PCR in Shiraz, Iran

Comparative study of enteric viruses, coliphages and indicator bacteria for evaluating water quality in a tropical high-altitude system

Surveillance of noroviruses in Rio De Janeiro, Brazil: occurrence of new GIV genotype in clinical and wastewater samples

Norovirus gene expression and replication

Emerging recombinant noroviruses identified by clinical and waste water screening

Quantification and molecular characterization of norovirus after two wastewater treatment procedures

Standardized multiplex one-step qRT-PCR for hepatitis A virus, norovirus GI and GII quantification in bivalve mollusks and water

Prevalence of norovirus and factors influencing virus concentrations during one year in a full-scale wastewater treatment plant

Detection and molecular characterization of noroviruses from five sewage treatment plants in central Italy

One-year monthly survey of rotavirus, astrovirus and norovirus in three sewage treatment plants in Beijing, China and associated health risk assessment

Progress on norovirus vaccine research: public health considerations and future directions

Hepatitis A: epidemiology and prevention in developing countries

Hepatitis A virus subgenotyping based on RT-qPCR assays

Type A viral hepatitis: a summary and update on the molecular virology, epidemiology, pathogenesis and prevention

Quantitative PCR detection and characterisation of human adenovirus, rotavirus and hepatitis A virus in discharged effluents of two wastewater treatment facilities in the Eastern Cape

Fate of viruses in water systems

Incidence of human adenoviruses and Hepatitis A virus in the final effluent of selected wastewater treatment plants in Eastern Cape Province, South Africa

Monitoring of adenovirus serotypes in environmental samples by combined PCR and melting point analyses

Hepatitis A virus: essential knowledge and a novel Identify-Isolate-Inform tool for frontline healthcare providers

Molecular characterization of hepatitis A virus isolates from environmental and clinical samples in Greece

Public health risks associated with food-borne parasites

Prevalence of protozoa species in drinking and environmental water sources in Sudan

Comparative study on waterborne parasites between Malaysia and Thailand: a new insight

Antibiotics in the aquatic environment-a review-part I

Dissemination of antimicrobial resistance in microbial ecosystems through horizontal gene transfer

Functional metagenomics reveals diverse β-lactamases in a remote Alaskan soil

Antibiotic resistance is prevalent in an isolated cave microbiome

Functional characterization of bacteria isolated from ancient arctic soil exposes diverse resistance mechanisms to modern antibiotics

The role of aquatic ecosystems as reservoirs of antibiotic resistance

Environmental dissemination of antibiotic resistance genes and correlation to anthropogenic contamination with antibiotics

Occurrence of antibiotics and antibiotic resistance genes in hospital and urban wastewaters and their impact on the receiving river

Urban wastewater treatment plants as hotspots for antibiotic resistant bacteria and genes spread into the environment: a review

Levels of antibiotic resistance genes in manure, biosolids, and fertilized soil

Epidemiology of resistance to antibiotics: links between animals and humans

Detection of antibiotic resistance genes in source and drinking water samples from a first nation community in Canada

Prevalence of antibiotic resistance in drinking water treatment and distribution systems

Antimicrobial Resistance: Global Report on Surveillance, World Health Organization

Society's failure to protect a precious resource: antibiotics

Hospital effluents are one of several sources of metal, antibiotic resistance genes, and bacterial markers disseminated in Sub-Saharan urban rivers

Identifying antimicrobial resistance genes with DNA microarrays

Evidence of increasing antibiotic resistance gene abundances in archived soils since 1940

Antibiotic resistance genes in water environment

Prevalence of antibiotic resistance genes and their relationship with antibiotics in the Huangpu River and the drinking water sources

Multiple drug resistance and biocide resistance in Escherichia coli environmental isolates from hospital and household settings

Wastewater treatment plant resistomes are shaped by bacterial composition, genetic exchange, and upregulated expression in the effluent microbiomes

Tackling antibiotic resistance: the environmental framework

Screening methods for the detection of antimicrobial resistance genes present in bacterial isolates and the microbiota

Culture-based methods for detection of antibiotic resistance in agroecosystems: advantages, challenges, and gaps in knowledge

Insights into antibiotic resistance through metagenomic approaches

Molecular evaluation of antibiotic susceptibility: Tropheryma whipplei paradigm

Molecular methods for detection of antimicrobial resistance

Multi-centre evaluation of real-time multiplex PCR for detection of carbapenemase genes OXA-48

Molecular detection of hepatitis A virus in urban sewage in Rio de Janeiro, Brazil

Simultaneous detection of antibiotic resistance genes on paper-based chip using

Molecular detection of antimicrobial resistance

Detection of antibiotic-resistant bacteria and their resistance genes in wastewater, surface water, and drinking water biofilms

Occurrence and characterization of mcr-1-harbouring Escherichia coli isolated from pigs in Great Britain from 2013 to

Co-occurrence of extended spectrum β lactamase and MCR-1 encoding genes on plasmids

Detection of mcr-1 encoding plasmid-mediated colistin-resistant Escherichia coli isolates from human bloodstream infection and imported chicken meat

Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study

Antibiotic resistance elements in wastewater treatment plants: scope and potential impacts, Wastewater Reuse and Current Challenges

Molecular epidemiology of extended-spectrum β-lactamases among Escherichia coli isolates collected in a Swedish hospital and its associated health care facilities from

The tetracycline resistance determinant Tet 39 and the sulphonamide resistance gene sulII are common among resistant Acinetobacter spp. isolated from integrated fish farms in Thailand

Usefulness of multiplex real-time PCR for simultaneous pathogen detection and resistance profiling of staphylococcal bacteremia

Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance, Microbiol

Class 1 integrons and tetracycline resistance genes in Alcaligenes, Arthrobacter, and Pseudomonas spp. isolated from pigsties and manured soil

Molecular Methods for Detection of Antibiotic Resistance, Antimicrobial Resistance in Bacteria of Animal Origin

DNA microarray detection of antimicrobial resistance genes in diverse bacteria

Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria

A novel universal DNA labeling and amplification system for rapid microarray-based detection of 117 antibiotic resistance genes in Gram-positive bacteria

Evaluation of an expanded microarray for detecting antibiotic resistance genes in a broad range of gram-negative bacterial pathogens

Development of a miniaturised microarray-based assay for the rapid identification of antimicrobial resistance genes in Gramnegative bacteria

Metagenomic and network analysis reveal wide distribution and co-occurrence of environmental antibiotic resistance genes

Identification of acquired antimicrobial resistance genes

The comprehensive antibiotic resistance database

ARG-ANNOT, a new bioinformatic tool to discover antibiotic resistance genes in bacterial genomes

ARDB-antibiotic resistance genes database

CARD 2017: expansion and model-centric curation of the comprehensive antibiotic resistance database

Genetic methods for detection of antimicrobial resistance

Bacterial communities and antibiotic resistance communities in a full-scale hospital wastewater treatment plant by high-throughput pyrosequencing

Metagenomic analysis reveals the impact of wastewater treatment plants on the dispersal of microorganisms and genes in aquatic sediments

Overexpression of antibiotic resistance genes in hospital effluents over time

Using the whole genome sequence to characterize and name human adenoviruses

Characterization of metagenomes in urban aquatic compartments reveals high prevalence of clinically relevant antibiotic resistance genes in wastewaters

Diversity and antibiotic resistance profiles of Pseudomonads from a hospital wastewater treatment plant

Trends and barriers to lateral gene transfer in prokaryotes

Plasmid encoded antibiotic resistance: acquisition and transfer of antibiotic resistance genes in bacteria

Detection of 140 clinically relevant antibiotic-resistance genes in the plasmid metagenome of wastewater treatment plant bacteria showing reduced susceptibility to selected antibiotics

Phylogenetic analysis shows that the OXA b-lactamase genes have been on plasmids for millions of years

Antimicrobial-resistant bacteria in the community setting

Genetic exchange between bacteria in the environment

Large-scale analysis of plasmid relationships through genesharing networks

CTX-M enzymes: origin and diffusion

Mechanisms of, and barriers to, horizontal gene transfer between bacteria

Two steps away from noveltyÀprinciples of bacterial DNA uptake

Bacterial transformation: distribution, shared mechanisms and divergent control

Persistence of extracellular DNA in river sediment facilitates antibiotic resistance gene propagation

Composite mobile genetic elements disseminating macrolide resistance in Streptococcus pneumoniae

Transfer of antibiotic-resistance genes via phagerelated mobile elements

Horizontal gene transfer: building the web of life

Characterization of a P1-like bacteriophage encoding an SHV-2 extended-spectrum β-lactamase from an Escherichia coli strain

Phage φC2 mediates transduction of Tn6215, encoding erythromycin resistance, between Clostridium difficile strains

Bacteriophage-mediated transduction of antibiotic resistance in enterococci

Efficient transfer of antibiotic resistance plasmids by transduction within methicillin-resistant Staphylococcus aureus USA300 clone

Gene transfer agents: phage-like elements of genetic exchange

Genetic recombination in Rhodopseudomonas capsulata

High frequency of horizontal gene transfer in the oceans

Cell fusion and hybrids in Archaea: prospects for genome shuffling and accelerated strain development for biotechnology

A multicopy plasmid of the extremely thermophilic archaeon Sulfolobus effects its transfer to recipients by mating

Current infection control recommendations for prion disease; a difficult problem for the deployed personnel of the armed forces

Kinetics of ozone inactivation of infectious prion protein

Epidemiological characteristics of human prion diseases

Early preclinical detection of prions in the skin of prion-infected animals

Prions in the environment: occurrence, fate and mitigation

Persistence of pathogenic prion protein during simulated wastewater treatment processes

Subviral agents

Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses

Detection of Coconut cadang-cadang viroid (CCCVd) in oil palm by reverse transcription loop-mediated isothermal amplification (RT-LAMP)