key: cord-0828078-93yxh0y5
authors: Gwenzi, Willis
title: The ‘thanato-resistome’ - The funeral industry as a potential reservoir of antibiotic resistance: Early insights and perspectives
date: 2020-07-25
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2020.141120
sha: d530e29a2289172c3f59a9d15047e80dfb9eed40
doc_id: 828078
cord_uid: 93yxh0y5

Abstract The funeral industry is a potential reservoir of antibiotic resistance. The occurrence, human exposure and health risks of antibiotic resistance in the funeral industry were examined. The funeral industry harbours antibiotic resistance to multiple common and last-resort antibiotics, hence constitutes the ‘thanato-resistome’. Hydrological processes, air-borne particulates and vectors disseminate antibiotic resistance, while horizontal gene transfer circulates antibiotic resistance among resistomes, forming a complex network. Ingestion, inhalation of air-borne particulates, dermal intake and clothes of workers contribute to human exposure. Human health risks include; development of drug resistance in previously susceptible pathogens, and increased morbidity and mortality caused by increased pathogenicity and outbreaks of multi-drug resistant infections. Ecological risks include the proliferation of resistant organisms at the expense of susceptible ones, thereby disrupting ecosystem structure and function, including biogeochemical cycles. Barring inferential data, quantitative evidence linking antibiotic resistance to human infections is weak. This reflects the lack of systematic quantitative studies, rather than the absence of such health risks. Quantitative risk assessment is constrained by lack of quantitative data on antibiotic resistance in various reservoirs and exposure routes. A framework for risk assessment and mitigation is proposed. Finally, ten hypotheses and emerging tools such as genomics, in silico techniques and big data analytics are highlighted.

Antibiotic resistance is a global issue posing human and animal health risks. Antibiotic resistance and its human health risks have received significant research attention. To date, antibiotic resistance has been detected in several bacteria, including the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species), Clostridium difficile, Escherichia coli and Proteus (De Oliveira et al., 2020; Ma et al., 2020) . The widespread use of antibiotic s for human and animal health care is recognized as the key factor contributing to the development of antibiotic resistance.

Existing literature, including reviews, is dominated by studies investigating the occurrence and behaviour of antibiotic resistance in municipal and industrial wastewaters (Rizzo J o u r n a l P r e -p r o o f 6 cases where human death occurs, thanatopraxy care involving the use of embalming fluids then follows. Embalming fluids and other thanatopraxy care chemicals include synthetic chemicals such as antimicrobials, pharmaceuticals, personal care products, as well as disinfectants, perfumes and moisturisers and penetrating agents (Varlet et al., 2019) . Human cadavers may contain residual pharmaceuticals including antibiotics used for the treatment of infectious diseases. Pharmaceuticals and antibiotic s co-selecting for antibiotic resistance (Knapp et al., 2016; Zhao et al., 2017) . Hence, solid wastes (e.g., used bandages, wipes, gloves), wastewaters and air-borne particulates from thanatopraxy care facilities may contain a complex mixture of contaminants, including pharmaceuticals and antibiotic resistant bacteria and their resistance genes.

The discharge of solid wastes and wastewaters transfers a complex mixture of contaminants into the environmental systems ( Figure 1) . Similarly, the burial and subsequent decomposition of human cadavers in cemeteries and release of leachates including pharmaceuticals and microbial agents into the environment (Abia et al., 2018) . First, thanatopraxy care facilities and cemeteries receive and accumulate complex mixtures of emerging contaminants and antibiotic resistance in embalming fluids, human cadavers, wastes and wastewaters. Subsequently, the contaminants, including pharmaceuticals and antimicrobial resistance genes are released into the environment via waste and wastewaters, and leachates.

To this point, it has been highlighted that, the funeral industry is a potential hotspot of emerging contaminants and antibiotic resistant bacteria. Whereas antibiotic resistance has received a lot of research attention, it is well-recognized that environmental research in the funeral industry has attracted very limited attention relative to other industries (Ucisik and J o u r n a l P r e -p r o o f 7 Rushbrook; Fiedler et al., 2016; Paíga and Delerue-Matos, 2016) . This then raises the question, 'Why has the funeral industry attracted less research attention than other industries?' To date, limited attempts have been made to answer this pertinent question and identify the reasons behind this trend. Here, it is argued that, a combination of policy and regulatory frameworks, socio-cultural factors, and attitudes and perceptions of the research community and funders may account for this trend.

It is tempting to think that the accreditation and practice of professionals in the funeral industry is governed by regulations in the health care industry, but this is not the case in most countries. In the USA, the Bureau of Labour Statistics Standard Occupational Classification System classify funeral service providers among commercial service providers rather than as health care professionals (Davidson and Benjamin, 2006) . In environmental sciences and other disciplines, the decision to investigate particular environmental phenomena, in this case, antibiotic resistance in the funeral industry, is based on whether such phenomena have been widely investigated in earlier studies. This is referred to as the 'Matthew' or 'bandwagon' effect, an emerging phenomenon in research (Daughton et al., 2014; Baveye, 2020a, b) . The bandwagon effect implies that trending research areas tend to be over-subscribed, while other equally relevant topics are neglected. The 'bandwagon/Matthew effect may unduly over-estimate or inflate the significance of certain topics in a field, thereby creating a 'hyperbole' (Baveye, 2020a, b) . The potential causes, detrimental effects of the 'bandwagon' and 'hyperbole' phenomena on research, and the need to overcome them have been discussed (Baveye, 2020a, b) .

Others may also argue that, the lack of research attention on AR in the funeral industry reflect the lack of unequivocal evidence on the human health risks. A qualitative comparison of research on AR in wastewater and drinking water versus that in the funeral industry does not support this notion. For example, AR in wastewater and drinking water systems, and even soils has attracted significant research attention, as evidenced by several research articles and including reviews Sanganyado and Gwenzi, 2019) . By contrast, only a handful of papers are available on AR in the whole funeral industry, spanning several compartments, including autopsy, thanatopraxy and cemeteries (Carstens, 2012; Carstens et al., 2014; Abia et al., 2018 Abia et al., , 2019 . Yet similar to AR in the funeral industry, quantitative data directly linked the occurrence of AR in wastewaters and drinking water to human health risks are scarce. This suggests that, the lack of evidence linking AR in the funeral industry cannot be the key reason for the lack of research on the topic. Rather, this lack of research on AR in the funeral industry could reflect an interplay of several factors, including socio-cultural factors.

J o u r n a l P r e -p r o o f 10 Socio-cultural factors, and attitudes and perceptions among both researchers and funders may also explain the limited research in the funeral industry. The fear and risk of contracting pathogenic organisms and infectious diseases may also act as deterrents to environmental researchers who often lack prior training in managing the human health risks associated with human cadavers. Research on thanatopraxy care and cemeteries may not be considered as attractive, partly because, besides the funeral service professionals, most people rarely come into contact with human cadavers. Anecdotal evidence shows that, in some socio-cultural settings particularly in Africa, strong resentment exists with respect to dealing with human cadavers, unless one is related to the deceased person. In such socio-cultural settings, any activities, including research dealing with human cadavers (e.g., in thanatopraxy care and cemeteries) is highly stigmatized, and considered a taboo that brings bad omen, including mental health problems. The current author is no exception; this manuscript was conceived about 10 years ago, but collaborators were unwilling to contribute citing socio-cultural reasons. During the same period, no research student has expressed interest to study emerging contaminants and antibiotic resistance in wastewaters and leachates from thanatopraxy care facilities and cemeteries compared to other topics. In view of the foregoing reasons, it is not surprising that literature documenting emerging contaminants and antibiotic resistance in thanatopraxy care facilities and cemeteries remain limited. It is likely that this trend will persist in the foreseeable future.

In summary, the human cadavers, autopsy, thanatopraxy and cemeteries, which form the funeral industry can be considered as a resistomes or hotspot reservoirs of antibiotic resistance and their resistance genes. Collectively, these antibiotic resistance pools or reservoirs constitute J o u r n a l P r e -p r o o f 11 the 'thanato-resistome'. 'Thanato-resistome' is derived from 'thanatos', the Greek term for death, and 'resistome'. The term antibiotic resistome or simply resistome, which is widely used in literature on antibiotic resistance, refers to the complete collective assemblage of antibiotic resistant bacteria and their resistance genes in a microbial ecosystem (Ho et al., 2020) . An antibiotic resistome acts as a reservoir that receives, harbours, and subsequently transfers antibiotic resistance. The term 'thanato-resistome' is related to 'thanatomicrobiome', an emerging term increasingly used to refer to the study of post-mortem microbial communities in human internal organs and orifices after death (Can et al., 2014; Javan et al., 2016 Javan et al., , 2017 

Human cadavers are putative hotspot reservoirs of intrinsic or induced antibiotic resistance derived from the human resistome. The human resistome consists of various subcompartments, including the gut, oral, urogenital and skin resistomes (Pal et al., 2016; Ho et al., 2020) . Evidence shows that he human resistome is a hotspot reservoirs of a broad spectrum of antibiotic resistant bacteria and their resistance genes (Pal et al., 2016; Ho et al., 2020; McInnes et al., 2020) . For example, studies show that the urogenital and oral antibiotic resistomes harbour resistance genes to macrolides and tetracyclines (Pal et al., 2016) . The human skin and airway antibiotic resistomes contained several resistance gene classes, including; those encoding for resistance to multiple drugs, tetracycline, macrolide, Amphenicol and Aminoglycoside (Pal et al., 2016) .

Several studies, including reviews, have reported antibiotic resistant bacteria and their resistance genes in both healthy and unhealthy populations (Forslund et al., 2014; Ho et al., 2020; Yokohama et al., 2020) . To date, several resistance genes have been detected, including those encoding resistance to the following antibiotics, among others: (1) macrolides (ermB, ereA), (2) aminoglycosides (strA, strB), (3) β-lactamases (bla TEM ), (4) sulfonamides (sul2), trimethoprim (dfrA14), bacitracin and tetracyclines (tetM, tetQ, tetW) (Ho et al., 2020) .

Antibiotic resistant bacteria detected in the human gut include the ESKAPE pathogens such as Escherichia coli, and Clostridium difficile (De Oliveira et al., 2020) . Accordingly, AR has been reported in faecal and body fluids specimens and from both healthy and unhealthy human beings (Yokohama et al., 2020) .

The antibiotic resistant bacteria and their resistance genes persist even after human and cell death. For example, one study showed that, extracellular and intracellular sul1, sul2, tetW and tetT antibiotic resistance genes frequently located on plasmids persisted in the environment J o u r n a l P r e -p r o o f 13 for over 20 weeks (Mao et al., 2013) . Other studies show that DNA from inactivated Enterococcus faecalis was still detectable using polymerase chain reaction one year after cell death, while the spontaneous degradation of hydrated DNA to short fragments may occur over several thousand years (Young et al., 2007) . The maximum longevity of DNA following cell death depends on spontaneous DNA decomposition caused by oxidation, hydrolysis, alkylation and UV irradiation, which depends environmental conditions such as temperature, mechanical stress and redox conditions (Young et al., 2007) . Moreover, biofilms, which occur in the human body and the environment, are well-known to promote the persistence and proliferation of ARGs by providing protection against antibiotics and other stresses (Stalder et al., 2020) . Hence, antibiotic resistant bacterial and their resistance genes derived from the human resistome are putatively carried over in human cadavers, and further propagate in the environment via various mechanisms. This notion is corroborated by autopsy studies detecting antibiotic resistance in human cadavers.

A number of autopsy studies have detected AR in specimens from human cadavers (Cohen et al., 2010; Bates et al., 2015; Liebermann et al., 2016; Burcham et al., 2019 value could be attached to understanding antibiotic resistance profiles in human cadavers. In addition, the limited data may also point to the difficulties associated with conducting autopsy studies. These challenges include; (1) strong cultural objections in some geographical and sociocultural settings, (2) a critical shortages of qualified experts to conduct such studies, and (3) under-developed and poorly equipped pathology infrastructure and services (Bates et al., 2016) .

However, recent studies suggest that analysis of post-mortem human microbiome or thanatomicrobiome provides some insights on the health conditions in living populations (Pechal et al., 2017) .

Funeral homes or thanatopraxy care facilities are potential hotspots of antibiotic resistance derived from human cadavers, and that induced through excessive use of antimicrobials. Yet very limited data exists on antibiotic resistance in funeral homes or thanatopraxy care facilities. The few studies available report extended-spectrum beta-lactams (ESBL), and multi-drug resistant hepatis virus, human immunodeficiency virus (HIV), tuberculosis and pathogenic fungi (Demiryurek et al., 2002; Davidson and Benjamin, 2006) .

Helicobacter and Enterobacteriaceae have also been detected in human cadavers in thanatopraxy care facilities (Davidson and Benjamin, 2006; Serefhanoglu et al., 2009 ).

One study conducted in an anatomy laboratory determined pathogens on laboratory coats before and after the dissection of human cadavers until the gastrointestinal tract was exposed (Kabadi et al., 2013) . Results showed that laboratory coats of 19 out of 67 students (i.e., 28.4%) had S. aureus, 8 (11.9%) had S. pyogenes, while 4 (6.0%) had E. faecalis. These values were significantly higher than those observed for S. aureus (13 of 67 students: 19.4%), S. pyogenes (5 out of 67: 7.46%) and E. faecalis (none) in the pre-dissection swab. However, Kabadi and coworkers (2013) did not test the isolates to assess their antimicrobial resistance profiles. The few available studies suggest that antibiotic resistant bacteria, including those causing human infections occur in the thanatopraxy care. However, there is a paucity of data on the occurrence and nature of antibiotic resistance in solid wastes, wastewaters and air-borne particulates in thanatopraxy care facilities.

Putrefied human cadavers can release leachate containing bacteria, viruses, and organic and inorganic contaminants, which may enter groundwater systems via seepage (Ucisik and Rushbrook, 1998) . The transport of contaminants into groundwater systems via seepage depends of various factors, including geology, soil texture, depth to groundwater and existence of preferential flow pathways (Ucisik and Rushbrook, 1998) . The risk of groundwater contamination is high on sandy and gravely soils with shallow groundwater systems such as wetlands. There are suggestions to consider cemeteries as a special type of landfill based on leachate chemistry (Dent and Knight, 1998; Fiedler et al., 2012) . This is problematic because J o u r n a l P r e -p r o o f Abia et al. (2018) also classified those isolates that were resistant to three or more antibiotic classes as multi-drug resistant. The results showed that 86 out of 87 isolates, equivalent to about 98.9% were resistant to at least two antibiotics. Only one isolate (i.e., 1.1%) was resistant to a single antibiotic Ceftolozane/Tazobactam (C/T), which is a cephalosporinlactamase inhibitor (Table 1) . A total of 72 out of the 87 isolates (i.e., 82.8%) were classified as multi-drug resistant, while 22 out of 87 (i.e., 25.3%) were resistant to four different antibiotics. Table 1 shows that a total of four isolates were resistant to all eight antibiotics tested by Abia et al. (2018) . The most abundant multi-drug resistant phenotypic group belonged to S-T-TM-CIP with 13 isolates, and 4 of them were from the borehole water samples (Abia et al., 2018) . The remaining four resistant isolates from the borehole water samples were two each presenting S-T and S-T-TM-CIP-C/T phenotypes. In addition, 41 out of 87 isolates (i.e., 47.1%) tested positive for at least one of the virulence genes, while 36 out of 87 isolates were pathogenic. However, both studies by Abia et al. (2018 Abia et al. ( , 2019 were limited to water samples from surface water bodies and boreholes in cemeteries, but did not include a control samples from outside the cemeteries.

Hence, the degree of enrichment of antibiotic resistance due to cemeteries relative to background values could not be estimated.

In another study conducted on cemeteries in Cape Town and Mpumalanga (South Africa), Abia et al (2019) investigated bacterial diversity and function in cemetery soils at the surface (0 cm) and below the depth of burial (2 m) using 6S rRNA-based metagenomic analysis.

Significant differences were observed between the two depths, with one cluster of surface samples dominated by Prauserella and Staphylococcus, while Pseudomonas and Rhodococcus were dominant in the 2-m depth cluster. The 2- m depth had a lower alpha diversity, but higher abundances of human diseases functional groups than surface samples (Abia et al., 2019b) . The J o u r n a l P r e -p r o o f human disease functional profiles were associated with the following; (1) cancers, (2) cardiovascular diseases (e.g., hypertrophic cardiomyopathy), (3) immune system diseases (e.g., systemic lupus erythematosus), (4) infectious diseases (e.g., cholera, tuberculosis), (5) metabolic diseases (e.g., Type I and II diabetes), and (6) neurodegenerative diseases (e.g., Alzheimer's disease). The infectious human disease signatures (e.g., for cholera) were the most prevalent (Abia et al., 2019b) .

In an independent study conducted in aquifers in two catchments in the North Western province in South Africa, six isolates of Pseudomonas, Leifsonia, Comamonas, and

Brevundimonas spp from surface and groundwater samples were tested for resistance to eight antibiotics (Carstens, 2012; Carstens et al., 2014) . In a study using Bacillus subtilis and E. coli as tester strains, Lesly and Scott (2020) identified antibiotic-producing isolates in soils collected near cemeteries in Illinois. The study by

Lesly and Scott (2020) did not investigate antibiotic resistance profile and identify the specific antibiotic-producing bacterial strains. The occurrence of antibiotics points to the possible existence of antibiotic resistant bacterial strains in such soils. Moreover, several studies have detected inorganic and microbiological contaminants of anthropogenic origin in groundwater from cemeteries (Rodrigues and Pacheco, 2003; Ucisik and Rushbrook, 1998; Oliveira et al., 2013) . Although these studies did not investigate antibiotic resistance, a possibility exists for the co-occurrence and co-transport of such microbial contaminants harbouring antimicrobial resistance.

Overall, existing evidence demonstrates that antimicrobial resistance occurs in environmental media impacted by cemeteries (Carstens, 2012; Carstens et al., 2014; Abia et al., 2018 Abia et al., , 2019 , suggesting that virulent antimicrobial resistant pathogens may lurk in the subsurface in cemeteries following the burial of the human cadaver for a longer period than often expected, thereby posing human health risks for years to come. The few available studies highlighted here provide only a snapshot on antibiotic resistance in cemeteries, highlighting the need for further research. It is well-established that, antibiotic resistant bacteria co-exist with their corresponding resistance genes (Tenover, 2006; Gillings, 2014; Gullberg, 2014) . Intuitively, one expects that, the resistance genes corresponding to the antibiotic resistant phenotypes detected in literature also exist. Based on the available evidence on cemeteries (Abia et al., 2018 (Abia et al., . 2019 Carstens, 2012; Carstens et al., 2014) and thanatopraxy care facilities (Sammit et al., 2019) the following antibiotic resistance genes may occur, among others: (1) Methicillin resistance (mec) genes (e.g., mecA), (2) Tetracycline resistance (tet) genes (e.g., tetC, tetA) and beta-lactamase genes (e.g., blaTEM and blaCTX-M). These inferences are corroborated by data from studies conducted in health care facilities showing the prevalence of these resistance genes (Klingenberg et al., 2001; Boyd et al., 2004; Mulvey et al., 2011) . However, further research focusing on solid wastes, wastewaters, air-borne particulates and aerosols is required to substantiate these inferences.

Hydrological and wind-driven processes control the mobilization and dissemination of antibiotic resistance Lunenberg et al., 2018) . Specifically, wastewater discharges, surface and sub-surface runoff, infiltration, leachates and groundwater recharge mobilize and disseminate antibiotic resistance from various hotspot reservoirs into other environmental compartments, including aquatic systems (Figure 1 ). In addition, surface watergroundwater interactions via baseflow and interflow allow the exchange of contaminants between surface aquatic systems and groundwater systems. Aerosols, and air-borne particulate such as dust have been reported to transmit antibiotic resistance (Davidson and Benjamin, 2006) .

In soils, sediments and groundwater systems, antibiotic resistant bacteria may undergo adsorption on solid matrix (Ucisik and Rushbrook, 1998; Wang et al., 2018) .

Insects, rodents, beetles and houseflies are attracted to human cadavers ( Davidson and Benjamin, 2006) . Seroconverison refers to the period when the immune system starts responding to HIV by producing detectable levels of antibodies. Although bacteria harbouring antibiotic resistance will ultimately die, antibiotic resistance persists and continue circulating in the various resistomes (Pinto et al., 2010; Vittecoq et al., 2016) . This persistence and continuous circulation is driven by horizontal gene transfer mediated by mobile gene elements and various dissemination processes (Figure 1 ).

Horizontal gene transfer mediated by mobile genetic elements disseminate antibiotic resistance into various resistance pools often known as resistomes (Gillings, 2014; Gullberg, 2014) . These mobile genetic elements include; transposons, plasmids, insertion sequences, integrative and conjugative elements, integrons and prophages and gene cassettes (Gillings, 2014 (Gillings, , 2016 . In horizontal gene transfer, the term mobilome is often used to refer to mobile genetic elements and their cargo resistance genes (Gillings, 2014; Gullberg, 2014) . Mobilomes transfer antibiotic resistance within and between bacterial species, and even to different species (von Winterdorff et al., 2016) . The role of the mobile genetic elements in horizontal gene transfer are discussed elsewhere (Joy et al., 2013; Gillings, 2014) .

Horizontal gene transfer implies that antibiotic resistance in the gut system of human cadavers (i.e., human cadaver resistome) and cemeteries can be transferred into other resistomes.

These resistomes include previously susceptible microorganisms in terrestrial and aquatic ecosystems, food animals, wastewaters, vectors, ambient air, air-borne particulates and even wildlife ( Figure 1 ). Other processes facilitating the dissemination of antibiotic resistance include human interactions and movements, vectors and globalization (Osborn and Bolter, 2002) . The continuous circulation of antibiotic resistance among various resistomes via horizontal gene J o u r n a l P r e -p r o o f 24 transfer and other dissemination processes results in a complex network of antibiotic resistance and resistomes (Figure 1 ). These complex interactions imply that the health risks associated with antibiotic resistance in the 'thanato-resistome' are not restricted to such environments. Instead, the antibiotic resistance is widely disseminated into other resistomes, hence posing wider health risks. Hence, addressing antibiotic resistance and its health risks calls for a 'one health'

approach. The 'one health' approach is an integrative framework that considers the interactions of antibiotic resistance in the environment, livestock, wildlife and humans, and even food safety (Institute of Medicine, 2012).

Antibiotic resistance is ubiquitous in both natural and human-impacted environmental resistomes. Further, the continuous exchange of antibiotic resistance among resistomes via horizontal gene transfer and anthropogenic processes adds another layer of complexity to its behaviour ( Figure 1 ). The partitioning of antibiotic resistance in the 'thanato-resistome' among the various reservoirs covering mortuaries, funeral homes and cemeteries is a daunting task.

Thus, besides literature documenting the occurrence and characterization of antibiotic resistance (Davidson and Benjamin, 2006) , evidence tracing antibiotic resistance to specific sources is scarce. This reflects the lack of systematic studies profiling antibiotic resistance in solid wastes, wastewaters, air-borne particulates, ambient air and even various occupational groups. In fact, most of the studies, including case reports lack control groups, robust statistical experiment design, and subsequent data analysis (e.g., Nyberg et al., 1990; Johnson et al., 1997; Sterling et al., 2000; Lauzardo et al., 2001) . The lack of evidence tracking the source of antibiotic resistance is also evident in studies focusing on cemeteries (Abia et al., 2018 (Abia et al., , 2019 Carstens, 2012; Carstens et al., 2014) . These studies focused on occurrence and characterization of antibiotic resistance profiles of isolates using antibiotic susceptibility tests, but the origins remain unclear. Possible sources of antibiotic resistance in cemeteries include: (1) residual antibiotic resistance in human cadavers and its subsequent dissemination via horizontal gene transfer, (2) can be directly induced by residual antibiotic s in human cadavers (Paíga and Delerue-Matos, 2016; Fiedler et al., 2017) and those biosynthesized in-situ (Abia et al., 2018) , and (3) induced by inorganic and organic contaminants in leachates from human cadavers such as nutrients and metals, which are well-known to coselect for antibiotic resistance (Zhao et al., 2017) . Nutrients and trace metals also promote the growth and proliferation of antibiotic resistant bacteria. Further research is required to determine the exact sources of antibiotic resistance detected in environmental samples from cemeteries.

Such research may entail the validation and application of emerging tools such as genomics, in silico techniques and network analysis to establish the sources and relationships among antibiotic resistances in various environmental media. High-risk workers in the funeral industry include autopsy pathologists, medical researchers and students, assistants and cleaners, and those involved in body bagging, washing, embalming and dressing of human cadavers. Other workers at risk include funeral directors, undertakers, grave diggers, funeral attendants, gardeners and researchers (Abia et al., 2018) . Data show that cemeteries contaminate surface and groundwater systems with antibiotic resistance (Carstens, 2012; Carstens et al., 2014; Abia et al., 2018; . Hence, non-occupational exposure may occur via ingestion of contaminated drinking water and aquatic foods, inhalation and dermal contact.

Occupational exposure to antibiotic resistance is not limited to the funeral industry, but may also occur in health care facilities, livestock production, and wastewater industry, among others. However, comparative data on the human exposure risks in the funeral industry to that of health care system and other industries are unavailable. Such data are critical for prioritizing research, surveillance systems and mitigation measures. Thus, quantitative health risk assessments comparing occupational exposure and health risks are required to address this gap.

Such information will provide direct evidence on whether the health risks in the funeral industry are higher or lower than those in other industries. Once such evidence is available, the scientific community, policy makers and the public will avoid relying on speculations and perceptions.

The global burden of antibiotic resistance from the funeral industry is currently not known with certainty. However, the United Nations (2019) estimate that more than 58.4 million human deaths occurred in 2019 alone, while more than 11 million human deaths occur due to parasitic and infectious diseases (WHO, 2002) . The total global deaths are expected to be higher J o u r n a l P r e -p r o o f 27 than this estimate because several human deaths in developing countries are often unreported.

The number could be particularly higher during periods of global pandemics such as COVID-19.

Existing global data on human deaths are not disaggregated by funeral practices, such as embalming, cremation and burial in cemeteries. However, data from the National Funeral Directors Association (NFDA, USA) show that, in the USA, approximately 2.5 million people were embalmed in 2003 (Green, 2003; Chiappeli and Chiappeli, 2008) . Thus, the global antibiotic resistance burden from the funeral industry, and the corresponding number of people exposed to antibiotic resistance could be considerably higher than currently perceived. For example, globally, it is estimated that antibiotic resistance will account for 10 million deaths per

year by 2050 (de Kraker et al., 2016) . Thus, further research is required to provide quantitative estimates of the global burden of antibiotic from the funeral industry.

To date, no compelling quantitative evidence exists directly linking human exposure to antibiotic resistance in the 'thanato-resistome' to specific human health risks. Moreover, no global estimates exist on health risks and antibiotic resistance acquired via occupational and nonoccupational exposure in the funeral industry. However, inferential evidence shows that human exposure to antibiotic resistance may induce resistance to single and multiple drugs in human pathogens (Tenover, 2006; Gillings, 2014 . Hence, the relative contribution of the various pathways to human exposure remains unclear. The human exposure and health risks associated with antibiotic resistance in the funeral industry could be particularly high in developing countries due to the following factors among others; (1) weak and poorly enforced environmental regulations, regulatory agencies lacking expertise and resources, and severe environmental pollution (K'oreje et al., 2020) , and (2) over-reliance on untreated drinking water from unprotected water sources such surface water and shallow wells (Potgieter et al., 2020) .

As more evidence on antibiotic resistance in the funeral industry becomes available, scope exists for comprehensive reviews focussing on specific aspects, including the occurrence, behaviour, fate, and human exposure and health risks. Such systematic reviews based on bibliometric and meta-analytic analyses could provide further insights into the human exposure and health risks, which are currently not evident given the limited evidence base.

Risk assessment is a critical first step in health risk mitigation, and it involves hazard characterization and evaluation (Gwenzi and Chaukura, 2018; US EPA, 2020) . The first step seeks to identify and characterize the nature of the hazards associated with various tasks involved in thanatopraxy care and cemeteries. In the current case, this involved the determination of the nature and occurrence of antibiotic resistance along the thanatopraxy care-cemetery pathway. In addition, the human population at risk (e.g., cleaners, assistants), exposure pathways (e.g., inhalation, dermal intake), and the amount of the various contaminants taken by via the various intake routes are determined.

The second step is risk evaluation, involving a qualitative or quantitative estimation of the likelihood or probability of occurrence of a health hazard, and the human and ecological consequences of the hazard if it occurs (US EPA, 2020). In qualitative risk assessment, the likelihood of occurrence and consequences, and the overall health risk can be ranked qualitatively as 'extremely high', 'high', 'moderate' and 'low/negligible'. In quantitative risk assessment, hazard characterization data are used to calculate a risk quotient or its variants (US EPA, 2020). In both qualitative and quantitative risk assessment, the resulting overall risk is compared to a set threshold conditions which is considered to be acceptable.

A key challenge in health risk assessment for antibiotic resistance is that the minimum threshold concentrations required to trigger adverse ecological and human health risks are not yet established. A heuristic approach could entail using data for non-impacted environments (control) as the baseline or acceptable value. In cases where ecotoxicological data exist for specific types of antibiotic resistance, threshold values such as the no observable effect concentrations (NOEC) can be used. Ultimately, the resulting health risk is used to identify health risks that require mitigation, and for prioritization of allocation of resources. The overall goal of risk assessment is to ensure that the level of risk in the funeral industry is understood so that unacceptable health risks are mitigated.

The 'Hard' engineering solutions include the removal of antibiotic resistance in aqueous systems through the application of conventional and advanced water and wastewater treatment methods . Conventional water treatment methods such as filtration have limited capacity to remove antibiotic resistance, while chlorination forms carcinogenic disinfection by-products (Hiller et al. 2019; Sanganyado and Gwenzi, 2019; Smyth et al., 2020) . Thus, advanced oxidation methods with high removal efficiencies are often used. (Michael et al., 2020; Zhou et al., 2020) . including those in biosand filters may act as reservoirs of antibiotic resistance, and promote its proliferation and persistence (Balcázar et al., 2015; Bleich et al., 2015) . Hence, further research is required to better understand the removal capacity and fate of antibiotic resistance such biofilms in biosand filters. (Nwaiwu and Aduba, 2020; Valli et al., 2020) . Omics tools are ideal for understanding complex interactions such as metabolic networks and ecosystems functions such as pathogen-host and trophic interactions (Valli et al., 2020) . Moreover, metagenomics are also critical for investigating several unculturable pathogens including antibiotic resistant bacteria and their metabolic functions and networks (Bodor et al., 2020; Gan et al., 2020; Perdigão et al., 2020) . Other recent advances include computed X-ray tomography for non-invasive 2-D and 3-D imaging of the internal structure of complex matrices (Mehrian et al., 2020) . The integration of conventional research approaches in as ecotoxicology, epidemiology and health risk assessment, and emerging tools could improve our understanding of the health risks of antibiotic resistance.

The integration of conventional and emerging tools in data acquisition will potentially generate large quantities of data on: (1) nature and concentrations of antibiotic resistant bacteria and their resistance genes in various environmental media, (2) ecotoxicology, and ecological health effects, and (3) human toxicology and epidemiology. The analysis, synthesis and visualization of such dataset using conventional statistical tools is a non-trivial task. Big data analytical tools such as machine learning, artificial intelligence and artificial neutral networks are ideal for that purpose (Hyun et al., 2020; Kim et al., 2020) . Moreover, in silico techniques or J o u r n a l P r e -p r o o f 35 computational (eco)toxicology and epidemiology and network analysis can be used for analysis, integration and synthesis of large dataset from various sources (Vuorinen et al., 2013; Raies and Bajic, 2016) . Together, the application of these tools is critical for addressing the knowledge gaps highlighted and advance our understanding of the health risks of antibiotic resistance.

The current review investigated the occurrence, behaviour and health risks associated with antimicrobial resistance in the 'thanato-resistome' consisting of human cadavers, autopsy, thanatopraxy and cemeteries. Evidence shows that the 'thanato-resistome' is a hotspot reservoir of multi-drug resistant bacteria and their resistance genes. The resistance genes conferred resistance to several common and last-resort multiple drugs, including Methicillin and betalactams. Hydrological processes, air-borne particulates, and rodents and insects harbouring antibiotic resistance disseminate antibiotic resistance. Horizontal gene transfer mediated by mobile genetic elements promote the persistence, and dissemination of antibiotic resistance into various resistomes, culminating into a complex network. Human exposure in occupational settings occurs via the oral route, inhalation and dermal intake via wounds and cuts in thanatopraxy care facilities and cemeteries. Non-occupational exposure occurs via ingestion of contaminated water and food, inhalation of air-borne particulates and clothes of occupational workers. However, currently, there is no compelling quantitative evidence directly linking antibiotic resistance in the 'thanato-resistome' to specific human health effects. The lack of evidence does not necessarily indicate lack of health risks. Rather, it reflects the lack of quantitative estimation of health risks caused by the corresponding lack of quantitative data partitioning antibiotic resistance among sources and exposure pathways. Thus, based on inferential evidence, human exposure to antibiotic resistance via the various intake pathways J o u r n a l P r e -p r o o f 36 may induce the development of antibiotic resistance against common and last-resort therapeutic drugs. This in turn, increases the pathogenicity and virulence of multi-drug resistant pathogens, leading to potential outbreaks of human infections. Potential ecological health risks include proliferation of resistant organisms at the expense of susceptible ones. This may in turn, disrupts ecosystem structure and function, including biogeochemical cycles. A framework for assessing and mitigating the health risks of antibiotic resistance in the 'thanato-resistome' was presented.

Ten hypotheses/propositions were formulated to guide future research, and opportunities offered by emerging tools such as metagenomics, in silico techniques and big data analytics were highlighted. 

Where did they come from-Multi-drug resistant pathogenic Escherichia coli in a cemetery environment?

Detection of antibiotic-resistant bacteria and their resistance genes from houseflies

Hospital waste management in developing countries: A mini review

Insect species colonized indoor and outdoor human corpses in Riyadh, Saudi Arabia

A case of flying insects visiting an anatomy and embalming laboratory in Medellín, Colombia

Evolution of antibiotic resistance at non-lethal drug concentrations

Seasonal characterization of municipal wastewater and performance evaluation of a constructed wetland system in Addis Ababa

Environmental impacts of an advanced oxidation process as tertiary treatment in a wastewater treatment plant

The role of biofilms as environmental reservoirs of antibiotic resistance

Burden of tuberculosis at post mortem in inpatients at a tertiary referral centre in sub-Saharan Africa: a prospective descriptive autopsy study. The Lancet infectious diseases

Bypass and hyperbole in soil research: A personal view on plausible causes and possible remedies

Bypass and hyperbole in soil research: Worrisome practices critically reviewed through examples

Thiopeptide antibiotics stimulate biofilm formation in Bacillus subtilis

Challenges of unculturable bacteria: environmental perspectives

Complete nucleotide sequence of a 92-kilobase plasmid harboring the CTX-M-15 extended-spectrum beta-lactamase involved in an outbreak in long-term-care facilities in Toronto

Detection of critical antibiotic resistance genes through routine microbiome surveillance

Distinctive thanatomicrobiome signatures found in the blood and internal organs of humans

Evaluation of microbiological and physico-chemical quality of water from aquifers in the North West Province, South Africa. MSc thesis

Antibiotic-resistant heterotrophic plate count bacteria and amoeba-resistant bacteria in aquifers of the Mooi River

Risk assessment for antibiotic resistance in South East Asia

Drinking Grandma: The Problem of Embalming

The prevalence and drug sensitivity of tuberculosis among patients dying in hospital in KwaZulu-Natal, South Africa: a postmortem study

Natural disasters, corpses and the risk of infectious diseases

Infection risks and embalming. Institute of Occupational Medicine

The Matthew Effect and widely prescribed pharmaceuticals lacking environmental monitoring: Case study of an exposure-assessment vulnerability

Risk of infection and tracking of work-related infectious diseases in the funeral industry

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Infective agents in fixed human cadavers: a brief review and suggested guidelines

Cemeteries: A special kind of landfill. The context of their sustainable management. Groundwater: Sustainable Solutions

Antimicrobial Resistance in ESKAPE Pathogens

The increase in serious staphylococcal infections as shown by post-mortem investigation

Towards a multi-faceted vadose zone assessment protocol: cemetery guidelines and application to a burial site located near a seasonal wetland

Long-lasting postmortem viability of human immunodeficiency virus: a potential risk in forensic medicine practice

Graveyards-special landfills

Do cemeteries emit drugs? A case study from southern Germany

Metagenomic insights into the human gut resistome and the forces that shape it

Methicillin-resistant Staphylococcus aureus disease in three communities

First genomic insights into carbapenem-resistant Klebsiella pneumoniae from Malaysia

Aerial dispersal of meticillin-resistant Staphylococcus aureus in hospital rooms by infected or colonised patients

Tuberculosis risk in funeral home employees

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Lateral gene transfer, bacterial genome evolution, and the Anthropocene

Antibiotic resistant enterococci and staphylococci isolated from flies collected near confined poultry feeding operations

Worry-free wastewater. The Director

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Sources, behaviour and health risks of antimicrobial resistance genes in wastewaters: A hotspot reservoir

Organic contaminants in African aquatic systems: current knowledge, health risks, and future research directions

Recurrent Cholera Outbreaks in Sub-Saharan Africa: Moving beyond Epidemiology to Understand the Environmental Reservoirs and Drivers

The infection hazards of human cadavers

Antibiotic microbial resistance (AMR) removal efficiencies by conventional and advanced wastewater treatment processes: A review

Final report on literature search on the infectious nature of dead bodies for the Embalming Chemical Manufacturers Association. Embalming Chemical Manufacturers Association

Systematic review of human gut resistome studies revealed variable definitions and approaches

Horizontal gene transfer in the human gastrointestinal tract: potential spread of antibiotic resistance genes. Infection and drug resistance

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Improving food safety through a One Health approach

Human thanatomicrobiome succession and time since death

Cadaver thanatomicrobiome signatures: the ubiquitous nature of Clostridium species in human decomposition

Autopsy risk and acquisition of human immunodeficiency virus infection: a case report and reappraisal

Fate and transport of antimicrobials and antimicrobial resistance genes in soil and runoff following land application of swine manure slurry

Potential pathogen transmission on medical student anatomy laboratory clothing

VAMPr: VAriant Mapping and Prediction of antibiotic resistance via explainable features and machine learning

Rapid PCR detection of the methicillin resistance gene, mecA, on the hands of medical and non-medical personnel and healthy children and on surfaces in a neonatal intensive care unit

Relationship between antibiotic resistance genes and metals in residential soil samples from Western Australia

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Methicillinresistant-Staphylococcus aureus hospitalizations, United States

Transmission of Mycobacterium tuberculosis to a funeral director during routine embalming

Identification of uncommon antibiotic-producing Illinois soil isolates. In: Identifying and characterizing novel antibiotic producing microbes from the soil

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

Occupational health epidemiology in Africa: Drought on fertile grounds

Methicillin and vacomycin resistant S. aureus in hospitalized patients

Water flow paths are hotspots for the dissemination of antibiotic resistance in soil

New insights into antibiotic resistome in drinking water and management perspectives: a metagenomic based study of small-sized microbes

A waterborne outbreak of Shigella sonnei with resistance to azithromycin and third-generation cephalosporins in China in 2015

Considerations and caveats in combating eskape pathogens against nosocomial infections

Infective endocarditis caused by multidrug-resistant Streptococcus mitis in a combined immunocompromised patient: an autopsy case report

Horizontal transfer of antibiotic resistance genes in the human gut microbiome

Computerised tomography scan in multi-drug-resistant versus extensively drug-resistant tuberculosis

The ecological risks of antibiotic resistance in aquatic environments: a literature review

Investigating the impact of UV-C/H 2 O 2 and sunlight/H 2 O 2 on the removal of antibiotics, antibiotic resistance determinants and toxicity present in urban wastewater

Beta-lactamases in Enterobacteriaceae-an ever-present threat

Infectious disease risks from dead bodies following natural disasters

New Delhi metallo-βlactamase in Klebsiella pneumoniae and Escherichia coli

An in silico analysis of acquired antimicrobial resistance genes in Aeromonas plasmids

Isolation of human immunodeficiency virus (HIV) at autopsy one to six days postmortem

Burial grounds' impact on groundwater and public health: an overview

When phage, plasmids, and transposons collide: genomic islands, and conjugative-and mobilizable-transposons as a mosaic continuum

Determination of pharmaceuticals in groundwater collected in five cemeteries' areas (Portugal)

Isolation and identification of amoeba-resisting bacteria from water in human environment by using Acanthamoeba polyphaga coculture procedure

The structure and diversity of human

A large-scale survey of the postmortem human microbiome, and its potential to provide insight into the living health condition

Using genomics to understand the origin and dispersion of multidrug and extensively drug resistant tuberculosis in Portugal

Genetic detection of extended-spectrum β-lactamases Escherichia coli in birds of prey from Serra da Estrela Natural Reserve of Portugal

Human Enteric Pathogens in Eight Rivers Used as Rural Household Drinking Water Sources in the Northern Region of South Africa

In silico toxicology: computational methods for the prediction of chemical toxicity

An observational study of safe and risky practices in funeral homes in South Africa

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

Groundwater contamination from cemeteries cases of study

Antibiotic resistance in drinking water systems: Occurrence, removal, and human health risks

Bloodstream infections caused by ESBL-producing E. coli and K. pneumoniae: risk factors for multidrug-resistance

Antibiotic resistant and extended-spectrum β-lactamase producing faecal coliforms in wastewater treatment plant effluent

Concentration of Heavy Metals in Soils under Cemetery Occupation

Soil and Sediment Contamination

Antimicrobial resistant enteropathogenic Escherichia coli and Salmonella spp. in houseflies infesting fish in food markets in Zambia

Inorganic soils contamination from cemetery leachate

Genomic comparison of multi-drug resistant invasive and colonizing Acinetobacter baumannii isolated from diverse human body sites reveals genomic plasticity

Evolving populations in biofilms contain more persistent plasmids

Transmission of Mycobacterium tuberculosis from a cadaver to an embalmer

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

Mechanisms of antimicrobial resistance in bacteria

The impact of cemeteries on the environment and public health: an introductory briefing

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Groundwater contamination sourced from the main cemetery of Tabriz

There is no hiding if you Seq: recent breakthroughs in Pseudomonas aeruginosa research revealed by genomic and transcriptomic next-generation sequencing

Toward safer thanatopraxy cares: formaldehyde-releasers use

A laboratory study of the leachate composition of selected metals in cemeteries (South Africa)

Antimicrobial resistance in wildlife

Dissemination of antimicrobial resistance in microbial ecosystems through horizontal gene transfer

In silico methods in the discovery of endocrine disrupting chemicals

Sulfadiazine/ciprofloxacin promote opportunistic pathogens occurrence in bulk water of drinking water distribution systems

Horizontal transfer of antibiotic resistance genes on abiotic touch surfaces: implications for public health

Burden of disease estimates by region in 2002

The effect and mechanism of urban fine particulate matter (PM2. 5) on horizontal transfer of plasmid-mediated antimicrobial resistance genes

Using metagenomics to investigate the impact of hospital stay and the ARK intervention on the human gut resistome

Bacterial DNA persists for extended periods after cell death

Methicillin resistant S. Aureus in autopsy cases

Nutrients, heavy metals and microbial communities co-driven distribution of antibiotic resistance genes in adjacent environment of mariculture

Removal of antibiotic resistant bacteria and antibiotic resistance genes in wastewater effluent by UV-activated persulfate

Impact of cemeteries on groundwater chemistry: A Review

Impact of cemeteries on groundwater contamination by bacteria and viruses -a review

I thank the two anonymous reviewers whose comments greatly improved the manuscript.I am also grateful to two other anonymous reviewers who made comments on an earlier version of the manuscript.