Water bodies are potential hub for spatio-allotment of cell-free nucleic acid and pandemic: a pentadecadal (1969–2021) critical review on particulate cell-free DNA reservoirs in water nexus

In recent times, there had been report of diverse particulate nucleic acid-related infections and diseases which have been associated with endemic, sporadic, and pandemic reports spreading within water nexus. Some of such disease cases were seldom reported in earlier years of technological advancement and research based knowledge-scape. Although the usefulness of water, wastewater treatment systems, water regulatory organizations and water re-use policy in compliant regions remains sacrosanct, it has been implicated in diverse gene distribution. A cosmopolitan bibliometric and critical assessment of cell-free DNA reservoir in water bodies was determined. This is done by analysing retrieved pentadecadal scientific publications in Scopus and Pubmed centre database, determining the twelve-monthly publication rates of related articles, and a content-review assessment of cell-free nucleic acids (cfNAs) in water environment. Our results revealed thirty-eight metric documents with sources as journals and books that conform to the inclusion criteria. The average reports/publication rate per year shows 16.7, while several single and collaborating authors are included with a collaboration index of 4.31. A zero average citation per document and citation per year indicate poor research interest and awareness. It is important to note that a redirected interest to studies on cfNAs in water environments would encourage advancement of water treatment strategies to include specific approaches on the removal of cfNAs, membrane vesicles or DNA reservoirs, plasmids or extra-chromosomal DNA and other exogenous nucleic acids from water bodies. It may also lead to a generational development/improvement of water treatment strategies for the removals of cfNAs and its members from water bodies.

chemical agents, bacteria, virus, protozoan, fungus and plankton of all kinds in wastewater release or effluents (Igere et al. 2021;Aminov 2011). Such treated/filtered and untreated water specimens have also revealed presence of particulate cell-free nucleic acids (PCFNAs) including plasmids or extra-chromosomal DNA, exogenous DNA (exDNA), phage and membrane vesicles (Abe et al. 2020;Woegerbauer et al. 2020). With the increasing reports of pandemics associated with PNAs from diverse surveillance data, the call to source track the origin of such PNA-borne infectious agents becomes eminent. PcfNAs are seen to have risen from incomplete chemical (chemical agents and antibiotics) breakdown of cellular components (organism) and worn-out tissues which gain their presence into water nexus via waste release (Woegerbauer et al. 2020;Toyofuku et al. 2019Toyofuku et al. , 2017. These cell-free nucleic acid (cfNA)-based components have been linked with various health depleting concerns including resistance to antibiotics, evolving strains and virulence determinants which are probably shared via horizontal gene transfer (HGT) (Abe et al. 2020;Aminov 2011). Over the years, one notable area, where such cfNA has been reported in the literatures with relevant implications, is antibiotic resistant genes (ARGs) (Igere et al. 2020;Hashiguchi et al. 2019). These non-cellular nucleic acid materials which float in natural water nexus (particle) are become increasingly implicated in PNA related diseases which are expanding across multiple countries with varying severity amongst population of bacteria, viruses, protozoan, etc. (Woegerbauer et al. 2020;Hong et al. 2018). Such cfNA components, although in particulate state, are been acquired by most environmental organism which instil in them the capacity to survive harsh conditions of their respective environments. Investigators of nucleic acid in water bodies have frequently reported exchange of genes via HGT using microbial genetic cassette including mobile genetic elements, biofilms, vesicles mediated DNA, phage, transposon, cell-free/released DNA and other mobile integrative and conjugative elements (Abe et al. 2020;Woegerbauer et al. 2020;Calero-Caceres et al. 2019;Partridge et al. 2018;Carattoli 2013;Wozniak and Waldor 2010). It is important to note that such genetic components reside in regions that are not necessarily involved in genetic mobility (Partridge et al. 2018), but to elicit health-related concerns (Woegerbauer et al. 2020;Norton et al. 2013). As a matter of verity, there is a non-negligible quantity of nucleic acids carried as cell-free deoxyribonucleic acids (cfDNAs) in the form of extra-chromosomal DNA or plasmids, exDNA, phage, and membrane vesicle DNA which are probably been involve in disease situation or emergence of disease cases (Woegerbauer et al. 2020). The question of mechanism of sharing of exogenous nucleic acids in water bodies is another aspect of interest, which has arouse questions as water reuse policy and the fate of cfNAs impact other lives in both water bodies and environment. Although much attention has not been given to potential alternative mechanism of exogenous nucleic acid acquisition in water bodies, it has encouraged difficulties in estimating their true implications. In spite of these aforementioned cases of cfNAs in water bodies, studies on its dissemination has received poor research-based attention as well as its potential health associated risk or concern.
Aims It is to this end our study determines a critical assessment of cfNAs in water bodies as potential hub for emergence of PNA diseases and microbes with a view to appraising related studies and arousing interest on the removal of cfNAs from water bodies.

Reporting and protocol registration
This investigation applied the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) protocols (Moher et al. 2009) which is submitted to the International Prospective Register of Systematic Reviews (PROSPERO). The study retrieved various reports of cellfree nucleic acids, membrane vesicles or MV, exogenous DNA and extra-chromosomal DNA in water bodies.

Search strategy
In PubMed and Scopus databases, the search phrases (Wastewater AND Cell free nucleic acid OR extra-chromosomal DNA OR exogenous DNA OR membrane vesicles) were used to find datasets. Most often, various journals publish studies that were not extensively described and conducted. To eliminate such concerns, IBE and OH applied the Scopus and PubMed database after adequate consultations with UUN. The application of Scopus and PubMed database as source retrieval database was associated with verity of information, datasets with reputable study relevance and the public health relevance of studies in the specified database. All downloaded information was documents that conform with the condition for retrieval and inclusion such as research articles, editor letter, articles proceedings, and abstract review articles, whereas documents such as book chapters, book reviews news documents, opinions, and adverts were not included since they are not constituents of primary sources. In addition, the content search documents were also retrieved as PDF documents on articles titles and abstract focused on exDNA, cfNAs, membrane vesicles, extra-chromosomal DNA, wastewater effluent and water nexus. Authors collaboration, countries of study, countries collaboration and collaboration index of studies on cfNAs components in wastewater and water nexus were also accessed.

Main text Inclusion and exclusion criteria
Only articles that contain any of the search term or word(s); (Wastewater AND Cell free nucleic acid OR extra-chromosomal DNA OR exogenous DNA OR membrane vesicles) in the title/abstract were retrieved from the databases for a period of January 1969 to July 2021. Duplicate documents were also removed, while author's articles and other nonconforming documents to the applied inclusion criteria were not selected.

Data analysis
Prior to data analysis, authors keywords, names, spelling errors and the appropriate Boolean were employed to extract relevant documents, and data were also normalised by IBE and OH. The datasets were saved in CSV format, combined in the excel file, and duplicate were removed. The clustered metric networks studied were built using a VOSviewer 1.6.13 optimized algorithm and the visualizing of similarity (VOS) protocol (Eck and Waltman 2007;Van Eck and Waltman 2007).
Whereas descriptive statistical methods were emp loyed to examine the retrieved data, the results were presented in tables and charts as ranges, percentages, and distribution/frequencies.

Investigators report and publication on cell-free nucleic acid in water nexus
Between the 50-year/jubileean period of studied articles (1969-2021), we collated annual scientific publication of diverse authors and journals which were grouped into 5 decadal period for the distribution of reports. It also reveals the frequency of related studies on cell-free nucleic acid (membrane vesicles, exogenous/extra-chromosomal nucleic acids) from diverse investigators, journals and interest-based personnel.

Reports on the various author's interest and actions on
Cell-free nucleic acid in water nexus were also retrieved to reveal the progress of studies and corrective steps to removing such non-cellular components from water bodies. It also shows the various countries that have embarked on related studies globally and the outcome of such studies. The study time span across 1969-2021, while unretrieved and nonconforming documents to the specified inclusion criteria were removed.

Issues on results and discussion
A total of two hundred and sixty documents were retrieved from Scopus and PubMed database as shown in Fig. 1 and Table 1 while the various datasets were analysed, respectively. Thirty-eight metric documents which conform to the inclusion criteria were both used for analysis and content review. These include: Sources (journals, books etc.) (34), average publication year and reports (16.7), included authors in related studies (157), single authors document (2), collaborating authors  (0) while funding and supporting organization includes nih, US govt, non-phs, non-US govt, phs, etc. Figure 1 shows the PRISMA strategy and flow chat for searching, reviewing and selecting of articles. From the forgoing, it is clear that the rate of production of articles/books as well as the numbers of studies on cellfree nucleic acids, exogenous nucleic acids, membrane vesicles, etc., in wastewater release into the environment remains few. The fewness of reported publications on the subject was further affirmed by a null average citation per document and per year. This has also resulted very low single author's index of collaboration, as well as multiple authors and countries collaboration index. The observed research supporting and funding organizations interest on related studies were also low affirming the low interest on studies of cell-free nucleic acids in water bodies.

Investigators report and publication on cell-free nucleic acid in water nexus
The annual metrics of investigators and authors study on Cell-free nucleic acid (membrane vesicles, exogenous/ extra-chromosomal nucleic acids) and water bodies globally is shown in Fig. 2. It can be observed from the above that a high proportion of such studies were conducted in 2010. The 5th decadal assessment and analysis of published articles showed that a high proportion of the documents were recorded/reported in the fourth decade (4), while the fifth decade ranked second in the publication frequency or reports (3). Also observed in the figure above is the silence in reports within some of the years under investigation, indicating that after the first report of cell-free nucleic acids in water bodies, interest on the study was not encouraged by research organizations; hence, research reports were not recorded in diverse countries and authors. This poor and/or low reporting state possess potential implications on the environments which are receiving the burden of cell-free nucleic acids release, since there are few recorded stride towards the removal of released cell-free nucleic acids. The various authors and countries productivity or publication frequency on Cell-free nucleic acid (membrane vesicles, exogenous/extra-chromosomal nucleic acids) and water bodies globally are reported in Table 2. USA ranked the highest country with the highest frequency or numbers of articles published on the subject under review with a percentage publication rate of 34.21% (13), while France ranked the second with a percentage publication rate of 5.26%. Amongst the authors investigative studies on the subject, it was observed that Deutsch DR ranked the highest in the numbers of articles (13), while other authors queue under the decreasing ranking ( Table 2). The sources of information were journals of life sciences and Biotechnology including ACS Synthetic biology, Biochemical and Biophysical Research Communications, Acta Neuropathologica, Biotechnology and Bioengineering, Animal Biotechnology etc. This has  shown that in addition to the low or few studies on cellfree nucleic acids, the release of such nucleic acid continues to burden environmental wellness with low removal tendency and research-based interest. From the analysis of keywords co-occurrence as shown in Fig. 3, 20 items met the threshold with a minimum number of occurrences of 3 for each, out of the 295 keywords. However, for each of the 20 keywords, the total strength of co-occurrence links with other keywords was calculated and the keywords with the greatest total linked strength were selected. These include plasmid, extrachromosomal inheritance, DNA circular, DNA bacterial, based sequence, DNA replication, messenger RNA, etc. It can be deduced that between 1996 and 2004, there has been research focus on such particulate nucleic acid from DNA to messenger RNA although low. The potential trend of studies on released cell-free nucleic acid members today is directed at human, organisms, plasmid, messenger RNA, etc., where there had been potential implications. The network and link of these cell-free nucleic acid members may be associated with pandemics or epidemics if their removal from the environment is not encouraged. Suffice to say that some earlier studies have reported the presence of such cell-free nucleic acid members in water bodies (Woegerbauer et al. 2020). Such non-negligible quantities of cell-free nucleic acids possess potential health implications as well as disease emergence in any environment (Woegerbauer et al. 2020;Norton et al. 2013). Table 3 shows the statistical distribution and occurrences of the keywords, their total linkage strength and the keywords co-occurrence analysis. It is observed that Human occurs the most relevant keyword (12) and a second position rank of linkage strength (22) indicating that human is the basic source to exogenous DNA or cell-free nucleic acids. The plasmid as a keyword ranked the second in occurrence (10), but it ranked the highest on the linkage strength (24) indicating that its linkage encourages transfer and/or sharing of exogenous nucleic acids. Suffice to say that plasmids are extra-chromosomal DNA which has both self-replicating tendency and antibiotic resistance genes. It also possesses the potential for horizontal sharing of its genetic constituents with other living organisms in water nexus and other environment (Ganesan et al. 2020;Ganesan and Vasudevan 2021;Duetsch et al. 2016). Other previous studies of some investigators have also revealed the acquisition of such relevant genetic characteristics via plasmids mediation by horizontal gene transfer (HGT). This is a potential source to the emergence of antibiotic resistance amongst microorganism as well as diverse pandemic particulate nucleic acids in the water nexus. With plasmid arising as the prevailing linkage strength, it is also supporting the previous studies as well as a potential indicator to the emerging nature of microbes within water bodies. Table 4 shows the general overview of studies on cellfree nucleic acid, authors, publishing journals, year of publication and PubMed citation ID. It may be adjudged that the few/low studies on cell-free nucleic acids in the environment and water bodies are attributable to poor interest or low awareness of the subject.
It is important to note that the various activities of man including the various strategies on the control of diverse pathogenic organisms from systemic and superficial infections using biocidal agents encourage the release of cell-free nucleic acids/exogenous nucleic acids into the water bodies. The low research interest, low publications and/or poor awareness on the continuous release of cellfree nucleic acids from man's activities into the environment is a potential time bomb. It is an explosive-based hotspot which may result disease as well as outbreak if appropriate research-based attention is not initiated.

Conclusions
The knowledge on cell-free nucleic acids, its diverse members and knowledge-based interest appears to be dearth amongst researchers and investigators. This is revealed in the few reports and publications observed in the forgoing assessment. From the first report in man in 1948 by Mandel and Metais, to its first report in the environment in1969 by Cave and Allen, and in extension till 2021, there had been very few reports, yet there is a non-negligible release of such cell-free nucleic acids into the water milieu. This study has further revealed the poor interest and/or unawareness of such noxious components which are present in the water bodies and may be linked with emerging diverse disease cases. It is important to note that a redirected interest in cfNAs in water environment would encourage advancement of water treatment strategies to include specific approach to the removal of cell-free nucleic acids, membrane vesicles or DNA reservoirs, plasmids or extrachromosomal DNA and other exogenous nucleic acids from water bodies. The interest on removal of cfNAs would also reduce the potential sharing of nucleic acids by diverse microbial strains in the environment which may also help to control/reduce occurrence of pandemic. In addition, it would also result in a reduction in HGT, metagenomic detection of exogenous nucleic acids and reduction in the mechanism of nucleic acids acquisition by cells and may lead to generational development of water treatment strategies.