The World Health Organization (WHO) recognizes antimicrobial resistance (AMR) as an urgent global issue with impending increases in mortality rates, hospitalization length and cross-contamination risks, as well as overall economic losses (World Health Organization [WHO], 2015). In spite of this problem, global antibiotic gross consumption and consumption per capita increased 65 and 39%, respectively, from 2000 to 2015. The broad-spectrum penicillins presented the highest increase, followed by cephalosporins, quinolones, and macrolides (Klein et al., 2018). In the current scenario, that antimicrobials are present in hospitals, animal production and communities, as well as, disposed of as wastewater in sewers, water and soil, the WHO emphasizes the need of a multidisciplinary, “One Health” approach to tackle this issue (World Health Organization [WHO], 2015).

Fortunately, ever since the seminal work of John Snow, pointing to a public water pump as a source of a cholera outbreak (Snow, 1855), several technologies in microbiology have been developed, from bacterial isolation and culture to next generation sequencing platforms (NGS). As such, different methodologies, in particular the so-called genomic surveillance, have become important to track the dissemination of infectious microbes and their genes of relevance among humans, humans and animals and these two and the environment. NGS can be particularly useful in order to track the ever-increasing dissemination of antimicrobial resistance genes (ARGs) (Mitchell and Simner, 2019; Ransom et al., 2020).

Among the most common ARGs, there are the beta-lactamase genes. Two of the most important classes of beta-lactamases are the extended-spectrum beta-lactamases (ESBLs) and the carbapenemases. The CTX-M enzymes are members of the ESBLs, able to hydrolyze expanded-spectrum cephalosporins and monobactams (Cantón et al., 2012). Prevalent worldwide, the CTX-M beta-lactamases are the most common ESBLs, especially CTX-M-15 (Bevan et al., 2017; Bush and Bradford, 2020). Infections with ESBL producers often drive the prescription of carbapenems, which may promote the selection and spread of potentially untreatable carbapenemase-producing Enterobacterales (Bevan et al., 2017). Among carbapenemases, the KPC-2 and KPC-3 are the most widespread and most commonly reported genes (Stoesser et al., 2017; Zhang et al., 2020). These genes are frequently associated with mobile genetic elements (MGEs), such as plasmids and transposons. Thus far, the most common mobile element associated with blaKPC, at least for the species Klebsiella pneumoniae, is the transposon Tn4401 (Yang et al., 2021). However, different transposable elements, broadly termed non-Tn4401 genetic elements (NTE_KPCs) were first described by Shen et al. (2009). Since then, several NTE_KPCs sequences can be found deposited in GenBank, but the majority has not been formally described in articles (Yang et al., 2021). Thus, it becomes important for the study of blaKPC dispersal in hospitals and the community to describe them. There are reports of these elements in association with high-risk clonal group 258 lineages in Brazil (Cerdeira et al., 2019) as well as found in hospital-associated carbapenemase-resistant bacterial outbreaks in Colombia and Chile (Rada et al., 2020; Wozniak et al., 2020) and in wastewater (Gomi et al., 2018).

In an environmental perspective, Gillings et al. (2018) have coined the term xenogenetic DNA to represent novel gene arrangements, such as (MGEs) with ARGs, whose assembly and dispersion have been promoted by human activity, in analogy to xenobiotic chemical pollutants. In another analogy, xenogenetic DNA behave like invasive species in as much as their abundance is determined not only by release and transport, but also by replication. Thus, xenogenetic DNA could be understood as a novel type of pollutant, being able to replicate, but also being generated as a consequence of human activity.

Klebsiella pneumoniae is a gram-negative rod belonging to Enterobacterales. It is also part of the ESKAPEE group of pathogens (Enterococcus faecium, Staphylococcus aureus, K. pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp. and Escherichia coli) known for their capacity of causing nosocomial infections and ability to resist to multiple classes of antibiotics (Rice, 2008; Partridge et al., 2018). Moreover, some strains of K. pneumoniae are also known as hypervirulent, are community-acquired and capable to promote metastatic infections (Shon et al., 2013). Many of such strains belong to the Cluster groups (CGs) 258 and CG11. Of note, there are also strains whose phenotype has converged into acquiring both multidrug resistance and hypervirulence traits (Wyres et al., 2020).

Wyres and Holt (2018) have shown that K. pneumoniae have a tendency to harbor more AMR genes and plasmids than other ESKAPEE pathogens. Furthermore, the species has a broad ecological distribution, ranging from gut colonization in mammals to insects, plants, water bodies and soil. Lastly, clinically relevant lineages have been isolated from non-clinical contexts, such as domestic animals. Hence, the authors suggest that K. pneumoniae may serve as an important hub for acquisition and transmission, via horizontal gene transfer, of AMR genes to microbes in different environments. Because of this capacity of colonizing the animal gut and the external environment (water and soil) and of being frequent carriers of acquired resistance, Berendonk et al. (2015) propose K. pneumoniae as a primary bacterial indicator of AMR spread in the environment.

Taking the context of water bodies in Brazil, Oliveira et al. (2014) have isolated a KPC-2 producing K. pneumoniae belonging to the Sequence type (ST)11 clonal complex from rivers, which is frequently associated with hypervirulent strains. Dropa et al. (2016) have described a, at the time, new K. pneumoniae ST harboring CTX-M-8 beta-lactamase from a wastewater treatment plant (WWTP). Moreover, carbapenemase resistant K. pneumoniae have also been found in recreational waters in Rio de Janeiro (Montezzi et al., 2015; de Araujo et al., 2016; Paschoal et al., 2017) and carbapenemase resistant Enterobacteriales have been found in Santos Bay (Andrade et al., 2020).

In the present study we describe a multidrug resistant (MDR) NTC_KPC-bearing K. pneumoniae strain belonging to a novel ST that has been found during a prospection for xenobiotic-degrading bacteria in an artificial urban lake, next to a WWTP.

In the present study we have identified an environmental K. pneumoniae isolate belonging to a novel sequence type, ST 5236 and displaying capsular serotype (K) KL45 and LPS serotype (O) O1v2. With respect to the K antigens, there are currently 78 serotypes, despite having more than 130 allelic combinations in its biosynthetic locus (KLs) (Wick et al., 2018; Wyres et al., 2020). Among these serotypes, K1 and K2 are both most commonly isolated from patients, more virulent in mice experiments and more resistant to phagocytosis and intercellular killing by phagocytes (Paczosa and Mecsas, 2016). On the other hand, regarding the O antigens, there are nine serotypes and 12 O-loci, with serotypes O1 and O2 being the most common for clinical isolates (Wyres et al., 2020). Storey et al. (2020) have recently characterized a novel T6SS effector for K. pneumoniae, termed VgrG4. The presence of this gene in bacterial strains contributed to toxicity against bacterial and fungal species. Within this gene, the portion coding for the DUF2345 domain was responsible for the intoxication. In our study, we have found three genes with DUF2345 conserved regions, alongside a nearly complete T6SS gene cluster. The presence of multiple copies of DUF2345-bearing genes could represent a competitive edge for K. pneumoniae isolates directly against microbiotas from different environments or a strategy for quickly acquiring genes from them.

Although this notion has been long known by Brazilian indigenous people (Krenak, 2020), the “One World-One health” concept was established in 2004 as a form to understand human health. As this concept goes, human health is dependent on animal health, both domestic and wild, and on environmental health. Increasing, modern anthropic pressures on the environment, such as pollution, habitat destruction and others, promote changes in its composition, which ultimately leads to the increase in frequency and intensity of disease-states in humans (Destoumieux-Garzón et al., 2018).

Nevertheless, the sequencing efforts for K. pneumoniae are mainly focused on clinical samples, a bias reflected in the phylogenetic analysis presented in Figure 3 where KpV3 has been placed in an undivided branch and the majority of bacterial isolates in the tree come from human samples. Besides, the samples derived from the sewer were actually taken from hospital effluents. Not only the AMR gene profile found in WWTPs reflects that of clinical settings (Pärnänen et al., 2019), but surveillance in WWTPs could represent a more accurate perspective of AMR spread at a populational level (Sims and Kasprzyk-Hordern, 2020). Moreover, a similar pattern was also observed in the MLST tree analysis (Figure 3) where the branch length represents the distance between groups. The analysis shows that the ST 5236 (KpV3) has a reasonable distance to its closest ST (ST 1041), which means that closer STs might exist, but are not yet identified. It is important to observe that this new ST is not related to other worldwide threat STs such as ST 11 and 258, meaning that this new ST might not be a threat in terms of hypervirulence.

It is disturbing that we have found an environmental K. pneumoniae strain containing both an ESBL (CTX-M-15) and a carbapenemase (KPC-2) enzyme, which raises concern about the selective pressure applied in the region. Of special note, both genes have been found inside or very near MGEs. The bla-CTX-M-15 was found in the bacterial chromosome, close to a putative Siphoviridae prophage sequence. This viral family has been associated with human fecal pollution in water bodies and with beta-lactamase gene transfer (Colomer-Lluch et al., 2011). Furthermore, the blaKPC2 gene was found in association with a transposase gene, forming a small gene cluster with two other genes. Klebsiella spp. ISs are often associated with AMR genes (Razavi et al., 2020) and transposition events can be an important factor in spreading these genes in contexts of positive selection among different plasmids, strains and species (Sheppard et al., 2016).

When comparing the genomes of the environmental KpV3 and the clinical isolate KpBSB31, we have detected that the ColRNAI plasmid is identical between the samples, which indicates a putative genetic flow of mobile elements between clinical and environmental isolates in the lake near the hospital. Similar observations of gene flows from hospitals water bodies have been made elsewhere (Ekwanzala et al., 2019; Lepuschitz et al., 2019; Bleichenbacher et al., 2020). However, it is unclear whether KpV3 isolate represents a clonal dispersion of hospital K. pneumoniae or if it has acquired genetic features from hospital-borne isolates via HGT. The extent of this genetic flow should be addressed by sampling different parts of the hospital system wastewater treatment. Thorough analyses of microbes discharged from the WWTPs into Paranoá lake are required to access the extent of their contribution to dissemination of AMR. Furthermore, the quantification of antibiotics and heavy metals in WWTPs, which may select/co-select resistant microbes (Hernando-Amado et al., 2019), is also of relevance, though fecal pollution alone may be the main factor in AMR spread (Karkman et al., 2019). Finally, Ekwanzala et al. (2019) have shown that in a river which receives WWTP effluent, a larger ratio of carbapenem resistant versus susceptible K. pneumoniae strains was found in its sediment, in contrast to surface waters. Thus, different portions of a given water body could weigh more in AMR maintenance. Danko et al. (2021) have analyzed >4,000 metagenomic samples from 60 cities around the world, as to characterize their urban microbiomes. Even with these many samples, the rarefaction curves for microbial species and ARGs did not saturate, indicating there is a considerable amount of diversity to be explored, particularly when considering the selective pressures for the formation of new ARGs and MGEs.

Brasília is located in a morphoclimatic domain, termed Cerrado, with two well-defined seasons, wet and dry. As a consequence of the dry season, the city has undergone into situations of water scarcity in recent years. As a response, Paranoá lake, which receives treated effluent from two WWTPs, has been turned into an environmental buffer for water reuse, including drinking water (Sodré and Sampaio, 2020). Hence, the discovery of an ESBL producer carbapenem-resistant K. pneumoniae isolate in this lake is of great concern, as regions undergoing water scarcity might become more susceptible to AMR spread, despite the presence of downstream water treatment processes. Additionally, climate change models based upon Intergovernmental Panel on Climate Change (IPCC) scenarios predict increases in temperature and dry season duration as well as precipitation decreases for the Cerrado biome (Bustamante et al., 2012). MacFadden et al. (2018) have shown that increases in minimal local temperatures are associated with higher frequency of infections caused by antimicrobial-resistant E. coli, S. aureus, and K. pneumoniae. Also, Anderson et al. (2008) have shown that K. pneumoniae blood stream infections occur more commonly with higher temperatures and dew point. There are many, non-excluding hypotheses for the causes of this correlation, from bacterial physiology to human societal changes (Rodríguez-Verdugo et al., 2020). Collignon et al. (2018) point that socioeconomical factors such poor infrastructure and governance, low health expenditure and high GDP and education were associated with higher AMR levels around the world. As the authors discuss, while temperature was positively correlated with AMR, this could be a correlation by proxy (Such as poor infrastructure) or a direct correlation. As water reuse may become more common as a result of climate change (Tram et al., 2014), this phenomenon may play a key role in AMR spread and warrants further inquiry.

Altogether, we have little evidence in favor of KpV3 as an isolate with high virulence. However, our observations suggest that there is a gene flow from hospitals into the lake, likely through WWTPs. While our findings point toward this hypothesis, it has been based so far on a single isolate. Therefore, the extent of ARG dissemination into the lake and how it may be represented in other isolates are a matter of future studies. Furthermore, there may be a positive selective pressure being applied in the Paranoá lake that may promote the selection and spread of potentially untreatable carbapenemase-producing bacteria while turning the region as a possible reservoir for ARGs. Thus, genomic surveillance and quality assessment programs with the wastewaters that are dumped in the lake are required to control and mitigate such pressure.

The dataset whole genome sequencing data for the KpV3 isolate is available under the NCBI Bioproject PRJNA738490, and for the Kp-BSB-A (KpBSB31) is available under the NCBI Bioproject PRJEB24576 (de Campos et al., 2018).

LJ and TD performed the strain isolation and phenotypic characterizations. LJ, FA, and RB performed the genome sequencing and bioinformatics analyses. GP, TC, and VM conceived and supervised the study. LJ, FA, GP, TC, and VM wrote the manuscript and analyzed the data. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.