Expansion of healthcare-associated hypervirulent KPC-2-producing Klebsiella pneumoniae ST11/KL64 beyond hospital settings

The spread of carbapenemase-producing Klebsiella pneumoniae beyond hospital settings is a global critical issue within a public health and One Health perspective. Another worrisome concern is the convergence of virulence and resistance in healthcare-associated lineages of K. pneumoniae leading to unfavorable clinical outcomes. During a surveillance study of WHO critical priority pathogens circulating in an impacted urban river in São Paulo, Brazil, we isolate two hypermucoviscous and multidrug-resistant K. pneumoniae strains (PINH-4250 and PINH-4900) from two different locations near to medical centers. Genomic investigation revealed that both strains belonged to the global high-risk sequence type (ST) ST11, carrying the blaKPC-2 carbapenemase gene, besides other medically important antimicrobial resistance determinants. A broad virulome was predicted and associated with hypervirulent behavior in the Galleria mellonella infection model. Comparative phylogenomic analysis of PINH-4250 and PINH-4900 along to an international collection of publicly available genomes of K. pneumoniae ST11 revealed that both environmental strains were closely related to hospital-associated K. pneumoniae strains recovered from clinical samples between 2006 and 2018, in São Paulo city. Our findings support that healthcare-associated KPC-2-positive K. pneumoniae of ST11 clone has successfully expanded beyond hospital settings. In summary, aquatic environments can become potential sources of international clones of K. pneumoniae displaying carbapenem resistance and hypervirulent behaviors, which is a critical issue within a One Health perspective.


Introduction
The environmental dissemination of carbapenem-resistant Klebsiella pneumoniae (CRKp) is a critical public health and One Health issue [1]. In fact, due its clinical impacts and therapeutic challenges, CRKp has been recently classified as a critical priority pathogen by the WHO [2,3], with carbapenem resistance being mostly associated with the production of carbapenemases such as KPC-2, NDM-1, and OXA-232 [4]. In the last years, hypervirulent K. pneumoniae (hvKp) strains displaying hypermucoviscosity (hmKp) have emerged in Asia, Europe and South America, being associated to capsular serotypes (K) and sequence type (ST) combinations K1/ST23, K2/ST86, K2/ST65, K16/ST685, K20/ ST268, K20/ST420, K54/ST29, K57/ST41 and K57/ST218 [4][5][6][7], all of them causing a variety of infections with significant rates of morbidity and mortality [8]. It is noteworthy that, while most hvKp strains exhibit an antibiotic-sensitive profile [9], CRKp strains display a relatively low virulent behavior. However, convergence of hypervirulence and carbapenem-resistance in K. pneumoniae has emerged in hospital settings [4,7], which requires close monitoring, in order to avoid its environmental spread. Utilizing a microbiological and whole genome sequencing methodology, we have undertaken a surveillance investigation to examine the prevalence of pathogenic bacteria harboring clinically relevant resistance genes in anthropogenically-impacted urban rivers in Brazil. In this regard, we report that healthcareassociated hypervirulent KPC-2-producing K. pneumoniae ST11/KL64 has successfully expanded beyond hospital settings, which constitute a critical issue within a One Health perspective.

Water samples collection, bacterial identification, and antimicrobial susceptibility testing
During a Brazilian surveillance study (OneBR project), we investigated the spread of WHO critical priority pathogens circulating in urban rivers in São Paulo, the most populated city in South America. In this regard, from 2016 to 2018, 500 mL of surface water samples were collected from three different locations at the Pinheiros River (− 23.702500: − 46.673889;-23.664722: − 46.709722; and − 23.531111, − 46.748333), and kept refrigerated until being processed (within 6 h after collection). In brief, 100 mL of each sample was filtered by 0.45 μm Millipore membrane. Subsequently, the membranes were placed onto MacConkey agar plates supplemented with meropenem (2 μg/mL) or ceftriaxone (2 μg/mL) [10].

Hypermucoviscosity test and genomic background of K-locus
Hypermucoviscosity phenotype of K. pneumoniae strains were analyzed by the string test, which is the ability of bacterial colonies grown on agar plates to form viscous strings of >5 mm when stretched is strongly associated with hypervirulence (hypermucoviscous) in K. pneumoniae pathogens [6,[16][17][18]. The inference of capsule structure was performed using BLASTn, followed by manual curation using Geneious Prime version. 2022.1.1 (Biomatters, New Zealand), against the K64 K. pneumoniae NCTC 8172 reference genome (GenBank accession number: AB924600.1) [19].

In vivo virulence assays of environmental K. pneumoniae ST11
The virulence potential of environmental K. pneumoniae strains were assessed by using the greater wax moth (Galleria mellonella) infection model [16,20]. In this respect, G. mellonella is a low-cost invertebrate infection model that presents an innate immune system highly similar with that of mammals, including cellular and humoral response [21]. In brief, groups of ten G. mellonella larvae of nearly 250 to 350 mg were infected with a10 μL aliquot containing10 6 CFU/mL of each strain, and survival was monitored for 96 h. Two biological replicates and two technical replicates were conducted to ensure experimental reproducibility. The hypervirulent K. pneumoniae (hvKP) K1/ST23 strain A58300 and the non-virulent K. pneumoniae ATCC 13883 were used as comparative controls [22]. Statistical analysis was conducted using the log-rank test, while the Kaplan-Meier method was employed for plotting survival curves. Through Graph Pad Software (San Diego, CA, USA) [16,20].

Results and discussion
In this study, two carbapenem-resistant K. pneumoniae strains (PINH-4250 and PINH-4900) were recovered from two different locations (− 23.664722: − 46.709722 and − 23.531111: − 46.748333) at the Pinheiros River, which runs 25 km (16 miles) through the city of São Paulo. Pinheiros River has been subjected to persistent anthropogenic pollution stemming from the continuous discharge of untreated domestic sewage into its various tributaries. Furthermore, inadequate street sweeping practices and the contamination of soils by industrial runoff and discharges contribute to the contamination of this river. In accordance with Resolution 357/2005 of the Brazilian Environment National Council (CONAMA), which classifies water quality into five categories ranging from pristine to polluted, according to the qualities that are required for their preponderant use, the Pinheiros River falls under class 4 classification, indicating a high level of pollution [23].
Both environmental strains were assigned to ST11, which has been recognized as a globally widespread lineage, being recovered from human and animal hosts, in several countries, including Brazil, Colombia, Egypt, Switzerland, Spain, Pakistan, Japan, Taiwan, and China. In this context, among the 60 K. pneumoniae ST11 genomes selected, phylogenomic analysis revealed a SNP range difference between 0 and 2313 (Supplementary Table 1); whereas ANI ranging varied between 99,3145 and 99,9762% ( Fig. 1A; Supplementary Table 2).
It is noteworthy that, environmental PINH-4250 and PINH-4900 strains were closely related to 14 clinical strains [24], and another environmental strain isolated from a water sample from an urban lake [25], which were recovered between 2006 and 2018, in Brazil (Fig. 1A). These findings are worrisome, since they support an anthropogenic origin of the spread of high-risk clones of K. pneumoniae for aquatic environments, impacting the human-environmental connectivity.
Capsule structure analysis revealed a conserved genetic organization involved with core assembly machinery (galF to wzc genes), at the 5′ end of the cps locus; whereas the wzc-gnd region was consist to genes associated with flippase (wzx), piruvyl tranferase (wcoV), polymerase (wzy), non-initial (wcoUT, wcsF, wcuK and wbaZ) and initial (wcaJ) glycosyltransferase, as previously reported [19]. Furthermore, the gnd-ugd region encompassed manB and manC genes, which are associated with the biosynthesis of GDP-D-mannose; as well as rmlA, rmlB, rmlC and rmlD genes, that are responsible for deoxythymidine diphosphate dTDP-L-rhamnose synthesis (Fig. 2). In this regard, D-mannose and L-rhamnose saccharides plays an important role in the synthesis of adhesins involved in the process of pathogen-pathogen and pathogen-host interaction and immune evasion of different pathogenic bacteria and fungi [29][30][31][32][33].
Although G. mellonella is not a natural host for K. pneumoniae, it has been effectively employed as an infection model to evaluate the pathogenicity of K. pneumoniae strains, owing to its resemblance to vertebrates in terms of the innate immune system [16,20,21]. In this regard, the hypervirulent behavior of K64/ST11 PINH-4250 and PINH-4900 strains was proven in the greater wax moth infection model. Indeed, both strains were capable to kill 100% larvae at 24 h post-infection, which was identically to the hypervirulent K1/ST23 control strain A58300 (Fig. 3) [22]. This hypervirulent behavior of K64/ST11 clones has been previously reported in human infections related to outbreaks in China [6,36].
In summary, we report the successful expansion of the healthcareassociated and hypervirulent KPC-2-producing K. pneumoniae ST11/ KL64 to an anthropogenically impacted river in Brazil. Historically, this river has suffered for a long time from several sources of pollution caused by anthropogenic activities, such as solid waste discharges, industrial runoff, and daily released non-treated domestic sewage on the various tributaries [23]. Strikingly, previous studies have reported the occurrence of CTX-M-15 or KPC-2-producing K. pneumoniae isolates belonging to global clones ST15, ST437, ST11, ST340 or ST321, in urban lakes and rivers, in Brazil Portugal and Switzerland [25,[37][38][39]. Currently, aquatic environments are recognized as important reservoirs and hot spots for clinically significant MDR bacteria and antibiotic resistance genes [40]. In fact, the WHO and the International Water Association (IWA) have classified the aquatic environments as vehicles for the sharing and acquisition of bacteria carrying medically important resistance genes, such as the bla KPC-2 gene [41]. Specifically, for the dissemination of carbapenemase-positive bacteria, several studies have demonstrated an epidemiological link with anthropogenic activities, including discharge of domestic and/or hospital wastewater effluents that did not receive appropriate treatment [38,[41][42][43][44][45]. Therefore, the assessment of water quality must include the examination of the multidrug resistance of clinically relevant bacterial species, providing an important link regarding the spread of MDR pathogens in a One Health context. In this regard, in this study, we demonstrate a One Health link based on a genomic approach, which reveal phylogenomic relatedness (55-93 SNP differences) between environmental and hospitalassociated KPC-2-poducing K. pneumoniae strains of ST11, recovered between 2013 and 2017, and stability and adaptation of this lineage to impacted urban rivers. Interestingly, lowest SNP differences are observed among environmental and nosocomial strains isolated in the same year, confirming hospital origin of environmental ST11.
Our findings highlight the role of aquatic environments in spreading hypervirulent and carbapenem-resistant K. pneumoniae, since water systems could favor and escalating the emergence of other pathogens with such coexistence profiles, associated with untreatable invasive human and non-human infections, which constitutes an unprecedented major public health challenge under a One Health perspective. Therefore, efforts to expand and strengthen genomic investigation of WHO critical priority pathogens in aquatic environments subjected to

Declaration of Competing Interest
All authors declare no conflicts of interest.

Data availability
The datasets presented in this study can be found in online repositories. Both PINH-4250 and PINH-4900 genome shotgun data have been deposited at GenBank database under the accession JAE-DYS000000000 and JAECUX000000000, respectively. Additionally, genomic information of PINH-4250 and PINH-4900 K. pneumoniae strains are available on the OneBR -KpBr platform (http://onehealthbr. com/) under the number ID ONE211 and ONE271, respectively.