Genomic Analysis of Carbapenem-Resistant Pseudomonas aeruginosa Isolated From Urban Rivers Confirms Spread of Clone Sequence Type 277 Carrying Broad Resistome and Virulome Beyond the Hospital

The dissemination of antibiotic-resistant priority pathogens beyond hospital settings is both a public health and an environmental problem. In this regard, high-risk clones exhibiting a multidrug-resistant (MDR) or extensively drug-resistant (XDR) phenotype have shown rapid adaptation at the human-animal-environment interface. In this study, we report genomic data and the virulence potential of the carbapenemase, São Paulo metallo-β-lactamase (SPM-1)-producing Pseudomonas aeruginosa strains (Pa19 and Pa151) isolated from polluted urban rivers, in Brazil. Bioinformatic analysis revealed a wide resistome to clinically relevant antibiotics (carbapenems, aminoglycosides, fosfomycin, sulfonamides, phenicols, and fluoroquinolones), biocides (quaternary ammonium compounds) and heavy metals (copper), whereas the presence of exotoxin A, alginate, quorum sensing, types II, III, and IV secretion systems, colicin, and pyocin encoding virulence genes was associated with a highly virulent behavior in the Galleria mellonella infection model. These results confirm the spread of healthcare-associated critical-priority P. aeruginosa belonging to the MDR sequence type 277 (ST277) clone beyond the hospital, highlighting that the presence of these pathogens in environmental water samples can have clinical implications for humans and other animals.


INTRODUCTION
Carbapenem-resistant Pseudomonas aeruginosa are a leading cause of hospital-acquired infections and have become a health priority (Tacconelli et al., 2018). Efforts have been made to prevent colonization, infection, and decrease mortality. Based on that, the WHO proposed a global priority pathogen list of multidrug-resistant (MDR) bacteria to drive research, discovery, and development of new antibiotics. Along with MDR P. aeruginosa, the critical pathogens WHO list included Acinetobacter baumannii and bacteria from Enterobacterales group (Tacconelli et al., 2018). They were categorized as critical priority through the use of multi-criteria, including being resistant to a large number of antibiotics, such as carbapenems and third generation cephalosporins, the best available options for treating MDR pathogens (Babu et al., 2020). Worryingly, carbapenem-resistant P. aeruginosa can cause severe and often deadly infections such as bloodstream infections, pneumonia, and osteomyelitis (Fernández-Barat et al., 2017;Pliska, 2020;Jean et al., 2020;Bobrov et al., 2021;Rosales-Reyes et al., 2021). Carbapenem resistance is usually multifactorial, including overexpression of efflux pumps (i.e., mexAB-oprM), deficiency or repression of the porin gene (oprD), alterations in the penicillin-binding proteins (PBPs), and chromosomal overexpression of cephalosporinase gene ampC (Van Nguyen et al., 2018;Gajdács, 2020;Xu et al., 2020). Moreover, resistance may be acquired by the selection of mutations in chromosomal genes or horizontal uptake of resistance determinants. However, carbapenem resistance has been most associated with production of carbapenemases, which include serine β-lactamases and metallo-β-lactamases (MβLs) (Polotto et al., 2012;Lupo et al., 2018), whereas high-risk global clones have been associated with MDR or extensively drug resistant (XDR) phenotypes. Currently, global P. aeruginosa high-risk clones include sequence types (STs) ST235, ST111, ST175, ST233, ST244, ST277, ST298, ST308, ST357, and ST654 (Del Barrio-Tofiño et al., 2020;Kocsis et al., 2021). Specifically, the ST277 has been sporadically reported in Asian, North American, and European countries, whereas in Brazil is highly prevalent (Gales et al., 2003;Hopkins et al., 2016;Del Barrio-Tofiño et al., 2020;Silveira et al., 2020;Kocsis et al., 2021). The success of the Brazilian endemic clone ST277 is associated with carbapenem resistance due to production of the MβL SPM-1 (Gales et al., 2003;Cipriano et al., 2007;da Fonseca et al., 2010;Nascimento et al., 2016;Silveira et al., 2020). Worryingly, SPM-1-producing P. aeruginosa have been identified in hospital sewage and hospital wastewater treatment plants (Fuentefria et al., 2009;Miranda et al., 2015), denoting potential to spread throughout the aquatic environment, enabling human exposure and transmission. However, although whole genome sequencing (WGS) of human SPM-1-positive isolates have been performed (Nascimento et al., 2016;Galetti et al., 2019), sequence data from environmental isolates have not been provided for comparative genomic studies. Based on WHO list priority pathogens criteria, which included pathogen mortality, hospital and environment transmissibility and limited treatment options, recognition and genomic characterization of critical priority pathogens is an essential first step to understanding their dynamic of acquisition/dissemination and ultimately to development of preventive intervention strategies (Hendriksen et al., 2019). In this study, we report genomic data and the virulence potential of carbapenem-resistant SPM-1-positive P. aeruginosa strains isolated from polluted urban rivers, in Brazil.

Whole Genome Sequencing and Genomic Analysis
Genomic DNA of Pa19 and Pa151 were extracted using PureLink Quick Gel Extraction & PCR Purification Combo Kit (Life Technologies, Carlsbad, CA). The Illumina paired-end libraries were constructed using a Nextera XT DNA Library Preparation Kit (Illumina Inc.), according to the manufacturer's guidelines. Whole genome sequencing was performed using an Illumina MiSeq platform with 300-bp read lengths. Reads were de novo assembled using SPAdes 3.13, 1 and the resulting contigs were automatically annotated by NCBI Prokaryotic Genome Annotation Pipeline (PGAP) version 3.2. 2 Antibiotic resistance genes were predicted using ResFinder 4.1 3 and the Comprehensive Antibiotic Resistance Database (CARD). 4 Multi-locus Sequence Typing prediction was performed using MLST v.2.0. 5 Heavy metal (HM) resistance genes were manually identified using the NCBI database 6 and Geneious Prime version 2020.04 (Biomatters, New Zealand). Additionally, phage prediction was performed by Genome Detective Virus Tool software. 7 The rmtD gene was detected and aligned by BLASTn 1 https://github.com/ablab/spades Frontiers in Microbiology | www.frontiersin.org 3 September 2021 | Volume 12 | Article 701921 (Alikhan et al., 2011) against the rmtD1 allele of the P. aeruginosa (PA0905 strain), recovered from a human patient (GenBank accession number. DQ914960). Genetic context analysis of bla SPM-1 and rmtD1 resistance genes of Pa151 were performed with BLASTn algorithm and manually curated using Geneious Prime version 2020.04 (Biomatters, New Zealand). Moreover, virulence genes, efflux systems, and regulators were determined through the Virulence Factor Database. 8 Serotype was predicted using Past 1.0. 9 SNP-based phylogenetic analysis was performed by using Prokka 1.13.4 10 for pangenome annotation, followed by Roary 3.13.0 11 for core genome analysis. SNP-sites tool 12 was used for SNPs extraction from the core gene alignment; whereas RAxML-NG version 0.9.0 13 for phylogenetic construction and a maximum likelihood tree based on SNP alignment. Additionally, comparative genomic analysis of P. aeruginosa sequences was performed by BRIG v.0.95 using the BLASTn algorithm and Island viewer 4.0.

Virulence Potential of Carbapenem-Resistant P. aeruginosa Strains in the Galleria mellonella Larvae Model
The virulence potential of P. aeruginosa Pa19 and Pa151 strains was evaluated using the Galleria mellonella infection model (Tsai et al., 2016). In brief, groups of G. mellonella containing 10 larvae of nearly 0.25-0.35 g (supplied by the Institute of Biomedical Sciences of the University of São Paulo, Brazil) were infected with 10 4 CFU/ml of each strain per larvae, by injecting a 10 μl aliquot in PBS, into the body of the larvae via the last left proleg, using a sterile ultra-fine needle syringe (Fuentes-Castillo et al., 2019). Survival was monitored every hour, for 96 h. Two biological replicates and two experimental replicates were performed with a group of 10 larvae per strain, in each replicate. SPM-1-producing P. aeruginosa clinical strain PA1088 was used as comparative control (Toleman et al., 2002). Moreover, a control group inoculated with sterile PBS was used in each biological and experimental replication assay, in order to verify that the larvae would not be killed by physical trauma. Survival curves were plotted using the Kaplan-Meier method, whereas statistical analyses were performed by the log rank test with p < 0.05 indicating statistical significance (OriginLab Software, Northampton, Massachusetts, United States).

RESULTS
In this study, two carbapenemase (SPM-1)-producing P. aeruginosa ST277 (Pa19 and Pa151 strains) isolated from impacted urban rivers in São Paulo, Brazil, were sequenced. As this clone has been endemic in Brazilian hospitals, being also identified in migratory birds (Figure 1), we have additionally performed a comparative analysis with publically available genomes obtained from ST277 lineages from human infections.
Genome sequencing yielded a total of 968,818 and 473,825 paired-end reads assembled into 395 and 337 contigs, with 305 and 299x of coverage, to Pa19 and Pa151 strains, respectively. The genome size of Pa19 was calculated at 6,927,007 bp, with a GC content of 67.8%, comprising 6,956 total genes, 60 tRNAs, three rRNAs, four ncRNAs, and 155 pseudogenes (accession number: PHST00000000.1). On the other hand, genome size of Pa151 was calculated at 6,799,801 bp, with a GC content of 66.9%, comprising 6,747 total genes, 59 tRNAs, three rRNAs, four ncRNAs, and 123 pseudogenes (accession number: PHSS00000000.1). Genomic information of P. aeruginosa Pa19 and Pa151 strains are available on the OneBR platform 15 under ONE609 and ONE610 ID numbers, respectively.
Schematic representations of the genetic context surrounding bla SPM-1 genes in the environmental P. aeruginosa PA151 strain is presented in Figure 5A. The bla SPM-1 was flanked by a ~4.8 kbp region composed of the IS91-bla SPM-1 -groEL-IS91 array. The presence of IS elements is related to horizontal gene transfer, whereas the groEL encodes for a heat-shock chaperon. Additionally, we also detected the traG (encoding a conjugal transfer protein), eexN (encoding the entry exclusion protein), traR (transcriptional regulator), bcr1 (bicyclomycin resistance), virD2 (gene encoding a relaxase), and hypothetical proteins. In Figure 5B is presented the genetic context surrounding rmtD1 gene in PA151 strain. The rmtD1 was flanked by a ~7.3 kbp region composed of the IS91-rmtD1-tgt-groEL-IS91 array. In addition, aacA4, bla OXA-56 , aadA7, and qacEΔ1 genes were located on a class 1 integron. Moreover, cmx and sul1 resistance genes, that encodes for chloramphenicol and sulphonamide resistance, respectively, were also identified along with genes encoding hypothetical proteins, transposase, IS110, IS481, and IS3 mobile elements.

DISCUSSION
Carbapenem-resistant P. aeruginosa are critical-priority pathogens associated with high mortality and morbidity (Georgescu et al., 2016;Tacconelli et al., 2018;Pang et al., 2019). In this regard, one of the major concerns has been the successful expansion and rapid spread of high-risk clones. In Brazil, the metalloβ-lactamase (SPM-1)-producing P. aeruginosa ST277 clone has gained significant attention, due to its endemicity status and further identification in migratory birds and polluted environments (Gales et al., 2003;Nascimento et al., 2016;Turano et al., 2016;Martins et al., 2018). Worryingly, previous studies have also reported the occurrence of carbapenemase (KPC-2)-producing Klebsiella pneumoniae belonging to the clonal group CG258 and OXA-23-positive A. baumannii ST79 in the Tietê River (Oliveira et al., 2014;Turano et al., 2016), supporting an anthropogenic trend, most likely due to hospital wastewater discharge and domestic wastewaters effluents (Nascimento et al., 2017;Bartley et al., 2019;Böger et al., 2021;Popa et al., 2021). Therefore, aquatic environment could play an important role in the widespread of critical pathogens (Devarajan et al., 2017). In fact, polluted rivers could be contributing for colonization of local and migratory fauna (Martins et al., 2018;Narciso et al., 2020).
Although, oral antibiotics have been successfully used in the treatment of bacterial infection, for P. aeruginosa few therapeutic options are available, being restricted to some fluoroquinolones, including ciprofloxacin, levofloxacin, and prulifloxacin, which are given alone or in combinations with a second intravenously or inhaled anti-pseudomonal antibiotic such as β-lactams (piperacillin/tazobactam, ceftolozane/ tazobactam, ceftazidime, cefepime, or carbapenems) and/or aminoglycosides (tobramycin, amikacin, or gentamicin) (Tümmler, 2019;Ibrahim et al., 2020;Nisly et al., 2020). However, under a clinical perspective, even co-resistance to carbapenems and aminoglycosides in ST277 have already been reported and limited therapeutic options. This resistance profile is mediated by bla SPM-1 and rmtD genes, respectively (Doi et al., 2007). Strikingly, in some ST277, including environmental (Pa19) and human (PA7790) lineages, the rmtD gene was not found. On the other hand, the rmtD1 identified in the environmental Pa151 strain, displayed 100% identity to the rmtD1 gene from P. aeruginosa PA0905 strain, recovered from a human patient in 2005, in Brazil (Doi et al., 2007). The rmtD1 was subsequently identified in K. pneumoniae and other Enterobacterales in Latin America, Europe, and North America (Bueno et al., 2016). Since acquisition of this gene has been linked to transposition events (Doi et al., 2007;Nascimento et al., 2016), most likely genomic plasticity of P. aeruginosa has led to the dissemination of rmtD + and rmtD-ST277 lineages For each strain, groups containing 10 G. mellonella larvae in each replicate were evaluated in two biological and experimental independent assays. (Silveira et al., 2020). In Brazil, occurrence of rmtD has also been documented in Escherichia coli and K. pneumoniae (Yamane et al., 2008;Leigue et al., 2015).
The bla SPM-1 , IS91-bla SPM-1 -groEL-IS91 gene array has been previously identified within a Tn4371-like integrative and conjugative element (ICE Tn4371 6061) considered stable in the chromosome loci of P. aeruginosa ST277 strains recovered from humans and animals (Fonseca et al., 2015;Nascimento et al., 2016). Since ICEs are genetic mobile platforms that play an important role during bacterial evolution, they are overlooked as vectors in the spread and resistance emergence in many bacterial species (Fonseca and Vicente, 2016). Moreover, the genetic context of rmtD1 (IS91-rmtD1-tgt-groEL-IS91) identified in the environmental strain was similar to previous descriptions, where the presence of the rmtD gene in clinical isolates was associated to the TnAs3 transposon (Fonseca et al., 2015;Nascimento et al., 2016).
In human and aquatic P. aeruginosa ST277 isolates the resistome was not restricted to antibiotics, and the presence of genes conferring tolerance to copper and QAC biocides was further detected. Currently, there is a growing concern about biocides that pollute aquatic environments, especially QACs, since these compounds are widely used in domiciliary and hospital settings, as disinfectants, soaps, toothpastes, and mouthwash formulations (Zubris et al., 2017;Fuentes-Castillo et al., 2020). Consequently, ecosystems impacted by HM and biocides could favor the selection and persistence of high-risk clones harboring a broad resistome (Baker-Austin et al., 2006;Zhao et al., 2012;Kim et al., 2018).
Although a limitation of this study was the lack of a known highly virulent P. aeruginosa to be used as a positive control in the in vivo assay; we observed that the virulent behavior of environmental strains was identical to clinical strains. Indeed, a wide virulome was also predicted in human and environmental P. aeruginosa ST277 lineages, denoting a pathogenic potential, as demonstrated in the G. mellonella infection model. Lipopolysaccharide (LPS) O-antigen, type IV pili, and flagella are components of the external cell wall structure of P. aeruginosa and play important roles in the early stage of colonization, persistence, and bacterial pathogenesis (Hauser, 2011;Behzadi et al., 2021). Furthermore, O-antigen is an important virulence factor in P. aeruginosa used for the detection of MDR/XDR high-risk clones (Del Barrio-Tofiño et al., 2019). Strikingly, among clinical strains were identified the serotypes O5 and FIGURE 4 | Circular genome maps of SPM-1-producing P. aeruginosa belonging to ST277. Circular maps were built by BLAST Ring Image Generator (BRIG) using seven P. aeruginosa genomes. All genomes were represented as individual rings and compared against the reference genome PA1088 (GenBank accession number: CP015001.1). The bla SPM-1 gene is indicated by a black arrow. Furthermore, several genes associated with DNA replication/repair/regulatory/defense and membrane proteins were identified in the major genomic island, indicated as GI-I. O2. The latter was also identified among environment strains. Both serotypes have been associated with acute and chronic infections (Lu et al., 2014;Li et al., 2018). Type secretion systems (TSSs) are mechanisms by which bacteria translocate a set of toxins into the cytosol of host cells and/or to the extracellular medium (Abby et al., 2016). Pseudomonas aeruginosa is known to have five TSSs, of which Types I (T1SS), II (T2SS), and III (T3SS) are involved in the virulence of this pathogen. Several studies have linked these TSSs with poor outcomes of patients with acute respiratory diseases (i.e., pneumonia), with T3SS being one of the most clinically relevant virulence determinants (Hauser, 2011;McMackin et al., 2019;Sarges et al., 2020). In this context, we detected ExoTSY exotoxins-encoding genes in both clinical and environmental strains. ExoTSY exotoxins are secreted by T3SS and reported to be involved in lung injury, pulmonaryvascular barrier disruption, and end-organ dysfunction in chronic infections, mainly in CF patients; as well as with mortality in animal models (Lu et al., 2014;Sarges et al., 2020;Jurado-Martín et al., 2021). Interestingly, the toxA gene (exotoxin A), which is present in the most clinically P. aeruginosa strains (Khosravi et al., 2016) was also identified in environmental strains. Exotoxin A has been associated with tissue damage related to poor outcomes of burn patients (Khosravi et al., 2016). In fact, the broad virulome harbored by P. aeruginosa ST277 seems to be associated with a remarkable ability to adapt to different human and non-human conditions (Jurado-Martín et al., 2021).
In brief, from comparative analysis, our data revealed that Pa19 and Pa151 environmental strains presented slight variations when compared against clinical strains, suggesting a high degree of genetic conservation, regardless isolation data and exposition to contaminants (antibiotics and biocides residues) present in the polluted aquatic environments.

CONCLUSION
In summary, we report genomic comparative data of antimicrobial-resistant P. aeruginosa isolated from aquatic environments in Brazil. The presence of SPM-1-producing P. aeruginosa ST277 in urban rivers could be associated with hospital effluents, since SNP-based phylogenomics showed high nucleotide sequence similarity between clinical and environmental genomes. Additionally, wide resistome and virulome have been conserved in environmental isolates, denoting that critical priority P. aeruginosa of the high-risk ST277 has successfully expanded beyond the hospital. Therefore, genomic surveillance is essential to rapidly identify and prevent the spread of WHO critical priority clones with One Health implications.

A B
FIGURE 5 | Overall comparison of genetic context of bla SPM-1 and rmtD1 genes carried by clinical and environmental P. aeruginosa strains belonging to ST277. (A) The bla SPM-1 was flanked by a ~4.8 kbp region composed of IS91-bla SPM-1 -groEL-IS91. (B) The rmtD1 was flanked by a ~7.3 kbp region composed of the IS91-rmtD1-tgt-groEL-IS91. In addition, aacA4, aadA7, bla OXA-56 , qacEΔ1, sul1, and cmx resistance genes were also identified along with genes encoding for hypothetical proteins, transposase; as well as IS110, IS481, and IS3 mobile genetics elements.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories.