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Article

Genomic Relationship Between High-Risk Pseudomonas aeruginosa Clone ST244 Serotypes O5 and O12 from Southeastern Brazil

by
Kayo Bianco
1,
Thereza Cristina da Costa Vianna
1,
Samara Santanna de Oliveira
1,
Kaylanne Montenegro
1,
Claudia Flores
1,
Ana Paula Alves do Nascimento
1,
Alexander Machado Cardoso
2 and
Maysa Mandetta Clementino
1,*
1
National Institute for Quality Control in Health, Oswaldo Cruz Foundation, Rio de Janeiro 21040-900, Brazil
2
Department of Biology, Rio de Janeiro State University (UERJ), Rio de Janeiro 20550-013, Brazil
*
Author to whom correspondence should be addressed.
Microbiol. Res. 2026, 17(1), 27; https://doi.org/10.3390/microbiolres17010027
Submission received: 3 December 2025 / Revised: 29 December 2025 / Accepted: 18 January 2026 / Published: 21 January 2026
(This article belongs to the Special Issue Host–Microbe Interactions in Health and Disease)
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Abstract

Pseudomonas aeruginosa is an opportunistic pathogen commonly associated with nosocomial infections and environmental dissemination. Among its high-risk clones, ST244 is notable for its global distribution and distinctive genomic traits. This study reports whole-genome sequencing of ten ST244 isolates from hospitalized patients and wastewater in a healthcare complex in Southeastern Brazil. Genomic comparisons revealed a highly conserved clonal group, with nine isolates forming a tight monophyletic cluster based on rMLST, SNP phylogeny, and average nucleotide identity (>99.5%). One isolate showed close phylogenetic proximity to strains from Asia and North America, suggesting international dissemination. Serotype analysis revealed both O5 and O12 variants, indicating intra-lineage antigenic diversity. Resistance profiling identified multidrug-resistant phenotypes carrying carbapenemase genes (blaOXA-494, blaOXA-396) and diverse insertion sequences (ISPa1, ISPa6, ISPa22, ISPa32, and ISPa37), facilitating horizontal gene transfer. Virulence gene analysis showed conserved elements related to adhesion, iron uptake, secretion systems, and quorum sensing, while the cytotoxin gene exoU was absent. These results highlight clonal persistence, possible intra-hospital transmission, and links to globally circulating ST244 sublineages. Our findings underscore the importance of genomic surveillance to track high-risk P. aeruginosa clones at the clinical–environmental interface.

1. Introduction

Hospital-generated effluents constitute a distinct category of hazardous waste exhibiting significant threats to public health and ecosystem equilibrium. These wastewaters contain elevated concentrations of pathogenic microorganisms and a complex array of antimicrobial compounds encompassing antibiotics, chemical disinfectants, heavy metal contaminants, and unmetabolized pharmaceutical residues [1,2].
Numerous bacterial species demonstrate survival capability within hospital wastewater environments, with Pseudomonas aeruginosa representing a particularly resilient organism capable of persistence across diverse ecological niches while exhibiting preferential colonization of moist environments [3]. This pathogenic microorganism reveals critical clinical significance, evidenced by its documented involvement in multiple nosocomial outbreaks, primarily attributed to intrinsic resistance mechanisms against numerous antimicrobial agents [4]. Subsequently, the World Health Organization (WHO) designated P. aeruginosa as a Priority 1 Pathogen, representing the most critical category requiring urgent research and development of novel antibiotic therapeutic interventions [5]. This classification underscores the clinical relevance and global threat posed by carbapenem-resistant P. aeruginosa, particularly in healthcare-associated infections, due to its intrinsic resistance mechanisms and remarkable ability to acquire additional resistance determinants.
In 2024, in the update of the WHO Bacterial Priority Pathogens List, P. aeruginosa continues to be recognized as a major public health concern. Although the revised categorization now includes critical, high, and medium priority groups, P. aeruginosa resistant to key antibiotics remains listed under the high priority category. This designation reflects its substantial global disease burden, therapeutic challenges, and the considerable difficulty associated with infection prevention and control [5]. The continued inclusion of P. aeruginosa among the highest-priority antibiotic-resistant bacteria highlights the persistent need for investment in antimicrobial research and development. Furthermore, it emphasizes the importance of implementing regionally tailored strategies to combat antimicrobial resistance and to ensure effective surveillance, stewardship, and infection control measures. Comprehensive characterization of sequence types (STs) associated with outbreak events across global geographical regions provides essential epidemiological data for containment strategies, enabling identification of infection sources, environmental reservoirs, and potential transmission pathways. High-risk clones represent those lineages posing the greatest global health threats, classified according to prevalence indices, geographical dissemination patterns, and antimicrobial resistance phenotypic profiles. The top ten worldwide P. aeruginosa clones comprise ST235, ST111, ST233, ST244, ST357, ST308, ST175, ST277, ST654, and ST298 [6,7].
Notably, ST244 demonstrates unique characteristics among high-risk clones, frequently exhibiting non-multidrug-resistant antimicrobial susceptibility profiles [8]. This observation indicates global prevalence independent of antibiotic resistance determinants. The adaptive versatility of P. aeruginosa correlates with extensive regulatory gene networks and genomic plasticity regions that confer selective advantages relative to other pathogenic microorganisms [9,10]. In the Brazilian context, the ST244 clone has been sporadically reported across different regions, primarily associated with clinical infections in tertiary care hospitals. Previous studies, notably those from São Paulo and Minas Gerais, have documented its presence since the early 2010s, often linked to outbreaks of multidrug-resistant P. aeruginosa [11]. However, these reports have largely focused on clinical isolates, leaving a significant gap in the understanding of the environmental reservoirs and the transmission dynamics at the clinical–environmental interface. Furthermore, the specific genetic mechanisms driving carbapenem resistance in the Brazilian ST244 sublineages, particularly the co-occurrence of blaOXA-494 and blaOXA-396, remain poorly characterized. Our study aims to bridge this gap by providing a high-resolution genomic comparison of ST244 isolates from both patient and wastewater sources within a single healthcare complex in Rio de Janeiro.
This study investigated the dissemination dynamics of high-risk P. aeruginosa ST244 sublineages from clinical infections to the municipal sewage infrastructure within the metropolitan region of Rio de Janeiro, Brazil. ST244 genomes isolated from hospitalized patients and the associated hospital wastewater treatment plant were subjected to whole-genome sequencing for high-resolution bacterial molecular typing, offering superior accuracy for epidemiological surveillance across both clinical and environmental settings.

2. Materials and Methods

2.1. Bacterial Isolates

Ten P. aeruginosa ST244 isolates were sourced from the One Health Surveillance Cultures Collection (Coleção de Culturas de Vigilância em Saúde Única—CCVSU) maintained by the National Institute for Quality Control in Health (INCQS/FIOCRUZ). The repository accession numbers of the isolates were CCVSU 3229, 3234, 3238, 3240, and 3252 (hospital effluent); CCVSU 3517 and 3889 (clinical samples); CCVSU 3550 (bronchial lavage); CCVSU 3611 (urinary tract infection); and CCVSU 3867 (blood). Isolate selection protocols were established based on predetermined criteria [12]. The CCVSU encompassed isolates (collected between 2018 and 2022) originating from hospital wastewater treatment plant effluent and isolates recovered from intensive care unit patients, both sources located within a single healthcare complex situated in the metropolitan region of Rio de Janeiro (22°59′4″ S/43°21′49″ O). Bacterial reactivation procedures were implemented according to standardized protocols, followed by taxonomic identification utilizing Matrix-Assisted Laser Desorption–Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF/MS) analytical methodology.

2.2. Whole-Genome Sequencing, Assembly Quality, and Genetic Profile

Genomic DNA extraction was performed using the PureLink™ Genomic DNA Mini Kit (Thermo Fisher Scientific, Waltham, MA, USA) in strict accordance with manufacturer-specified protocols. Library preparation procedures employed the Illumina DNA Prep Kit (Illumina, San Diego, CA, USA), while high-throughput sequencing was achieved using MiSeq Reagent Kit v3 (600-cycle) with a minimum coverage threshold of 200× on the Illumina MiSeq platform housed within INCQS facilities (Fiocruz Genomic Network). Quality control parameters included trimming of sequences exhibiting Phred quality scores below 20 using the Fastp bioinformatics tool. De novo assembly of sequenced reads was accomplished through Unicycler software v0.5.0 implementation [13]. Assembly quality assessment was conducted using BUSCO 5.5.0 [14] to evaluate genome completeness metrics. Misassembly identification and correction protocols employed the RagTag 2.1.0 correction module [15] with P. aeruginosa PAO1 (GCF_000006765.1) serving as the reference genome standard. Taxonomic verification of assembled genomes was performed through the Type (Strain) Genome Server (TYGS) database [16]. Antimicrobial resistance genomic assessment utilized the Resistance Gene Identifier (RGI) component of the Comprehensive Antibiotic Resistance Database (CARD) for identification of acquired antibiotic resistance genes (ARGs), supplemented by ResFinder analysis for resistance-inducing mutations. Mobile genetic element characterization was executed using MobileElementFinder v1.0.3 [17]. Pathogenicity determination and virulence factor (VF) identification employed PathogenFinder v 2-0.5.0 [18] and VirulenceFinder 2.0 [19], respectively. Serotype determination was performed in silico using the P. aeruginosa Serotyper tool (Past, version 1.0) on the assembled WGS data.

2.3. Phylogenomic Analysis

All assembled genomes underwent submission to the Multilocus Sequence Typing database within the PubMLST platform to establish Ribosomal MLST (rMLST) designations. The rMLST analytical framework indexes nucleotide sequence variation across 53 genes encoding bacterial ribosomal protein subunits (rps genes), facilitating integrated microbial taxonomy and molecular typing methodologies. Comparative analysis incorporated all 237 P. aeruginosa ST244 genomes deposited in the database through January 2025. A statistically representative subset (n = 134) was selected for multiple sequence alignment, neighbor-joining (NJ) phylogenetic tree construction, and average nucleotide identity (ANI) calculations using CLC Genomics Workbench version 25.0 (Qiagen, Germantown, MD, USA). Single-nucleotide polymorphism (SNP) identification and phylogenetic inference employed CSIPhylogeny (https://cge.food.dtu.dk/services/CSIPhylogeny/, accessed on 12 January 2025), available through the Center for Genomic Epidemiology platform (www.genomicepidemiology.org). The CSI Phylogeny pipeline executes sequential operations, including SNP calling, stringent filtering protocols, site validation procedures, and maximum likelihood phylogenetic inference based on concatenated alignments of high-quality polymorphic sites. Statistical analysis for phylogenetic inference was based on maximum likelihood (ML).

3. Results

3.1. General Genomic Features

Genomes of ten Pseudomonas isolates collected from intensive care unit patients and hospital wastewater treatment plant effluents were sequenced for comparative analysis. The strains exhibited remarkably homogeneous genomic characteristics, with draft genome sizes ranging from 6.6 to 6.85 Mb and GC content approximating 66%, values consistent with established P. aeruginosa genomic features. Given the draft nature of the assemblies, the estimated total gap size per genome is approximately 5000 bp. Contig numbers per assembled genome varied from 78 to 130, while N50 values exceeding 120 kilobases indicate acceptable assembly quality metrics. Protein-coding sequence (CDS) enumeration demonstrated relative stability across strains, ranging from 6335 to 7043 genes, while RNA gene counts exhibited minimal inter-strain variation (Table 1).

3.2. Phylogenomic Analysis and Global Context

A systematic genomic analysis of the ten local isolates using the comprehensive PubMLST reference database confirmed that P. aeruginosa sequence type 244 (ST244) is taxonomically subdivided into 13 distinct ribosomal sequence types (rSTs), with an empirically documented geographic distribution across 27 countries worldwide (Figure 1). Quantitative prevalence analysis of these rST variants revealed that rST 20964 exhibited statistically significant dominance in global frequency distribution, whereas rSTs 79892 and 75320 demonstrated marked geographical restriction, predominantly found in Brazil. The core genome analysis of the 134 P. aeruginosa ST244 isolates revealed 4500 core genes.
Phylogenetic reconstruction employing the ribosomal multilocus sequence typing (rMLST) methodological framework revealed nine genetically proximate P. aeruginosa isolates organized within a monophyletic cluster characterized by three discrete rST designations (79892, 20964, and 75320). This phylogenetic clustering pattern demonstrates statistically significant genetic cohesion among geographically disparate isolates, indicating shared evolutionary ancestry and potential clonal dissemination pathways. The network topology demonstrates the pronounced global distribution centrality of rST 20964 (represented as the primary node), with secondary phylogenetic clusters representing geographically constrained variants, specifically rSTs 79892 and 75320, which exhibit statistically significant preferential distribution within Brazilian populations. The phylogenetic architecture reveals distinct evolutionary lineages, with specific rSTs displaying geographical clustering patterns that provide empirical evidence for regional clonal expansion phenomena, while alternative lineages demonstrate conclusive evidence of international dissemination across multiple continental boundaries.
This comprehensive analytical approach provides critical quantitative insights into the molecular epidemiological dynamics and global circulation patterns of this high-risk clonal lineage within both clinical nosocomial environments and environmental reservoir systems. The data presented establishes a robust foundation for understanding the complex biogeographical distribution mechanisms governing this pathogenically significant bacterial clone across diverse ecological niches and geographical scales. This phylogenetic grouping, delineated in yellow within the constructed dendrogram, indicates substantial genetic relatedness among constituent strains (Figure 2). The phylogenomic analysis revealed two well-supported monophyletic clusters within the lineage. The larger cluster showed limited genetic diversity and a broad geographic distribution, consistent with a globally disseminated high-risk clone. In contrast, the smaller cluster exhibited greater internal diversity and a more restricted distribution, suggesting local diversification and limited clonal expansion.
Phylogenetic clustering architecture reveals discrete monophyletic assemblages corresponding to specific rST designations, with systematic color-coding annotations: yellow delineation for rST 20780, blue for rST 20964, green for rST 75320, and orange for rST 79892. Whole-genome-based circular phylogenetic reconstruction demonstrates comprehensive evolutionary relationships among 134 P. aeruginosa ST244 isolates sourced from PubMLST repositories. The conspicuous, yellow-highlighted clade demonstrates pronounced phylogenetic clustering of nine study-specific isolates (CCVSU 3229, 3234, 3238, 3240, 3252, 3517, 3550, 3611, and 3867), indicating significant phylogenetic proximity and shared evolutionary ancestry. This phylogenetic grouping, delineated in yellow within the constructed dendrogram, establishes substantial genetic relatedness among constituent strains with average nucleotide identity values exceeding 99.5%. Conversely, isolate CCVSU 3889 exhibited distinct phylogenetic positioning, clustering independently with strains originating from Asian and North American geographical regions rather than associating with the predominant South American assemblage. This divergent clustering pattern provides empirical evidence for potential international dissemination events, thereby distinguishing this isolate from the primary geographical cluster and suggesting complex transmission dynamics transcending continental boundaries.
The phylogenetic architecture demonstrates pronounced geographical clustering phenomena with South American isolates, particularly Brazilian strains, forming statistically robust monophyletic assemblages, while simultaneously revealing empirical evidence of intercontinental dissemination events. To assess genetic diversity parameters and establish precise evolutionary relationships among bacterial isolates, phylogenetic reconstruction was executed utilizing comprehensive single-nucleotide polymorphism (SNP) analytical methodologies across the two discrete phylogenetic clusters previously identified through rMLST protocols. The resulting phylogenetic architecture of the nine-strain assemblage by comprehensive single-nucleotide polymorphism (SNP) demonstrated systematic distribution across multiple monophyletic clades, exhibiting pronounced taxonomic differentiation between serotypes O5 and O12, as empirically demonstrated through systematic color-coded branch designations within the dendrogram (Figure 3).
Within the clade containing isolate CCVSU 3889, broader serotype dispersion was observed alongside enhanced phylogenetic clustering among constituent isolates, particularly those classified as serotype O5. Notably, CCVSU 3889 demonstrated greater genetic relatedness to the Chinese isolate compared to the Chicago isolate, corroborating previous findings derived from rST analysis (Figure 4). The phylogenetic topology reveals potential evidence of inter-regional transmission events, with genetically similar strains distributed across geographically distant locations, suggesting possible nosocomial dissemination patterns. Average nucleotide identity (ANI) similarity analysis demonstrated exceptional genomic homogeneity among CCVSU strains, with calculated values exceeding 99%, substantially surpassing the 95% threshold conventionally employed for species-level taxonomic assignment (Supplementary Figures S1 and S2) [20,21].

3.3. Resistance and Virulence Gene Profile

Comprehensive virulence factor genomic screening revealed the presence of genes associated with adhesion mechanisms, antimicrobial activity, antiphagocytic functions, biosurfactant production, enzymatic processes, iron acquisition systems, proteolytic activities, quorum sensing networks, regulatory mechanisms, secretion system apparatus, and toxin production (Supplementary Table S1). Notably, all isolates possessed the complete genetic machinery for the Type III Secretion System (T3SS), including structural components (pscB-U) and translocators (popB, popD, and popN). The T3SS is a major virulence mechanism that injects effector proteins directly into host cells, leading to cytotoxicity and immune evasion. Furthermore, the highly potent Exotoxin A (ETA), encoded by the toxA gene, was identified in all strains. ETA is a critical virulence factor that inhibits host protein synthesis, contributing significantly to tissue damage and systemic toxicity during infection. Genes for hydrogen cyanide (HCN) production (hcnA, hcnB, and hcnC), a potent respiratory toxin, were also conserved across the isolates. These conserved virulence elements, including those related to adhesion, iron acquisition (pyoverdine and pyochelin systems), and quorum-sensing networks. These conserved virulence elements are critical for host colonization and pathogenesis [1,2,3,4].
All isolates exhibited multiple antibiotic resistance genes, indicating a multidrug-resistant profile (Table 2). The identified genes included but were not limited to aph(3′)-IIb (aminoglycoside resistance), aac(6′)-Ib3 (aminoglycoside resistance), blaOXA-494, blaOXA-396, blaPAO (beta-lactam resistance), fosA (fosfomycin resistance), catB7 (chloramphenicol resistance), crpP, sul1 (sulfonamide resistance), and qacE (quaternary ammonium compound resistance). The presence of the two carbapenemase genes, blaOXA-494 and blaOXA-396, is particularly noteworthy, as these genes are associated with resistance to carbapenems, a class of last-resort antibiotics. The variability in resistance gene sets among isolates suggests differing selective pressures and mechanisms of resistance acquisition.
Regarding mobile elements, several insertion sequences (IS) were detected, such as ISPa1, ISPa6, ISPa22, ISPa32, ISPa37, ISPa52, ISPst5, ISPa79, ISPa100, ISPa1635, and ISPre3 (Table 2). The co-occurrence of multiple insertion sequences in a single isolate highlights the potential for mobility and dissemination of these resistance genes within and between bacterial populations. The presence of these elements is indicative of the genomic plasticity of P. aeruginosa and its capacity to acquire and rearrange resistance genes. The probability of being human pathogens was consistently high for all isolates, ranging from 0.9760 to 0.9928, confirming their relevance as etiological agents of human infections.

4. Discussion

This study examined for the first time the genomic and phylogeographic analysis of P. aeruginosa ST244 in Brazilian hospital and environmental settings. The empirical evidence generated through this comprehensive genomic investigation underscores the pervasive environmental distribution and sustained persistence of P. aeruginosa ST244 within nosocomial settings, thereby presenting substantial implications for environmental epidemiology and public health risk assessment. While the global literature often highlights the prevalence of high-risk clones such as ST235, ST111, and ST308 in multidrug-resistant P. aeruginosa outbreaks, our findings reinforce the significant, albeit often underreported, role of ST244 within the Brazilian epidemiological landscape. In Brazil, the spread of carbapenemase-producing P. aeruginosa has been largely attributed to the dissemination of ST277 and ST463, which frequently carry the blaSPM-1 and blaKPC-2 genes, respectively [21]. The identification of ST244 isolates carrying blaOXA-494 and blaOXA-396 in a major healthcare complex in Rio de Janeiro provides a crucial counterpoint, demonstrating that the local epidemiology of resistance is driven by a diverse array of high-risk clones and resistance mechanisms. This local context underscores the necessity of continuous, high-resolution genomic surveillance to accurately map the dominant circulating clones and their specific resistance determinants, which may differ substantially from global trends.
The implementation of the rST methodology facilitated precise phylogenetic differentiation of the P. aeruginosa isolates, revealing that nine specimens demonstrated pronounced genetic homology despite originating from disparate ecological niches (clinical versus environmental matrices). This finding substantiates the hypothesis of intra-institutional transmission dynamics and environmental persistence within aquatic systems. Furthermore, comprehensive geographical distribution analysis revealed that within this phylogenetic cluster, only a single strain exhibited Asian geographical origin, while the remaining isolates originated from South American regions, specifically the Southeast geographical zone. This distribution pattern suggests local dissemination processes or regional clonal expansion events, thereby emphasizing the critical importance of genomic surveillance protocols for understanding circulation dynamics of these pathogenic lineages. Conversely, one isolate demonstrated enhanced genetic similarity to strains from Asian and North American geographical regions, indicating probable international dissemination events. Its taxonomic classification within a discrete phylogenetic cluster further emphasizes the genetic heterogeneity inherent within ST244, potentially mediated by virulence factor repertoires, intrinsic resistance mechanisms, and remarkable genomic plasticity [22,23].
Serotype characterization of ST244 isolates revealed the presence of O5 and O12 lipopolysaccharide antigens, demonstrating the capacity of this clonal lineage to harbor distinct O-antigen variants. Although traditionally associated with the O2 serotype, the comprehensive genomic analysis based on 44 genomes deposited in PubMLST revealed diversified serotype distribution: 41% of isolates classified as O12, with the remainder distributed between O2 and O5 variants [24]. This antigen demonstrates exceptional adaptive capacity under selective pressures and participates in horizontal gene transfer events mediated by recombination of lipopolysaccharide (LPS) biosynthesis gene clusters, as documented in the replacement of ancestral O4 antigens in ST111 strains [25]. Analogous alterations have been characterized in alternative clonal lineages, such as ST253 (typically associated with O19), suggesting that ST244 may be subjected to similar molecular mechanisms, potentially explaining the absence of a dominant serotype within this clonal group [24].
The identification of O12 antigens in this epidemiological context possesses a particular clinical significance, as this serotype demonstrates frequent association with high-risk clones, including ST111, extensively isolated from nosocomial environments and associated with multidrug-resistant (MDR/XDR) phenotypes [26]. The O5 antigen demonstrates frequent association with ST244, particularly among clinical isolates [27], and its detection within wastewater matrices indicates potential environmental circulation of lineages possessing epidemiological relevance. This serotype is present in the reference strain PAO1, extensively utilized in virulence and adaptation studies, which reinforces the hypothesis that its lipopolysaccharide structure contributes to surface persistence and resistance to adverse environmental conditions [28].
Moreover, the phylogenetic organization into well-defined clades observed through SNP, rMLST, and ANI analyses reinforces the hypothesis that these isolates share recent evolutionary ancestry and circulate predominantly within national geographical boundaries. These findings corroborate empirical data, which emphasize the significance of associations between serotype, genomic profile, and geographical distribution patterns in understanding the evolution and dissemination of multidrug-resistant P. aeruginosa clones [29].
The increasing prevalence of multidrug-resistant P. aeruginosa represents a threat to global public health caused by the remarkable adaptive ability and genomic plasticity of bacteria [3,30]. Among the most alarming genes are genes such as BlaOXA-494 and BlaOXA-396. These genes encode class D carbapenemases, enzymes that hydrolyze antibiotics that are often used as a last resort against P. aeruginosa infections [31,32]. The dissemination of these genes, often associated with mobile genetic elements, significantly contributes to therapeutic failure and increased morbidity and mortality in clinical settings [33].
The insertion sequences (IS), such as ISPa1, ISPa6, ISPa22, ISPa32, ISPa37, ISPa52, ISPst5, ISPa79, ISPa100, ISPa1635, and ISPre3, are critical in the development and spread of antimicrobial resistance [34]. These elements can promote resistance gene transfer, integron and transposon creation, and homologous recombination, hence boosting the acquisition and expression of novel resistance determinants [35,36]. The diversity of IS identified in our isolates implies that P. aeruginosa uses a range of mechanisms to improve the acquisition and expression of its resistome, allowing for quick adaptability to settings with antibiotic selection pressure.
These bacterial species demonstrate exceptional adaptive capacity to hostile host environments, a capability facilitated by the production of multiple virulence factors that enable both infection initiation and disease progression [30]. This adaptive capacity is reflected in data obtained from our strain collection, which demonstrated significant genomic conservation regarding principal virulence genes, suggesting a common repertoire that potentially contributes to the maintenance of pathogenic phenotypes characteristic of these strains. Despite this genomic uniformity, specific variations were observed, primarily in genes associated with secretion mechanisms and antimicrobial factors. Such variations, although discrete, may exert significant influence on the infectious behavior of individual strains. A notable observation is the universal detection of the exoS gene, responsible for cytoskeletal alterations and apoptosis induction, while the exoU gene, encoding a potent cytotoxin associated with severe clinical manifestations, was absent in all specimens [37]. This suggests that these strains may employ virulence strategies focused on host persistence rather than immediate tissue destruction.
The recovery of near-identical ST244 isolates from both hospitalized patients and the hospital wastewater system highlights the critical role of the clinical–environmental interface as a reservoir and potential source for intra-hospital transmission. Hospital wastewater, rich in antibiotics and disinfectants, acts as a selective pressure environment, promoting the persistence and evolution of resistant strains before their discharge into the municipal sewage system. Our data strongly suggest that the wastewater infrastructure within the healthcare complex serves as a dynamic niche where the high-risk ST244 clone can persist and potentially re-enter the clinical setting, leading to nosocomial infections. This finding has direct implications for infection control and hospital waste management policies, emphasizing the need for effective pre-treatment of hospital effluents to mitigate the risk of resistant pathogen dissemination into the wider community. Among the analyzed variants, strain CCVSU 3238 exhibited distinct characteristics through the presentation of a more restricted virulent gene repertoire. This strain does not have secretion genes common to other strains (exoT and ppkA), maintaining only those associated with the type VI secretion system (T6SS) and sirigomycin toxin production. The T6SS, a recently characterized secretion apparatus, participates in inter-bacterial competition and biofilm formation, additionally facilitating direct toxin delivery into target cells, thereby promoting bacterial survival under adverse conditions [38,39]. Sirigomycin demonstrates antimicrobial and antifungal activity, potentially contributing to competitive organism elimination and colonization facilitation [40].
While the phylogenetic analysis revealed a close relationship between one local isolate (CCVSU 3889) and international ST244 sublineages from Asia and North America, suggesting a potential link to global dissemination events, it is imperative to interpret this finding within the limitations of our localized study. Our sampling was restricted to a single healthcare complex in Southeastern Brazil, and the observed global connectivity should be viewed as a signal of the international mobility of this clone, rather than a definitive conclusion about its global epidemiological impact. The primary conclusion remains the local clonal persistence and the urgent need for enhanced genomic surveillance within the Brazilian healthcare system to track the local evolution and spread of this high-risk, carbapenemase-producing ST244 clone. These observations emphasize the critical importance of continuous surveillance of virulence determinants in P. aeruginosa. Although the genetic landscape demonstrates substantial conservation, minor variations in critical genes can significantly impact virulence profiles, clinical outcomes, and antimicrobial therapy responses, indicating the necessity for individualized management strategies in treating these infections.

5. Conclusions

This study provides robust genomic evidence of the clonal persistence and intra-hospital dissemination of the high-risk P. aeruginosa ST244 clone within a Brazilian healthcare complex. The identification of this clone in both clinical and wastewater samples, coupled with its carriage of the carbapenemase genes blaOXA-494 and blaOXA-396, underscores a significant and ongoing public health threat. Our findings highlight the critical role of the hospital environment as a reservoir for multidrug-resistant organisms and emphasize the urgent need for enhanced genomic surveillance programs that integrate both clinical and environmental sampling. Such integrated surveillance is essential to accurately track the local evolution and global connectivity of high-risk clones like ST244, informing targeted infection control and waste management strategies to control the spread of carbapenem-resistant P. aeruginosa.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microbiolres17010027/s1, Table S1: Virulence-associated genes found; Figure S1: Average Nucleotide Identity (ANI) matrix demonstrating genomic homogeneity among P. aeruginosa isolates. This heatmap illustrates the pairwise ANI values calculated for a set of P. aeruginosa isolates, including the reference strain 3232_PAO1. Values exceeding 99% (highlighted in red) indicate exceptional genomic similarity, surpassing the 95% threshold typically used for species-level taxonomic assignment.; Figure S2: Average Nucleotide Identity (ANI) matrix for a subset of P. aeruginosa isolates, including global references. This heatmap displays pairwise ANI values for a selected group of P. aeruginosa isolates, providing further insight into their genomic relatedness. Values above 99% (highlighted in red) confirm high genomic homogeneity, reinforcing the clonal nature observed.

Author Contributions

Conceptualization, K.B., A.M.C. and M.M.C.; methodology, K.B., T.C.d.C.V., S.S.d.O., K.M., C.F. and A.P.A.d.N.; formal analysis, K.B., T.C.d.C.V., S.S.d.O., K.M., C.F., A.P.A.d.N., A.M.C., and M.M.C.; writing—original draft preparation, K.B.; writing—review and editing, K.B., A.M.C. and M.M.C.; supervision, M.M.C.; project administration, K.B. and M.M.C.; funding acquisition, M.M.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) (E-26/211.762/2021), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (409749/2021-4), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Institutional Review Board Statement

Not applicable. According to Brazilian ethical guidelines (National Health Council Resolutions No. 466/2012 and No. 510/2016) and internationally accepted standards for research ethics, studies based exclusively on microorganisms, anonymized secondary data, or environmental samples do not constitute research involving human subjects and therefore do not require Ethics Committee approval. Accordingly, this work is classified as an observational microbiological and genomic surveillance study and is exempt from ethical review.

Informed Consent Statement

Not applicable. Informed consent was not required for this study because patient samples were anonymized, and no personal data were collected.

Data Availability Statement

All sequence data generated in this study have been deposited in the NCBI BioProject database under accession number PRJNA1294175 https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1294175/, accessed on 25 November 2025).

Acknowledgments

We would like to express our sincere appreciation to the members of FIOCRUZ and UERJ for their valuable support and collaboration throughout this research.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
WHOWorld Health Organization
STsDirectory of Open Access Journals
CCVSUColeção de Culturas de Vigilância em Saúde Única
MALDI-TOF/MSMatrix-Assisted Laser Desorption–Ionization Time-of-Flight Mass Spectrometry
TYGSType (Strain) Genome Server
RGIResistance Gene Identifier
CARDComprehensive Antibiotic Resistance Database
ARGsAntibiotic Resistance Genes
VFVirulence Factor
rMLSTRibosomal Multilocus Sequence Typing
NJNeighbor-Joining
SNPSingle-Nucleotide Polymorphism

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Figure 1. Phylogenetic network analysis of P. aeruginosa ST244 ribosomal sequence type (rST) diversity and global geographical distribution. Minimum spanning tree based on rST profiles, illustrating the clustering of isolates according to their rSTs. Each node represents an rST, with node size proportional to the number of isolates and colors indicating the country of origin. Lines connecting nodes denote genetic relatedness between rSTs. Local isolates cluster closely around their corresponding rST designations, highlighting their genetic proximity and supporting the assignment of these isolates to specific rST lineages.
Figure 1. Phylogenetic network analysis of P. aeruginosa ST244 ribosomal sequence type (rST) diversity and global geographical distribution. Minimum spanning tree based on rST profiles, illustrating the clustering of isolates according to their rSTs. Each node represents an rST, with node size proportional to the number of isolates and colors indicating the country of origin. Lines connecting nodes denote genetic relatedness between rSTs. Local isolates cluster closely around their corresponding rST designations, highlighting their genetic proximity and supporting the assignment of these isolates to specific rST lineages.
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Microbiolres 17 00027 g002
Figure 2. Whole-genome-based circular phylogenetic reconstruction demonstrating evolutionary relationships among 134 P. aeruginosa ST244 isolates sourced from international genomic surveillance repositories. The dendrogram architecture was systematically constructed employing neighbor-joining (NJ) computational algorithms operating on complete genomic sequence datasets. Geographical distribution encoding employs standardized geometric node representation: triangular symbols (African continent), quadrilateral symbols (Asian continent), rhomboidal symbols (European continent), circular symbols (North American continent), hexagonal symbols (Oceanic regions), and inverted triangular symbols (South American continent).
Figure 2. Whole-genome-based circular phylogenetic reconstruction demonstrating evolutionary relationships among 134 P. aeruginosa ST244 isolates sourced from international genomic surveillance repositories. The dendrogram architecture was systematically constructed employing neighbor-joining (NJ) computational algorithms operating on complete genomic sequence datasets. Geographical distribution encoding employs standardized geometric node representation: triangular symbols (African continent), quadrilateral symbols (Asian continent), rhomboidal symbols (European continent), circular symbols (North American continent), hexagonal symbols (Oceanic regions), and inverted triangular symbols (South American continent).
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Microbiolres 17 00027 g003
Figure 3. Comprehensive SNP-based phylogenetic reconstruction of nine P. aeruginosa ST244 isolates demonstrating serotype-specific evolutionary clustering and source-environment distribution patterns. The circular dendrogram architecture was constructed utilizing single-nucleotide polymorphism (SNP) analytical methodologies, providing high-resolution phylogenetic inference based on genome-wide sequence variation across all analyzed specimens. The phylogenetic tree exhibits systematic color-coded branch designations delineating serotype classification: orange branches represent serotype O5 isolates, while purple branches indicate serotype O12 strains. Triangular markers designated Belo Horizonte (Brazil) isolates, square markers represented Kunming (China) specimens, circular markers indicated Rio de Janeiro (Brazil) strains, filled circular markers signified São Paulo (Brazil) isolates, while hollow circular markers denoted strains of undetermined geographical origin.
Figure 3. Comprehensive SNP-based phylogenetic reconstruction of nine P. aeruginosa ST244 isolates demonstrating serotype-specific evolutionary clustering and source-environment distribution patterns. The circular dendrogram architecture was constructed utilizing single-nucleotide polymorphism (SNP) analytical methodologies, providing high-resolution phylogenetic inference based on genome-wide sequence variation across all analyzed specimens. The phylogenetic tree exhibits systematic color-coded branch designations delineating serotype classification: orange branches represent serotype O5 isolates, while purple branches indicate serotype O12 strains. Triangular markers designated Belo Horizonte (Brazil) isolates, square markers represented Kunming (China) specimens, circular markers indicated Rio de Janeiro (Brazil) strains, filled circular markers signified São Paulo (Brazil) isolates, while hollow circular markers denoted strains of undetermined geographical origin.
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Figure 4. Phylogenomic reconstruction and source attribution analysis of cross-continental bacterial isolates. The dendrogram displays the phylogenetic relationships among bacterial isolates (n = 8) derived from diverse geographical locales and biological matrices. Genomic differentiation patterns are represented in a circular phylogenetic tree with integrated metadata visualization. Isolates exhibit serotype-specific stratification corresponding to O2 (red) and O5 (yellow-green) antigenic profiles. Geographic origin of isolates is denoted by standardized node symbology: Chicago, USA (cross); Hangzhou, China (square); Rio de Janeiro, Brazil (circle); and Zhejiang, China (diamond). Terminal branches are labeled with strain-specific alphanumeric identifiers (PAO1, PAL060, SRSH15, etc.), facilitating isolate tracking across epidemiological datasets. The outermost concentric ring (metadata layer #1) indicates biological source attribution through chromatic differentiation: blood specimens (red), clinical samples (magenta), catheter tip isolates (blue), sputum specimens (yellow), and wound isolates (green).
Figure 4. Phylogenomic reconstruction and source attribution analysis of cross-continental bacterial isolates. The dendrogram displays the phylogenetic relationships among bacterial isolates (n = 8) derived from diverse geographical locales and biological matrices. Genomic differentiation patterns are represented in a circular phylogenetic tree with integrated metadata visualization. Isolates exhibit serotype-specific stratification corresponding to O2 (red) and O5 (yellow-green) antigenic profiles. Geographic origin of isolates is denoted by standardized node symbology: Chicago, USA (cross); Hangzhou, China (square); Rio de Janeiro, Brazil (circle); and Zhejiang, China (diamond). Terminal branches are labeled with strain-specific alphanumeric identifiers (PAO1, PAL060, SRSH15, etc.), facilitating isolate tracking across epidemiological datasets. The outermost concentric ring (metadata layer #1) indicates biological source attribution through chromatic differentiation: blood specimens (red), clinical samples (magenta), catheter tip isolates (blue), sputum specimens (yellow), and wound isolates (green).
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Table 1. Key genomic features of isolated P. aeruginosa.
Table 1. Key genomic features of isolated P. aeruginosa.
StrainsSize
(bp)		GC Content (%)N50Number of Contigs (with PEGs)Number of CDSNumber of RNAs
32296,598,02966.0219,23878633562
32346,705,13966.0133,63995644762
32386,848,15766.0212,058108664861
32406,850,55466.0126,493123671262
32526,855,43066.0132,994119669261
35176,774,62866.0130,378110658161
35506,867,81566.0182,286101673662
36116,742,52466.0259,337102648263
38676,797,65366.0120,522122666960
38897,077,28665.7161,978130704361
Table 2. Comprehensive overview of the genetic determinants of antibiotic resistance and associated mobile genetic elements identified.
Table 2. Comprehensive overview of the genetic determinants of antibiotic resistance and associated mobile genetic elements identified.
IsolaterSTSourceSerotypeResfinderMobile ElementProbability *
322920964Hospital effluentO5aph(3′)-IIb; blaPAO; blaOXA-494; blaOXA-396; fosA; catB7ISPa1; ISPa6; ISPa22; ISPa32; ISPa52; ISPst50.9893
323420964Hospital effluentO5aph(3′)-IIb; blaPAO; blaOXA-494; blaOXA-396; fosA; catB7ISPa1; ISPa6; ISPa32; ISPa37; ISPa52; ISPst50.9901
324020964Hospital effluentO5aph(3′)-IIb; blaPAO; blaOXA-494; blaOXA-396; fosA; catB7ISPa1; ISPa6; ISPa22; ISPa32; ISPa37; ISPa52; ISPst50.9928
388920964ClinicalO5aph(3′)-IIb; aadA1; aac(6′)-Ib3; aac(6′)-Il; blaOXA-494; blaOXA-396; blaPAO; blaOXA-9; blaOXA-129; fosA; catB7; cmx; catB3; sul1; dfrA21; qacEISPa1; ISPa6; ISPa22; ISPa32; ISPa37; ISPa1000.9850
355075320Bronchial lavageO5aph(3′)-IIb; aadA6; ant(2′’)-Ia; blaPAO; blaOXA-494; blaOXA-396;fosA; catB7; cmx; crpP; sul1; qacEISPa1; ISPa6; ISPa22; ISPa32; ISPa37;0.9895
386775320BloodO5aph(3′)-IIb; aadA6; ant(2′’)-Ia; blaPAO; blaOXA-494; fosA; catB7; cmx; sul1; qacEISPa1; ISPa6; ISPa22; ISPa370.9802
323879892Hospital effluentO12aph(3′)-IIb; aac(6′)-Ib3; blaPAO; blaOXA-494; blaOXA-396; fosA; catB7; crpP; sul1; qacEISPa1; ISPa6; ISPa22; ISPa32; ISPa37; ISPa79; ISPre30.9856
325279892Hospital effluentO12aph(3′)-IIb; aac(6′)-Ib3; blaOXA-494; blaOXA-396; blaPAO; fosA; catB7; crpP; sul1; qacEISPa1; ISPa6; ISPa22; ISPa32; ISPa37; ISPa790.9885
351779892ClinicalO12aph(3′)-IIb; aac(6′)-Ib3; blaPAO; blaOXA-494; blaOXA-396; fosA; catB7; crpP; sul1; qacEISPa1; ISPa6; ISPa22; ISPa32; ISPa37; ISPa79; ISPa1635; ISPre30.9760
361179892Urinary tract
infection		O12aph(3′)-VIII; aadA1; aph(3′)-IIb; aac(6′)-Ib3; blaOXA-494; blaOXA-396; blaPAO; fosA; catB7; catB3; crpP; sul1; qacEISPa1; ISPa6; ISPa22; ISPa32; ISPa37; ISPa79; ISPa1635; ISPre30.9827
* Probability of being a human pathogen. Resistance genes were assigned based on established literature describing aminoglycoside-modifying enzymes, β-lactamases, fosfomycin, chloramphenicol, sulfonamide, trimethoprim, fluoroquinolone-associated, and quaternary ammonium compound resistance mechanisms according to the Comprehensive Antibiotic Resistance Database (CARD).
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Bianco, K.; Vianna, T.C.d.C.; Oliveira, S.S.d.; Montenegro, K.; Flores, C.; Nascimento, A.P.A.d.; Cardoso, A.M.; Clementino, M.M. Genomic Relationship Between High-Risk Pseudomonas aeruginosa Clone ST244 Serotypes O5 and O12 from Southeastern Brazil. Microbiol. Res. 2026, 17, 27. https://doi.org/10.3390/microbiolres17010027

AMA Style

Bianco K, Vianna TCdC, Oliveira SSd, Montenegro K, Flores C, Nascimento APAd, Cardoso AM, Clementino MM. Genomic Relationship Between High-Risk Pseudomonas aeruginosa Clone ST244 Serotypes O5 and O12 from Southeastern Brazil. Microbiology Research. 2026; 17(1):27. https://doi.org/10.3390/microbiolres17010027

Chicago/Turabian Style

Bianco, Kayo, Thereza Cristina da Costa Vianna, Samara Santanna de Oliveira, Kaylanne Montenegro, Claudia Flores, Ana Paula Alves do Nascimento, Alexander Machado Cardoso, and Maysa Mandetta Clementino. 2026. "Genomic Relationship Between High-Risk Pseudomonas aeruginosa Clone ST244 Serotypes O5 and O12 from Southeastern Brazil" Microbiology Research 17, no. 1: 27. https://doi.org/10.3390/microbiolres17010027

APA Style

Bianco, K., Vianna, T. C. d. C., Oliveira, S. S. d., Montenegro, K., Flores, C., Nascimento, A. P. A. d., Cardoso, A. M., & Clementino, M. M. (2026). Genomic Relationship Between High-Risk Pseudomonas aeruginosa Clone ST244 Serotypes O5 and O12 from Southeastern Brazil. Microbiology Research, 17(1), 27. https://doi.org/10.3390/microbiolres17010027

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