https://orcid.org/0009-0001-5733-3610
Figures
Abstract
This study marks the first identification and genomic characterization of Comamonas kerstersii isolates from diarrheal patients in Bangladesh. We carried out the whole genome sequencing of three C. kerstersii isolates to analyze genomic features using bioinformatics tools. We hypothesize that C. kerstersii can contribute to the diarrheal disease process through indirect mechanisms, potentially by interacting synergistically with other enteric pathogens such as Vibrio cholerae (both O1 and non-O1 serogroups). The presence of diverse virulence factors, including type IV pili, type VI secretion systems, chemotaxis proteins, and toxin genes such as zot and RTX, suggests a capacity for adhesion, motility, and immune evasion. Notably, genomic analyses indicate that C. kerstersii shares several offensive and defensive virulence factors with other pathogenic Comamonas spp, including mechanisms for biofilm formation, nutrient acquisition, and stress tolerance. These factors, combined with antimicrobial resistance genes identified genes – aph(6)-Id, aph(3”)-Ib, mph(E), mph(F), msr(E), sul2, and tet(A), may enhance survival and adaptability of C. kerstersii in the gut environment, potentially augmenting the pathogenicity of co-infecting diarrheal pathogens. These initial findings highlight the need for extensive genomic surveillance across diarrheal patients, along with further investigation into molecular interactions with co-pathogens that could reveal novel pathways influencing diarrheal disease outcomes.
Citation: Rib NI, Shams F, Khan F, Sharmin S, Hossain SA, Sadique A, et al. (2026) Genomic characterization of Comamonas kerstersii isolated from diarrheal patients in Bangladesh. PLoS One 21(4): e0346980. https://doi.org/10.1371/journal.pone.0346980
Editor: Babak Pakbin, Texas A&M University, UNITED STATES OF AMERICA
Received: December 11, 2025; Accepted: March 26, 2026; Published: April 10, 2026
Copyright: © 2026 Rib et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The 3 Comamonas kerstersii genome sequences and analysis from this study have been submitted to GenBank database, with accession numbers: JBEBZE000000000 (NG17), JBEBZF000000000 (NG13), and JBEBZG000000000(NG14).
Funding: This study was funded by the North South University Conference and Travel Research Grant (CTRG) (CTRG-20-SHLS-23), Bangladesh. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The increasing number of persistent infections caused by relatively unknown non-fermenting gram-negative rods is a crucial concern in healthcare environments [1]. These emerging pathogens, which include species from the Comamonas genus, are opportunistic and pose severe risk to global health [2]. One little-known species in the Comamonas genus is Comamonas kerstersii [3]. C. kerstersii was first recognized as a distinct species in 2003, alongside Comamonas aquatica and Comamonas terrigena [3–6].
This species belongs to rRNA homology group III and is characterized by the presence of a unique 23S rRNA gene and a distinct 16S rRNA gene sequence [7,8]. C. kerstersii was initially included in the C. terrigena complex [9]. Subsequent taxonomic studies by Willems et al. and Wauters, et al and demonstrated that C. terrigena contains 3 different DNA groups [4,10]. Based on the 16S rRNA sequencing and DNA-DNA hybridization analyses, DNA group I retained the classification C. terrigena, whereas DNA groups II and III were reclassified as C. aquatica and C. kerstersii, respectively [4].
C. kerstersi species are non-fermenting, gram-negative, rod-shaped bacillus [9]. The smooth-surfaced and non-pigmented colonies have a diameter of 1.5 mm on blood agar when grown under an ambient aerobic environment for 48 hours of incubation [9,11]. C. kerstersii has previously been considered nonpathogenic. It is extensively found in soil, plants, and water with the ability to withstand aquatic environments [3]. Although once considered nonpathogenic, C. kerstersii is now increasingly acknowledged as an environmental organism with opportunistic pathogenic potential, especially in polymicrobial intra-abdominal infections. In recent years, the species have been found in several clinical samples like abdominal fluid, peritoneal fluid, blood, urine, stool and recently in sputum [5,6,9,11–16]. Reported infections include conditions like intra-abdominal including appendicitis, peritonitis, diverticulosis, and a few extra abdominal cases, including urinary tract infection, bacteremia and pneumonia, have been reported [5,6,9,11–16]. Most infections associated with C. kerstersii have been polymicrobial in nature [2,17].
Species of the genus Comamonas are associated with certain human infections and exhibit diversified virulence factors [15,18]. Studies have shown that C. aquatica, C. terrigena, and C. kerstersii form a distinct cluster, sharing common virulence genes involved in bacterial motility, adhesion, and chemotaxis signaling [19]. These include adhesin biosynthesis genes like IlpA and Hsp60, biofilm-forming components, hemolysin genes, and stress response proteins such as ClpEP and SodB, which contribute to cellular defense [19].
In Bangladesh, diarrheal diseases are driven by a spectrum of enteric pathogens including V. cholerae, Shigella spp., Salmonella spp., rotavirus, entero-toxigenic Escherichia coli (ETEC), with ETEC and Vibrio cholerae O1 together accounting for nearly 50% of annual cases and exhibiting varying incidence and severity across age groups [20,21]. The studies report that mixed infections contribute to 12% to 26% of acute watery diarrhea cases [20,21]. Additionally, a significant proportion of diarrheal cases ranging from 36.5% to 51% across different surveillance periods have no identifiable enteric pathogen using conventional laboratory methods [20,22]. While this does not establish the presence of emerging or novel bacterial agents, it highlights the limitations of standard detection methods. Biswas et al. reported that over a period of two years, pure growth of C. kerstersii was isolated from 27 fecal samples submitted for diarrhea investigation and suggested that environmental exposure and subsequent carriage within the bowel may be more common than currently assumed [23]. Furthermore, Comamonas spp., including C. aquatica and C. testosteroni, have been detected in stool samples from patients with gastrointestinal symptoms like diarrhea and abdominal pain [24].
While direct causality remains unprovenit is still unclear if Comamonas spp. directly cause these symptoms, the repeated detection of C. kerstersii and related comamonas spp. in stool samples suggests a possible opportunistic role in diarrheal diseases specially in polymicrobial contexts. We hypothesize that C. kerstersii could participate in the pathogenesis of diarrheal diseases indirectly, possibly via synergistic relationships with other enteric pathogens. The presence of various offensive and defensive virulence factors including mechanisms for biofilm formation, nutrient acquisition, and stress tolerance in C. kerstersii and other Comamonas spp. [19], further indicates its capacity to survive and adapt in the gut environment and potentially augmenting the pathogenicity of co-infecting diarrheal pathogens.
Given its widespread distribution and increasing detection in clinical samples, it is important to investigate the genomic characteristics and pathogenic potential of C. kerstersii, particularly in regions like Bangladesh where data are limited. This study presents the first comprehensive genomic analysis of C. kerstersii isolates from diarrheal patients in Bangladesh. It provides insights into their antimicrobial resistance profiles and potential virulence factors, contributing to a better understanding of the role of this emerging organism in gastrointestinal infections.
Materials and methods
Sample collection, bacterial isolation and molecular identification
From 11 March 2020–12 November 2020, 17 stool samples were collected from patients presenting with acute gastroenteritis at Dhaka Central International Medical College & Hospital (DCIMCH) under institutional ethical approval (2020/OR-NSU/IRB/1205), with the initial aim of isolating V. cholerae. Prior to sample collection, each patient was informed of the study's purpose and the voluntary nature of participation. All individuals provided verbal informed consent prior to sample collection. The consent procedure was approved by North South University's Institutional Review Board (IRB). Verbal consent was documented by the attending clinician or research staff on the sample collection record, and the process was witnessed by a member of the clinical team. To protect the confidentiality of participants, no personally identifiable information was recorded.
Samples were transported in 5 mL Cary-Blair transport medium at 4°C. For enrichment, 3 mL of each sample was incubated in alkaline peptone water (APW) at 37°C for 6–8 hours. Following enrichment, aliquots were streaked onto thiosulfate-citrate-bile salts-sucrose (TCBS) agar and tellurite taurocholate gelatin agar (TTGA) plates, which were incubated overnight at 37°C. Yellow colonies on TCBS and black-centered colonies on TTGA—presumptively identified as V. cholerae based on colony morphology—were sub cultured onto gelatin agar (GA) for further screening.
Subsequent Gram staining and standard biochemical tests were performed for characterization of isolates. Of the 17 presumptive Vibrio isolates, 14 demonstrated gelatin hydrolysis on GA, consistent with V. cholerae characteristics. However, three isolates (NG13, NG14, and NG17) did not show gelatin hydrolysis and exhibited biochemical profiles inconsistent with V. cholerae. These three isolates were therefore subjected to 16S rRNA gene sequencing for definitive identification. Sequencing results confirmed that they belonged to the Comamonas genus, indicating initial misidentification based on conventional methods. A summary of their distinguishing biochemical profiles is provided in Table 2.
Genome sequencing, assembly, and annotation
Isolates were inoculated in lysogeny broth (LB) and grown at 37°C overnight for DNA extraction. Genomic DNA was extracted using QIAamp® DNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. Library preparation and sequencing of 3 selected strains were carried out at Genome Research Institute of North South University (NGRI), Bangladesh. High molecular weight genomic DNA of the strains was used to prepare libraries using a Nextera DNA Library Prep Kit and employed for 250-bp paired-end whole genome sequencing with Illumina® MiSeq platform following the instruction provided by the manufacturer. High-quality paired-end Illumina sequencing data (Q ≥ 30) were obtained for the bioinformatic analysis. Quality control was performed using Quast [25] on the sequencing data to ensure the integrity and accuracy of the information. SPAdes genome assembly software (version 3.15.5) [26] was used for the de novo assembly of the genome with 20x to 30x coverage and particular filters employed to lower the number of mismatches and short indels, improving the quality of the generated contigs. The PROKKA pipeline (version 45) [27] was employed for annotation to decipher the genetic content and functional elements encoded within the assembled contigs. Rapid Annotation using Subsystem Technology (RAST) [28], an automated service for annotating genomes of bacteria and archaea, was also employed for annotation.
Genomic feature determination
Nine genomes (S2 File) of C. kerstersii were obtained from the National Center for Biotechnology Information (NCBI). The obtained genomes were annotated using the PROKKA pipeline [27]. The core genome alignment was generated using Roary [29] and then used in FastTree [30] to construct a midpoint rooting phylogenetic tree and visualized using FigTree. Assembled genomes were visualized using the BLAST ring image generator (BRIG) [31]. Antimicrobial resistance genes were identified utilizing the Center for Genomic Epidemiology (CGE) based tool ResFinder (version 4.1) [32]. In this study, the Virulence Factor Database (VFDB) (http://www.mgc.ac.cn/VFs/) has been used to identify and analyze virulence factors associated with the studied strains [33]. Identification of plasmids was conducted employing PlasmidFinder [34] and phage regions using PHASTEST [35] and Phigaro [36] respectively.
Host pathogen protein-protein interaction (PPI) network construction
Amino acid sequences from the three C. kerstersii were clustered using MMseqs2 (Steinegger & Söding, 2017) (version 13.45111) with 90% identity and 80% coverage thresholds. To construct a host–pathogen interaction (HPI) network, 9,333 human–bacterial PPIs were retrieved from the PHISTO and 9,031 unique interactions were retained after removing duplicates. Corresponding bacterial protein sequences were then obtained from UniProt [37]. Clustered Comamonas proteins were aligned to these sequences using BLASTp [38] (NCBI BLAST+ v2.5.0) and the resulting predicted Comamonas–human PPI network was visualized and analyzed in NetworkX (version 2.8.8), identifying the top 10 hub proteins based on degree centrality. These hub proteins were subsequently functionally annotated using EggNOG-mapper v2 [39] to predict their biological roles through orthology-based inference. To further investigate host response, KEGG pathway enrichment analysis was performed on 1,360 human interacting proteins using the R package clusterProfiler (version 4.12.6) [40], applying a p-value and q-value cutoff of 0.05 with Benjamini-Hochberg correction. The top 15 significantly enriched pathways (FDR < 0.05) were selected, revealing key host cellular processes potentially targeted during Comamonas infection. It is important to note that the host–pathogen interactions reported here are computational predictions based on sequence similarity to experimentally validated interactions in the PHISTO database and have not been experimentally validated in this study.
Results
Clinical characteristics and bacterial identification
Three patients were found to carry bacterial isolates that were later identified as C. kerstersii (NG13, NG14, and NG17). All three presented with acute gastrointestinal symptoms, including watery diarrhea (three or more episodes per day) and vomiting, along with varying degrees of dehydration. Their demographic details, clinical history, presenting symptoms, and treatment are summarized in Table 1.
Patient 1 (sample NG13) was a 19-year-old male who consumed boiled water and reported probiotic intake in the form of curd. He experienced moderate dehydration accompanied by oliguria. He was treated with both oral and intravenous saline, as well as antibiotics. Patient 2 (sample NG14) was a 40-year-old female who consumed municipal (untreated) water and consumed curd. She presented with similar gastrointestinal symptoms but experienced more severe dehydration, including anuria lasting approximately 12 hours. She was managed with fluid and electrolyte replacement without antibiotic intervention. Patient 3 (sample NG17) was a 24-year-old female who consumed boiled water and curd. Her symptoms included diarrhea, vomiting, stomach pain, and oliguria, with signs of severe dehydration. Like Patient 1, she was treated with antibiotics in addition to oral and IV saline.
Initially, the bacterial isolates from these patients were presumptively identified as V. cholerae based on colony morphology on selective media. However, biochemical testing showed that all three isolates were oxidase- and catalase-positive, motile, and capable of reducing nitrate and hydrolyzing trypsin. Importantly, none of the isolates demonstrated gelatin hydrolysis, which is typically associated with V. cholerae. They also tested negative for acid production from glucose, urease activity, hydrogen sulfide (H₂S) production, and indole formation. Given the inconsistency with typical V. cholerae profiles, 16S rRNA gene sequencing was performed and confirmed the identity of all three isolates as Comamonas spp. The detailed biochemical characteristics of these isolates are provided in Table 2.
Genomics features
Draft genome assemblies of Comamonas strains—NG13, NG14, and NG17 yielded 50, 68, and 81 contigs respectively. The assembled genome sizes were approximately 3.48 Mbp for each strain, with a consistent GC content of around 59.6% (Fig 1).
Circular representation of the genomes of the strains NG13, NG14, and NG17 showcasing coding sequences (CDS), GC content and GC skew. The homology rate is indicated by color saturation; blanks indicate no resemblance. The blue circle represents the reference genome C. kerstersii 8943.
Gene prediction and annotation revealed a similar number of coding sequences (CDS) across all three isolates, ranging from 3,211–3,215. The number of tRNA genes varied slightly, with NG13 and NG17 containing 94 tRNAs and NG14 containing 97. All strains carried 7 rRNA genes and 1 transfer-messenger RNA (tmRNA). The N50 values, indicating assembly continuity, ranged from 181,861 bp (NG13) to 284,992 bp (NG14). A detailed summary of the genomic features is presented in Table 3.
Pan-core and phylogenetic analysis
A total of 5586 genes were used from all the strains of C. kerstersii (n = 12) for the pan genome analysis, of which 2079 comprised the core genome (99%−100% of strains). Among the accessory genome, 1825 genes were in the shell and 1682 genes were in the cloud. With the introduction of a new genome, the number of core genes declined and hit the plateau (Fig 2). While with each additional genome added, the size of the pan-genome increased steadily suggesting that the C. kerstersii has an open pan-genome. The pan-genome indicates that the strains under investigation possess unique genes that are not found in other strains, and that the size of the gene pool will only rise as more genomes are included in the analysis. But coming to such a conclusion should be taken with caution since these kinds of studies should ideally involve more than 100 genome sequences [41].
Box plots show the change in the number of genes with the sequential addition of genomes from 12 Comamonas kerstersii strains. (Left) Number of conserved (core) genes decreases as the additional genomes are incorporated and slowly approaches plateau. (Right) Number of genes in the pan-genome increases as the new genomes add up indicating open pan-genome.
Core genome analysis demonstrated close clustering among the three strains, indicating high genomic similarity and shared evolutionary lineage (Fig 3). The tree shows two distinct clusters – Cluster A and Cluster B. According to the phylogenetic tree, three C. kerstersii strains under study - NG13, NG14 and NG17, shared the same ancestor and belonged to Cluster A, with CP020121 being the closest relative. CP020121, also named C. kerstersii 8943, was isolated from the effluent of peritoneal dialysis, highlighting the close relationship among human-originated isolates. All these strains shared features like ampicillin resistance and sul2 and tetA genes conferring resistance to sulfonamide and tetracycline, respectively [42].
Mid-point rooted phylogenetic tree of publicly available C. kerstersii strains and sequenced isolates from Bangladesh shows relationship between the strains based on their core genome alignment. NG13, NG14 and NG17 belonging to CLADE A clustering together indicates their clonal character.
The CP060413 strain from the other cluster- Cluster B, was isolated from a rectal swab similar to the isolates of our study (Fig 3). Even being from the same clinical source, the CP060413 branched distantly. This strain had no resistance genes other than 2 class A and 1 class C beta-lactamase genes [43].
The C. kerstersii strains used in the phylogenetic analysis were isolated from human feces. The presence of various resistance genes across different strains underscores the role of horizontal gene transfer in enabling adaptation to diverse environments.
Identification of antimicrobial resistance genes (ARG)
Multiple antibiotic resistance genes were identified in all three C. kerstersii strains (Table 4). These included genes linked to resistance against β-lactams, aminoglycosides, tetracyclines, and sulfonamides, signifying a varied resistome profile. The tet(A) gene, potentially conferred resistance by exporting tetracycline out of the cell in exchange for protons, was identified [44].
The strains possessed mph(E), mph(F), and msr(E) genes. The mph(E) and mph(F) encode macrolide phosphotransferase enzymes that likely inactivate macrolides through phosphorylation [45]. The msr(E) gene may confer resistance to macrolides, lincosamides, and streptogramin B [46]. The presence of the aph(3”)-Ib and aph(6)-Id genes suggests resistance to streptomycin. These genes are often linked and encode enzymes that inactivate streptomycin. The presence of sul2 gene confer resistance to sulfonamides [47].
We found macA, macB and tolC encoding for macrolide export protein MacA, macrolide export ATP-binding/permease protein MacB and outer membrane protein TolC, respectively. This MacAB-TolC comprises a tripartite efflux system, contributing to resistance to the macrolide class of antibiotics in E. coli and other Gram-negative pathogenic bacteria [48]. Beyond antibiotic resistance, this complex is also involved in the transportation of protoporphyrin [49] lipopolysaccharides [50] and virulence factors, i.e., enterotoxin II [51]. Furthermore, several multidrug resistance protein-encoding genes are also present in the genomes of C. kerstersii strains (S1 File).
Adaptive mechanism and virulence
C. kerstersii can adapt to diverse environments, and annotating the genomes revealed some genes that enable them to withstand environmental stressors, including polyphosphate kinase (ppk). Polyphosphate (polyP) has previously been linked to osmotic and nutritional stress response in E. coli and bacterial growth, survival, and pathogenicity in other bacteria [52,53]. The C. kerstersii strains of the study possess osmY, a periplasmic protein strongly induced by hyperosmotic stimuli. RAST annotation of the isolates revealed an outer membrane protein A (ompA) precursor under the stress response category, also helping to survive a high osmolarity environment [54]. Additionally, a colicin V-producing gene (cvpA), crucial for gut colonization, is present in C. kerstersii strains [55,56].
Analysis using the Virulence Factor Database (VFDB identified key proteins potentially associated with pathogenicity in all three C. kerstersii strains (Table 5). Notable findings include proteins involved with type IV pili (T4P), type II secretion systems (T2SS), type VI secretion system (T6SS), general secretion pathways, and flagella. Both NG13 and NG14 strains encoded proteins involved in T4P biogenesis, including PilB and PilF. Strain NG17 possessed a PilJ protein. T4P plays a crucial role in pilus formation, adhesion, and bacterial motility, all of which contribute to pathogenesis. T4P is important in pilus formation, adhesion, and dynamics [57].
Genomic analysis of C. kerstersii identified two conserved Type IV pilus operons (Fig 4), each encoding essential components for pilus assembly, stability, and function. The first operon (Fig 4), associated with pilus assembly and adhesion, includes genes such as FimV, PilY1, PilC, and PilW. These proteins are critical for biogenesis, adhesion, and surface colonization [57–59]. The second operon encodes (Fig 4) the core machinery, including PilM, PilN, PilO, PilP, and PilQ, vital for pilus extrusion and stabilization [60]. Interestingly, PilA, the pilin subunit, was absent, indicating possible divergence or alternative pilin expression mechanisms. The presence of these operons underscores their roles in adhesion, motility, horizontal gene transfer, and biofilm formation, highlighting their ecological importance and adaptability in C. kerstersii [57–59].
The schematic diagram illustrates the organization of genes in two Type IV pilus assembly and adhesion operons. The top operon includes genes encoding FimV, PilY1, PilC, and PilW, while the bottom operon comprises PilM, PilN, PilO, PilP, and PilQ. These genes are involved in the assembly and function of the Type IV pilus, a structure critical for bacterial adhesion and motility. Each rectangle represents a gene, with the linear arrangement reflecting their genomic organization.
A thorough examination of the C. kerstersii genome, revealed several toxin coding sequences (CDS), including zona occludens toxin (zot) protein-encoding sequences that may play virulence-related activities. Other examples are apxIB, ratA, algC, algE, algE1, clpP and clpP1 (putative Clp proteolytic core), clpA, clpC, and clpX (Clp-ATPases), and clpS (adaptor protein). A cluster of genes involved in copper homeostasis was also found in the annotated genomes of C. kerstersii strains (Table 5).
C. kerstersii genomes were examined for the protein export and secretion system genes. We found SecA, SecB, and SecYEG encoding genes, although these genes are not present in a cluster. In contrast, TatABC genes encoding the components of the Tat apparatus are present in a cluster (S1 File).
Plasmids and prophages
In each of the three strains used in this investigation, we located a 157 bp-long fragment identical to plasmids of the IncQ family. 100% identity was found in the region between 5741 and 6536 base pairs (bp) in the plasmid when it was compared using BLAST against the E. coli plasmid RSF1010 (Accession no. M28829). The distinct strand-displacement replication mechanism of the IncQ-family of plasmids allows them to work in a broad range of bacterial hosts, and their high mobilization potential enables these plasmids to exhibit great promiscuity [61]. Several studies have mentioned plasmid IncQ to be multidrug-resistant, carrying tetA, Sul or carbapenem resistance gene blaKPC-2 [62,63] although the IncQ1 plasmid of the studied strain does not contain any resistance genes.
Prophage regions were identified in all three C. kerstersii strains (Figs 5 and 6). Each prophage region was approximately 63.6 kb in size with a GC content of ~58% and showed similarity to Pseudomonas phage F116 of the Podoviridae family. These regions encoded proteins were involved in phage replication (integrase, repressor, terminases), regulation (regulatory protein), and lysis proteins, tail proteins, holin) and contained a significant number of hypothetical proteins. Interestingly, Phigaro identified an additional prophage region belonging to the Siphoviridae family alongside the Podoviridae region in each strain (Fig 6a). This region encoded phage assembly proteins (tail, portal, coat proteins). Multiple prophage regions and many hypothetical proteins within them suggest potential roles in C. kerstersii fitness. Further investigation of these hypothetical proteins may provide valuable insights into their specific functions.
Genomic map of an identified intact prophage region where the arrows indicate the predicted open reading frames (ORFs). Each ORF is color coded according to predicted functional category including holin, phage-like protein, tail protein, lysis proteins. Uncharacterized proteins are depicted in grey.
Linear genomic maps of two distinct viral regions with annotated functional genes. (a) A phage region belonging to the Siphoviridae family with color-coded features highlighting genes encoding coat, tail, assembly, and portal proteins. (b) A phage region belonging to the Podoviridae family with color-coded features highlighting genes responsible for lysis, terminase, tail, and portal functions. Genes lacking specific functional predictions are classified as “Other” and shown in grey.
Comamonas kerstersii host-pathogen PPI network
Following the BLASTp alignment of clustered Comamonas protein sequences with bacterial proteins from the PHISTO database, 1,269 homologous Comamonas proteins were identified. These proteins corresponded to known human–bacterial interactions in PHISTO, leading to the prediction of 2,119 protein–protein interactions (PPIs) between Comamonas and human proteins (Fig 7a). The PPI network analysis identified the top 10 Comamonas hub proteins based on degree centrality. These proteins were Q5NF74, Q7CGS9, Q7CHZ0, Q5NGF1, Q5NFP9, Q8CZZ7, Q8D0D4, Q5NIP6, Q7CKZ7, and Q5NIP5. Together, these hub proteins interacted with 172 unique human proteins. This formed a subnetwork consisting of 184 predicted Comamonas–human protein–protein interactions (Fig 7b).
(a) Host-pathogen PPI network illustrates predicted interactions where green nodes represent C. kerstersii proteins, while orange nodes represent human proteins. Edges indicate predicted interactions based on sequence similarity to experimentally validated host–pathogen interactions. (b) Subnetwork of the top 10 C. kerstersii hub proteins and their human interaction partners. Green nodes represent the top 10 C. kerstersii proteins with the highest number of interactions. Orange nodes represent human proteins.
The top 10 C. kerstersii hub proteins (Table 6) are involved in a range of functions essential for bacterial survival and interaction with the host. TktA (Q5NF74) and PurC (Q5NGF1) are linked to core metabolic pathways, such as the pentose phosphate pathway and purine biosynthesis, which are important for energy production and nucleotide formation. PgaA (Q8D0D4) is associated with the export of exopolysaccharides, suggesting a role in biofilm formation and surface adhesion. RecN (Q8CZZ7), a DNA repair protein, likely helps the bacteria cope with genomic stress. Two proteins, GatA (Q5NIP6) and GatB (Q5NIP5), function in the transamidation pathway to maintain translation accuracy by correcting mischarged tRNAs—this is particularly important in organisms lacking specific tRNA synthetases. FliN (Q7CHZ0), a component of the flagellar motor switch, supports motility and may aid in navigating the host environment. StbC (Q7CKZ7), which contains a PapC domain, is possibly involved in pilus formation, contributing to host attachment. Another hub, PepP (Q7CGS9), belongs to the peptidase M24B family and may assist in protein degradation or processing. Overall, the identified hub proteins highlight C. kerstersii's reliance on metabolic adaptability, stress response, motility, and host interaction mechanisms—features that may play important roles in its potential pathogenic behavior.
Enrichment analysis showed significant representation of pathways associated with infectious diseases (Fig 8), including Epstein–Barr virus infection, Herpes simplex virus 1 infection, Human cytomegalovirus infection, and Tuberculosis, suggesting a shared host response mechanism to various pathogens. Notably, Salmonella infection, Shigellosis, and Leishmaniasis were also significantly enriched, highlighting parallels with known bacterial infections. Immune-related pathways such as Antigen processing and presentation, Endocytosis, and Human T-cell leukemia virus 1 infection were prominently represented, indicating a strong involvement of immune signaling in response to the bacterial proteins.
The bar plot shows the top 15 significantly enriched pathways based on adjusted p-values (Benjamini–Hochberg method). The color gradient represents the adjusted p-value, with lighter colors indicating higher significance. Count represents the number of human proteins involved in each pathway.
The pathway Hepatitis B showed the strongest statistical significance (lowest adjusted p-value), followed by Human T-cell leukemia virus 1 infection and Osteoclast differentiation, suggesting specific immunological and possibly inflammatory processes may be targeted by the pathogen. The Hepatitis B pathway had the lowest adjusted p-value among the enriched pathways, followed by Human T-cell leukemia virus 1 infection and Osteoclast differentiation. These results indicate statistically supported enrichment of immune-related pathways among the host proteins interacting with C. kerstersii. Additionally, the involvement of pathways such as Measles and Pertussis further supports the hypothesis that C. kerstersii may engage immune pathways common to viral and bacterial infections.
Discussion
C. kerstersii is becoming a prominent clinical and environmental pathogen due to its broad illness spectrum, ubiquity, adaptability in various environments and resistance capabilities. These characteristics reflect a high degree of genomic and functional diversity, underscoring the need for a deeper understanding of how C. kerstersii adapts to diverse ecological niches. This study marks the first identification of C. kerstersii in Bangladesh; thus, genomic analysis and understanding the behavior of this bacterium is crucial for mitigating potential public health risks associated with its infections.
In this study, we characterized three C. kerstersii isolates (NG13, NG14, and NG17) and compared our results with those from Jiang et al. regarding C. kerstersii 8943 [42]. Our isolates show genome sizes of about 3.48 Mb and a GC content of roughly 59.6%, which aligns well with the 3.55 Mb circular genome described by Jiang et al [42]. This similarity indicates that C. kerstersii has a relatively compact genome compared to other Comamonas species, as previously mentioned [42]. We noticed some significant differences in the antibiotic resistance gene profiles between our isolates and the strain in different studies. While Jiang et al. found genes like tetA, strB, sul1, blaOXA-1, strA, sul2, catB3, and floR, our isolates carry aph(6)-Id, aph(3”)-Ib, mph(E), mph(F), msr(E), sul2, and tet(A). The presence of aph(6)-Id and aph(3”)-Ib suggests a different way of developing streptomycin resistance compared to strA and strB. Other studies back up these findings; one noted resistance to ciprofloxacin [64], and another highlighted resistance to ampicillin [65]. Moreover, a genetic analysis of the C. kerstersii strain 3132976 uncovered three β-lactamase genes (CDSs H8N02_05890, H8N02_08740, and H8N02_17110), with H8N02_08740 and H8N02_17110 categorized as class A and C β-lactamases, respectively [66]. Most strikingly, a recent report on strain 121606 indicated resistance to nearly all β-lactam antibiotics, showcasing a broad drug-resistance profile [16]. All in all, these findings underscore the genetic diversity and changing resistance patterns in C. kerstersii, highlighting the importance of ongoing monitoring to better inform treatment strategies.
Several studies have mentioned the phylogeny of C. kerstersii, for instance, Wu et al., Zhang et al. and Jiang et al. constructed phylogenetic trees [19,42,63], however, the construction of these phylogenies were within the species of the genus Comamonas rather than on comparisons among different C. kerstersii strains [19,61]. In the present work, we constructed a phylogenetic tree including nine available C. kerstersii strains, which revealed clonal relationships alongside evidence of diversity and adaptation to various ecological niches.
One protein of particular interest in conferring niche adaptability is polyphosphate kinase (ppk). The presence of this enzyme explains how C. kerstersii is equipped to withstand osmotic stresses in various aquatic environments like blood, urine, or peritoneal fluid. In V. cholerae, polyphosphate reserves enhance resistance to environmental stressors, especially under phosphate-limited conditions [67]. The presence of ppk in C. kerstersii suggests a similar mechanism, enabling it to survive in low-phosphate environments (e.g., excrement). PolyP has also been implicated in virulence, including motility and biofilm formation, in a few studies [68–70]. Furthermore, our isolates harbor osmY, a periplasmic protein induced by hyperosmotic stimuli, indicating that C. kerstersii may tolerate osmotic pressure in environments such as the bladder or colon, as observed in Escherichia coli [71]. The outer membrane protein A (ompA) precursor of C. kerstersii may enable it to withstand high osmolarity as the protein equips E. coli to survive in an acidic environment, high osmolarity, and pooled human serum [54]. OmpA has also been associated with virulence factors like adhesion, invasion, intracellular survival, host defense evasion, and these activities are especially important in the urogenital, respiratory, or nervous system diseases [72]. The inner membrane protein required for strong colonization of the gut is encoded by the cvpA gene [55], and the presence of this gene in C. kerstersii strains of the study explains its adaptability in the gut of diarrheal patients.
Our three clinical isolates possess additional virulence factors beyond T4P and T6SS. Genes involved in flagellar morphogenesis, assembly, and chemotaxis (S1 File) emphasizes the role of mobility in bacterial pathogenic strategies. To get nutrition and adjust to changing and favorable environmental conditions, bacteria use motility and chemotaxis. In pathogenic bacteria, chemotaxis contributes to host invasion and infection by allowing the bacteria to sense and move towards host tissues and nutrients [73]. Several toxins (S1 File) were also identified in our analysis, including apxIB, which codes for the Toxin RTX-I translocation ATP-binding protein. RTX toxins are known for their rich content of glycine and aspartate, and they are secreted by the type I secretion system found in Gram-negative bacteria [74]. A well-known RTX protein, hemolysin, is produced by the tlyA gene in our isolates and has the potential to disrupt the intestinal barrier, leading to symptoms of diarrhea [75]. Additionally, we identified ratA gene, which encodes Ribosome association toxin RatA, a component of type II toxin-antitoxin (TA) system. In E. coli, the association of RatA protein with the 50S subunit of ribosome prevents the binding of small subunit 30S, thereby preventing the formation of 70S ribosomes, leading to a decrease in protein translation [76].
The C. kerstersii strains possess genes that encode proteins involved in the alginate biosynthesis pathway, Phosphomannomutase/phosphoglucomutase, Poly (beta-D-mannuronate) C5 epimerase 1, Poly (beta-D-mannuronate) C5 epimerase 5 encoded by algC, algE, algE1, respectively. A negative regulator of alginate synthesis called alginate biosynthesis sensor protein KinB, encoding gene kinB is also present in the genome. AlgC protein plays a vital role in producing alginate, LPS and rhamnolipid. Three important P. aeruginosa virulence-associated factors Alginate-producing P. aeruginosa strains have a mucoid phenotype and are protected from antibiotics and other host defense components [77]. The holin protein present in the phage region can also induce pathogenicity. The enhanced release of extracellular toxins in E. coli, including SheA and Stx1, has been linked to phage-encoded Holins [78]. According to several investigations, holins most likely also have a significant function in the production of biofilms [79]. For instance, it is known that the Staphylococcus aureus cid and lrg operons regulate cell lysis and the release of genomic DNA, which eventually becomes a structural element of the biofilm matrix and is thus implicated in the production of biofilms [79]. It is also plausible that these strains have acquired virulent characteristics from the other species that may have coexisted in the infection milieu.
Clp protease (caseinolytic protease; ClpP) is a highly conserved serine protease found in many bacteria. This ATP-dependent protease functions as a two-component complex consisting of a regulatory ATPase subunit and a proteolytic subunit. While the proteolytic subunit possesses some intrinsic catalytic activity, both components are essential for optimal enzymatic function in the presence of ATP [80,81]. We identified genes encoding various components of the Clp protease complex, including clpP and clpP1 (putative Clp proteolytic core), clpA, clpC, and clpX (Clp-ATPases), and clpS (adaptor protein). proteases play crucial roles in various cellular processes, including the degradation of misfolded proteins, regulation of short-lived proteins, and housekeeping removal of dysfunctional proteins. They also control cell growth and target DNA-binding proteins from starved cells. ClpP has also been linked to the tight regulation of virulence genes in Gram-positive and Gram-negative pathogens such as Staphylococcus aureus, Listeria monocytogenes and Salmonella typhimurium [80,81]. Therefore, the presence of Clp protease complex encoding genes indicates the contribution of this system to the pathogenic lifestyle of the C. kerstersii strains.
We examined the C. kerstersii genomes and found that several genes are present to encode enzymes and proteins involved in iron uptake mechanisms. Iron is often limited in host environments as part of the innate immune response, so bacteria must actively scavenge iron from sources like haemoglobin. This iron homeostasis is closely linked to bacterial virulence, stress resistance, and overall fitness. Enterobactin is a siderophore that can extract iron from host iron-binding proteins like transferrin. Haemoglobin-haptoglobin binding proteins allow bacteria to retrieve heme iron from haemoglobin, a major iron source in the host [82]. Iron acquisition regulators like Fur control the expression of iron uptake systems in response to iron availability, allowing bacteria to tightly regulate iron uptake to avoid toxicity while ensuring sufficient iron for growth [82]. All these proteins are key adaptations that enable C. kerstersii to efficiently scavenge iron from the host, a critical nutrient for growth and virulence. In addition, several genes encode key enzymes in the heme biosynthesis pathway that is essential for bacterial growth, metabolism, and virulence. Targeting these enzymes could be a potential antimicrobial strategy.
Alternatively, C. kerstersii may aid in the infection process through systems not generally linked to virulence but through resistance mechanisms like regulation of copper homeostasis. A cluster of genes involved in copper homeostasis was found in the annotated genomes of the strains (Table 5). Li et al. mentioned that a crucial factor in the pathophysiology of bacteria is copper homeostasis, which involves the complex interaction of bacterial survival strategies and hosts defenses [83]. Host macrophages deliberately create a copper overdose environment to fight invasive pathogens, exposing a host-driven defense mechanism [32]. Copper homeostasis-related genes, such as copA (efflux system), copZ (copper chaperone), cueO (copper oxidase), copS (sensor protein), and copR (transcriptional activator), were present in all three of the C. kerstersii strains included in this study. Notably, studies have shown that loss of the copper efflux pathway (CopA) in certain bacteria, such as S. pneumoniae, reduces virulence during infection [84]. This discovery implies that the pathogenic potential of C. kerstersii may depend on its capacity to maintain copper homeostasis.
Protein export systems regulate the secretion of different types of proteins across the cell envelope and thus have an important role in colonizing bacteria living in various niches. In Comamonas species, the Sec (secretion) and Tat (twin-arginine translocation) pathways are essential for protein transport across the cytoplasmic membrane [85]. The Sec pathway typically handles the translocation of unfolded proteins, whereas the Tat pathway is specialized for transporting fully folded proteins. Most proteins transported by the Sec and Tat pathways remain within the periplasm inner membrane and may be transported in the extracellular environment through type II or type V secretion systems [86]. The genes epsE, gspF and out encode components of the type II secretion system and genes to encode the Chaperone-Usher pathway (S1 File). The chaperone–usher (CU) pathway is the most widespread method for assembling adhesive surface pili on the surface of Gram-negative bacteria. For uropathogenic Escherichia coli and Pseudomonas aeruginosa, these adhesive pili-mediated localization and host colonization and biofilm formation, are considered as important virulence factors [86].
Genomic analysis of C. kerstersii has identified two conserved Type IV pilus (T4P) operons crucial to the bacterium's pathogenicity. The first operon is responsible for pilus assembly, adhesion, and surface colonization, and the other operon provides the machinery required for pilus extrusion and stabilization. However, the lack of PilA, the typical pilin subunit, suggests unique evolutionary adaptations. For example, divergence of operon or alternative pilin mechanisms, whereas variability in PilC points to potential strain-specific adaptations. In many Gram-negative pathogens, including V. cholerae and Escherichia coli type IV pili are widely recognized as critical virulence factors. For V. cholerae, the toxin-coregulated pilus (TCP) facilitates adherence to intestinal epithelial cells, which is a vital step in infection and disease development [59]. In E. coli O157:H7, hemorrhagic coli pili (HCP) are essential for adherence to host cells, with mutations in the hcpA gene, significantly reducing colonization [87]. Identifying these two conserved T4P operons in C. kerstersii highlights their pivotal role in adhesion, motility, biofilm formation, and horizontal gene transfer [88], emphasizing their importance in ecological adaptability and pathogenic potential.
The virulence genes, including the toxin proteins zot and RTX, responsible for diarrhea, coupled with the biofilm-producing protein holin, allow C. kerstersii to spread infections and fend off host immunological reactions. Although the limited number of samples of C. kerstersii is one of the notable limitations of this study and other investigations to fully understand the true nature and genetic diversity of this species. To acquire a comprehensive knowledge of the genetic diversity of this species at the spatial-temporal level and to obtain a more accurate picture of the genetic variety of C. kerstersii circulating across the country, it is crucial to increase the number of samples from various sources and places.
The predicted host–pathogen PPI network between C. kerstersii and human proteins uncovers mechanisms involved in both host manipulation and bacterial survival. The top 10 hub proteins uncovered metabolic enzymes, DNA repair proteins, motility apparatuses, along with adhesion-related proteins which point toward C. kerstersii's reliance on stress tolerance, metabolizing energy, exhibiting motility, as well as interacting with a host, all of which could link to its opportunistic pathogenic capability. Importantly, KEGG pathway enrichment for human interactors had an important overlap with immune-related and infectious disease pathways, which include those implicated in viral infections (e.g., Hepatitis B, EBV, HTLV-1), bacterial infections (e.g., Salmonella, Shigella), and antigen presentation. According to a trend, C. kerstersii may exploit host immune responses like other pathogens do. Pathways implicated in inflammatory responses and immune modulation are participating, so C. kerstersii may disrupt host defense systems. More research is justified into its role in human disease, especially in hosts with immunocompromised or microbiome disruption.
As a relatively new opportunistic pathogen, C. kerstersii has received little attention in scientific research. Only two papers have explored and reported the genomic analysis of this species thus far suggesting that the genomic microbial databases might have incomplete information on C. kerstersii. This lack of information may have made it difficult to identify every gene connected to the organism. The presence of many hypothetical proteins has been discovered by the annotated data of the studied strains. These proteins are hypothetical gene products, the roles of which are neither entirely understood nor confirmed by experiments. Even though they are speculative, these potential proteins can provide important information about the genetic composition of C. kerstersii and could be useful research targets to clarify their functions in pathogenicity and antibiotic resistance.
Conclusion
Most researchers focus on well-known pathogens like V. cholerae and E. coli frequently when isolating microorganisms from diarrheal patients. However, recent advances in high throughput genomics facilities have identified other potential pathogens from diarrheal samples. It is imperative that we adopt state-of-the-art genomics technologies and expand our research focus to consider other possible pathogens like C. kerstersii. Significant insights into the role of C. kerstersii in diarrheal disorders can be gained from an understanding of its pathophysiology and genetic features. By uncovering previously unknown infections, this widened focus may contribute to developing more thorough diagnostic procedures and focused therapeutic methods. Researchers can improve public health responses and our understanding of diarrheal disorders by investigating the genomes of C. kerstersii to identify its virulence factors, resistance mechanisms, and potential epidemiological consequences.
Supporting information
S1 File. Prokka annotated genes of the strains NG13, NG14, NG17.
https://doi.org/10.1371/journal.pone.0346980.s001
(XLSX)
S2 File. Nine publicly available genomes of C. kerstersii obtained from (NCBI) used for core-pan genome analysis and phylogenetic tree construction.
https://doi.org/10.1371/journal.pone.0346980.s002
(XLSX)
Acknowledgments
We thank Dhaka Central International Medical College & Hospital (DCIMCH) for the assistance with sample and data collection. We are grateful to NSU Genome Research Institution (NGRI) for their support to carry out lab experiments and genomic analyses. We note that an earlier version of this manuscript has been deposited as preprint on MedRxiv (Doi: 10.1101/2024.07.02.24309841) and Authorea (Doi: 10.22541/au.172115040.03950879/v1).
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