Insights into the taxonomy and virulence-related genetic profiles in Cupriavidus by comparative genomic analysis

doi:10.21203/rs.3.rs-4291589/v1

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Insights into the taxonomy and virulence-related genetic profiles in Cupriavidus by comparative genomic analysis

https://doi.org/10.21203/rs.3.rs-4291589/v1

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Background: Cupriavidusspecies have significant potential for environmental and industrial applications. Some have been recognized as opportunistic pathogens and are increasingly reported to be related to human infections. However, the taxonomic classification of Cupriavidus at the species level remains challenging. This may partly limit the understanding of pathogenesis in Cupriavidus.Here, we aim to clarify the Cupriavidus taxonomy and elucidate virulence-related genetic profiles using comparative genomic analysis.

Results:This study used eight Cupriavidus species to investigate the taxonomy of Cupriavidus. Of these, C. alkaliphilus, C. basilensis, C. gilardii, C. oxalaticus, C. necator, and C. taiwanensis are species complex, and the first four species are first reported as the species complex. More importantly, C. alkaliphilus and part of C. taiwanensis mixed, suggesting that some strains of these two species were probably mislabeled. The open pan-genome and prevalence of mobile genetic elements indicated high genetic variation in Cupriavidus. A total of 47 genes related to virulence factors were identified in Cupriavidus. Virulence factors in almost all Cupriavidus strains were related to the antimicrobial activity (acrB), biofilm (adeG, algU), stress survival (clpP, katA, sodB, ureA, ureB, and ureG), adherence (htpB,kdsA) and others (icl); the opportunistic pathogens, C. gilardii, and C. metallidurans, contained extra virulence genes (plc-2). Furthermore, six types of secretion systems (T1SS-T6SS) were identified in Cupriavidus. T2SS was conserved across all eight species; the other secretion systems presented diverse distribution. Interestingly, C. gilardii possesses two different T3SS clusters. Finally, emre, responsible for the efflux pump of aminoglycoside antibiotics, is a major antibiotic resistance gene of Cupriavidus strains. Other genes related to aminoglycosides, β-lactam, fosfomycin, and multidrug resistance are species-specific among opportunistic pathogens individually.

Conclusions: This study for the first time investigated the taxonomic classification ofCupriavidus andvirulence-related genetic profiles by comparative genomic analysis. Findings from this study lead us to propose updating the taxonomy of the six identified Cupriavidusspecies containing species complex. The virulence-related information, including virulence factors, secretion systems, and antibiotic resistance genes, provides insights into understanding the pathogenesis of Cupriavidus and may facilitate the diagnosis of infections caused by pathogenic Cupriavidus spp.

Cupriavidus pauculus

Species complex

Virulence factors

Secretion system

Antibiotic resistance

Bacterial species taxonomy is crucial to basic microbial research and applications [1]. An indisputable fact, however, is that bacterial identification and classification seem more puzzling in the molecular era with unprecedented genome sequences available [2]. The bacterial species complex, closely related microbial species sharing similar phenotypic features but with different genomic characteristics, has become not uncommon since genome-based methods were introduced into prokaryotic taxonomy [3, 4]. The issue of species complex has been recognized in some bacteria, for example, Burkholderia cepacia, Acinetobacter calcoaceticus, and Klebsiella oxytoca etc [3, 5]. Nonetheless, bacterial species remain largely underexplored compared to the similar problem described as "cryptic species" in other life entities, such as fungi, algae, and animals [6].

Cupriavidus, Gram-negative, belonging to the family Burkholderiaceae with the class Betaproteobacteria, is found in various environmental niches (soil, waters, plants, and clinical sources) [7, 8]. The species of the genus Cupriavidus have multiple implications for industry, biotechnology, and environmental sciences [9–11]. More importantly, some Cupriavidus species emerged as opportunistic pathogens and were resistant to many antibiotics [12, 13]. Nonetheless, studies on the opportunistic pathogens of Cupriavidus were commonly limited to case reports. The difficulty in correctly classifying Cupriavidus species using biochemical and genetic approaches partly limits understanding of the pathogenicity of Cupriavidus [14–18]. To date, two Cupriavidus species, namely C. necator and C. taiwanensis, have been identified as species complex, not a single species, by average nucleotide identity (ANI) and in silico DNA–DNA hybridization (isDDH) [19, 20]. Given the fact that Cupriavidus species shared a high degree of similarity of 16S rRNA sequences (> 97.5%), the 16S rRNA sequences are considered to have limited taxonomic resolution for some Cupriavidus species. [21–23]. Accordingly, we hypothesize that there are likely to be present other species complex within the Cupriavidus genus except for C. necator and C. taiwanensis.

In addition to the species complex, another critical issue regarding Cupriavidus species is that some Cupriavidus species, i.e., C. gilardii, C. metallidurans, and C. pauculus, have been increasingly reported as opportunistic pathogens involved in human diseases, for instance, bacteremia, meningitis, and sepsis [18, 24–27]. Worse still, a growing number of Cupriavidus species, including C. basilensis, C. gilardii, C. metallidurans, C. pauculus, C. respiraculi, and C. taiwanensis, have been isolated from specimens of cystic fibrosis (CF) patients and have gained increasing attention [25, 28–30]. Nevertheless, the pathogenicity of the genus Cupriavidus in human infections remains unclear. [12, 31]. To date, the virulence-related genetic profiles of Cupriavidus, like virulence factors and secretion systems, have not been reported. Moreover, there are few studies involving antibiotic resistance genes of C. gilardii by genomic analysis so far [13, 32]. Thus, more knowledge about the accurate taxonomy and virulence-related aspects of Cupriavidus is urgently needed.

In this study, we aim to (1) validate our hypothesis that there is likely to be another species complex within the Cupriavidus genus except for C. necator and C. taiwanensis and clarify the taxonomy of Cupriavidus, (2) elucidate virulence-related genetic profiles contributing to understanding the pathogenesis of Cupriavidus using comparative genomic analysis.

Accessing genomes of Cupriavidus strains

The present study used 97 genomes of Cupriavidus for comparative analysis. Of these, one genome of C. gilardii CR3 was sequenced in our previous study [33]; other genomes of Cupriavidus species were collected from the National Center for Biotechnology Information (NCBI). To ensure the reliability of the results of the comparative genomic analysis, a species should have at least three genomes available in NCBI. As such, 97 genomes belonging to eight species (C. alkaliphilus, C. basilensis, C. gilardii, C. pauculus, C. metallidurans, C. oxalaticus, C. necator, and C. taiwanensis) were satisfied. The detailed information on the genomes used in this study was summarized in Table. S1.

Phylogenetic tree, ANI and isDDH analysis

To validate our hypothesis that there is likely to be another species complex within the Cupriavidus, the 97 genomes of Cupriavidus strains were used to construct the phylogenetic tree to determine their phylogenetic relationship. The maximum likelihood (ML) phylogenetic tree was established based on core single nucleotide polymorphism (SNP) by MEGA11 with 1000 bootstraps. Specifically, Kchooser was used to select the reference kmer size (kmer value=23). The SNP matrix was generated by kSNPv3.0 [34]. ANI and isDDH analysis were employed to ensure precise species assignments at the species level [35]. ANI was calculated using FastANI with default settings [36]. isDDH was performed using the genome-to-genome distance calculator (Recommend formula 2) [37]. The genomic similarity between strains was determined by either the ANI cutoff value (> 95%) or isDDH cutoff value (> 70%) [38, 39]. The visualization of ANI and isDDH was used by pheatmap R packages.

Pan-genome analysis

Pan-genome analysis was carried out by BPGA v1.3 [40] using default parameters, in which 50% sequence identity as the cutoff for clustering identity was applied to USEARCH.

Gene function category

We used COG databases to analyze the functional classes of core gene families. Specifically, genomic protein annotation was performed on the eggNOG database using the eggNOGmapper (v2) [41] with "diamond blastp" at default settings.

Identification of mobile genetic elements

This study identified the mobile genetic elements in the tested Cupriavidus species from three facets: genomic islands, prophages, and clustered regularly interspaced short palindromic repeats (CRISPRs). Genomic islands of Cupriavidus were predicted using IslandViewer 4 [42]. Prophages were identified using PHASTER [43]. The CRISPRs were determined using the CRISPR recognition tool (CRT1.2) with default parameters [44].

Pathogenicity prediction and TA system identification

The PathogenFinder v1.1 [45] was employed to assess the potential pathogenicity in humans of the tested Cupriavidus species. TAfinder [46] was used to identify the Type I-VIII toxin-antitoxin (TA) system in tested Cupriavidus species.

Identification of secretion systems

Secretion systems of tested Cupriavidus species were analyzed using MacSyFinder by default parameters [47]. We calculated the Codon Adaptation Index (CAI) value and GC content by condW. Furthermore, the T6SS was also classified using the T6SS classification tool in SecReT6 [48].

Identification of virulence and antibiotic resistance genes

To identify virulence-related genes and antibiotic resistance genes in the tested Cupriavidus species, the protein sequences from all the selected 97 genomes were aligned with a dataset from the virulence factors database (VFDB) and comprehensive antibiotic resistance database (CARD), respectively. The parameters were as follows: E-value threshold <1e-6, identity threshold >60%, and coverage > 60% [49, 50].

Phylogenetic, ANI and isDDH analysis

In the eight tested Cupriavidus species, we found that six species of C. alkaliphilus, C. basilensis, C. gilardii, C. oxalaticus, C. necator, and C. taiwanensis are species complex based on phylogenetic analysis coupled ANI and isDDH (Fig. 1). Among the six species complex, C. taiwanensis and C. alkaliphilus are the most challenging problems because all C. alkaliphilus strains (7 strains) mixed with parts of C. taiwanensis (11 of 42 strains) and formed two new groups (group19, group20). Strains in each new group shared a high value of genomic similarity. For example, in group 19, genomic similarity (value of either ANI or isDDH) between C. alkaliphilus MLR2-44 and C. taiwanensis LMG 19430 was all over 99.8%. Thus, we inferred that some C. alkaliphilus and C. taiwanensis strains might be mislabeled.

Notedly, the ANI cutoff of 95% could not completely differentiate 17 C. taiwanensis strains and 7 C. alkaliphilus strains. Accordingly, the ANI cut-off value for these strains was adjusted from 95% to 96%, whereas the ANI cut-off value for other strains was maintained at 95%. As a result, 17 C. taiwanensis strains and 7 C. alkaliphilus strains were successfully separated into three groups (groups 18-20). In this way, six species (C. alkaliphilus, C. basilensis, C. gilardii, C. oxalaticus, C. necator, and C. taiwanensis) of the tested eight Cupriavidus species were identified as species complex, which included 18 genomic groups.

Characterization of pan-genome and core genome

The pan-genome in the eight species of Cupriavidus contained 37991 gene families (Fig. 2a). The pan-genome data fitted a power law curve (y = ax^b, b=0.37). These results showed that the pan-genome of Cupriavidus was open due to the value of b > 0 [51]. In the case of the core genome, the number of core gene families was 1616; the core genome accounts for 4.25% of the pan-genome. The remaining gene families (accessory and unique genome) amounted to 36375 (95.75%); they formed the variable genome of Cupriavidus, suggesting that Cupriavidus strains have a high degree of genetic variation. COG assignments were employed further to determine the function categories of the core gene families of Cupriavidus (Fig. 2b). A total of 1578 core gene families were annotated. A larger number of core gene families of Cupriavidus were involved in the cell wall/membrane/envelope biogenesis (category M: 98 gene families); translation, ribosomal structure and biogenesis (category J: 134 gene families); transcription (category K: 121 gene families); energy production and conversion (category C: 135 gene families); and amino acid transport and metabolism (category E: 168 gene families). Notably, function unknown (category S: 308 gene families) is the most significant amount of the core genome, indicating that the current knowledge of Cupriavidus is limited.

Mobile genetic elements, TA system, and pathogenicity prediction

The features of mobile genetic elements in Cupriavidus were described from three facets: genomic islands, prophages, and CRISPRs (Table. S2). Genomic islands are among the most abundant mobile genetic elements. All the 97 Cupriavidus strains have genomic islands; the number of genomic islands ranged from 2 to 162, with an average of 59. Prophages take the second place. Most strains (71/97) possessed between 1 to 8 prophages. CRISPRs were the least type of mobile genetic elements; only nine strains had CRISPRs. Overall, genomic islands are the most significant mobile genetic elements in Cupriavidus, which might be responsible for a high degree of genetic variation and promote the Cupriavidus strain's adaptation to various harsh environments and stresses.

The TA system, widely spreading in bacterial genomes, comprises a gene pair encoding a toxin and its corresponding antitoxin [52]. It is crucial in various biological processes, especially stress response and antimicrobial persistence [53]. Unexpectedly, no TA system was found in all strains of Cupriavidus (Table. S2).

To assess the pathogenic potential of Cupriavidus, we employed PathogenFinder, a computational tool for predicting pathogenicity. The probability of being a human pathogen range for this prediction was estimated to be 0.183-0.325 (Table. S2).

Virulence factors identification

A total of 47 genes related to virulence factors associated with virulence genetic profiles were identified in the eight species of Cupriavidus (Fig. 3), in which 12 major virulence factors exist in almost all the 97 selected Cupriavidus strains. These 12 virulence factors are associated with antimicrobial activity (acrB), biofilm (adeG, algU), stress survival (clpP, katA, sodB, ureA, ureB, and ureG), adherence (htpB, kdsA) and others (icl). Additionally, several species-specific virulence factors occur in particular species. The species-specific virulence factors include membrane-acting toxin, immune modulation, inflammatory signaling pathway, and iron uptake. Of these, a virulence factor associated with a membrane-acting toxin (plc-2) is present in almost all strains of C. gilardii, C. metallidurans, C. necator, and part of C. taiwanensis but is absent in the other four tested species. Virulence factors related to immune modulation and inflammatory signaling pathways are mainly present in C. taiwanensis (wbtL), C. basilensis (rfbF, acpXL, and rfbD), and C. gilardii (acpXL). Most iron uptake virulence factors were mainly identified in C. oxalaticus (pvdH), C. taiwanensis (pvdA, mbtH-like), and C. gilardii (mbtH-like). Overall, the differential distribution of virulence factors suggested that the pathogenicity of Cupriavidus might vary from species to species.

Detection of the secretion systems

Cupriavidus strains contain six types of secretion systems (T1SS-T6SS, Fig. 4). T1SS is present in seven Cupriavidus species (C. alkaliphilus, C. basilensis, C. gilardii, C. pauculus, C. oxalaticus, C. necator, and C. taiwanensis). The number of T1SS clusters in Cupriavidus strains ranges from 1 to 5. The genetic organization of the T1SS clusters in Cupriavidus is highly diverse (Fig. 5a), and the components of different T1SS showed a low protein sequences identity threshold (20%), indicating that the presence of T1SS is strain-specific. The phylogenetic tree based on shared sequences of T1SS was not congruent to the genome-based phylogenetic tree (Table. S1a, Fig. 1), suggesting that T1SS occurs in multiple horizontal gene transfer (HGT) events during divergent evolution in Cupriavidus strains.

T2SS was identified in all eight Cupriavidus species (96 strains; strain NDB4MOL1 was the only exception, Fig 4). T2SS shared similar genetic organization in the genomes of 96 Cupriavidus strains (Fig. 5b), and protein sequences of T2SS components indicated a high degree of identity threshold (82%). The phylogenetic tree of the shared sequences of T2SS showed a similar topology to the core SNPs phylogenetic tree (Fig. S1b, Fig. 1). Accordingly, T2SS is conserved in all eight species of Cupriavidus.

T3SS was identified as present in the four species of Cupriavidus, C. alkaliphilus, C. gilardii, C. taiwanensis, and one strain of C. necator (Fig 4). The genetic organization of T3SS clusters was very similar (Fig. 5c); the protein sequences of T3SS components also kept a high protein identity threshold (92%). The phylogenetic tree based on the shared sequences of T3SS showed a similar topological structure compared with the core SNPs phylogenetic tree (Fig. S2a, Fig. 1), suggesting that the strains lacking T3SS clusters probably result from earlier deletion events during divergent evolution. Additionally, it is interesting to note that all of C. gilardii strains were identified to possess an extra T3SS clusters with different genetic organization, while the other seven Cupriavidus species are absent (Fig. 5d). The extra T3SS clusters in C. gilardii strains displayed evident deviation in CAI with their host genomes (Fig. S2b), indicating that the extra T3SS clusters are likely acquired through HGT events. To further identify whether the extra T3SS in C. gilardii has putative homologous with the other bacteria, we performed blastp searches in the NCBI non-redundant protein database. The results showed that the extra T3SS in C. gilardii species is closely related to the T3SS clusters in Yersinia enterocolitica (protein identity with approximately 52%, Fig. 5d).

T4SS was identified with five types (T, I, F, G, and pT4SSt) in 33 strains of eight species of Cupriavidus (Fig. 4). Interestingly, we found that T4SS clusters predominately occurred in pathogenic Cupriavidus species (C. gilardii, C. metallidurans, and C. pauculus) instead of other species. Specifically, 78% (21/27) of pathogenic strains possess T4SS clusters, while 22% (13/60) of the other strains contain T4SS clusters. The genetic organization of T4SS clusters is different from each other (Fig. 6). The phylogenetic tree based on shared sequences of T4SS differed from the tree based on the core SNPs (Fig. S3). As such, T4SS probably results from the multiple HGT events.

T5SS was present in all 97 strains of the eight species of Cupriavidus, in which T5SS could be classified into three distinct types: T5aSSs, T5bSSs, and T5cSSs (Fig. 4). These results showed that the T5SS clusters in Cupriavidus are highly strain-specific because the type and the number of T5SS clusters vary considerably from strain to strain. For example, the maximum number of T5SS clusters in strain KF709 was up to 12, while the least T5SS clusters were as low as 1.

T6SS was identified in 61 strains of six Cupriavidus species, including C. alkaliphilus, C. gilardii, C. pauculus, C. metallidurans, C. necator, and C. taiwanensis (Fig 4). The T6SS clusters in Cupriavidus strains are present in four different genetic organizations (Fig. 7a). To further investigate the diverse origins of T6SS, a phylogenetic tree using the shared TssB proteins of Cupriavidus strains in combination with the data of the SecReT6 database. The T6SS clusters in 61 Cupriavidus strains were clearly classified into four types: i2 (C. metallidurans NA4), i3 (C. pauculus KF709), i4a (C. necator NBRC 102504), and i4b (58 strains) (Fig. 7b). The four different Cupriavidus T6SSs clusters scatter in different locations, showing high homology to other T6SS clusters from other species, such as Escherichia coli(i2) and Yersinia pseudotuberculosis(i3), Pseudomonas syringae (i4a), and Ralstonia solanaacearum (i4b). Thus, the divergent reason for the T6SS clusters in Cupriavidus is probably due to various donor species' HGT events.

Antibiotic resistance gene in Cupriavidus

Forty antibiotic resistance genes were identified in 97 strains of Cupriavidus (Fig. 8). There were 12 major antibiotic resistance genes identified in all strains, which all are related to five types of system of efflux pumps, namely ATP-binding cassette system, resistance-nodulation-cell division system, kdpDE system, major facilitator super-family system, and small multidrug resistance system. Efflux pumps are known as either single-component transporters or multiple-component systems [54]. However, we found that the 12 antibiotic resistance genes do not encode the complete efflux pump except for a small multidrug resistance system (emre).

The opportunistic pathogens of Cupriavidus, C. gilardii, C. metallidurans, and C. pauculus possess more specific-species antibiotic resistance genes compared with the other Cupriavidus species. For example, C. gilardii contain aminoglycoside resistance genes ant(3'')-Ib, aac(3)-Ivb, aac(3)-IV, and a β-lactam resistance gene oxa-837; C. metallidurans contain three β-lactam resistance genes och-4, och-8, and och-1, a fosfomycin efflux pump AbaF, and a multidrug efflux pump MexAB-OprM; C. pauculus has a multidrug efflux pump MexAB-OprM. In the case of the other species, C. taiwanensis is the most distinctive because some strains of C. taiwanensis (group17,18, and 19) have six genes associated with aminoglycoside resistance (ant(3'')-IIa, ant(3'')-IIb, ant(3' ')-IIc, ant(3'')-IId, ant(3'')-IIe, ant(3'')-Iig).

Identify six species complex among the Cupriavidus species

Species complex, a nomenspecies containing more than one genomic group, is a new challenge in prokaryotic taxonomy due to the availability of vast microbial genome data with the rapidly growing sequencing technology [3]. ANI and isDDH provide a viable solution for accurate species identification and can further address this issue [5, 35]. In the case of the eight Cupriavidus species, we found two worthy taxonomic results. Firstly, we found that six species among the eight tested Cupriavidus species, C. alkaliphilus, C. basilensis, C. gilardii, C. oxalaticus, C. necator, and C. taiwanensis, are species complex, individually, i.e., each of the six species composing several closely related genomic groups (Fig. 1). Of these, the four species (C. alkaliphilus, C. basilensis, C. gilardii, and C. oxalaticus) were identified as species complex for the first time in the present study because C. necator and C. taiwanensis have been identified as species complex in previous works [19, 20]. Hence, the results of phylogenetic analysis coupled with ANI and isDDH validated our hypothesis that there is likely to be another species complex within the Cupriavidus genus except for C. necator and C. taiwanensis. As defined by Ursing et al., each genomic group in the species complex probably represents a new distinct species [55]. As such, each of the six species, C. alkaliphilus, C. basilensis, C. gilardii, C. oxalaticus, C. necator, and C. taiwanensis, might contain several unnominated new species.

Moreover, we found that C. alkaliphilus and C. taiwanensis were the most distinctive among the six identified species complex in Cupriavidus. Unlike the other identified species complex, C. alkaliphilus mixed with parts of C. taiwanensis and formed two groups (groups 19 and 20, Fig. 1). Then, a question will be asked why only the two species display such phylogeny. This might be related to the taxonomic identification of C. alkaliphilus using C. taiwanensis LMG 19424 as a type strain of C. taiwanensis. In 2001, Chen et al. first reported C. taiwanensis as a new species and defined strain LMG 19424 as the type strain of C. taiwanensis [30]. After this, C. taiwanensis LMG 19424 has been used as a type strain of C. taiwanensis until now. Subsequently, in 2012, C. alkaliphilus was first described as a new species, in which the 16S rRNA sequence of strain LMG 19424 was used as the type strain of C. taiwanensis to compare with the four new isolates [21]. Ultimately, these new isolates were formally delineated as new species of C. alkaliphilus, although the new isolates exhibited high similarity (∼98.9%) in 16S rRNA sequences to strain LMG 19424. Nonetheless, C. taiwanensis has been identified as a species complex in 2018 [20]. Namely, it is not reasonable anymore to use C. taiwanensis LMG 19424 as a type strain of C. taiwanensis because of the presence of multiple novel species within C. taiwanensis. Thus, the above dated taxonomic identification history of C. alkaliphilus well explains our results that all C. alkaliphilus strains mix with parts of C. taiwanensis.

Accurate species classification and identification are crucial for discovering and applying valuable microbial resources and dealing with pathogenic microorganisms [1, 56]. This study found six Cupriavidus species contained species complex, including C. alkaliphilus, C. basilensis, C. gilardii, C. oxalaticus, C. necator, and C. taiwanensis. Notably, some strains of C. alkaliphilus and C. taiwanensis were mislabeled. Thus, we suggested to update the taxonomy of the above six Cupriavidus species based on further phenotypic characterization confirmation.

Virulence factors of Cupriavidus

Virulence factors are molecules encoded by virulence genes that enable microbial pathogens to evade host defense mechanisms and induce disease in a host organism [57]. Identification of virulence factors is imperative for gaining comprehensive insights into microbial pathogenesis [58, 59]. From the perspective of the Cupriavidus genus, few studies available have addressed virulence factors of C. gilardii specie and a strain of C. pauculus so far [32, 60]. This lack of studies associated with virulence factors of Cupriavidus strains impedes the understanding of the pathogenesis of Cupriavidus.

Our results showed that virulence factors (Isocitrate lyase, ClpP, and SodB) were identified across almost all the investigated Cupriavidus species (Fig 3). The virulence factor isocitrate lyase, encoded by the icl gene, has been recognized as one of the virulence determinants of Pseudomonas aeruginosa in CF [61]. Isocitrate lyase (icl) is required for the persistent infection in CF, which allows bacteria to utilize fatty acids as carbon and energy sources [62]. In the case of ClpP and SodB, these two virulence factors are encoded by the clpP and sodB gene, respectively, playing crucial roles in the stress survival of pathogenic microorganisms [63, 64]. Additionally, we found that phospholipase C, encoded by gene plc-2, is only present in four Cupriavidus species: C. gilardii, C. metallidurans, C. necator, and C. taiwanensis. In the case of the functions of gene plc-2, it is known for encoding phospholipase C, which has been reported to facilitate the invasion of Acinetobacter baumannii pathogen by lysing and degrading phospholipids of host cells [65, 66].

Overall, the genetic information related to virulence factors from this study will facilitate bridging the gap in the pathogenesis of Cupriavidus. More experiments related to the virulence factors of Cupriavidus should be implemented to verify the results of genome-based analysis.

Secretion systems of Cupriavidus

Bacterial secreted proteins by secretion protein systems are one of the important strategies utilized by most bacterial pathogens to tackle host cells [67]. To date, nine types of secretion systems (T1SS–T9SS) have been identified in bacteria according to protein secretion apparatuses [68]. Although the distribution of secretion systems in many pathogenic bacteria (Klebsiella pneumoniae, A. baumannii, and Hafnia genus) has been elucidated [67, 69, 70], little is known about the secretion systems contained in Cupriavidus strains.

T2SS is essential for bacterial pathogenicity by secreting effector proteins [71, 72]. Notably, the virulence factor phospholipase C is secreted via T2SS in some essential human bacterial pathogens (e.g., A. baumannii and P. aeruginosa). It thereby damages membrane phospholipids and sphingomyelin of host cells [73, 74]. A most important case is that the phospholipase C secreted through T2SS contributes to bacterial pathogenicity in lung colonization, inflammation, and CF [73–75]. Coincidentally, we found that T2SS is the only conserved secretion protein among all the six secretion systems (T1SS–T6SS) occurring in all the eight Cupriavidus species (Fig. 4). Moreover, previous studies have reported that multiple Cupriavidus species, including C. basilensis, C. gilardii, C. metallidurans, C. pauculus, and C. taiwanensis, are isolated from the respiratory specimens of CF patients [25, 28, 30]. Nonetheless, the role of these species in contributing to lung disease in CF was unclear [28]. More causation studies on phospholipase C and T2SS are worthy of performing in the pathogenesis of Cupriavidus strains.

T3SS is another important secretion system that can inject effector proteins directly into the cytosol of host cells [76]. It can manipulate the cellular activities of the host for the benefit of the bacterial pathogens. [76]. T3SS is widely possessed in most gram-negative pathogenic bacteria (Ralstonia, Salmonella, Shigella, Yersinia, E. coli, and P. aeruginosa), including those causing disease in humans and animals [77–79]. In Cupriavidus, we found that T3SS clusters are presented in all the strains of C. alkaliphilus, C. gilardii, and C. taiwanensis and one strain of C. necator (Fig. 4). Interestingly, C. gilardii possesses two sets of T3SS (Fig. 5c, Fig. 5d), which is the only exception because the other species only have one set of T3SS. The additional T3SS clusters in C. gilardii (extra T3SS) are very close to the T3SS clusters in the pathogen Y. enterocolitica, in which the protein sequence identity of T3SS components between the two bacterial species reaches as high as 52% (Fig. 5d). Thus, the set of extra T3SS in the C. gilardii is probably as a result of the HGT events. The T3SS in Y. enterocolitica has been reported to play an important role in colonizing gastrointestinal tissue in mice [80]. Therefore, the extra T3SS clusters probably have the potential to contribute to the pathogenicity of C. gilardii strains.

Antibiotic resistance profiles of Cupriavidus

The emerging case reports about opportunistic pathogens Cupriavidus spp., especially C. gilardii, C. metallidurans, and C. pauculus, underline the importance of investigating Cupriavidus antibiotic resistance. However, information on antibiotic resistance of Cupriavidus species, either by culture-based antimicrobial susceptibility testing or genome-based analysis, is very scarce [13, 24, 26]. So far, only a few works have addressed the antibiotic resistance genes of C. gilardii species by comparative genomic analysis [13, 32].

In the present study, we characterize antibiotic resistance profiles in Cupriavidus for the first time using comparative genomic analysis. The emre gene encoded efflux pump EmrE was identified in all the eight tested strains (Fig. 8). The EmrE efflux pump is a small multidrug resistance (SMR) family, which was first found in P. aeruginosa and has been shown to contribute to resistance against aminoglycoside antibiotics via expelling aminoglycoside from bacterial cells [81]. More importantly, previous clinical case studies have reported that the mentioned three opportunistic pathogens, Cupriavidus species, C. gilardii, C. metallidurans, and C. pauculus, are resistant to aminoglycosides [7, 13, 16, 82]. Thus, we inferred that the EmrE efflux pump encoded by an emre gene might play a crucial role in Cupriavidus spp. resistance to aminoglycosides.

Pathogens are known to acquire new antibiotic resistance genes from other species [83–85]. Coincidentally, the number of antibiotic resistance genes in opportunistic species C. gilardii, C. metallidurans, and C. pauculus are the highest among the eight species; these species specifically contain more species-specific antibiotic resistance genes (Fig. 8). Thus, we infer that the antibiotic resistance genes of pathogenic Cupriavidus spp. are most likely to acquire by HGT events. Indeed, cases associated with the infection of C. gilardii have proved that this species can acquire new antibiotic resistance during patient treatment [86]. Hence, the infections caused by pathogenic Cupriavidus spp. should be a concern because they may acquire new antibiotic resistance genes.

The present study for the first time investigated the taxonomy of Cupriavidus by comparative genomic analysis using 97 genomes belonging to eight representative species: C. alkaliphilus, C. basilensis, C. gilardii, C. pauculus, C. metallidurans, C. oxalaticus, C. necator, and C. taiwanensis. Of these, C. alkaliphilus, C. basilensis, C. gilardii, and C. oxalaticus were first reported as species complex. More importantly, C. alkaliphilus and part of C. taiwanensis mixed. We suggested that the existing taxonomy of Cupriavidus should be updated. Additionally, we first elucidated the virulence-related profiles of Cupriavidus from aspects of virulence factors, secretion systems, and antibiotic resistance genes. The presence of virulence factors (Isocitrate lyase, ClpP, SodB, and phospholipase C) and secretion systems (T2SS) might be associated with the pathogenicity of Cupriavidus. Finally, the detected antibiotic resistance genes in C. gilardii, C. metallidurans, and C. pauculus were largely linked to aminoglycosides, β-lactam, fosfomycin, and multidrug resistance. Together, findings from this study shed new light on taxonomic relationships in Cupriavidus and facilitated a better understanding of virulence-related profiles in Cupriavidus to provide insights into Cupriavidus pathogenesis.

ANI Average Nucleotide Identity

isDDH In silico DNA–DNA hybridization

ML Maximum Likelihood

NCBI National Center of Biotechnology Information

COG Cluster of Orthologous Group

CRISPRs Clustered Regularly Interspaced Short Palindromic Repeats

TA system Toxin-antitoxin system

CAI Codon Adaptation Index

VFDB Virulence Factors Database

CARD The Comprehensive Antibiotic Resistance Database

HGT Horizontal Gene Transfer

ORF Open Reading Frame

T1SS-T9SS Type I - IX secretion

CF Cystic Fibrosis

Acknowledgments

Not applicable.

Authors' contributions

CSL and WXY designed the research. CSL performed bioinformatics analyses and wrote the original draft. WXY reviewed the manuscript and revised it. ZLL contributed to both the conceptual and technical aspects of bioinformatics. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (no. 52070037).

Availability of data and materials

All genomes analyzed in the manuscript are publicly available on the NCBI Database at the assembly accession provided (Table S1).

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Insights into the taxonomy and virulence-related genetic profiles in Cupriavidus by comparative genomic analysis

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Abstract

Figures

Background

Materials and Methods

Accessing genomes of Cupriavidus strains

Phylogenetic tree, ANI and isDDH analysis

Pan-genome analysis

Gene function category

Identification of mobile genetic elements

Pathogenicity prediction and TA system identification

Identification of secretion systems

Identification of virulence and antibiotic resistance genes

Results

Phylogenetic, ANI and isDDH analysis

Characterization of pan-genome and core genome

Mobile genetic elements, TA system, and pathogenicity prediction

Virulence factors identification

Detection of the secretion systems

Antibiotic resistance gene in Cupriavidus

Discussion

Identify six species complex among the Cupriavidus species

Virulence factors of Cupriavidus

Secretion systems of Cupriavidus

Antibiotic resistance profiles of Cupriavidus

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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