Skip to main content
Download PDF

Genomic characteristics, virulence potential, antimicrobial resistance profiles, and phylogenetic insights into Nocardia cyriacigeorgica

Annals of Clinical Microbiology and Antimicrobials volume 24, Article number: 22 (2025) Cite this article

Abstract

Background

Nocardia cyriacigeorgica, an opportunistic pathogen, is increasingly implicated in human infections. This pathogen predominantly causes pulmonary infections, leading to acute, subacute, or chronic necrotizing suppurative lesions, in severe cases, may progress to disseminated infections. Effective clinical diagnosis, prevention, and treatment strategies require a thorough understanding of its biological characteristics and pathogenic mechanisms. However, despite the rising incidence of nocardial diseases, research on the pathogenicity of N. cyriacigeorgica remains limited, primarily focusing on case reports and epidemiological studies. This study aimed to provide a comprehensive analysis of the genomic features, phylogenetic relationships, antimicrobial resistance profiles, and candidate virulence factors of N. cyriacigeorgica strains to inform future investigations into its pathogenesis.

Methods

Whole-genome sequencing was conducted on five N. cyriacigeorgica strains isolated from patients with pulmonary infection at our hospital. This analysis utilized a combination of second-generation Illumina HiSeq and third-generation PacBio sequencing technologies. Additionally, publicly available genomic data from 58 strains in the National Center Biotechnology Information database were integrated, resulting in a dataset of 63 genomes. These genomes were subjected to comparative genomic analyses, including phylogenetic reconstruction, pan-genome evaluation, and gene distribution assessments.

Results

Phylogenetic analysis identified five major clades within N. cyriacigeorgica. ANI analysis further subdivided clade B into five distinct subgroups. Pan-genome analysis revealed clade-specific orthogroups in the distribution of genes assigned to Clusters of Orthologous Groups, with clade A containing the highest number of clade-specific gene families. Comparative genomic analysis uncovered several potential pathogenic genes implicated in host cell invasion, phagosomal maturation arrest, and intracellular survival within macrophages, which were conserved across all analyzed strains. Notable differences in the distribution of enterobactin-encoding genes were observed among the clades. The mce3C gene also displayed variable distributions across clades; however, no correlation was established between its presence and strain source. Among the 63 strains, 27 were found to harbor both mce3C and mce4F genes, which were categorized into five distinct patterns. Furthermore, antibiotic resistance genes, including VanSO, VanRO, erm(O)-Irm, srmB, ermH, bcl, bla1, and cmIR, demonstrated clade-specific distribution patterns. Notably, the genes erm(O)-Irm, srmB, and ermH were associated with the isolation origin of the strains.

Conclusions

This study provides a comprehensive evaluation of the genomic characteristics, potential virulence factors, antimicrobial resistance genes, and phylogenetic relationships of N. cyriacigeorgica. The findings offer valuable insights into the mechanisms underlying intracellular survival, replication within macrophages, and pathogen-host interactions in N. cyriacigeorgica infections. These results establish a foundation for future research into the pathogenesis and clinical management of N. cyriacigeorgica.

Introduction

Nocardia, a genus of Gram-positive, weakly acid-fast branching bacilli, is widely distributed in various environmental niches globally, including soil, water, and aquatic ecosystems. It is classified as part of the aerobic actinomycetes group [1]. It is recognized as an opportunistic pathogen responsible for nocardiosis in both humans and animals. Nocardia spp. infect humans primarily through respiratory inhalation and injured skin [2]. To date, more than 100 Nocardia species have been identified (https://www.bacterio.net), with over 50 species implicated in human infections affecting both immunocompetent and immunosuppressed individuals [3]. Data from the Centers for Disease Control and Prevention (CDC) indicate that an estimated 500 to 1000 new cases of nocardiosis are reported annually in the United States (https://www.cdc.gov/nocardiosis/hcp/clinical-overview/). However, on a global scale, the precise incidence of nocardiosis remains uncertain due to the absence of standardized national reporting systems.

In contrast to the well-known and extensively studied ESKAPE pathogens—Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa, and Enterobacter species [4, 5], Nocardia species are clinically uncommon bacteria. Their diagnosis and treatment remain challenging in clinical practice, highlighting a significant gap in both recognition and management. Nocardia spp. are associated with a wide spectrum of human diseases, including pulmonary infections, superficial cutaneous and subcutaneous infections, and systemic infections resulting from hematogenous dissemination [6, 7]. Hematogenous dissemination is a critical factor that markedly elevates mortality rates, with figures reaching as high as 85% among immunocompromised individuals [8,9,10]. Additionally, Nocardia species exhibit intracellular pathogenicity, enabling them to invade and survive within host cells, thereby causing refractory infections that are often challenging to treat [11, 12].

Nocardia cyriacigeorgica was first definitively identified in 2001 through the isolation and 16S rRNA gene sequencing of strain DSM 44484 T from the bronchial secretions of a patient with chronic bronchitis [13]. This intracellular pathogen has been implicated in a variety of infections, including pneumonia, brain abscesses, and infections of the kidney, heart, and eye [14,15,16,17]. Among the various Nocardia species, N. cyriacigeorgica is recognized as the most prevalent cause of human nocardiosis in regions such as North America [18, 19], Spain [20, 21], and Iran [22]. In China, its prevalence ranges from18.3 to 40.2% among nocardial infections [23,24,25,26]. Likewise, N. cyriacigeorgica has been frequently reported as a commonly isolated Nocardia species in other countries, including Thailand, Japan, Belgium, Australia, and South Africa [27,28,29,30,31,32]. In recent years, the incidence of nocardiosis has notably increased, posing a significant threat to public health. Addressing this growing challenge necessitates the development of effective clinical diagnostic, therapeutic, and preventive strategies, which, in turn, require a deeper understanding of the virulence mechanisms and biological properties of N. cyriacigeorgica. Current research on N. cyriacigeorgica has predominantly focused on case reports and epidemiological studies [14,15,16,17, 23]. Notably, previous studies have identified distinct virulence of N. cyriacigeorgica strains [33] and highlighted the role of heparin-binding hemagglutinin (HBHA) in adhesive and immunoregulatory functions [34]. Despite these findings, the biological characteristics, virulence factors, antimicrobial resistance genotypes, and pathogenic mechanisms of N. cyriacigeorgica remains insufficiently understood.

Numerous molecular methods have been utilized to study Nocardia species, including 16S rRNA gene sequence analysis [35], sequencing of single copy gene such as secA1, gyrB, hsp65, and ropB [36,37,38,39], multilocus sequence analysis (MLSA) [40], matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis [41]. However, each of these techniques is associated with certain limitations. Whole genome sequencing (WGS), a high-throughput sequencing technology, provide a powerful platform for investigating bacterial pathogenicity, drug resistance mechanisms, evolutionary trends, and phylogenetic relationships. WGS serves as a valuable tool for investigating genetic factors associated with pathogenicity. Additionally, it supports the development of innovative genetic and molecular approaches, which can improve clinical diagnostics and aid in the implementation of effective control strategies.

In this study, we determined and annotated the complete genome sequences of five clinical N. cyriacigeorgica strains isolated from pulmonary infection patients. To gain deeper insights, we performed a comparative genomic analysis of 63 strains, which included 58 publicly available strains retrieved from the National Center for Biotechnology Information (NCBI) database and the five clinical strains sequenced in this study. The analysis focused on key aspects such as genomic features, phylogenetic relationships, mobile genetic elements (MGEs), potential virulence factors, and antibiotic resistance genes (ARGs). The overarching goal was to investigate the phylogenetic relationships, biological properties, antimicrobial resistance genotypes, and potential virulence mechanisms of N. cyriacigeorgica.

Materials and methods

This study employed a combination of microbiological, molecular, and bioinformatics approaches to investigate the genomic and functional characteristics of N. cyriacigeorgica. The methodology included bacterial isolation and identification, Antimicrobial susceptibility testing (AST), WGS, genome assembly and annotation, and comparative genomic analyses. The detailed steps for each phase are described below.

Bacterial isolation, species identification, and antimicrobial susceptibility testing

Clinical samples were inoculated on the blood-containing medium at 35 °C for 3–7 days under aerobic conditions. Suspected Nocardia species were identified through Gram staining, modified acid-fast staining, and acid-fast staining, followed by confirmation via 16S rRNA gene sequencing. Isolates of N. cyriacigeorgica were preserved in brain heart infusion (BHI) broth supplemented with 25% glycerol at – 80 ℃ for subsequent studies.

The AST was performed in accordance with the guidelines outlined by the Clinical and Laboratory Standards Institute (CLSI, M24S-Ed2) for Nocardia spp. [42]. Briefly, the Nocardia strains were grinded in sterile 0.9% sodium chloride water and subjected to repeated vortexing until no visible particles or deposits remained. The resulting uniform bacterial suspension was then adjusted to match the turbidity of the 0.5 McFarland standard. Antimicrobial susceptibility testing was conducted using first-line recommended drugs, including amoxicillin-clavulanate (AMC), imipenem (IPM), minocycline (MIN), tobramycin (TOB), amikacin (AMK), ciprofloxacin (CIP), trimethoprim/sulfamethoxazole (STX), linezolid (LZD), ceftriaxone (CRO), and clarithromycin (CCR). The broth microdilution (BMD) method was used to determine minimum inhibitory concentrations (MICs), which were interpreted based on CLSI susceptibility breakpoints. Quality control for AST was ensured by employing Escherichia coli ATCC 35218, Pseudomonas aeruginosa ATCC 27853, and Staphylococcus aureus ATCC 29213 as reference strains.

Library construction and whole-genome sequencing

Following the identification and preservation of clinical isolates, genomic DNA was extracted for WGS to enable detailed genomic analyses.

The clinical isolates were cultured in BHI broth (Hopebio Technology, Qingdao, China) at 37 °C for 48–72 h with constant shaking at 180 rpm. Genomic DNA was extracted using the TIANamp Bacteria DNA Kit (TIANGEN, China) following the manufacturer’s instructions. The extracted DNA was subjected to WGS using both the second-generation Illumina Novaseq 6000 platform (Illumina, San Francisco, CA, USA) and the third-generation PacBio Sequel IIe platform (Pacific Biosciences, San Francisco, CA, USA).

For Illumina sequencing, genomic DNA was fragmented into 400–500 bp segments using a Covaris M220 Focused Acoustic Shearer, following the manufacturer’s protocol. The fragmented DNA was subsequently prepared into Illumina sequencing libraries, which were used for paired-end sequencing (2 × 150 bp) on the Illumina Novaseq 6000 platform. For PacBio sequencing, DNA fragments were purified, end-repaired, and ligated with SMRTbell sequencing adapters to generate libraries with an average size of approximately 10 kb, in accordance with the manufacturer’s guidelines (Pacific Biosciences, CA). Raw sequence data were filtered to exclude reads containing more than 40 bp of low-quality bases, more than 10 bp of ambiguous bases (N), or adapter sequences overlapping by more than 15 bp. High-quality reads were subsequently de novo assembled into contigs using SPAdes v3.8.0 [43]. Raw reads from both sequencing platforms were subjected to quality assessment and trimming to ensure the reliability and accuracy of downstream analyses.

Genome assembly and annotation

Sequence data generated from the PacBio Sequel IIe and Illumina Novaseq 6000 platforms were utilized for bioinformatics analysis. Raw Illumina sequencing reads from the paired-end library were quality-filtered using fastp v0.23.0. Clean short reads and HiFi reads from the PacBio platform were assembled into complete genomes using Unicycler v0.4.8 [44]. The assembly was subsequently polished using Pilon v1.22, which used short-read alignments to reduce the occurrence of small errors [45]. Coding sequences (CDs) on chromosomes and plasmids were predicted using Glimmer 3.02 and GeneMarkS 4.3, respectively [46, 47]. Gene and open reading frame (ORF) annotations were performed using Prodigal and Prokka with default parameters.

Comparative genomics analyses

A dataset of 63 genomes, comprising 58 publicly available complete genome sequences of N. cyriacigeorgica from the NCBI database and five clinical isolates sequenced in this study, was analyzed. Pangenome analysis was conducted using Roary and FastTree to identify core and accessory genomic elements. Average Nucleotide Identity (ANI) values across all genomes were calculated using the ANI calculator, as previously described [48], to assess overall genome similarity.

To evaluate genetic relatedness, a high-quality maximum likelihood (ML) phylogenetic tree was constructed based on the concatenation of 1164 conserved single-copy genes present in 99–100% of the strains (bootstraps, 1000), following the methodology outlined by Li et al. [48]. Orthogroups among all tested genomes were identified using OrthoFinder v2.4.0 with default parameters [49]. Functional annotation of orthogroups was performed using emapper v2.0.1 against the eggNOG v5.0 database [50].

Insertion sequence (IS) elements were predicted using ISEScan v1.7.2.2 [51]. Antimicrobial resistance genes (ARGs) were identified using Diamond v0.9.14 with a cutoff E-value of 1 × 10–6, a minimum identity of 60%, and alignment against the Comprehensive Antibiotic Resistance Database (CARD) [52]. Virulence factors were identified using Diamond v0.9.14 with the same cutoff E-value, minimum identity, and coverage thresholds, utilizing the VFDB database (http://www.mgc.ac.cn/VFs/). Plasmid identification was performed using PlasFlow.

Results

Antimicrobial susceptibility profiles of clinical isolates

Five clinical isolates, identified as N. cyriacigeorgica through 16S rRNA gene sequencing, were obtained from patients diagnosed with pulmonary nocardiosis at Bethune International Peace Hospital between January 1, 2019, and December 31, 2020. The AST results for these isolates are presented in Table S1. All strains were susceptible to trimethoprim/sulfamethoxazole, amikacin, imipenem, linezolid, clarithromycin, tobramycin, and minocycline. However, resistance was observed against ciprofloxacin and amoxicillin-clavulanate. Additionally, 80% (4/5) of the isolates were susceptible to ceftriaxone.

Genomic characteristics of N. cyriacigeorgica strains

This comparative genomic study analyzed 63 N. cyriacigeorgica strains, including five clinical isolates sequenced in this study and 58 publicly available strains retrieved from the NCBI database. Among these, 50.8% (32/63) were identified as human pathogens, 12.7% (8/63) originated from environmental sources, and 36.5% (23/63) had no source information. The genomic features, including genome size, G + C content, number of CDSs, contig N50, rRNA, tRNA, and isolation sources, are summarized in Table 1.

Table 1 The genomic features of 63 Nocardia cyriacigeorgica strains
Full size table

The genome sizes of the analyzed strains ranged from 5.92 to 6.83 Mb, with an average size of approximately 6.42 Mb. The number of CDSs varied between 5,360 and 6,589, while the G + C content ranged from 66.92% to 68.47%, with an average of 68.2%. Notably, four strains had G + C contents below 68%. Members of clade B had more CDSs compared to other clades. Additionally, strains in clades A and B harbored more rRNA genes than those in other clades (Table 1 and Fig. 1).

Fig. 1
figure 1

Genomic features of the investigated strains in this study, including total length, CDS, GC content, N50, rRNA, and tRNA

Full size image

Phylogenetic relationships and average nucleotide identity analysis

To evaluate the genetic relatedness of N. cyriacigeorgica strains, a maximum likelihood (ML) phylogenetic tree was constructed using the concatenation of 1,164 conserved single-copy core genes (Figure 2). The phylogenetic analysis revealed five major clades, with clade B further subdivided into five subgroups. Notably, 84.4% (27/32) of strains isolated from human sources were distributed among clades A, B, and E, whereas clades C and D predominantly comprised strains with no detailed source information. Five strains (BJ06-0109, BJ06-0147, NBC-00369, 112071522, 760185010) did not cluster with any of the five major clades. Genes with a presence higher than 30% from the accessory genomes were further analysed by DPAC (Dynamic profile analysis for clusters) and hclust (Hierarchical clustering), and the clustering results were consistent with those based on core genes (Figure S1 and Figure S2).

Fig. 2
figure 2

The phylogenetic tree of N.cyriacigeorgica constructed based on the concatenation of the nucleotide sequence of 1164 conserved single-copy core genes

Full size image

Genomes are classified as belonging to the same species if their pairwise ANI value is ≥95%. The pairwise ANI comparisons for each genome are presented in Table S2. Clade B strains were further divided into five subgroups, consistent with the phylogenetic tree findings (Figure 3). ANI values between genomes within the five subgroups of clade B were below 95%, and clade B strains exhibited ANI values of < 95% when compared to strains in other clades. In contrast, ANI values among strains in clades A, C, D, and E exceeded 95%, indicating high genome sequence identity within these clades.

Fig. 3
figure 3

Heat map of pairwise ANI analyses for 63 N.cyriacigeorgica genomes

Full size image

Pan-genome analysis across five clades

The pan-genome analysis of 63 N. cyriacigeorgica strains identified a total of 33,900 genes, with only 1,164 (3.4%) classified as core genes shared by 99% of the strains. The remaining 32,736 genes (96.6%) were categorized as accessory genes, including 305 soft-core genes (present in 95% to < 99% of strains), 7253 shell genes (present in 15% to < 95% of strains), and 25,178 cloud genes (present in < 15% of strains) (Fig. 4).

Fig. 4
figure 4

Heat map of pan-genome analysis of 63 N.cyriacigeorgica

Full size image

The analysis further identified 2507 orthogroups distributed across all five clades. Among these, clade A contained the highest number of unique orthogroups (292), followed by clade B (34), clade D (13), clade E (13), and clade C (10). Clades A and B shared 48 orthogroups, suggesting some functional overlap (Fig. 5A). Functional annotation of clade-specific orthogroups, based on the eggNOG database, revealed that a significant proportion of genes in all clades were annotated as “function unknown” (Fig. 5B), highlighting the need for further functional studies.

Fig. 5
figure 5

A Distribution of the number of orthogroups in the N.cyriacigeorgica genomes. Clade-specific orthogroups are indicated in blue. B The associate functional COGs to specific clades

Full size image

The indicspecies R package was performed to statistically analyze the associate COGs in each clade, considering a P-value < 0.01 as statistically significant. As shown in Fig. 5B and Table S6, the distribution of genes assigned to Clusters of Orthologous Groups (COG) categories varied among the clades. Clade A, B C, and D exhibited the most diverse set of functional COG categories, with a notable enrichment of genes involved in “replication, recombination, and repair”, “transcription”, and “amino acid transport and metabolism”. However, compare to the clade A, B, C, and D, clade E contained fewer functional COGs associated with “amino acid transport and metabolism”. Clade D and E were enriched with genes linked to “replication, recombination and repair” and “inorganic ion transport and metabolism”. Notably, clade A harbored unique genes associated with “coenzyme transport and metabolism” which were absent in other clades, while lacking genes related to “signal transduction mechanisms” and “defense mechanisms”. These findings suggest that the functional gene repertoire of each clade reflects its potential ecological and pathogenic adaptations.

Distribution of mobile genetic elements in the N. cyriacigeorgica genome

The mobile genetic elements (MGEs), such as ISs and plasmids, play a pivotal role in shaping genome structure, driving bacterial evolution, and conferring adaptive advantages. The presence of MGEs was analyzed across the 63 N. cyriacigeorgica strains, with their distributions shown in Figs. 6 and 7.

Fig. 6
figure 6

Distribution of insertion sequences and plasmids in the 63 N.cyriacigeorgica genomes

Full size image
Fig. 7
figure 7

Members of the IS families identified in the analysed strains

Full size image

Plasmids were absent in 20.6% (13/63) of the strains, including 10 clinical isolates, 2 strains with no detailed source information, and 1 strain from soil (Fig. 6). Among the identified IS elements, 15 distinct IS families were detected, with the IS3 family being the most prevalent across the strains (Fig. 7). These findings highlight the variability in MGE content among N. cyriacigeorgica strains and highlight the potential role of IS elements in mediating genomic plasticity and adaptation.

Putative virulence-associated genes and their distribution in the N. cyriacigeorgica genome

To investigate potential virulence factors and assess the pathogenic characteristics of N. cyriacigeorgica, BLASTP searches of its CDSs were conducted against the Virulence Factor Database (VFDB) using stringent criteria (E-value < 1E-6, sequence length overlap > 60%, sequence similarity > 60%). A total of 208 candidate virulence-associated genes were identified, encompassing a wide range of functions critical for bacterial pathogenesis. These include roles in adherence and invasion, secretion systems, stress adaptation, nutrient acquisition, toxin production, modulation of phagocyte function, and intracellular survival within macrophages (Table 2).

Table 2 Virulence factor candidates in the Nocardia cyriacigeorgica according to the Virulence Factor Database (VFDB)
Full size table

The mammalian cell entry (mce) gene family, known for encoding proteins that facilitate Mycobacterium tuberculosis invasion and survival within macrophages, was represented by nine clusters in N. cyriacigeorgica. Notably, the mce1 and mce2 clusters were absent in all strains, while mce5, mce6, and mce9 were universally present. The remaining mce genes (mce3, mce4, mce7, and mce8) exhibited distinct distribution patterns across the 63 analyzed strains (Fig. 8 and Table S3).

Fig. 8
figure 8

Putative virulence-associated genes in the genomes of N.cyriacigeorgica. The distribution of the mce3 and mce4 gene family are shown on the right side. Each dot indicates the presence of the gene

Full size image

The mce4 gene was present in nearly all strains, except for three strains from oil sources in clade B. A specific subunit, mce4F, was detected in 95.2% (60/63) of strains. In contrast, mce3 was identified in 32 strains, of which only 10 were recognized as human pathogens. All strains in clade C carried mce3, compared to 43.5% (10/23) in clade B, 27.3% (3/11) in clade A, 25% (1/4) in clade E, and 36.4% (4/11) in clade D. Subunits mce3C and mce3D were detected in 100% (9/9) and 77.8% (7/9) of clade C strains, respectively, while mce3E was identified exclusively in three oil-derived strains from clade B.

The mce7 gene was predominantly detected in clades D and C, but it was absent in clades A and E. mce8 was identified in 11 strains, including 3 strains from clade B, 1 from clade D, and 7 from clade C. Analysis of the mce gene family revealed that 27 strains carrying both mce3C and mce4F were categorized into five distinct distribution patterns, as summarized in Table S4.

The fbpC gene, encoding antigen 85C (Ag85C), a protein involved in Mycobacterium tuberculosis cell wall assembly, was detected in only 6.3% (4/63) of strains. Genes associated with oxidative and nitrosative stress responses (katA, sodC, sodA, narG, narH, narI), iron import (ideR), and antioxidant defense (ahpC) were universally present across all strains. Similarly, genes encoding the Type VII secretion system (eccC4) and the effector delivery system (ricA) were also identified in all strains.

Genes critical for intracellular survival, such as ndk (nucleoside diphosphate kinase) and ptpA (protein tyrosine phosphatase A), which facilitate macrophage phagosomal arrest, were consistently detected in all strains. Interestingly, enterobactin-encoding genes (entB), responsible for siderophore-mediated iron acquisition, were found in 60.9% (14/23) of clade B strains but were absent in other clades. In contrast, yersiniabactin biosynthetic protein-related genes (irp5) were detected in all strains from clades A, C, D, and E, but were present in only 56.5% (13/23) of clade B strains. These findings highlight the conserved nature of key virulence-associated genes in N. cyriacigeorgica, while also revealing clade-specific variations that may influence pathogenicity and host interactions.

Comprehensive identification and distribution of antibiotic resistance genes in the N. cyriacigeorgica genome

A systematic screening for ARGs in the N. cyriacigeorgica pan-genome, conducted using BLASTP against the Comprehensive Antibiotic Resistance Database (CARD), identified 268 resistance genes spanning at least 12 distinct antibiotic classes. These genes encode a variety of resistance mechanisms, including efflux pumps, β-lactamases, and plasmid-mediated methyltransferases, which confer resistance through antibiotic inactivation, active efflux, and target modification (Table 3).

Table 3 Antibiotic resistance genes in the Nocardia cyriacigeorgica genome according to the Comprehensive Antibiotic Resistance Database (CARD)
Full size table

Resistance genes targeting multiple antibiotic classes were observed across all analyzed strains. These included cephalosporins (AST-1), aminoglycosides (kdpE), fluoroquinolones (SoxR, gyrB), glycopeptides (rpoC), macrolides (oleC, oleB, carA), tetracyclines (tetA(58), tetB(58)), sulfonamides (folC, folP), rifamycins (rbpA, rpoB2), and fosfomycin (murA, AbaF) (Fig. 9 and Table S5). Among these, β-lactamase-encoding genes, such as CTX-M and KPC, were highly prevalent, underscoring the widespread potential for β-lactam resistance in N. cyriacigeorgica.

Fig. 9
figure 9

Antibiotic resistance genes in the genomes of N.cyriacigeorgica. Each red dot indicates the presence of the gene

Full size image

While β-lactamase genes such as CTX-M and KPC were broadly distributed, certain β-lactamase genes displayed clade-specific patterns. For example, the β-lactamase genes bcl and bla1 were restricted to clade B, being present in 43.5% (10/23) and 21.7% (5/23) of strains, respectively. These genes were absent in strains from other clades, suggesting localized selective pressures influencing β-lactam resistance within clade B.

Resistance genes for vancomycin (VanSO and VanRO) were widely distributed across clades A, C, D, and E, but were present in only 43.5% (10/23) of clade B strains. Conversely, the macrolide resistance gene erm(O)-Irm was detected in 43.5% (10/23) of clade B strains but was absent in other clades.

The macrolide efflux pump gene srmB exhibited a distinct distribution, being present in 78.9% (30/38) of strains from clades A, B, and E, but absent in clades C and D. The plasmid-mediated macrolide resistance gene ermH was identified in 69.6% (16/23) of clade B strains but was not detected in clades A, C, D, or E. These findings highlight the heterogeneous distribution of macrolide resistance mechanisms among clades.

Rifamycin resistance genes (Rox-nf and Rox-sv) and transposon-mediated streptogramin resistance genes (VatF) were exclusively identified in four strains from clade B. Additionally, chromosome-encoded chloramphenicol resistance genes (cmlR) were detected in 21.7% (5/23) of clade B strains but were present in all strains from other clades. These clade-specific distributions suggest that localized environmental or clinical pressures may have driven the evolution of these resistance mechanisms.

Discussion

Consistent with previous research [53], our analysis revealed that N. cyriacigeorgica consists of five distinct clades, as determined by the concatenation of 1164 conserved single-copy core genes. Within clade B, five subgroups were further delineated using pairwise ANI analysis. However, inconsistencies between the phylogenomic tree and ANI values were noted for specific strains. Specifically, three strains isolated from oil samples (CNM20110649, CNM20110648, CNM20110639) within clade B demonstrated high homology with each other but exhibited low genetic relationships between other stains. This observation indicates a notable evolutionary divergence between these three isolates and other clade B strains.

Low ANI values between clade B strains and those from other clades suggest high genomic diversity within clade B, potentially representing distinct species. Interestingly, five strains (BJ06-0109, BJ06-0147, NBC-00369, 112071522, 760185010) could not be assigned to any clades in the phylogenomic tree. Despite this, ANI values exceeding 95% between these strains and strains from clades A, C, D, and E suggest high genomic similarity. These inconsistencies likely arise from methodological differences: phylogenomic trees rely on conserved single-copy core genes, whereas ANI analysis incorporates whole-genome comparisons, including rapidly evolving genes. To further investigate evolutionary characteristics, clade-specific orthologous groups were analyzed using the eggNOG database. Functional differences among unique genes in the five clades suggest their potential roles in environmental adaptation. However, the presence of numerous orthologous groups with “unknown function” highlights the need for additional research to elucidate the evolutionary features of N. cyriacigeorgica.

Nocardia species exhibit diverse antimicrobial resistance profiles. Previous studies [54, 55] have classified N. cyriacigeorgica within the type VI drug resistance pattern, characterized by susceptibility to amikacin, cefamandole, cefotaxime, ceftriaxone, and imipenem, but resistance to amoxicillin-clavulanic acid, ampicillin, ciprofloxacin, clarithromycin, and erythromycin. In this study, the antimicrobial susceptibility profiles of five clinical strains largely aligned with the type VI pattern, except for clarithromycin. Imipenem resistance has been reported at high rates in N. cyriacigeorgica, with one study documenting an 86.7% resistance rate (52/60 strains) [53]. Similarly, McGuinness et al. [56] observed elevated resistance to imipenem in N. cyriacigeorgica strains. In contrast, all five clinical strains analyzed in our study exhibited complete sensitivity to imipenem (100%, 5/5). This discrepancy may result from geographical variation or the limited sample size in our analysis.

Antibiotic resistance represents a critical global public health challenge, with the proliferation of ARGs contributing to the rise of multidrug-resistant pathogens. In the genomes of the analyzed N. cyriacigeorgica strains, numerous ARGs were identified, corresponding to four primary resistance mechanisms: (1) mutations or regulatory changes in target enzymes, (2) efflux pump-encoding genes, (3) inhibition of protein or cell wall synthesis, and (4) antibiotic inactivation.

Sulfonamides, particularly trimethoprim-sulfamethoxazole (TMP-SMX), remain the cornerstone of treatment for Nocardia infections [57]. However, prolonged or suboptimal antibiotic exposure increases the risk of resistance development. TMP-SMX resistance is often linked to mutations in the folP and folA genes, which encode dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR), respectively. A prior study demonstrated that adaptive mutations in folP and folP2 reduced TMP-SMX sensitivity in eight strains of Nocardia nova and two strains of N. cyriacigeorgica [58]. Furthermore, resistant folP variants have been identified on MGEs or plasmids, facilitating interspecies transmission [59,60,61]. Although detailed resistance phenotypes for all tested strains in our study remain unresolved, the detection of the folP gene in all strains suggests a widespread prevalence of TMP-SMX resistance genes. This finding underscores the importance of monitoring resistance mechanisms in N. cyriacigeorgica to ensure effective clinical management and mitigate the spread of resistance.

Plasmid-encoded resistance genes, such as ermH and ermR, which confer macrolide resistance, were identified in the majority of strains within clade B (69.6%, 16/23) and clade A (54.5%, 6/11), respectively. This observation suggests the potential for the evolution and dissemination of resistance genes among strains from diverse sources via plasmids. Horizontal gene transfer, mediated by genetic elements such as plasmids and ISs, has been shown to significantly contribute to the dissemination of antibiotic resistance among bacteria [62, 63]. In our study, most strains carried plasmids, and a wide variety of IS elements were detected. These findings underscore the importance of preventing the horizontal transfer of ARGs among Nocardia strains to mitigate the worsening of antimicrobial resistance.

Despite the presence of various resistance genes across all strains, differences in the distribution of specific genes, including VanSO, VanRO, erm(O)-Irm, srmB, ermH, bcl, bla1, and cmIR, were observed. Particularly noteworthy, we found that 80% (8/10) of strains carrying the macrolide resistance gene erm(O)-Irm in clade B, over 60% (19/30) of strains carrying srmB in clades B, A, and E, and 75% (12/16) of strains carrying ermH in clade B were isolated from human specimens. These findings suggest that the antimicrobial resistance genotypes of N. cyriacigeorgica strains may be associated with their isolation origin. The observed variation in resistance genes among strains from different sources highlights the potential for distinct resistance mechanisms in clinical strains, warranting further investigation.

The virulence mechanisms of N. cyriacigeorgica remain poorly characterized. Known virulence factors in Nocardia species include catalase, superoxide dismutase (SOD), secreted toxins, and cell wall proteins [64, 65]. However, the virulence factors vary significantly among different pathogenic Nocardia species [9]. In this study, 208 potential virulence genes were identified, which may contribute to N. cyriacigeorgica pathogenesis and its interactions with host cells. Among these, the Mce family proteins, recognized as key virulence factors in M. tuberculosis and Nocardia spp., plays a critical role in host cell invasion and intracellular survival within macrophages [66,67,68]. The mce gene family comprises nine clusters (mce1–9), with mce4F confirmed as essential for bacterial pathogenesis in M. tuberculosis [69]. For instance, Mce3C as a surface protein has been shown to promote mycobacterial adhesion to and invasion of macrophages [70]. Phylogenetic and pan-genome analyses of 141 Nocardia genomes revealed the absence of mce1 and mce2 in 27 N. cyriacigeorgica strains, while highlighting the pathogenic roles of mce3C and mce4F in N. keratitis [71]. Our findings demonstrated that mce3, mce4, mce5, mce6, mce7, mce8, and mce9 were present in most tested strains, whereas mce1 and mce2 were absent. Further analysis revealed that all strains, except for three environmental strains from oil, harbored mce4F. Additionally, 27 of the 63 strains carried both mce4F and mce3C. The widespread presence of mce4F suggests its potential as a reliable detection marker. Although mce3C exhibited varied distributions across the five clades, no correlation was observed between the mce3C gene and strain origin, possibly due to incomplete source information for some strains. Further investigation is required to clarify the roles of mce4F and mce3C in the pathogenic mechanisms of N. cyriacigeorgica.

Antioxidant proteins such as catalase and SOD are believed to counteract the oxidative killing mechanisms of phagocytes [64]. Consistent with previous studies on Nocardia species, including N. seriolae UTF1, N. brasiliensis HUJEG-1, and N. cyriacigeorgica GUH-2 [72,73,74], our study identified two catalase genes (katA and katG) and two SOD genes (sodA and sodC) in the majority of N. cyriacigeorgica genomes. Intracellular pathogens must also adapt to low-oxygen conditions to survive within host cells. In our study, nitrosative stress-related genes (narG, narH, and narI) were present in all analyzed strains. However, nirB and nirD were not detected, contrasting with previous genomic research on N. cyriacigeorgica GUH-2 [72]. The importance of catalase, SOD, and nitrate reductase genes in the pathogenesis of N. brasiliensis has been demonstrated in studies of genomic changes associated with the loss of virulence after 200 continuous subcultures in mice [75]. Based on our comparative genomic results, katA, katG, sodA, sodC, narG, narH, and narI may serve as potential targets for further investigation into the pathogenic mechanisms of N. cyriacigeorgica.

When engulfed by phagocytes, intracellular pathogens are enclosed within phagosomes, which subsequently fuse with vesicles to form phagolysosomes for the degradation of ingested particles [76]. Previous studies have shown that Nocardia can survive within host cells and evade immune responses by inhibiting phagosome-lysosome fusion and reducing intracellular acid phosphatase levels in macrophages [12]. PtpA and Ndk proteins have been implicated in arresting macrophage phagosomal maturation, enabling pathogens to evade immune clearance and establish persistent infections [77]. Our study identified the presence of the ptpA and ndk genes in all strains, which are associated with phagosome arrest. These findings underscore the necessity of further investigation into the roles of ptpA and ndk as potential candidate genes in phagosomal maturation arrest. Future studies could utilize gene knockout experiments, confocal microscopy analysis, and multi-omics approaches to investigate their functions.

The entB gene, responsible for enterobactin production, has been identified as a virulence factor in enterobacteria [78,79,80]. Notably, Han et al. demonstrated that the siderophore virulence gene entB plays a critical role in significantly enhancing the virulence of carbapenem-resistant K. pneumoniae (CRKP) strains [81]. In our study, entB was detected in over 60% (14/23) of strains within clade B, with the majority of these strains (64.3%, 9/14) originating from clinical sources. This observation indicates clade-specific variations in entB distribution. To date, limited research has been conducted on the role of entB as a siderophore virulence gene in intracellular pathogens. Further studies are warranted to elucidate its contribution to the virulence of N. cyriacigeorgica.

Conclusions

Phylogenetic analysis based on single-copy genes identified five major clades within N. cyriacigeorgica. This finding enhances our understanding of the species’ genetic diversity and evolutionary relationships. Additionally, plasmid-mediated ARGs were identified within the N. cyriacigeorgica chromosome, underscoring the potential risk of horizontal transmission of drug resistance genes via MGEs. Comparative genomic analysis identified several pathogenic genes that may play critical roles in the virulence of N. cyriacigeorgica. However, the precise functions and mechanisms of these genes in pathogenicity remain unclear. These findings emphasize the need for further comprehensive studies to investigate the molecular basis of N. cyriacigeorgica pathogenesis and its implications for clinical treatment strategies.

Limitations

This comparative genomic study has several limitations that warrant acknowledgment. First, the lack of detailed source information for some strains from the NCBI database may have introduced bias, potentially affecting the accuracy of the phylogenetic and genomic findings. Second, although ISs and plasmids within the N. cyriacigeorgica chromosome were analyzed, potential virulence or ARGs on plasmids were not investigated. Such an investigation would offer a more comprehensive assessment of the risks linked to horizontal gene transfer of these elements.

Third, while the mce3 and mce4 gene families were evaluated, the pathogenicity of strains carrying both mce3C and mce4F genes was not explored. This represents a significant gap in understanding the roles of these genes in virulence and host–pathogen interactions. Future research should prioritize functional studies, including gene knockout experiments and in vivo models, to clarify the roles of mce3C and mce4F in the pathogenic mechanisms of N. cyriacigeorgica.

Addressing these limitations will enhance our understanding of the genomic features, resistance mechanisms, and pathogenic potential of N. cyriacigeorgica. Such insights are essential for developing effective strategies to combat infections caused by this opportunistic pathogen.

Data availability

The sequencing data in the study are publicly available. These data can be found here: the National Library of Medicine (http://www.ncbi.nlm.nih.gov/bioproject/1214743) under the accession numbers PRJNA1214743.

References

  1. Brown-Elliott BA, Brown JM, Conville PS, Wallace RJ. Clinical and laboratory features of the Nocardia spp. based on current molecular taxonomy. Clin Microbiol Rev. 2006;19(2):259–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Traxler RM, Bell ME, Lasker B, Headd B, Shieh WJ, McQuiston JR. Updated review on Nocardia species: 2006–2021. Clin Microbiol Rev. 2022;35(4): e0002721.

    Article  PubMed  Google Scholar 

  3. Conville PS, Brown-Elliott BA, Smith T, Zelazny AM. The complexities of Nocardia taxonomy and identification. J Clin Microbiol. 2018. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/JCM.01419-17.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Roch M, Sierra R, Andrey DO. Antibiotic heteroresistance in ESKAPE pathogens, from bench to bedside. Clin Microbiol Infect. 2023;29(3):320–5.

    Article  CAS  PubMed  Google Scholar 

  5. Venkateswaran P, Vasudevan S, David H, Shaktivel A, Shanmugam K, Neelakantan P, Solomon AP. Revisiting ESKAPE Pathogens: virulence, resistance, and combating strategies focusing on quorum sensing. Front Cell Infect Microbiol. 2023;13:1159798.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Beaman BL, Beaman L. Nocardia species: host-parasite relationships. Clin Microbiol Rev. 1994;7(2):213–64.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Wang HK, Sheng WH, Hung CC, Chen YC, Lee MH, Lin WS, Hsueh PR, Chang SC. Clinical characteristics, microbiology, and outcomes for patients with lung and disseminated nocardiosis in a tertiary hospital. J Formos Med Assoc. 2015;114(8):742–9.

    Article  PubMed  Google Scholar 

  8. Martinez-Barricarte R. Isolated Nocardiosis, an unrecognized primary immunodeficiency? Front Immunol. 2020;11:590239.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Mehta HH, Shamoo Y. Pathogenic Nocardia: a diverse genus of emerging pathogens or just poorly recognized? PLoS Pathog. 2020;16(3): e1008280.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. McNeil MM, Brown JM. The medically important aerobic actinomycetes: epidemiology and microbiology. Clin Microbiol Rev. 1994;7(3):357–417.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Lerner PI. Nocardiosis. Clin Infect Dis. 1996;22(6):891–903.

    Article  CAS  PubMed  Google Scholar 

  12. Davis-Scibienski C, Beaman BL. Interaction of Nocardia asteroides with rabbit alveolar macrophages: association of virulence, viability, ultrastructural damage, and phagosome-lysosome fusion. Infect Immun. 1980;28(2):610–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Yassin AF, Rainey FA, Steiner U. Nocardia cyriacigeorgici sp. nov. Int J Syst Evol Microbiol. 2001;51(4):1419–23.

    Article  CAS  PubMed  Google Scholar 

  14. Saunier F, Grange S, Rigaill J, Lutz MF, Gagneux-Brunon A, Botelho-Nevers E. Bacteremia and adrenal gland abscess due to Nocardia cyriacigeorgica: a case report and review. BMC Infect Dis. 2022;22(1):966.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Gabay S, Yakubovsky M, Ben-Ami R, Grossman R. Nocardia cyriacigeorgica brain abscess in a patient on low dose steroids: a case report and review of the literature. BMC Infect Dis. 2022;22(1):635.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Akcaglar S, Yilmaz E, Heper Y, Alver O, Akalin H, Ener B, Tore O, Ersoy C, Imamoglu S. Nocardia cyriacigeorgica: pulmonary infection in a patient with Basedow-Graves disease and a short review of reported cases. Int J Infect Dis. 2008;12(3):335–8.

    Article  CAS  PubMed  Google Scholar 

  17. Alshammari K, Al Hothaly B, Alrabiah F. Case of Nocardia cyriacigeorgica infection of the eye in a granulomatosis with polyangiitis patient. Cureus. 2020;12(10): e11178.

    PubMed  PubMed Central  Google Scholar 

  18. Schlaberg R, Huard RC, Della-Latta P. Nocardia cyriacigeorgica, an emerging pathogen in the United States. J Clin Microbiol. 2008;46(1):265–73.

    Article  CAS  PubMed  Google Scholar 

  19. Gupta S, Grant LM, Powers HR, Kimes KE, Hamdi A, Butterfield RJ, Gea-Banacloche J, Vijayvargiya P, Hata DJ, Meza Villegas DM, et al. Invasive Nocardia infections across distinct geographic regions United States. Emerg Infect Dis. 2023;29(12):2417–25.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Ercibengoa M, Camara J, Tubau F, Garcia-Somoza D, Galar A, Martin-Rabadan P, Marin M, Mateu L, Garcia-Olive I, Prat C, et al. A multicentre analysis of Nocardia pneumonia in Spain: 2010–2016. Int J Infect Dis. 2020;90:161–6.

    Article  CAS  PubMed  Google Scholar 

  21. Valdezate S, Garrido N, Carrasco G, Medina-Pascual MJ, Villalon P, Navarro AM, Saez-Nieto JA. Epidemiology and susceptibility to antimicrobial agents of the main Nocardia species in Spain. J Antimicrob Chemother. 2017;72(3):754–61.

    CAS  PubMed  Google Scholar 

  22. Hashemi-Shahraki A, Heidarieh P, Bostanabad SZ, Hashemzadeh M, Feizabadi MM, Schraufnagel D, Mirsaeidi M. Genetic diversity and antimicrobial susceptibility of Nocardia species among patients with nocardiosis. Sci Rep. 2015;5:17862.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Wang H, Zhu Y, Cui Q, Wu W, Li G, Chen D, Xiang L, Qu J, Shi D, Lu B. Epidemiology and antimicrobial resistance profiles of the Nocardia species in China, 2009 to 2021. Microbiol Spectr. 2022;10(2): e0156021.

    Article  PubMed  Google Scholar 

  24. Yang J, Ren HT, Wang J, Dong AY, Chen YL, Hu DX, Wang H, Ji HM, Wen HN, Chen Y, et al. Clinical characteristics, susceptibility profiles, and treatment of nocardiosis: a multicenter retrospective study in 2015–2021. Int J Infect Dis. 2023;130:136–43.

    Article  CAS  PubMed  Google Scholar 

  25. Han Y, Cheng M, Li Z, Chen H, Xia S, Zhao Y, Wang Y, He W, Wang W. Clinical characteristics and drug resistance of Nocardia in Henan, China, 2017–2023. Ann Clin Microbiol Antimicrob. 2024;23(1):23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Wei M, Xu X, Yang J, Wang P, Liu Y, Wang S, Yang C, Gu L. MLSA phylogeny and antimicrobial susceptibility of clinical Nocardia isolates: a multicenter retrospective study in China. BMC Microbiol. 2021;21(1):342.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Kageyama A, Hoshino Y, Yazawa K, Poonwan N, Takeshita N, Maki S, Mikami Y. Nocardia cyriacigeorgica is a significant pathogen responsible for nocardiosis in Japan and Thailand. Mycopathologia. 2005;160(1):15–9.

    Article  PubMed  Google Scholar 

  28. Kageyama A, Yazawa K, Ishikawa J, Hotta K, Nishimura K, Mikami Y. Nocardial infections in Japan from 1992 to 2001, including the first report of infection by Nocardia transvalensis. Eur J Epidemiol. 2004;19(4):383–9.

    Article  CAS  PubMed  Google Scholar 

  29. Poonwan N, Mekha N, Yazawa K, Thunyaharn S, Yamanaka A, Mikami Y. Characterization of clinical isolates of pathogenic Nocardia strains and related actinomycetes in Thailand from 1996 to 2003. Mycopathologia. 2005;159(3):361–8.

    Article  PubMed  Google Scholar 

  30. Wauters G, Avesani V, Charlier J, Janssens M, Vaneechoutte M, Delmee M. Distribution of nocardia species in clinical samples and their routine rapid identification in the laboratory. J Clin Microbiol. 2005;43(6):2624–8.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Tan YE, Chen SC, Halliday CL. Antimicrobial susceptibility profiles and species distribution of medically relevant Nocardia species: results from a large tertiary laboratory in Australia. J Glob Antimicrob Resist. 2020;20:110–7.

    Article  PubMed  Google Scholar 

  32. Lowman W, Aithma N. Antimicrobial susceptibility testing and profiling of Nocardia species and other aerobic actinomycetes from South Africa: comparative evaluation of broth microdilution versus the Etest. J Clin Microbiol. 2010;48(12):4534–40.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Han L, Ji X, Xu S, Fan S, Wang C, Wei K, Wang X, Song H, Zheng N, Sun L, et al. Microbiological profile of distinct virulence of Nocardia cyriacigeorgica strains in vivo and in vitro. Microb Pathog. 2020;142:104042.

    Article  CAS  PubMed  Google Scholar 

  34. Ji X, Zhang X, Sun L, Hou X, Song H, Han L, Xu S, Li H, Qiu X, Li M, et al. The heparin-binding hemagglutinin of Nocardia cyriacigeorgica GUH-2 stimulates inflammatory cytokine secretion through activation of nuclear factor kappaB and mitogen-activated protein kinase pathways via TLR4. Front Cell Infect Microbiol. 2020;10:3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Kong F, Chen SC, Chen X, Sintchenko V, Halliday C, Cai L, Tong Z, Lee OC, Sorrell TC. Assignment of reference 5’-end 16S rDNA sequences and species-specific sequence polymorphisms improves species identification of Nocardia. Open Microbiol J. 2009;3:97–105.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Kong F, Wang H, Zhang E, Sintchenko V, Xiao M, Sorrell TC, Chen X, Chen SC. secA1 gene sequence polymorphisms for species identification of Nocardia species and recognition of intraspecies genetic diversity. J Clin Microbiol. 2010;48(11):3928–34.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Carrasco G, Valdezate S, Garrido N, Medina-Pascual MJ, Villalon P, Saez-Nieto JA. gyrB analysis as a tool for identifying Nocardia species and exploring their phylogeny. J Clin Microbiol. 2015;53(3):997–1001.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Carrasco G, Valdezate S, Garrido N, Villalon P, Medina-Pascual MJ, Saez-Nieto JA. Identification, typing, and phylogenetic relationships of the main clinical Nocardia species in spain according to their gyrB and rpoB genes. J Clin Microbiol. 2013;51(11):3602–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Yin X, Liang S, Sun X, Luo S, Wang Z, Li R. Ocular nocardiosis: HSP65 gene sequencing for species identification of Nocardia spp. Am J Ophthalmol. 2007;144(4):570–3.

    Article  CAS  PubMed  Google Scholar 

  40. McTaggart LR, Richardson SE, Witkowska M, Zhang SX. Phylogeny and identification of Nocardia species on the basis of multilocus sequence analysis. J Clin Microbiol. 2010;48(12):4525–33.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Toyokawa M, Ohana N, Ueda A, Imai M, Tanno D, Honda M, Takano Y, Ohashi K, Saito K, Shimura H. Identification and antimicrobial susceptibility profiles of Nocardia species clinically isolated in Japan. Sci Rep. 2021;11(1):16742.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Woods GL, Brown-Elliott BA, Conville PS, Desmond EP, Hall GS, Lin G, Pfyffer GE, Ridderhof JC, Siddiqi SH, Wallace RJ, et al. In Susceptibility testing of mycobacteria, Nocardiae, and other aerobic actinomycetes. 2nd ed. Wayne: CLSI Standards: Guidelines for Health Care Excellence; 2011.

    Google Scholar 

  43. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19(5):455–77.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Wick RR, Judd LM, Gorrie CL, Holt KE. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol. 2017;13(6): e1005595.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, Cuomo CA, Zeng Q, Wortman J, Young SK, et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS ONE. 2014;9(11): e112963.

    Article  PubMed  PubMed Central  Google Scholar 

  46. Hyatt D, Chen GL, Locascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinform. 2010;11:119.

    Article  Google Scholar 

  47. Besemer J, Borodovsky M. GeneMark: web software for gene finding in prokaryotes, eukaryotes and viruses. Nucleic Acids Res. 2005;33:W451-454.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Li Z, Lu X, Wang D, Liang WL, Zhang J, Li J, Xu J, Pang B, Kan B. Genomic comparison of serogroups O159 and O170 with other Vibrio cholerae serogroups. BMC Genom. 2019;20(1):241.

    Article  Google Scholar 

  49. Emms DM, Kelly S. OrthoFinder: solving fundamental biases in whole genome comparisons dramatically improves orthogroup inference accuracy. Genome Biol. 2015;16(1):157.

    Article  PubMed  PubMed Central  Google Scholar 

  50. Huerta-Cepas J, Szklarczyk D, Heller D, Hernandez-Plaza A, Forslund SK, Cook H, Mende DR, Letunic I, Rattei T, Jensen LJ, et al. eggNOG 5.0: a hierarchical, functionally and phylogenetically annotated orthology resource based on 5090 organisms and 2502 viruses. Nucleic Acids Res. 2019;47(D1):D309–14.

    Article  CAS  PubMed  Google Scholar 

  51. Xie Z, Tang H. ISEScan: automated identification of insertion sequence elements in prokaryotic genomes. Bioinformatics. 2017;33(21):3340–7.

    Article  CAS  PubMed  Google Scholar 

  52. Alcock BP, Raphenya AR, Lau TTY, Tsang KK, Bouchard M, Edalatmand A, Huynh W, Nguyen AV, Cheng AA, Liu S, et al. CARD 2020: antibiotic resistome surveillance with the comprehensive antibiotic resistance database. Nucleic Acids Res. 2020;48(D1):D517–25.

    CAS  PubMed  Google Scholar 

  53. Xu S, Wei M, Li G, Li Z, Che Y, Han L, Jia W, Li F, Li D, Li Z. Comprehensive analysis of the Nocardia cyriacigeorgica complex reveals five species-level clades with different evolutionary and pathogenicity characteristics. mSystems. 2022;7(3): e0140621.

    Article  PubMed  Google Scholar 

  54. Conville PS, Witebsky FG. Organisms designated as Nocardia asteroides drug pattern type VI are members of the species Nocardia cyriacigeorgica. J Clin Microbiol. 2007;45(7):2257–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Wallace RJ Jr, Steele LC, Sumter G, Smith JM. Antimicrobial susceptibility patterns of Nocardia asteroides. Antimicrob Agents Chemother. 1988;32(12):1776–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. McGuinness SL, Whiting SE, Baird R, Currie BJ, Ralph AP, Anstey NM, Price RN, Davis JS, Tong SY. Nocardiosis in the tropical northern territory of Australia 1997–2014. Open Forum Infect Dis. 2016;3(4):ofw208.

    Article  PubMed  PubMed Central  Google Scholar 

  57. Wilson JW. Nocardiosis: updates and clinical overview. Mayo Clin Proc. 2012;87(4):403–7.

    Article  PubMed  PubMed Central  Google Scholar 

  58. Mehta H, Weng J, Prater A, Elworth RAL, Han X, Shamoo Y. Pathogenic Nocardia cyriacigeorgica and Nocardia nova evolve to resist trimethoprim-sulfamethoxazole by both expected and unexpected pathways. Antimicrob Agents Chemother. 2018;62(7):e00364-e418. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/AAC.00364-18.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Metcalf BJ, Chochua S, Gertz RE Jr, Li Z, Walker H, Tran T, Hawkins PA, Glennen A, Lynfield R, Li Y, et al. Using whole genome sequencing to identify resistance determinants and predict antimicrobial resistance phenotypes for year 2015 invasive pneumococcal disease isolates recovered in the United States. Clin Microbiol Infect. 2016;22(12):1002 e1001-1002 e1008.

    Article  Google Scholar 

  60. Huovinen P. Resistance to trimethoprim-sulfamethoxazole. Clin Infect Dis. 2001;32(11):1608–14.

    Article  CAS  PubMed  Google Scholar 

  61. Cornick JE, Harris SR, Parry CM, Moore MJ, Jassi C, Kamng’ona A, Kulohoma B, Heyderman RS, Bentley SD, Everett DB. Genomic identification of a novel co-trimoxazole resistance genotype and its prevalence amongst Streptococcus pneumoniae in Malawi. J Antimicrob Chemother. 2014;69(2):368–74.

    Article  CAS  PubMed  Google Scholar 

  62. Yao S, Yu J, Zhang T, Xie J, Yan C, Ni X, Guo B, Cui C. Comprehensive analysis of distribution characteristics and horizontal gene transfer elements of bla(NDM-1)-carrying bacteria. Sci Total Environ. 2024;946:173907.

    Article  CAS  PubMed  Google Scholar 

  63. Yin Y, Chen H, Li S, Gao H, Sun S, Li H, Wang R, Jin L, Liu Y, Wang H. Daptomycin resistance in methicillin-resistant Staphylococcus aureus is conferred by IS256 insertion in the promoter of mprF along with mutations in mprF and walK. Int J Antimicrob Agents. 2019;54(6):673–80.

    Article  CAS  PubMed  Google Scholar 

  64. Beaman BL, Black CM, Doughty F, Beaman L. Role of superoxide dismutase and catalase as determinants of pathogenicity of Nocardia asteroides: importance in resistance to microbicidal activities of human polymorphonuclear neutrophils. Infect Immun. 1985;47(1):135–41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Black CM, Beaman BL, Donovan RM, Goldstein E. Intracellular acid phosphatase content and ability of different macrophage populations to kill Nocardia asteroides. Infect Immun. 1985;47(2):375–83.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Arruda S, Bomfim G, Knights R, Huima-Byron T, Riley LW. Cloning of an M. tuberculosis DNA fragment associated with entry and survival inside cells. Science. 1993;261(5127):1454–7.

    Article  CAS  PubMed  Google Scholar 

  67. Ji X, Tan X, Hou X, Si C, Xu S, Tang L, Yuan X, Li Z. Cloning, expression, invasion, and immunological reactivity of a mammalian cell entry protein encoded by the mce1 operon of Nocardia farcinica. Front Microbiol. 2017;8:281.

    Article  PubMed  PubMed Central  Google Scholar 

  68. Ji X, Zhang X, Sun L, Hou X, Song J, Tan X, Song H, Qiu X, Li M, Tang L, et al. Mce1C and Mce1D facilitate N. farcinica invasion of host cells and suppress immune responses by inhibiting innate signaling pathways. Sci Rep. 2020;10(1):14908.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Joshi SM, Pandey AK, Capite N, Fortune SM, Rubin EJ, Sassetti CM. Characterization of mycobacterial virulence genes through genetic interaction mapping. Proc Natl Acad Sci U S A. 2006;103(31):11760–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Zhang Y, Li J, Li B, Wang J, Liu CH. Mycobacterium tuberculosis Mce3C promotes mycobacteria entry into macrophages through activation of beta2 integrin-mediated signalling pathway. Cell Microbiol. 2018;20(2): e12800.

    Article  Google Scholar 

  71. Guo X, Zhang Z, Chen Q, Wang L, Xu X, Wei Z, Zhang Y, Chen K, Wang Z, Lu X, et al. Whole genome sequencing highlights the pathogenic profile in Nocardia keratitis. Invest Ophthalmol Vis Sci. 2024;65(3):26.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Zoropogui A, Pujic P, Normand P, Barbe V, Belli P, Graindorge A, Roche D, Vallenet D, Mangenot S, Boiron P, et al. The Nocardia cyriacigeorgica GUH-2 genome shows ongoing adaptation of an environmental Actinobacteria to a pathogen’s lifestyle. BMC Genom. 2013;14:286.

    Article  CAS  Google Scholar 

  73. Yasuike M, Nishiki I, Iwasaki Y, Nakamura Y, Fujiwara A, Shimahara Y, Kamaishi T, Yoshida T, Nagai S, Kobayashi T, et al. Analysis of the complete genome sequence of Nocardia seriolae UTF1, the causative agent of fish nocardiosis: The first reference genome sequence of the fish pathogenic Nocardia species. PLoS ONE. 2017;12(3): e0173198.

    Article  PubMed  PubMed Central  Google Scholar 

  74. Vera-Cabrera L, Ortiz-Lopez R, Elizondo-Gonzalez R, Ocampo-Candiani J. Complete genome sequence analysis of Nocardia brasiliensis HUJEG-1 reveals a saprobic lifestyle and the genes needed for human pathogenesis. PLoS ONE. 2013;8(6): e65425.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Gonzalez-Carrillo C, Millan-Sauceda C, Lozano-Garza HG, Ortiz-Lopez R, Elizondo-Gonzalez R, Welsh O, Ocampo-Candiani J, Vera-Cabrera L. Genomic changes associated with the loss of Nocardia brasiliensis virulence in mice after 200 in vitro passages. Infect Immun. 2016;84(9):2595–606.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Vieira OV, Botelho RJ, Grinstein S. Phagosome maturation: aging gracefully. Biochem J. 2002;366(Pt 3):689–704.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Forrellad MA, Klepp LI, Gioffre A, Sabio y Garcia J, Morbidoni HR, de la Paz Santangelo M, Cataldi AA, Bigi F. Virulence factors of the Mycobacterium tuberculosis complex. Virulence. 2013;4(1):3–66.

    Article  PubMed  PubMed Central  Google Scholar 

  78. Hu D, Li Y, Ren P, Tian D, Chen W, Fu P, Wang W, Li X, Jiang X. Molecular epidemiology of hypervirulent carbapenemase-producing Klebsiella pneumoniae. Front Cell Infect Microbiol. 2021;11:661218.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Hwang JH, Park JS, Bae TW, Hwang JH, Lee J. Community-acquired solitary brain abscesses caused by Hypervirulent Klebsiella pneumoniae in a healthy adult. Microorganisms. 2024;12(5):894.

    Article  PubMed  PubMed Central  Google Scholar 

  80. May T, Okabe S. Enterobactin is required for biofilm development in reduced-genome Escherichia coli. Environ Microbiol. 2011;13(12):3149–62.

    Article  CAS  PubMed  Google Scholar 

  81. Han R, Niu M, Liu S, Mao J, Yu Y, Du Y. The effect of siderophore virulence genes entB and ybtS on the virulence of Carbapenem-resistant Klebsiella pneumoniae. Microb Pathog. 2022;171:105746.

    Article  CAS  PubMed  Google Scholar 

  82. Carrasco G, Monzón S, San Segundo M, et al. Molecular characterization and antimicrobial susceptibilities of Nocardia species isolated from the soil; a comparison with species isolated from humans. Microorganisms. 2020;8(6):900. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/microorganisms8060900.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Xu S, Li Z, Huang Y, et al. Whole genome sequencing reveals the genomic diversity, taxonomic classification, and evolutionary relationships of the genus Nocardia. PLoS Negl Trop Dis. 2021;15(8): e0009665. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pntd.0009665.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Vautrin F, Bergeron E, Dubost A, et al. Genome sequences of three Nocardia cyriacigeorgica strains and one Nocardia asteroides strain. Microbiol Resour Announc. 2019;8(33):e00600-e619. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/MRA.00600-19.

    Article  PubMed  PubMed Central  Google Scholar 

  85. Jorgensen TS, Mohite OS, Sterndorff EB, et al. A treasure trove of 1034 actinomycete genomes. Nucleic Acids Res. 2024;52(13):7487–503. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/nar/gkae523.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Not applicable.

Funding

This research was funded by the Hebei Natural Science Foundation (No.H2022505004), the National Natural Science Foundation of China (No.31200142), and the Hebei Medical Science Research Project (No.20211116).

Author information

Author notes

  1. Chen Yang, Yue-xin Zheng, Hong-yi Gu and Hong Chen have contributed equally to this work.

Authors and Affiliations

  1. National Engineering Research Center of Immunological ProductsDepartment of Microbiology and Biochemical PharmacyCollege of Pharmacy, Army Medical University, Chongqing, 400038, China

    Chen Yang & Jiang Gu

  2. Department of General Surgery, The Sixth Medical Center of PLA General Hospital, Beijing, 100048, China

    Yue-xin Zheng

  3. Department of Public Affairs Management, Tianjin Medical University, Tianjin, 300203, China

    Hong-yi Gu

  4. Department of Clinical Pharmacy, Bethune International Peace Hospital, Shijiazhuang, 050081, China

    Hong Chen

  5. Department of Clinical Laboratory, Bethune International Peace Hospital, Shijiazhuang, 050081, China

    Wei Li, Fang Li, Jing Chen, Fu-kun Wang, Zuo-hao Wu & Yan Cheng

  6. Department of Information, Bethune International Peace Hospital, Shijiazhuang, 050081, China

    Yu-wang Bi

  7. Department of Basic Medical Laboratory, Bethune International Peace Hospital, Shijiazhuang, 050081, China

    Qing-qing Sun & Han-bing Meng

  8. Department of Laboratory Medicine, People’s Hospital of Chongqing Hechuan, Chongqing, 401520, China

    Shu Yu

Authors
  1. Chen Yang
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Yue-xin Zheng
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Hong-yi Gu
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Hong Chen
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Wei Li
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Fang Li
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Yu-wang Bi
    View author publications

    You can also search for this author inPubMed Google Scholar

  8. Jing Chen
    View author publications

    You can also search for this author inPubMed Google Scholar

  9. Fu-kun Wang
    View author publications

    You can also search for this author inPubMed Google Scholar

  10. Qing-qing Sun
    View author publications

    You can also search for this author inPubMed Google Scholar

  11. Han-bing Meng
    View author publications

    You can also search for this author inPubMed Google Scholar

  12. Zuo-hao Wu
    View author publications

    You can also search for this author inPubMed Google Scholar

  13. Shu Yu
    View author publications

    You can also search for this author inPubMed Google Scholar

  14. Jiang Gu
    View author publications

    You can also search for this author inPubMed Google Scholar

  15. Yan Cheng
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

All authors contributed to this work. All authors have read and agreed to the published version of the manuscript. YC, JG, and SY designed the study. YC, JG, SY, CY, Y-x Z, H-y G, HC, WL, FL, Y-w B, JC, F-k W, Q-q S, H-b M, and Z-h W performed the experiments and interpreted the data. YC, JG, SY, CY, Y-x Z, H-y G, and HC wrote the first draft of the paper. YC, JG, and SY reviewed and approved the final report.

Corresponding authors

Correspondence to Shu Yu, Jiang Gu or Yan Cheng.

Ethics declarations

Ethics approval and consent to participate

This study was approved by the Ethics Committee of Bethune International Peace Hospital (No.2024-KY-409).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

12941_2025_791_MOESM1_ESM.pdf

Supplementary material 1: Figure S1. Genes with a frequency higher than 30% from the accessory genomes were further analysed by hclust (Hierarchical clustering).

12941_2025_791_MOESM2_ESM.pdf

Supplementary material 2: Figure S2. Genes with a frequency higher than 30% from the accessory genomes were further analysed by DPAC (Dynamic profile analysis for clusters).

Supplementary material 3.

Supplementary material 4.

Supplementary material 5.

Supplementary material 6.

Supplementary material 7.

Supplementary material 8.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yang, C., Zheng, Yx., Gu, Hy. et al. Genomic characteristics, virulence potential, antimicrobial resistance profiles, and phylogenetic insights into Nocardia cyriacigeorgica. Ann Clin Microbiol Antimicrob 24, 22 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12941-025-00791-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12941-025-00791-x

Keywords

Annals of Clinical Microbiology and Antimicrobials

ISSN: 1476-0711

Contact us