Isolation and Characterization of a Novel Achromobacter Strain from a Diarrheal Stool Specimen

Introduction

Achromobacter species, which belong to the family Alcaligenaceae, are non-fermentative gram-negative bacteria that have garnered attention because of their environmental resilience and clinical significance. These organisms are ubiquitous in various environments, including soil and water, and are known to be opportunistic pathogens, particularly in immunocompromised individuals.¹ The incidence of Achromobacter infections has been increasing, particularly among patients with cystic fibrosis, chronic lung disease, and those undergoing invasive procedures. The clinical management of Achromobacter infections poses significant challenges, as these bacteria often exhibit multidrug resistance, complicating treatment options and leading to increased morbidity and mortality rates.²

The classification of Achromobacter species is evolving, with several identified species including Achromobacter xylosoxidans and others.³ However, the taxonomy of the genus is complex and unresolved, and further research is required to clarify the species distinctions and pathogenic potential. While traditional taxonomy relies on 16S rRNA gene sequencing, whole-genome sequencing (WGS) offers more precise species delineation using metrics, such as average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH).^4,5 These genomic tools are essential for distinguishing closely related species in genera such as Achromobacter, where phenotypic similarities often obscure taxonomic boundaries. Understanding Achromobacter’s biological traits and clinical implications of Achromobacter is vital for developing effective diagnostics and treatments given the rising prevalence of related infections.

This study details the isolation of strain L3024hy from a patient with diarrhea and its precise classification within the Achromobacter genus using whole-genome sequencing, ANI/dDDH analysis, and core-genome phylogenetics. We also evaluated its antimicrobial resistance and virulence genes to understand its clinical significance and clarify the genomic traits and potential impact of this new Achromobacter strain.

Methods

Strain Isolation

During routine checks for carbapenem-resistant bacteria at a tertiary teaching hospital, fecal samples were collected from diarrheal patients. On November 16, 2020, a sample from a 19-year-old Crohn’s disease patient with a surgically treated perianal fistula revealed Achromobacter sp. strain L3024hy. The samples were incubated overnight in Brain Heart Infusion Broth and screened on MacConkey agar containing meropenem to detect resistant strains.

Whole-Genome Sequencing (WGS) and in silico Analyses

Genomic DNA was extracted using the SteadyPure Kit and sequenced on Illumina Novaseq 6000 and Nanopore PromethION platforms. For Illumina, DNA was sonicated to 350 bp, end-polished, A-tailed, and adapter-ligated, Fastp (0.23.1) assessed short-read sequencing quality by removing paired reads with adapter contamination, over 10% unknown bases, or more than 50% low-quality bases (Phred quality < 5), using default machine settings unless specified otherwise. The hybrid assembly strategy was implemented with Unicycler v0.4.8, which utilizes short reads to correct long reads, to obtain a high-quality, circularized genome sequence, and annotation was done with Prokka v1.14.^6,7 Abricate identified antibiotic resistance and virulence genes in the L3024hy strain using CARD and VFDB databases.^8,9

Average Nucleotide Identity (ANI) and Digital DNA-DNA Hybridization (dDDH)

The genetic similarity of the isolated Achromobacter spp. strains was assessed using ANI and dDDH analyses. The complete genome sequences of other Achromobacter spp. in the NCBI database were used for comparison. ANI values were calculated using PyANI and dDDH estimates using the GGDC 2.1. These analyses aimed to evaluate whole-genome genetic similarity and determine taxonomic relationships between Achromobacter spp. isolates and related species, based on species delineation thresholds.

Phylogenetic Analysis

To determine the phylogenetic relationships between the isolated Achromobacter strains and the reference species, a phylogenetic tree was created using the complete genome sequences of 23 Achromobacter species from the NCBI database. The Snippy pipeline (v1.0) facilitated multiple sequence alignment and SNP calling. Core genome SNPs were used to construct a maximum-likelihood phylogenetic tree with Fasttree (v2.1), using the best substitution model from ModelFinder. The tree was visualized and annotated using iTOL (https://itol.embl.de/) and its robustness was assessed with 1000 bootstrap replicates.

Results and Discussion

On November 16, 2020, Achromobacter sp. strain L3024hy was isolated from a 19-year-old Crohn’s disease patient with a perianal fistula. The carbapenem-resistant strain was identified as A. xylosoxidans by MALDI-TOF. Whole-genome sequencing, employing both long-read and short-read technologies (Table 1), was conducted for precise species identification. Long-read sequencing yielded 243,062 reads totaling 2.15 Gb, with an average read length of 8839.7 bp and N50 of 11,580 bp, indicating high continuity and reliability. Short-read sequencing generated 9.41 million raw reads (1.41 Gb), with 99.79% effective reads and excellent quality scores (Q30 = 89.95%, GC% = 66.21), ensuring the accuracy of further analyses. The hybrid assembly produced a single circular chromosome of 6,905,297 bp (GC% = 66) with 311× coverage, resulting in a fully closed genome (one contig, L50 = 1).

Table 1 Features of the Complete Whole-Genome Sequences of Strain L3024hy

Based on the KmerFinder database, the strain L3024hy was classified as A. insolitus, however, classification based on the nrdA gene sequence (https://pubmlst.org/bigsdb?db=pubmlst_achromobacter_seqdef) identified it as A. aegrifaciens [n = 10], and this discrepancy was further compounded by variations in the mass spectrometry profiles. To accurately determine the phylogenetic position of strain L3024, we constructed a core-genome phylogenetic tree using an expanded dataset of 78 genomes. This included the 23 type strains of the genus Achromobacter and up to three additional genomes per species, offering a detailed view of phylogenetic relationships. (`ure 1A). Strain L3024hy clustered within the A. aegrifaciens clade but formed a distinct branch with high bootstrap support. This divergence was further quantified through ANI and dDDH analysis. Strain L3024hy had the highest ANI (94.76%) and dDDH (60.1%) values with the type strain of A. aegrifaciens (SAMEA6797398) (Table 2 and Figure 1B). An ANI value below 95% and a dDDH value below the 70% threshold confirmed that strain L3024hy represents a distinct and previously unrecognized species within the genus Achromobacter. Furthermore, an extensive ANI analysis of 583 Achromobacter genomes from NCBI database showed that only seven strains had an ANI value over 95%, confirming strain L3024hy as a distinct species (Table S1).

Table 2 Average Nucleotide Identity (ANI) and Digital DNA–DNA Hybridization (dDDH) Values Between Strain L3024hy and Type Strains of the Genus Achromobacter

Figure 1 (A) Maximum likelihood phylogenomic tree based on the concatenated nucleotide sequence of core genes of strain L3024hy and type strains of the genus Achromobacter. (B) Average nucleotide identity analysis of strain L3024hy and type strains of the genus Achromobacter. (C) Maximum-likelihood phylogenetic tree based on core-genome SNPs of Novel Achromobacter strains.

To understand the origins and clinical significance of the Achromobacter strain L3024hy, we conducted phylogenomic and comparative genomic analyses (Figure 1C). A phylogenetic tree showed that L3024hy is closely related to environmental strains from Chinese soil, indicating possible environmental sources for this pathogen. Despite minor genomic differences, L3024hy contained more antimicrobial resistance genes than its environmental counterparts (Table S1). Both strains shared the virulence gene, brkB, the few virulence factors identified are probably due to databases that may not fully cover the specialized or unknown virulence mechanisms of Achromobacter species. This suggests that clinical settings drive resistance gene acquisition, making L3024hy a threat to multidrug resistance. The close relationship between clinical and environmental strains highlights the need for monitoring zoonotic transmission and antibiotic resistance.

In summary, extensive phylogenomic analysis utilizing Average Nucleotide Identity and digital DNA-DNA Hybridization values substantiated that strain L3024hy constitutes a distinct and novel species within the genus Achromobacter. The pronounced genotypic divergence, along with its unique genomic characteristics, justifies its taxonomic distinction from other recognized species. This discovery underscores the emergence of clinically significant Achromobacter lineages and emphasizes the critical role of integrating genomic taxonomy into pathogen surveillance systems to enhance monitoring and response strategies against the evolving threats of antimicrobial resistance.