Strain isolation and characteristics

Strain 3007 was isolated from a patient with proteinuria and septic shock. Strain 23021821 came from a septic shock patient's ascites sample, linked to intra-abdominal infection. The two strains were isolated from patients in two geographically distinct hospitals, located in Henan and Shaanxi provinces, respectively. Moreover, the strains were collected at different time points, with no temporal overlap between the two cases. Both Strain 3007 and Strain 23021821 were identified as Providencia rettgeri by MALDI-TOF MS, scoring 9.254 and 9.596 points, respectively. Both strains are resistant to multiple antibiotics: ampicillin, ceftazidime, tetracycline, tigecycline, polymyxin B, colistin, trimethoprim/sulfamethoxazole, and ciprofloxacin. However, they are susceptible to aztreonam (Table 1).

Table 1 Antimicrobial susceptibilities of the strain 3007 and 23021821

The genetic basis for the resistance observed in both strains includes the tet(59) gene (tetracycline resistance), the dfrA1 gene (trimethoprim/sulfamethoxazole resistance), lnu(F) (lincosamide resistance), and arr-3 (rifamycin resistance)(Table S1). Beta-lactam resistance (ampicillin, ceftazidime) is apparent, with Strain 23021821 additionally resistant to cefepime, imipenem, and meropenem due to the presence of blaNDM-1. Conversely, Strain 3007 exhibits sensitivity to cefepime and meropenem, attributable to its sole possession of the blaDHA-1 gene, lacking the capacity to hydrolyze carbapenems. Notably, Strain 3007 demonstrated unexpected imipenem resistance, possibly related to blaDHA-1's weak false-positive AmpC expression [16]. Additionally, Strain 3007's resistance to gentamicin is associated with the aac(3)-IVa gene. Both strains exhibit significant colistin resistance (MIC > 128 mg/L), consistent with previous findings of inherent resistance in this genus [17].

Phylogeny represents a unique branch in genus Providencia

Phylogenetic analysis, including 16S rRNA gene sequencing, multilocus sequence analysis (MLSA) of five housekeeping genes (fusA, gyrB, ileS, lepA, and leuS), and core single nucleotide polymorphism (SNP) analysis derived from whole genome, consistently supported the classification of Strains 3007 and 23021821 as a novel species within the Providencia genus. The 16S rRNA gene sequences of both strains were identical (99.5% identity to Providencia rettgeri) and confirmed their placement in the Providencia genus (Fig. 1A). The MLSA phylogeny, based on 11,739 bp of concatenated gene sequence alignments, distinctly separated Strains 3007 and 23021821 into a unique evolutionary branch with high bootstrap support (90–100%) (Fig. 1B). The core SNP phylogenetic tree displayed a structure largely congruent with the MLSA tree (Fig. 1C), further reinforcing the novel species status of these strains.

Fig. 1

Maximum-likelihood phylogenetic trees of the Providencia genus, represented in three parts. A depicts the tree constructed from 16S rRNA gene sequences. B presents the tree based on concatenated sequences of five housekeeping genes. C illustrates the tree derived from whole-genome SNPs. All parts are rooted using Proteus mirabilis ATCC 29906 as the outgroup. Branch nodes with bootstrap values above 50%, ascertained from 1,000 resamplings, are marked on each tree. Nodes exceeding 80% bootstrap support are distinctly indicated with black circles. The strains studied are highlighted with red circles

Genotypic identification

To further validate the taxonomic classification, Strains 23021821 and 3007 were subjected to whole-genome sequencing, resulting in draft genomes of 4.88 Mb and 4.35 Mb with GC contents of 40.3% and 40.2%, respectively. The average nucleotide identity (ANI) values comparing Strain 23021821 with type strains of all known Providencia species and Strain 3007 revealed a high similarity to strain 3007 (99.28%) but significantly lower similarities to other Providencia species (79.84–84.20%, Table 2). Similarly, the in-silico DNA-DNA hybridization (dDDH) values showed the highest correspondence between strains 23021821 and 3007 (95.1%), with markedly lower values when compared with other Providencia species (21.1–25.6%, Table 2). These results strongly suggest that strains 3007 and 23021821 belong to the same, novel species within the Providencia genus.

Table 2 ANI and dDDH values between strains 23021821 and the type strains of Providencia speciesPhenotypic characterization

Strain 23021821, chosen as the representative for a detailed description, demonstrated biochemical characteristics consistent with Strain 3007. The biochemical profile of Strain 23021821, in comparison to other known Providencia species, is thoroughly detailed in Table 3. Microscopic analysis confirmed these bacteria as Gram-negative, motile, and facultatively anaerobic rods. Growth experiments demonstrated Strain 23021821's adaptability to various culture media (TSA, LB, BHI, and MH agar), particularly thriving at 37 °C in aerated conditions. Colony morphology featured circular, raised, yellow, opaque, and smooth textures (Fig. 2). Temperature tolerance ranged from 22–42 °C, peaking at 35–37 °C. The strain tolerated a pH spectrum of 5–10, optimally at 7.0–8.0, and grew in 0–7% NaCl solutions. Additionally, it grew under both aerobic and anaerobic conditions, showing catalase positivity but oxidase negativity.

Table 3 Biochemical characteristics of strain 23021821 and type strains of other Providencia speciesaFig. 2

Morphological characteristics of strain 23021821. The morphology of the strain 23021821 grown on different culture media is shown in A-D. A: Müller-Hinton agar, B: Luria-Bertani agar, C: Tryptic soy agar, D: Brain Heart Infusion agar. All bacteria were cultured at 37 °C in an aerobic environment

Description of Providencia xianensis sp. nov.

Providencia xianensis (xi.an.en'sis. N.L. fem. adj. xianensis, of Xi'an, referring to Xi'an City, Shaanxi Province, where the organism was isolated).

The cells are Gram-negative, motile, facultatively anaerobic rods, positive for catalase, and negative for oxidase. They exhibit robust growth on various culture media, including TSA, LB, BHI, and MH agar, especially at an optimal temperature of 37 °C in aerated conditions. The colonies are circular, raised, yellow, opaque, and smooth-textured. Growth is supported in a pH range of 5 to 10, with an optimum at pH 7–8, and in 0–7% (w/v) NaCl concentrations. Biochemical tests indicate positive results for deaminase activity and the fermentation of D-glucose, D-mannitol, inositol, amygdalin, aesculin, arbutin, glycerol, and salicin, as well as for urea hydrolysis and citrate utilization.

The type strain, 23021821T (CCTCC AB 2023264T = NBRC 116615T), was isolated from a sample of ascites specimen of Xi'an City, Shaanxi Province, China. It has a DNA G + C content of 40.3%.