Genomic and phenotypic characterization of Chryseomicrobium imtechense from canine pyometra

Abstract

Chryseomicrobium imtechense is a bacterium primarily isolated in environment samples. Here we demonstrated the virulent and antimicrobial resistant profile of the C. imtechense LBV029/19 strain, which was isolated in a pyometra infection in canines, being related as causal agent of that disease. The bacterium was recovered from purulent content of a uterus with pyometra and the identity was confirmed by both, biochemical tests followed MALDI-TOF MS and 16S-rDNA gene sequencing. Antimicrobial susceptibility test, biofilm formation assessment, genomic sequencing with Illumina MiSeq platform followed by the genome characterization and phylogenetic relationships were conducted with C. imtechense LBV029/19 strain. The phylogenetic analysis revealed a close evolutionary relationship between the C. imtechense LBV029/19 and the others publicly available 16S-rDNA genes of C. imtechense. The in vitro assays shown that C. imtechense LBV029/19 has a strong ability for biofilm formation. Additionally, the antimicrobial susceptibility tests revealed a smaller inhibition zone for novobiocin, cefuroxime, and erythromycin. To the best of our knowledge the C. imtechense LBV029/19 genome is the first genome sequenced and available of C. imtechense. Regarding the genotypic profile, resistance genes to tetracyclines, fluoroquinolones, beta-lactams, and multidrug resistance were present in this genome. Additionally, genes encoded to virulence factors, including biofilm formation, motility, and sporulation were identified. The phenotypic profile associated with the genotypic description indicates the potential of C. imtechense LBV029/19 as a primary agent of animal infectious diseases. The genomic description will contribute to the understanding of the biology of the bacterium and the pathogenesis process involved in the disease.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-025-01635-8.

Introduction

Canine pyometra is a reproductive disease that affects adult female dogs. The disease characteristics include the inflammatory exudate accumulation in the uterus due to hormonal disturbances and bacteria presence. As the disease progresses, bacteremia and death of the animals may occur [1]. Therefore, treatment involves ovariohysterectomy surgery and/or the use of antimicrobials [2].

The primary microorganism involved in the pyometra is Escherichia coli [3–5]. However, other microorganisms, in a lowest frequency, may also be involved, such as Pseudomonas spp. [4, 6], Klebsiella spp. [1], Staphylococcus pseudintermedius [6, 7], Enterococcus spp., Streptococcus spp., and Bacillus spp. [6]. Considering the bacteriological nature of the illness, detecting the causative bacterium and performing bacterial susceptibility tests are essential for favorable prognosis [4].

Members of the genus Chryseomicrobium are gram-stain-positive, rod-shaped, non-motile, non- spore-forming, strictly aerobic bacterias. In this context, Chryseomicrobium imtechense is an environmental bacterium first isolated from seawater sample [8]. The bacterium was also found in fabric dyeing wastewater [9], cementitious environment [10], and plastic degradation [11]. However, there are no description in the literature of neither the C. imtechense involved in clinical infections nor C. imtechense genomic sequencing and characterization. Thus, the study aimed to describe the phenotypic and genotypic profile of C. imtechense LVB029/19 isolated from a purulent content as causal agent of canine pyometra.

Materials and methods

Strain identification

Microbiological culture of uterine content from a bitch with suspected pyometra was performed. The sample was collected during the ovariohysterectomy at a private veterinary hospital in Porto Alegre, Rio Grande do Sul State (South Brazil). The collection procedures were approved by the UFRGS Ethical Committee on Animal Use (N^o. 31874). Briefly, microbiological culture was performed on MacConkey agar (Biolog, Brazil) and Blood Agar Base (Biolog, Brazil) supplemented with 5% sheep blood and incubated at 37 °C for 48 h in aerobic, microaerobic, and anaerobic conditions.

A pure bacterium isolate was obtained on aerobic condition, and subjected to gram staining and several biochemical tests: O-Nitrophenyl-β-D-Galactopyranoside (ONPG), arginine dihydrolase (ADH), lysine decarboxylase (LDC), ornithine decarboxylase (ODC), citrate (CIT), hydrogen sulfide (H₂S), urease (URE), tryptophan deaminase (TDA), indole (IND), Voges-Proskauer (VP), gelatinase (GEL), glucose (GLU), mannose (MAN), inositol (INO), sorbitol (SOR), rhamnose (RHA), sucrose (SAC), melibiose (MEL), amygdalin (AMY), arabinose (ARA), reduction of nitrates to nitrites (NO₂) and N₂ gas, catalase and motility.

Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) was used to confirm the identification, employing the Microflex LT instrument and MALDI Biotyper 3.1 software (Bruker Daltonik, Bremen, Germany). Furthermore, molecular identification based on 16S-rDNA gene sequencing was performed. For this, total DNA extraction from colonies was conducted using the PureLink® Genomic DNA Mini Kit (Invitrogen, Van Allen Way, Carlsbad, CA, USA) according to the manufacturer's instructions, and the polymerase chain reaction (PCR) was performed as described by [12]. The PCR products were analyzed on 0.9% agarose gel stained with Unisafe Dye (Uniscience, Miami Lakes, FL, USA).

Both strands from the PCR reaction were purified and sequenced using the ABI-PRISM 3500 Genetic Analyzer (Applied Biosystems Inc., Norwalk, CT, USA). The partial 16S-rDNA gene sequence data were inspected, trimmed, and aligned using Geneious v11.1.5 (Biomatters Ltd., Auckland, New Zealand) and submitted to the BLAST-NCBI database. The phylogenetic analysis was performed in MEGAv11.0.9 [13] by neighbor-joining method using 16S-rDNA sequence of our isolated (Chryseomicrobium imtechense LBV029/19), the available Chryseomicrobium spp., and species of the family Planococcaceae in the public database of GenBank (April, 2024) (Supplementary Table 1). The evolutionary distances were calculated using the Kimura 2-parameter model with a bootstrap value of 1,000.

In vitro antimicrobial susceptibility test

The susceptibility profile to antibiotics of the isolate bacterium was tested using Kirby & Bauer disc diffusion method [14]. The tested antimicrobial drugs were: carbenicillin (100 μg), vancomycin (30 μg), clindamycin (2 μg), chloramphenicol (30 μg), rifampicin (5 μg), streptomycin (300 μg), cefuroxime (30 μg), gentamicin (10 μg), amoxicillin (10 μg), neomycin (30 μg), erythromycin (15 μg), ampicillin (10 μg), tetracycline (30 μg), polymyxin B (300 μg), and novobiocin (5 μg), which were obtained from Laborclin (Pinhais, Paraná/Brazil). The drugs choice was based on the previous literature [15, 16], to provide a broader range of tested antibiotics.

Biofilm formation and quantification

The strain Chryseomicrobium LBV029/19 was initially cultured in a Blood Agar Base (Biolog, Brazil) plate and incubated for 24 h at 37 ºC. Then, the culture was transferred to Tryptone Soya Agar (TSA) and incubated for 24 h. Pure cultures of C. imtechense were inoculates in 3 mL of sterile saline solution 0.85%. The turbidity is adjusted to 0.5 McFarland standard (10⁸ CFU/mL). Finally, the biofilms were cultured on sterile 96-well flat-bottom microtiter plates using a previously described method [17]. Biological and technical triplicates were performed. Wells were filled with 180 uL of Tryptic Soy Broth (TSB) and 20 uL of the bacterial suspension. For the negative control, the wells were filled with 200 uL of TSB. The microtiter plate was then incubated at 37ºC for 72 h. Biofilms were quantified by washing microtiter plates with saline solution (0.85% w/v NaCl) post-incubation to remove non-adherent cells. Following, biofilms were fixed with 99% v/v methanol for 20 min at room temperature and air-dried overnight. Next, 200 μL of 0.5% v/v crystal violet solution was added to each well and incubated for 15 min. After rinsing with distilled water, 96 plates were dried at 55 °C for 25 min. Biofilms were then dissolved in 200 μL of 95% v/v ethanol, incubated at room temperature for 30 min, and quantified using a spectrophotometer equipped with a microtiter plate reader (SpectraMax 340PC, Molecular Devices, Sunnyvale, CA, USA) at 570 nm wavelength. The results were analyzed according to Stepanovic et al. [17]. The average optical density at 570 nm (OD) was calculated for Chryseomicrobium imtechense LBV 029/19 (ODs) and the control (ODc).

Genome sequencing and characterization

The genomic DNA was extracted using PureLink® Genomic DNA Mini Kit (Invitrogen, Van Allen Way Carlsbad, CA / USA) according to the manufacturer's instructions. Subsequently, the DNA quality and quantity were measured by NanoDrop Lite (Thermo Fischer Scientific, Massachusetts, USA) and Qubit (Life Technologies, Grand Island, NY), respectively. The paired-end (2 × 250 bp; coverage ∼100 ×) library was constructed with the DNA Library Prep Kit (Illumina) sequencing with Illumina MiSeq System (Illumina, California, USA) following the manufacturer´s instruction (MiSeq® Reagent Kit v2, 500 cycles).

The evaluation of raw reads quality was performed by FastQC v0.11.9. Trimmomatic v0.33 software was used to remove Illumina adapter sequences and Phred quality score < 30. Then, Spades v3.11.s1 [18] was used to assemble the paired-end sequence reads into contigs. MOB-Suite v3.0.0 [19] and Platon v1.6 [20] were used to remove plasmid sequences. Subsequently, IMAGE v2.4.1 (https://sourceforge.net/projects/image2/) was used to identify and close residual chromosome gaps. Assembly statistical evaluation was performed using QUAST v4.0 [21]. Genome annotation was conducted using the software Prokka v1.14.5 [22]. For general genome characterization, Resfinder v4.4.2 [23], VirulenceFinder v2.0.3 [24], RGI-CARD [25], and web service through RASTtk were utilized, considering 80% of identity. Moreover, genes of interest had their annotation manually refined.

Data availability

The 16S-rDNA gene and genome sequence of C. imtechense LBV029/19 were deposited in GenBank database accession numbers PQ046938 (https://www.ncbi.nlm.nih.gov/nuccore/PQ046938.1/) and PRJNA1140640 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1140640/), respectively.

Results and discussion

After the incubation period of the content derived from canine pyometra, the culture on blood agar incubated in aerobic conditions exhibited the growth of medium-sized, shiny, orange and non-hemolytic colonies. Gram staining revealed the presence of Gram-positive bacilli. The bacterium biochemical tests indicated the presence of tryptophan deaminase and catalase enzyme, and the absence of several enzymes: β-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, and urease. Gelatin was not hydrolyzed. H₂S production, indole formation, citrate test, Voges-Proskauer test, and motility test were negative. Glucose, mannose, inositol, sorbitol, rhamnose, sucrose, melibiose, and amygdalin were not fermented, while arabinose was fermented. Moreover, nitrate was not reduced to nitrite. Some of these biochemical results, together with gram-stain, spore-formation, and colonies aspect, can help to direct the identification of C. imtechense and differentiate it from other species, especially from Planococcus spp. (Supplementary Table 2).

The MALDI-TOF MS software was not able to identify the bacterium, still the 16S-rDNA gene sequencing data showed 99.86% similarity with Chryseomicrobium imtechense sequences available in the NCBI database. In addition, phylogenetic analysis with the partial 16S-rDNA gene sustained the division of the family Planococcaceae into two specific clades, for the genus Chryseomicrobium and for the genus Planococcus (Fig. 1). The studied strain C. imtechense LBV029/19 was positioned in the Chryseomicrobium genus clade, with a closely relationship to the species C. imtechense, which supports the identification of the isolate (Fig. 1).

Fig. 1.

Phylogenetic relationship of Chryseomicrobium imtenchense LBV029/19. * Strain of the study

Currently (July/2024), no genome sequence of C. imtechense was sequenced and deposited in public databases. Therefore, the C. imtenchense LBV029/19 strain represent the first genome for this bacteria specie. The draft genome comprises 2.6 Mb and overall G + C content of about 42.4% (Table 1). The genome contains 2,653 coding sequences (CDSs), of which 1,651 have known functions and 1,002 are annotated as hypothetical. C. imtenchense LBV029/19 genome presents four genes encoding the ribosomal RNAs (rRNAs) 16S and 23S, three genes encoding rRNA 5S, and 48 genes encoding the transfer RNAs (tRNAs) representing all 20 amino acids. The general genome features were listed in Table 1.

Table 1.

Features of Chryseomicrobium imtenchense LBV029/19 genome

Characteristics
Total length (bp)	2,618,756
GC Content (%)	42.4
Number of Contigs	64
Contig N50	213,035
Total n. CDSs	2,653
Known proteins	1,651
Hypothetical proteins	1,002
Number of rRNA	7
Number of tRNA	48
CRISPR	3
Putative Virulence Factors	44
Antimicrobial Resistance markers	35

To provide insights on virulence and antimicrobial resistance profile of the C. imtenchense LBV029/19, a deep accurate search for putative genes was performed. In summary, 44 genes associated with putative virulence potential (Supplementary Table 3), highlighting the genetic machine for motility and sporulation of this bacteria specie. Additionally, 35 genes were associated with antibiotic resistance (Supplementary Table 4), linked to resistance to tetracyclines, fluoroquinolones, beta-lactams, and multidrug resistance. Moreover, ten mobile genetic elements (MGEs) (Supplementary Table 5) corresponding to transposases and plasmids sequences were identified, which strengthens the possibility of genomic plasticity in this bacterium strain.

Despite an exhaustive literature research, no indicative data regarding halo sizes to antimicrobial susceptibility tests interpretation for Chryseomicrobium spp. was identified. Therefore, we presented the halo sizes (after 18 h of incubation), for each drug, as follows: carbenicillin – 36 mm; vancomycin – 25 mm; clindamycin – 30 mm; chloramphenicol – 32 mm; rifampicin – 44 mm; streptomycin – 32 mm; cefuroxime – 17 mm; gentamicin – 31 mm; amoxicillin – 40 mm; neomycin – 26 mm; erythromycin – 14 mm; ampicillin – 41 mm; polymyxin B – 20 mm; novobiocin – 8 mm; and, tetracycline – 37 mm.

According to European Committee on Antimicrobial Susceptibility Testing (EUCAST) [26], the inhibition zones for cefuroxime (17 mm) and erythromycin (14 mm) indicated resistance profile. Then, considering the results of both, the in vitro antimicrobial susceptibility test and the genotypic analysis, the genomic presence of cephalosporinase related to the blaB gene [27], which confer resistance to cephalosporin, might be influencing the lower inhibition zone for cefuroxime (17 mm) that we identified. Additionally, the in vitro inhibition zone for novobiocin (8 mm) could be associated with a mutation in the gyrB gene, similar to the observed in Staphylococcus saprophyticus [28]. Furthermore, the inhibition zone for erythromycin (14 mm) may be related to the occurrence of efflux pumps [29], as ATP-binding cassette (ABC) family multidrug resistance efflux pumps are present in the C. imtenchense LBV029/19 genome (Supplementary Table 4). On the other hand, tetracycline exhibited an in vitro inhibition zone of 37 mm despite the presence of resistance genes for this antimicrobial in the C. imtenchense LBV029/19 genome (Supplementary Table 4). This result could be explained by laboratory conditions, which may not be promoting the expression of all resistance genes [30]. The ‘silent’ genes – those who are present in the bacteria’s genome but are not phenotypically expressed – can revert and restore the resistance profile, complicating treatment protocols [31].

Despite the lack of spore formation and the expected absence of motility in C. imtechense [8], our genomic analysis revealed the genetic potential for expressing these characteristics – spore and motility. As the synthesis of flagella and, consequently, motility, are associated with high energy expenditure [32], the expression regulation of these genes by C. imtechense may be a bacteria mechanism under the conditions faced by the bacterium. Regarding spore formation, sporulation is a complex process that can affect the expression of different genes [33], posing a limitation for spore formation in C. imtechense LBV029/19 due to either the absence of some essential genes in its genome or activation conditions.

C. imtechense LBV029/19 has a high in vitro capacity of biofilm formation. This ability was sustained by the genomic presence of the ymcA and ydfK genes, which are related to biofilm regulation and formation [34, 35]. Additionally, other genomic characteristics, such as the presence of genes related to the Type IV pilus and Type II secretion system protein F, were also found in the C. imtechense LBV029/19. These factors are known to enhance bacteria virulence and facilitate the infection process [36, 37]. Biofilms can contribute to the persistence of bacteria in the uterus and complicate the treatment [38]. In addition, adaptive and innate immune defenses can be avoided by biofilms [39] and this increase the difficulty of both clinical treatments and biofilms elimination. Thus, the description of the phenotypic and genotypic characteristics presents in the C. imtechense LBV029/19 strain contributes to the understanding of the bacterium and, consequently, take new insights in the comprehension of canine pyometra pathogenesis.

Conclusion

The identification of C. imtechense as a causative agent of canine pyometra underscores the necessity of precise diagnosis to guide the treatment of the disease. Furthermore, the phenotypic and genotypic characterization of C. imtechense LBV029/19 may reveal the species’ potential to act as a primary agent in animal infectious diseases. Additionally, the study will provide approaches to future comparisons with other strains, enabling the monitoring of this bacterium's evolution and enhancing the understanding of the pathogenesis, considering the genotypic characteristics and the capacity of biofilm formation of C. imtechanse LBV029/19, in canine pyometra.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

The authors [Mariana Costa Torres] and [Laura Cadó Nemitz] contributed equally. Study conception and design were performed by [Mariana Costa Torres], [Laura CadóNemitz], and [Franciele Maboni Siqueira]. Material preparation, data collection and analysis were performed by [Mariana Costa Torres], [Laura Cadó Nemitz], [Maria Eduarda Rocha Jacques da Silva], [Andreza Francisco Martins], and [Franciele Maboni Siqueira]. Resources were provided by [Andreza Francisco Martins], [Afonso Luis Barth], and [Franciele Maboni Siqueira]. The first draft of the manuscript was written by [Mariana Costa Torres] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Declarations

Conflict of interest

None to declare.