Genome analysis and safety assessment of Achromobacter marplatensis strain YKS2 strain isolated from the rumen of Yaks in China

doi:10.21203/rs.3.rs-2949948/v1

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Genome analysis and safety assessment of Achromobacter marplatensis strain YKS2 strain isolated from the rumen of Yaks in China

https://doi.org/10.21203/rs.3.rs-2949948/v1

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published 26 Jul, 2023

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Achromobacter marplatensis strain YKS2 isolated from the Yak rumen has a feature of producing cellulose. This study aims to analyze the genome and safety of strainYKS2 in vivo considering its future research and application prospects. The genome of strain YKS2 was sequenced and used for genomic in silico studies. The administration of strain YKS2 with three doses was carried out on mice for 3 days of oral and 7 days of clinical observation tests. The BW, FI, organ indices, gut microbiota, and histological appearances of organs & intestines, along with hematological parameters and serum biochemistry were measured on mice. The chromosome size of strain YKS2 was 5 918 133 bp with GC content of 89.82%. The strain was component of 6 058 coding genes and had multiple functional genes, but no plasmids. The mice in all groups were alive, with good mental states and functional activities. Compared with the control group, there was no significant difference in the three dose groups on BW, FI, hematological parameters (WBC, LYM, etc.) and serum biochemistry (ALB, ALT, etc.). No abnormalities were observed in main visceral organs, intestinal tissue and V/C value in groups. However the IEL number of duodenum and gut microbiota diversity (Shannon index) in the high-dose group was significantly higher than in the control group (p < 0.05). Besides, the low dose of strain YKS2 also significantly affected the bacterial abundance of firmicutes, actinobacteria and desulphurizing bacteroidetes at the phylum. There was no significant effect at genus levels in groups. In a word, the study provided the genome and potential functional genes of strain YKS2, which is beneficial to understand the features of the A. marplatensis strain, and proved train YKS2 without acute toxicity to mice. However, a long-term feeding toxicity experiment in vivo should be performed to further ensure its potential application value strain in the animal industry.

Achromobacter marplatensis strain YKS2

genome

safety

Yak

mice

As a rare species living in the plateau area of China, the yak (Bos grunniens) has the production value of meat, milk, fur, and fuel. It plays a vital role in the plateau ecosystem, significantly impacting the local society, ecology, and economy. Their ability to adapt to such harsh environmental factors is facilitated by their large lungs, large hearts, efficient foraging and energy metabolism, and their gut microbiota which contributes significantly to living at a high altitude [1]. Metataxonomic analysis revealed that the rumen microbiota was significantly different among plateau yak, Qaidam yellow cattle (Bos taurus), and dzomo (hybrids of cattle and yak), and yak rumen microbiota elevated fiber degradation ability and altered rumen fermentation pattern, which provided sufficient nutrition for a yak to resistant the harsh environment [2] The yak rumen microbiota comprises bacteria, fungi, and archaea [3]. Bacteria and fungi play a major role in cellulose degradation, and their interaction can more effectively secrete cellulase for cellulose degradation [4]. However, bacteria produced more cellulase and species than fungi (Makkar & McSweeney., 2005). Microbial additives have recently become common in ruminant nutrition and can replace antibiotics as growth promoters [5]. Therefore, the isolation and characterization of microbes with high cellulase production from yak rumen fluids is a reasonable method, especially for potential use as additives in ruminant feeding. However, there are few related reports. Two cellulase-producing Enterococcus species were isolated from Tibetan yak rumen, identified as E. faecalis (EF85) and E. faecium (EF83) by 16S rRNA sequencing, and their combined inoculation to various forages (Napier grass ensiling, whole-crop corn and sweet sorghum) markedly reduced the fiber content of the resulting silages [6]. A total of 23 strains of cellulase Bacillus were screened from high-temperature resistant Bacillus strains collected from 10 yak rumen samples, including 16 Bacillus cereus strains and 7 Bacillus thuringiensis strains [7].

In our previous study, the rumen contents of 5-7-year-old yaks (mainly grazed and without any supplementary feed) were collected in the Gannan Tibetan Platea area, and 17 anaerobic bacteria strains with fiber degradation were isolated and numbered 1–17, respectively. Moreover, one of the strains showed superior enzyme activity, including cellulose endoglucanase, filter paper enzyme, and β-glucosidase, and showed high activity in the degradation of straw and cellulose powder [8]. Therefore, we named it the YKS2 strain and hypothesized that the YKS2 strain might be a potential alternative for commercial inoculants in silage production and microbial additives in the ruminant diet. Therefore, the molecular biological identification and phenotypic characteristics of the YAK2 strain were analyzed. The results showed that the YKS2 strain belonged to Achromobacillus sp and displayed 99.0% similarity with the sequence of A. marplatensis XG49 (Accession: MZ707567). For the phenotypic characteristics, it is gram-negative, white, non-motile, non-flagellate, rod-shaped, and facultative aerobic[9]. It has been reported that A. marplatensis could metabolize important organic pollutants such as pentachlorophenol, an environmental toxin that few microorganisms can degrade [10, 11]. However, until now, there has been limited knowledge of the genome characteristics of A. marplatensis.

As previously stated, before being used as a microbial additive in animal feed, the bacteria must be safe for hosts and have no toxic side effects on the body [12]. Moreover, it should have the ability to withstand adverse conditions, including tolerance to gastric acid, bile salt, various proteases, and digestive juices [13, 14]. Previous studies have shown that the A. marplatensis YKS2 strain is a non-drug resistant strain with no hemolysis, no acid production, good proliferation performance, and has the properties of bile salt resistance, gastric fluid resistance, self-condensation, and surface hydrophobicity[9]. In order to provide more information about the safety of the A. marplatensis YKS2 strain, the genome information, drug resistance gene, and virulence genes were analyzed by silica studies, and its acute toxicity in mice was evaluated in this study.

2.1 Bacteria source and culture conditions

A. Marplatensis YKS2 strain (GenBank accession No. PRJNA881767, CGMCC Strain Preservation Center, China, No. 24678) was isolated from yak rumen fluid by the Chinese Veterinary innovation team of Lanzhou Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agricultural Sciences. A. Marplatensis YKS2 strain was received as single colonies on plates. They were grown in small flasks containing 10 mL of Luria-Bertani (LB) broth (Huonkai Microbe, China) at 37 ℃ in a shaking incubator (Yiheng, China) under anaerobic conditions for 24 h and stored in 50% glycerol solutions at 80 ℃. The strains were passaged at least two times in LB broth before use.

2.2 Genome analysis

The suspension of A. Marplatensis YKS2 strain from yak rumen cultured overnight was centrifuged at 8000 r/min for 10 min to obtain the bacteria. The bacterial pellet was washed with sterile phosphate buffer and centrifuged at 8000 r/min for 10 min. The bacteria were then placed in a dry ice freezer, and Beijing Novo Gene Technology Co., LT was commissioned to sequence the entire genome of the strain.

The genome components of A. Marplatensis YKS2 strains were predicted using six software: GeneMarkS (version 4.17), RepeatMasker (version 4.0.5), rRNAmmer (version 1.2), tRNAscan-SE (version 1.3.1), IslandPath-DIOMB (version 0.2), phiSpy (version 2.3), and Rfam (version 11.0). The GO database [15] was used to predict and assign functions to genes from the YKS2 strain. In order to further study the relevant biological characteristics and safety, the CARD database [16] and VFDB database [17] were used to detect the presence of antibiotic resistance genes and genes related to bacterial pathogenesis. Data of CARD matching > 75% were selected for further analysis. Data of VFDB matching > 60% were selected for further analysis of the NR database [18]. Moreover, the CAZy database [19] annotated gene species information and carbohydrate-active enzyme analysis.

2.3 Test animals and Ethics

SPF Kunming mice were obtained from the Lanzhou Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences (Lanzhou, China). The animals in the animal holding room were controlled in a pathogen-free environment (temperature: 20 ℃, humidity: 50 ± 10%, light / dark cycle: 12 h) and were fed and watered freely. As a result, the water and food reached the standard laboratory level.

In the acute toxicity study, animals were fasting for 12 h before gavage bacteria suspension for three consecutive days. Feed was provided to the fasted animals after 3 h of dose administration. Animals were fasted overnight prior to necropsy for clinical pathology investigations.

All the studies were performed following the requirements of the Guide for the Care and Use of Laboratory Animals, Lanzhou Institute of Husbandry and Pharmaceutical Sciences of the Chinese Academy of Agricultural Sciences. Furthermore, the experiment was approved by the Ethical Committee of Lanzhou Institute of Husbandry and Pharmaceutical Sciences of the Chinese Academy of Agricultural Sciences (the permission number: SYXK(Gan) 2019-0002).

2.4 Acute toxicity study

The study was carried out to evaluate the toxicity of A. Marplatensis YKS2 strain after 3-day repeated administration through oral route in SPF Kunming mice for 7 days. For this study, 40 mice (20 males and 20 females) of about 20 g were randomly divided into 4 groups (n = 10/group) after adaptive feeding for a week. The mice in the experimental groups were fed with high concentration (1.0×10⁹ CFU/mL), medium concentration (1.0×10⁸ CFU/mL), and low concentration (1.0×10⁹ CFU/mL) of bacteria suspensions at the doses of 0.2 mL/10 g by gavage. The mice in the control group were fed with an equal volume of sterile saline solution by gavage.

2.4.1 Evaluation indicators

Clinical signs and mortality

Clinical signs/symptoms were observed daily after 1–2 h of dosage. The mice were randomly checked for mortality and morbidity in the morning and afternoon of each day. If the mice died during the observation period, the organs were dissected immediately to observe the pathological changes.

Body weight and feed consumption

Animals were weighed on the day before and days 3, 5, and 7 after the oral route period. Additionally, fasting body weights were recorded on the day of the necropsy to calculate relative organ weight. Finally, animals were fed quantitative feed daily, and the leftover was measured the next day to calculate the mice's average daily feed consumption (g/day). The formula is as follows.

Average feed consumption of animal (g/day) = Feed provided (g) to cage – feed leftover (g) in the cage / No of Animals per cage × No of days.

Hematological analysis

Mice were anesthetized with isoflurane, and approximately 0.4 mL of blood was collected from each mouse by cardiac puncture. The parts were collected into EDTA anticoagulant tubes. The fresh blood collected from the EDTA anticoagulant tube was measured within 2 h by a blood cell analyzer (Zoetis, USA), and the blood cell index data of the control and experimental groups were collated and analyzed. Another part of the collected blood was collected into a heparin lithium anticoagulant tube. The whole blood samples were analyzed for albumin (ALB), alanine transphosphatase (ALT), total bilirubin (TBI), urea nitrogen (BUN), creatinine (CRE), and blood glucose (GLU) by the blood biochemical instrument (Zoetis, USA). However, samples with hemolysis were excluded from the analysis.

Pathological evaluation

The heart, liver, spleen, lung, and kidney were weighed, and those organs and duodenum segments were all immersed in 4% paraformaldehyde (WELLBI, China) for fixation. After fixation, they were embedded in paraffin and continuously sliced to 5 µm thick. Complete segments were taken, dried, dewaxed, hydrated, and stained with hematoxylin-eosin (H&E). The morphology and structure of organs and intestines were observed under the optical microscope (Olympus, Japan) with a magnification of 200 times. Typical images were selected for micrographs and histopathology evaluation.

2.4.2 Histological evaluation of small intestine.

After the intestinal segment imaging of the duodenum was completed by the above steps, using Image-ProPlus 6.0 (Media Cybernetics, USA) analysis software, taking millimeters as the standard unit and selecting 100 times visual field, the length of 5 intact intestinal villi in each section and the corresponding depth of 5 crypts were measured respectively. Villus: crypt ratios (V/C values) were calculated. At the same time, 400 times the visual field was selected to measure the length of intestinal villus epithelium and count the number of goblet cells (GCs) and lymphocytes (IELs) in this length to calculate Gcs number per 1 mm length of the villi epithelium and IELs number per 1 mm length of the villi epithelium [20].

2.4.3 DNA extraction and 16S rRNA sequencing of the intestinal microbiota.

About 4–5 g of small intestinal fecal samples were collected by gently pressurizing the abdomen of mice and stored in sterile tubes at -80 ℃ for further isolation of total DNA. The released genomic DNA was used as the template. The V3-V4 regions of the 16S rRNA gene were amplified by the PCR (ABI GeneAmp® 9700, USA) using primers 515F: 5’-GTGCCAGCMGCCGCGGTAA-3'; 806R: 5’-GGACTACHVGGGTWTCTAAT-3'. The sequence data were stored in the NCBI Sequence Read Archive (SRA) database (Accession No.PRJNA884875) and analyzed on the free online platform of Illumina MiSeq Platform by Beijing Novo Gene Technology Co., LT.

2.4.4 Bioinformatics analysis

Paired-end reads from the same original DNA were assembled using FLASH (v1.2.7, http://ccb.jhu.edu/software/FLASH/). Then the high-quality sequences were clustered into operational taxonomic units (OTUs) according to a 97% threshold of pairwise identity using the Uclust function in QIIME (v1.8.0, http://qiime.org/). The classify-sklearn algorithm of QIIME2 (v0.21.3, https://docs.qiime2.org/) was further used as a reference database to annotate the sequences. The community diversity (α-diversity) was analyzed using Mothur (v.1.30.1, http://www.mothur.org/wiki/ Schloss_SOP#Alpha_diversity) via calculating Simpson, Chao1, and Shannon indexes. Variation in community composition (β-diversity) was measured by calculating the principal component analysis (PCA), the principal coordinate analysis (PCoA), weighted and unweighted uniFrac nonmetric multidimensional scaling (NMDS) analysis, and hierarchical clustering analysis based on weighted UniFrac distances. Dominant bacterial relative abundance at the phylum and genus levels was also analyzed. They were all performed by QIIME and R software.

2.5 Statistical analysis

All the values in the figures and tables are presented as mean ± SD. The data were analyzed by IBMSPSS 22.0 software, single factor analysis of variance (One-Way ANOVA), LSD multiple comparisons, Tukey's test, and plotted by Graphpad prism software. Different letter superscripts in the charts and figures indicate significant differences (P < 0.05). The same or no superscript means no significant difference (P > 0.05).

3.1 Genome features of YKS2

The whole genome of A. Marplatensis YKS2 strain is composed of a circular 6 588 568 bp chromosome, with GC contents of 65.27%. The cyclization sequence forms a ring structure, but no plasmid exists (Fig. 1). The chromosome contains 6 058 predicted coding sequences, and the total gene length was 5 918 133 bp, with an average gene length of 977 bp. In addition, the strain contained 58 tRNAs and 13 rRNAs (including 5 5S rRNA, 4 16S rRNA, and 4 23S rRNA), and no sRNA was found. There were 48 scattered repeats (20 long terminal repeats, 5 DNA transposons, 12 long scattered repeats, 11 genetic islands, and 11 short scattered repeats), 269 tandem repeats, 229 small satellites, 5 microsatellites, 11 genomic islands (Gls), and 5 prophages (Table 1).

Table 1

General features of the *A. Marplatensis* YKS2 genome.
Feature	Chromosome
Size (bp)	6 588 568
GC content (%)	65.27
predicted coding sequences	6 058
Average gene length	977
Scattered repetitive sequences	48
Tandem repeat sequence	269
Small satellite	229
microsatellite	5
tRNA	58
rRNA	13
sRNA	0
Gls	11
Prophage	5

3.2 Functional analysis of A. Marplatensis YKS2 strain genome

3.2.1 NR database annotation

NR is a non-redundant protein database characterized by its comprehensive content. At the same time, the annotation results contain species information, which can be used for crop species classification. Genomic information of A. Marplatensis YKS2 strain was compared per the NR database (Fig. 2). The results showed that the vast majority of the genes were classified into A. marplatensis, which was consistent with 16S rDNA assay results in the previous study [9], and the number of non-redundant proteins was 4 834.

3.2.2 GO database annotation

GO database divided the genes of A. Marplatensis YKS2 strain into three categories: cell component, molecular function, and biological process for description (Fig. 3). Among them, the number of genes describing the biological process was the highest, reaching 9 573. The number of genes describing molecular function was 5261. The number of genes describing cellular molecules was 3807. In terms of molecular function, most genes were related to adhesion, nucleic acid-binding transcription factor activity, catalytic activity, etc. Regarding cell components, most genes constitute the structure of cells and organs and contribute to the structure and function of cells. For biological processes, most genes are related to cell and cellular metabolic processes, localization, and establishment of localization, biology, and regulation of biological processes.

3.2.3 COG database annotation

Figure 4 shows a total of 5796 genes of A. Marplatensis YKS2 strains were annotated compared to the COG gene bank, which could be divided into 26 categories according to their functions. Among them, genes with amino acid transport and metabolism function had the highest number (801 genes), followed by genes with transcription function (628 genes), genes with energy metabolism (453 genes), and genes with carbohydrate transport and metabolism (321 genes), respectively. The least number of genes were those with RNA processing and modification, chromatin structure and dynamics, and cellular scaffold function.

3.2.4 Cazy database annotation

The Cazy database contains the enzyme families that catalyze carbohydrate degradation, modification, and biosynthesis. The genomic information of A. Marplatensis YKS2 strain was compared with the Cazy database. In Fig. 5, 148 genes were annotated in the strain total, including 4 genes annotated as auxiliary activities (AAs) and 13 genes annotated as carbohydrate esterases (CEs). In total, 40 genes were annotated as glycoside hydrolases (GHs), 63 genes were annotated as glycosyl transferases (GTs), 3 genes were annotated as polysaccharide lyases (PLs), and 25 genes annotated as carbohydrate-binding Module (CBMs).

3.2.5 Antibiotic resistance and virulence-related genes

The genes related to antibiotic resistance in A. Marplatensis YKS2 strains were further screened by referencing the CARD database. As shown in Table 2, there were 22 antibiotic genes with a high matching degree. Their antibiotic-resistant classes include amino coumarin, macrolides, monocyclic lactam, tetracycline, aminoglycosides, cephalosporins, fluoroquinolones, carbapenems, florfenicol, diaminopyrimidine, cephamycin, penicillins, and penem. The adeF, Axy, and OprZ genes may be resistant to multiple antibiotics (≥ 4 classes). The result suggests that the genes may contribute to the strain's resistance to antibiotics in the environment. However, it needs to be verified by an antibiotic resistance test.

Table 2

Putative antibiotic resistance genes identified in the genome of *A. Marplatensis* YKS2
Gene ID	Gene	Antibiotics	Mechanism	Identity (%)
GM005484	adeF	fluoroquinolone antibiotic;tetracycline antibiotic	antibiotic efflux	97
GM001205 GM001206	AxyY AxyX	aminoglycoside antibiotic;cephalosporin;fluoroquinolone antibiotic;macrolide antibiotic;	antibiotic efflux	91.3/84.1
GM004349	OXA-243	carbapenem;cephalosporin;penam	antibiotic inactivation	88.4
GM005248	DfrA43	diaminopyrimidine antibiotic	antibiotic target replacement	86.9
GM001204	OprZ	aminoglycoside antibiotic;cephalosporin;fluoroquinolone antibiotic;macrolide antibiotic	antibiotic efflux	85.2

Seven predicted virulence factors in the genome of A. Marplatensis YKS2 strains were predicted by the VFDB database (Table 3). Hsp60, Polar flagella, and Brk genes are associated with bacterial adhesion, complement resistance, and invasion. Moreover, the SodB gene is important for intracellular survival and transmission. The clpP gene is associated with bacterial metabolism, and the acinetobacter gene has the function of competing with host cells for iron. The Cya gene associated with hemolytic activity was also found among the predicted genes. However, in the previous study, the strain did not show any hemolytic ability after 48 h incubation on a blood agar medium [9].

Table 3

Putative virulence factors in the *A. Marplatensis* YKS2 genome.
Gene ID	Gene	Predicted functions	Identity (%)
GM005154	Brk	Adherence and resists complement	80.9
GM005153	Hsp60	adherence	73.2
GM003777	SodB	Survival、transmission	68.4
GM001297	Polar flagella	motility,adhesion and invasion	62.8
GM005149	Acinetobactin	competing with host cells for iron	62.6
GM003676	ClpP	associated with bacterial metabolism	60.8
GM002232	Cya	Bifunctional toxin harboring both adenylate cyclase and hemolytic activities; anti-inflammatory	47

3.3 Acute toxicity of A. Marplatensis YKS2 strain on mice

3.3.1 Effects of A. Marplatensis YKS2 strain on the overall health of mice

During the experiment, no animals were recorded to have disease, death, or diarrhea symptoms. The fur, diet, drinking water, and mental state of mice in each group were good, and no acute poisoning and related toxic reactions were observed. The anatomy showed that the organ structure of mice was generally normal, and the naked eye observed no pathological changes. Figure 6 and Table 4 show no significant differences in body weight, feed intake, and organ index among all groups (P > 0.05).

Table 4

Relative organ index of groups after intragastric administration for 7 days
Index	Control	YKS2-Low	YKS2-Middle	YKS2-High
Heart / BW (%)	0.71 ± 0.17	0.62 ± 0.06	0.69 ± 0.10	0.61 ± 0.10
Liver / BW (%)	4.57 ± 0.56	4.45 ± 0.34	4.72 ± 0.26	4.66 ± 0.54
Spleen / BW (%)	0.34 ± 0.08	0.32 ± 0.03	0.34 ± 0.09	0.37 ± 0.08
Lung / BW (%)	0.79 ± 0.12	0.78 ± 0.09	0.80 ± 0.11	0.77 ± 0.11
Kidney / BW (%)	1.39 ± 0.23	1.36 ± 0.20	1.40 ± 0.23	1.35 ± 0.16

3.3.2 Effects of A. Marplatensis YKS2 strain on blood hematology and biochemical analytes

As shown in Table 5, white blood cell count (WBC), lymphocyte count (LYM), monocyte count (MON), and red blood cell count (RBC) of the experimental groups (YKS2-High group, YKS2-Middle group, and YKS2-Low group) showed a slight increase compared with the control group. However, all indicators had no significant difference (P > 0.05). Furthermore, as shown in Fig. 7, there were no obvious variations present in albumin (ALB), alanine transphosphatase (ALT), total bilirubin (TBI), alkaline phosphatase (ALP), total protein (TP), urea nitrogen (BUN), creatinine (CRE), and blood glucose (GLU) between the groups.

Table 5

Effect of feeding *A. Marplatensis* YKS2 on hematological parameters of mice
Blood cell parameters	Groups
Blood cell parameters	Control	YKS2-Low	YKS2-Middle	YKS2-High
WBC (10⁹/L)	6.31 ± 1.80	6.35 ± 1.71	6.85 ± 1.53	7.05 ± 1.77
LYM (10⁹/L)	4.33 ± 1.99	4.38 ± 1.52	4.95 ± 1.41	5.10 ± 1.82
MON (10⁹/L)	0.32 ± 0.09	0.33 ± 0.07	0.31 ± 0.09	0.36 ± 0.13
RBC (10¹²/L)	9.44 ± 0.41	9.58 ± 0.86	9.73 ± 0.98	9.48 ± 0.90
HCT (%)	47.43 ± 3.39	48.56 ± 2.34	48.49 ± 3.43	48.94 ± 1.71
MCV (fL)	47.25 ± 1.75	48.80 ± 2.30	46.90 ± 1.52	48.2 ± 1.62
HGB (g/L)	15.10 ± 1.39	14.64 ± 1.71	15.11 ± 2.06	15.61 ± 0.78
MCH (pg)	15.24 ± 0.8	14.52 ± 1.83	14.91 ± 0.79	15.37 ± 0.55
MCHC (g/dL)	32.25 ± 0.87	29.79 ± 3.37	31.77 ± 1.12	31.88 ± 1.08
PLT(10⁹/L)	298.5 ± 35.29	319.40 ± 29.12	320.20 ± 37.83	313.40 ± 40.18

3.3.3 Effects of A. Marplatensis YKS2 strain on tissue histology

No apparent histopathological damage was observed in the heart, liver, spleen, lung, or kidney tissues of the mice treated with A. Marplatensis YKS2 strain (Fig. 8). The myocardial tissues of the mice in all groups were evident, the myocardial fibers were slender, cylindrical, arranged regularly and tightly, and there was a small amount of connective tissue between the myocardial fibers. The hepatic lobules of the mice in all groups were clear and complete, the hepatocytes were arranged radially around the central vein, and the morphological structure was normal. The spleens of mice in all groups were clear and complete, the boundary between the white and red pulp was clear, the central artery of the lymph node and the lymphatic sheath around the artery could be seen, and the lymphocytes were dense. The lung alveoli tissues of mice in groups were complete and clear, the alveolar and bronchial cavities were very clean, and there were a few red blood cells. The kidney of the mice in all groups was normal, with the cortex and medulla divided, a large number of urinary tubules, and renal interstitium composed of a small amount of connective tissue, blood vessels, nerves, etc., and renal tubular epithelial cells were intact.

3.3.4 Effects of A. Marplatensis YKS2 strain on intestinal tissue

Intestinal villi and crypts

As shown in Fig. 9A, High, middle, and low concentrations of the bacterial suspension did not cause damage to the morphology of intestinal mucosa, and all groups showed normal physiological morphology. The intestine's villi were neatly arranged and dense, without swelling or shortening, crypts of comparable depth or deepening, and no signs of inflammation or epithelial cell damage. V/C value is an important indicator for measuring intestinal structure and morphology and digestive and absorption capacity [21]. It was shown in Fig. 9B that the intestinal villus length and crypt depth of the YKS2-Low, YKS2-Middle, and YKS2-High groups were increased compared to the control group, with no statistical differences among the groups (P > 0.05). The experimental groups' V/C values increased slightly compared to the control group, but there was no significant difference (P > 0.05). The results proved that the A. Marplatensis YKS2 strain had no adverse effects on the physical barrier of the small intestine.

Small intestinal immune cells

The mucins secreted by GCs can repair mucosal damage and prevent external opportunistic pathogens from adhering, making the intestinal mucosa smooth and stabilizing the intestinal chemical barrier. IELs are the primary immune cells in the gut that is in contact with antigens in the intestinal immune response and can reflect the status of the intestinal chemical barrier. As shown in Fig. 10A, GCs in the groups were mainly located between columnar cells, which were white vacuolar cells (red arrows) according to the staining morphology. IELs were mainly located in the lamina propria of the intestinal epithelium, a small, round cell with purplish-blue nuclei (yellow arrows). As shown in Fig. 10B, the IELs number per 1 mm length of the villi epithelium in the experimental groups was increased compared with the control group, and the data in the YKS2-High group was significantly increased (P < 0.05). For the GCs number per 1 mm length of the villi epithelium, the YKS2-Middle group was significantly higher than YKS2-High (P < 0.05) and higher than the control group (P > 0.05). The result showed that the A. Marplatensis YKS2 strain had no adverse effects on the chemical barrier of the small intestine and could increase the IEL number on the duodenal villi of mice.

Intestinal microbiota composition

We used Miseq sequencing technology to reveal differences in the composition of the fecal microbial community between the experimental and control groups. A total of 1 918 718 reads were obtained from all 24 samples. After quality control and chimera filtering, 1 848 811 reads were generated. The box chart (Fig. 11A) showed a sharp rise with the increase in sample size, indicating that many species were found in the community. The dilution curve (Fig. 11B) of all samples tended to be flat, indicating that the amount of new and conventional species detected in the sample was sufficient to meet the demand of this experiment.

As shown in Table 6, the coverage indexes in all samples were greater than 0.999, indicating that all samples' diversity could be measured. For the Chao1 index (representing the number of microorganisms), the YKS2-Middle group was significantly higher than that in the YKS2-Low group (P < 0.05). However, there was no significant difference between the experimental and control groups (P > 0.05). There is a positive correlation between the dose of A. Marplatensis YKS2 strain suspension and Shannon index (representing microbial diversity) and YKS2-High group were significantly higher than that of the control group (P < 0.05). Simpson index (representing the diversity and evenness of species distribution in the community) result showed that there was no statistical significance between the control group and the experimental groups (P > 0.05).

Table 6

Evaluation of the representative abundance, diversity, evenness, coverage and other indicators in the fecal samples of groups
Groups	Chao1	Shannon	Simpson	Coverage
Control	405.3 ± 85.27^ab	5.98 ± 0.55^b	0.94 ± 0.04^a	0.9998 ± 0.0004^a
YKS2-Low	356.41 ± 85.07^b	6.28 ± 0.29^ab	0.97 ± 0.01^a	1.0000 ± 0.0000^a
YKS2-Middle	477.64 ± 81.82^a	6.36 ± 0.47^ab	0.96 ± 0.02^a	0.9998 ± 0.0004^a
YKS2-High	422.05 ± 115.01^ab	6.54 ± 0.23^a	0.97 ± 0.01^a	0.9998 ± 0.0004^a

The Venn diagram in Fig. 12A displays the common and unique ASVs among the different samples. We identified 1 584 ASVs in all samples, and 395 ASVs were shared by the four groups, suggesting a similarity in their bacterial composition. The unique ASVs percentages of the groups were 32.4% (control group), 28% (YKS2-Low group), 46.4% (YKS2-Middle group), and 26.9% (YKS2-High group). Overall, the results showed similarities and differences between the groups. The beta diversity analysis results are presented in the form of PCA, PCoA, and NMDS dimension reduction (Figs. 12B, 12C, and 12D). PCA analysis was performed based on ASV levels. In the PCA analysis, all samples in four groups were clustered within a range except for 2 samples that deviated far. PCoA and NMDS were analyzed based on UniFrac evolutionary distance to evaluate the similarity between the samples. Except for two samples that deviated significantly from the norm, all samples from the other groups were clustered within a range in the PCoA analysis. In the NMDS analysis, stress equaled 0.085 (< 0.2) based on the unweighted UniFrac distance. All the species diversity analyses indicated no significant differences between the experiment and control groups.

Phylum and genus levels of intestinal microbiota

At the phylum level (Fig. 13A), the top 10 species with an enormous abundance are selected to generate a columnar accumulation chart of the relative abundance of species. Bacteroidota, Firmicutes, and Desulfobacterota are the most dominant phyla in the intestinal microbiota, and the sum proportion of the three phyla was more than 90%. For the Bacteroidetes abundance, there was no significant difference between the control and experimental groups (P > 0.05). For the Firmicutes abundance, the experimental groups were higher than the control group, and the increasing trend of the YKS2-Low group was significant (P < 0.05). For the Desulphurobacterium abundance, the experimental groups were lower than the control group, and the decreasing trend of the YKS2-Low group was significant (P < 0.05). In addition, the abundance of non-dominant phyla showed that the Actinobacteriota abundance in the YKS2-Low group was significantly higher than in other groups (P < 0.05). There was no significant change in the Proteobacteria abundance among the groups (P > 0.05).

At the genus level (Fig. 13B), the top 10 species with the largest abundance were Muribaculaceae, Alloprevotella, Eubacteriµm_coprostanoligenes_group, Bacteroides, Clostridia_UCG-014, Enterorhabdus, Lactobacillus, Parabacteroides, Lachnospiraceae_NK4A136_ group, and Desulfovibrio genus. For the Bacteroide abundance, YKS2-Middle and YKS2-High groups were higher than the control group, but not significantly (P > 0.05), YKS2-Low group was significantly lower than that in the YKS2-Middle groups (P < 0.05). For the Clostridium abundance, there was no significant change between the experimental groups and the control group (P > 0.05), but YKS2-Middle and YKS2-High groups were significantly lower than that of the YKS2-Low group (P < 0.05). In the other 8 genes, there was no significant difference among the groups (P > 0.05). In conclusion, these data further revealed that the A. Marplatensis YKS2 strain could regulate the microbial community in murine intestinal tracts.

The rumen microbiota is generally recognized to play a vital role in feed efficiency [22], milk protein yield [23], and health status [24] for the ruminants. As a unique kind of ruminant, yak feed on grasses rich in cellulose, and their special digestion mode and the special environment in the rumen are highly conducive to the growth of microorganisms producing cellulose [25]. In a previous study, a strain with high cellulase activity was isolated from the rumen of yak in the Gannan Tibetan area and was identified as A. Marplatensis by 16S rRNA sequencing assay. This paper's annotation results of the NR database also confirmed that the YKS2 strain was A. Marplatensis. However, until now, there has been limited knowledge of the genome characteristics of A. Marplatensis isolated from plateau animals. The genome component result of A. Marplatensis YKS2 strain showed that the total size of the whole gene was 6 588 568 bp, and the GC content was 64.26%. The 5s, 16s, and 23 sRNA in the coding genes were 5, 4, and 4, respectively. This is similar to the genome component of A. spanius strains studied by Li [26]. However, it was found that the GC content of the A. marplatensisis strain isolated from a pneumonia patient was 54–56% [27]. We speculate that this may be related to the differences in bacterial reproduction environment and host factor. For the result of COG database annotation, we found that the function of the five most abundant genes was transport and metabolism of amino acids (801), transcription (628), energy production and conversion (453), inorganic ion transport and metabolism function prediction (450), general (436), which was similar to the results of Li's study [26]. It has been reported that carbohydrate-active enzymes are indispensable for the body's nutrient absorption [28]. These specific enzymes deconstructed the cellulose from the plant cells to short oligo-/disaccharides, which then can be translocated into the bacteria cells and further processed to release energy, and GHs are essential enzymes required for the breakdown of the polysaccharides [29]. Together with other carbohydrate-active enzymes (e.g., CEs, PLs, GTs, and CBMs ), these proteins support key processes across ecosystems [30]. More recently, polysaccharide monooxygenases (PMOs) were found to degrade various polysaccharides via a copper-dependent hydroxylation oxidatively and re-classified in the CAZY database as AA families [31]. We found 148 genes annotated in the CAZY database, and the number of genes encoding GHs and GTs was the most in the strain genome. These enzymes are responsible for the cellulose degradation of A. Marplatensis YKS2 strain.

The research showed that antibiotic resistance in probiotic strains was observed against antibiotics as an innate ability found among all species of the same genus [32]. Moreover, the lactobacilli isolated from commercial products in Europe comprised certain strains resistant to tetracycline (29.5%), chloramphenicol (8.5%), and erythromycin (12%), and overall, more than 68% of the isolates exhibited resistance to two or more antibiotics [33]. A total of 7 resistance genes with a match rate of > 75% in the CARD database screened A. Marplatensis YKS2 strain, and each gene corresponds to two or more resistant antibiotics. Furthermore, the previous antibiotic sensitivity tests showed that the YKS2 strain was sensitive to 11 antibiotics in the list of 14 tested antibiotics and was only tolerant to cephalexin (cephalosporins) and amtriaxnam (carbapenems) [9]. Therefore, we speculate that the partly drug-resistant genes in the strain genome were not expressed or weakly expressed. Hemolysins produced by bacteria contribute to the direct lysis of a variety of cell types and damage in vascular permeability, which lead to intercellular propagation of bacteria and inhibition of the proliferation of lymphocytes and the production of lymphokines and immunoglobulins (Hillman et al., 1993). Although there was a hemolysin-associated gene-Cya gene with low matching (47%) in the YKS2 strain genome, the previous experiments had confirmed that the strain is negative hemolytic. So, it is supposed that the Cya gene is unlikely to be expressed in the YKS2 strain genome. However, the other 6 virulence factors in the VFDB database, including the genes of Hsp60, Polar flagella, Brk, clpP, and acinetobacter, might exist in the genome of the YKS2 strain. In addition, no plasmid was detected in the genome of the YKS2 strain, preventing the possibility of transferring the potential characteristics of virulence or antibiotic resistance to other intestinal commensals.

In general, the safety of the strain should be evaluated in vivo experiments [34]. However, 20 days old, mice are still in the developmental stage and have an unmatured intestinal epithelial barrier. Therefore, they are susceptible to toxic stimulation and are currently used as biological and toxicological models [35]. After being gavaged with the A. Marplatensis YKS2 strain at three doses for 3 days, mice in all groups presented no symptoms of illness, death, or infection recorded in a 7-day in vivo experiment. In addition, the HE staining result did not show inflammatory infiltration, tissue sclerosis, lymphoid hyperplasia, and other pathological changes in the tissue sections. Body weight and relative organ indices are good indicators of the adverse effects of a substrate in animal studies [36]. However, the statistical analysis showed that the YKS2 strain does not affect mice's body weight, feed intake, organ index, and hematological parameters in 3 days of gavage.

The small intestine is an important organ for material digestion and absorption. Therefore, the changes in the villus length, crypt depth, and V/C value of the small intestine are important indicators of the digestive tract's ability to digest and absorb nutrients[37]. Given the above, we further investigated the structure and barrier function of the small intestine. A. Marplatensis YKS2 strain had no toxic effects on the small intestine of mice when HE staining results and V/C values of jejunum were combined. Moreover, IELs and Gcs are currently considered the most significant parameter for the evaluation of small intestinal mucosal immunity, and an increase in the number of IELs was often observed in individuals with enhanced intestinal immune function[38, 39]. In this research, we did not observe a significant change between groups for Gcs quantity per 1 mm length of the villi epithelium but found that IELs quantity of the 1 mm length of the villi epithelium in the YKS2-High group was significantly higher than the control group. So, it indicates that A. Marplatensis YKS2 strain suspension (the concentration less than 1.0×10⁸ CFU/mL) does not affect the immune barrier function of the small intestine.

The intestinal microbial system works in synergy with the immune system to form a common functional network, which plays an important role in nutrient processing, absorption, and energy intake and is closely related to the homeostasis of the intestinal barrier [40]. In order to verify whether the YKS2 strain can interfere with the ecological balance of mice intestinal microflora, 16S rRNA sequencing technology was used to study the changes in intestinal microflora after feeding the bacterial solution. α- diversity analysis showed no significant change in the Chao and Simpson index in the YKS2 treatment group compared with the control group. In contrast, the Shannon index in the YKS2-H group was significantly higher than in the control group, indicating that YKS2 could enhance the overall microbial diversity when the YKS2 strain reached a certain dose concentration. It had no significant effect on the diversity, evenness, and richness of species distribution in the community. Multiple analyses of the β-diversity analysis showed that the microflora of each group was in a common range, and the YKS2 treatment groups did not change the composition of intestinal microflora. Therefore, the results showed that the YKS2 strain could not change the diversity, evenness, and richness of the whole community, But high doses of YKS2 can increase the diversity of unknown microbes. Furthermore, we looked at changes in bacterial community composition at the phylum and genus levels in the control and YKS2 treatment groups. At the phylum level, the results showed that Firmicutes, Bacteroidetes, and Bacteroidetes accounted for more than 90% of all microorganisms in each group. Firmicutes and Bacteroidetes could produce carbohydrate-degrading enzymes related to the degradation of difficult-to-digest sugars such as cellulose. Bacteroides mainly degrade complex carbohydrates, while Firmicutes prefer oligosaccharides [41]. Desulphurizing bacteria, also known as sulfate-reducing bacteria (SRB). H₂S produced by the bacterium, although it may be associated with some diseases, can act as signaling molecules and energy sources of mitochondria and play a dual role in the gut. Its harmful role has not yet been determined [42–45]. This study found that the abundance of Bacteroidetes and Proteobacteria was not significantly affected in all test groups. However, the abundance of Firmicutes and Actinobacteria was significantly increased in the YKS2-Low group, while the abundance of desulphurizing bacteroidetes was significantly decreased. At the genus level, we found that YKS2 bacterial solution in high-dose and medium-dose groups could increase the abundance of Bacteroides, but not significantly. Furthermore, there was no significant difference between the experimental and control groups for the abundance of Muribaculaceae,Alloprevotella,Eubacteriµm_coprostanoligenes_group, Clostridia_UCG-014, Enterorhabdus, Lactobacillus, Parabacteroides, Lachnospiraceae_NK4A136_ group, and Desulfovibrio genus.

These results indicated that YKS2 strains in the high-dose, medium-dose, and low-dose groups had a certain regulatory effect on the intestinal microbial community of mice, but this effect did not cause toxicity in mice. However, given the experimental animals' short feeding time, whether the YKS2 strain can cause toxicity by interfering with the ecological balance of host intestinal microbes will need to be tested in a long-term in vivo toxicity test.

This study provides an overview of the genome and reports on the unique genetic properties of A. Marplatensis strain YKS2 isolated from yak rumen. The genomic analysis of this strain is beneficial to understand the function of the A. marplatensis strain in Yak rumen and the interactions between the resistance to the harsh environment of yak and rumen bacteria. High, middle, and low doses of A. Marplatensis strain YKS2 showed no adverse effects on feed intake, body weight gain, tissue development, blood indexes, intestinal tissue, and physical barrier in mice, suggesting that the strain was not of acute pathogenicity. However, long-term feeding toxicity experiments in vivo should be performed before the animal industry uses the strain. We hope that future research will help understand whether its effect on the IELs number and microbiome ecological balance in the duodenum has a positive or negative effect on host health.

Author contributions

LDP and ZJY provided the research idea. WL, LQ, GZT, XGW, ZH, WL, GZT and ZK contributed reagents, materials, and analysis tools. LDP and HSW performed the experiments and wrote the manuscript. LJX and ZJY revised the manuscript. All authors participated in writing and reviewing the manuscript.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Ackonwledgements

This work was supported by the National Key Research and Development Program of China (2022YFD1801104), National Natural Science Foundation of China (31602101), International Cooperation Program of Gansu Province (22YF7WA032), Technical innovation guidance plan of Gansu Province (22CX8NA013).

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Genome analysis and safety assessment of Achromobacter marplatensis strain YKS2 strain isolated from the rumen of Yaks in China

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Abstract

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1. Introduction

2. Materials and Methods

2.2 Genome analysis

2.3 Test animals and Ethics

2.4 Acute toxicity study

2.4.1 Evaluation indicators

2.4.2 Histological evaluation of small intestine.

2.4.3 DNA extraction and 16S rRNA sequencing of the intestinal microbiota.

2.4.4 Bioinformatics analysis

2.5 Statistical analysis

3. Results

3.1 Genome features of YKS2

3.2 Functional analysis of A. Marplatensis YKS2 strain genome

3.2.1 NR database annotation

3.2.2 GO database annotation

3.2.3 COG database annotation

3.2.4 Cazy database annotation

3.2.5 Antibiotic resistance and virulence-related genes

3.3 Acute toxicity of A. Marplatensis YKS2 strain on mice

3.3.1 Effects of A. Marplatensis YKS2 strain on the overall health of mice

3.3.2 Effects of A. Marplatensis YKS2 strain on blood hematology and biochemical analytes

3.3.3 Effects of A. Marplatensis YKS2 strain on tissue histology

3.3.4 Effects of A. Marplatensis YKS2 strain on intestinal tissue

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

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