Isolation and Identification of Bacterial Pathogens of Proteus vulgaris and Chryseobacterium meningo

Guomin JIANG Li LIU Wuying CHU Zhigang HE Li ZOU Dongwu WANG Shiming DENG

Isolation and Identification of Bacterial Pathogens of Proteus vulgaris and Chryseobacterium meningosepticum from Peodiscus sinensis and Observation on the Histopathology

Abstract Two dominant strains of bacteria Lb18-01 and Lb18-02 were isolated and purified from scabies, liver and intestine of diseased Peodiscus sinensis. By artificial infection test, the two strains were verified as pathogenic strains with similar characters to that of natural infectious cases. The strains Lb18-01 and Lb18-02 showed strong pathogenicity to healthy P. sinensis in the artificial infection experiment, so they were the pathogenic strain of the disease. According to the morphology, physicochemical characteristics, 16 S rDNA sequence analysis and phylogenetic tree clustering, the pathogenic strains Lb18-01 and Lb18-02 were identified as Proteus vulgaris and Chryseobacterium meningosepticum which were tolerant to 20 drugs such as penicillin, tetracycline and ampicillin. Histopathological observation on diseased P. sinensis showed the pathological symptoms of sepsis such as hemorrhage and congestion of liver, spleen and intestine, and glomerular disintegration.

Key words Peodiscus sinensis; Proteus vulgaris; Chryseobacterium meningosepticum; Drug susceptibility test; Histopathology

Received: November 23, 2020  Accepted: January 21, 2021

Supported by Hunan Provincial Key Laboratory of Nutrition and Quality Control of Aquatic Animals (2018TP1027); Key R&D Projects of Hunan Province (2018NK2074).

Guomin JIANG (1976-), male, assistant research fellow, devoted to research about aquaculture.

Li LIU (1973-), female, assistant researcher, devoted to research about animal nutrition.

# These authors distributed equally to this work.

*Corresponding author.E-mail:1309128968@qq.com; E-mail:dsmjgm@126.com.

Chinese soft-shelled turtles (Peodiscus sinensis) have a high protein content, are rich and comprehensive in nutrition, and can be used as a medicine. They are a favorite aquatic food for people, as well as an important aquaculture economic animal in China［1］. However, with the  rapid development of Chinese soft-shelled turtle breeding industry, changes in the breeding environment factors, nutrition imbalance and other factors have led to the frequent occurrence of various diseases, causing significant economic losses to farmers and bringing great resistance to the sustainable and healthy development of the Chinese soft-shelled turtle breeding industry in China.

Chinese soft-shelled turtle scabies (also called caverned disease［2］ in the later stage of disease) is a common bacterial disease which is highly infectious with high incidence and great harm in cultured Chinese soft-shelled turtles, According to a lot of research reports, there are many types of pathogens of "scabies", including Aeromonas hydrophila, Aeromonas sobria, Aeromonas punctata, and Proteus vulgaris, and there are single infection, mixed infection or secondary infection of pathogens with high incidence and mortality rates［3-6］. From June to October 2018, those farmed young turtles in Hunan showed pathological features of "scabies" (Fig. 1), such as slow movement, loss of appetite, some soybean-sized white spots appeared on the carapace and plastron of diseased P. sinensis and gradually expanded with the disease progresses, and the contents of white spots looked like bean curd. At the same time, there were no food in gut, some white spots or lumps in the liver which looked like a flower and the mortality rate was as high as 100%.

In view of the phenomenon of "the same symptoms, different pathogens" for Chinese soft-shelled turtle diseases, on the basis of excluding parasite and virus infections, the pathogenic bacteria were isolated and identified, the pathological sections were observed and analyzed, and the drug sensitivity test was carried out in the experiments to correctly diagnose and control the disease and provide reference for healthy breeding of Chinese soft-shelled turtles.

Materials and Methods

Materials

The diseased juvenile Chinese soft-shelled turtles came from Hanshou County, Hunan Province, with a mass of (90-100) g/turtle. The healthy juveniles were from the aquatic seed farm of Hunan Province, with a mass of (100-120) g/turtle. They were healthy and undamaged in appearance.

Isolation and purification of pathogenic bacteria

Under aseptic conditions, the contents of body surface lesions, liver and intestine were streaked and inoculated on nutrient agar plates, and incubated at 28 ℃ for 24 h, and then the single typical and suspicious dominant colonies were selected to purify and culture for 18-24 h at 28 ℃.The purified bacreria were preserved in a refrigerator at -60 ℃ for later use.

Morphological identification of isolates

Isolates Lb18-01 and Lb18-02 cultured for 18-24 h were picked out for Gram staining and physiological and biochemical tests. The preliminary identification was carried out combining with Common Bacterial System Identification Manual［7］, Bergeys manual of determinative bacteriology［8］, Pharmacological Experiment Methodology［9］.

Sequence analysis of 16S rDNA of isolates

The bacterial genomic DNA extraction kit was used to extract the total DNA of the isolated bacteria, and the general primers were used for PCR amplification. The amplification products were purified and recovered and subjected to sequencing analysis. Using Clustalw and DNAstar software, the obtained 16S rDNA sequence was analyzed for homology in GenBank/Blast of NCBI, and the neighbor-joining method was employed to construct a phylogenetic tree.

Artificial infection test

The healthy juvenile soft-shelled turtles were subjected to an artificial infection test after 7 d of temporary culture. They were randomly divided into 7 groups, of which one was a control, and 6 groups were test groups, each with 10 turtles. The isolated bacteria Lb18-01 and Lb18-02 cultured at a constant temperature of 28 ℃ for 16-18 h were prepared into 1.0×108, 5.0×108, 1.0×109 CFU/ml bacterial suspensions with saline. A certain amount of corresponding bacterial suspension (0.1 ml) was injected from the hind limbs of young turtles, and the control group was injected with the same amount of saline. During the test, the turtles were fed properly and observed continuously for 15 d. The incidence of juvenile soft-shelled turtles was recorded, and the dying diseased turtles were dissected and used for isolation and identification of pathogenic bacteria.

Drug susceptibility test of pathogenic bacteria

The drug susceptibility test was carried out by the paper disk method［7］, in which each kind of drug susceptibility paper (6 mm in diameter) was tested in 5 replicates. The test results were judged according to Standard of Antibiotics Susceptibility Test (Kirby-Bauer Method) of the Ministry of Health of the Peoples Republic of China (WS/T125).

Visceral pathology observation

The liver, spleen, kidney and intestine tissues of healthy and diseased Chinese soft-shelled turtles were fixed. The fixed samples were subjected to dehydrate with gradient ethanol,hyalinize with xylene, embed in paraffin, slice inseries stain with H.E staining, and observe and  photograph with Olympus optical microscope, so as to analyze and study the pathological changes of the Chinese soft-shelled turtle tissue structures.

Results and Analysis

Morphological characteristics of isolates

One dominant purified bacterium was obtained from the body surface lesions and intestines of diseased soft-shelled turtles, named Lb18-01, and one dominant purified bacterium was obtained from the liver, named Lb18-02. The Lb18-01 strain grew diffusely on the common nutrient medium, covering the surface of the medium, like a cloud, and had a rancid smell. It was negative by Gram staining, purplish red, rod-shaped or filamentous, and had different lengths. The Lb18-02 strain was round or oval on the culture medium, light yellow, translucent, moist, smooth, and had no rancid smell. It was negative by Gram staining, purple-red, short rod-shaped, and the both ends were blunt.

Physical and chemical properties of isolates

According to physiological and biochemical analysis tests, the Lb18-01 strain could migrate and decompose glucose, maltose, etc.; and the Lb18-02 strain had no motility, did not ferment rhamnose, maltose, and was positive for contact enzymes, as shown in Table 1. According to references［7-8］, it was preliminarily determined that the Lb18-01 strain was P. vulgaris, and the Lb18-02 strain was P. vulgaris.

Analysis of 16S rDNA gene phylogeny

The lengths of the gene sequences of the isolates Lb18-01 and Lb18-02 were determined to be 1 416 and 1 387 pb, respectively, and the GenBank accession numbers were MN685224 and MN685235, respectively. Through the online comparison analysis of Blast in NCBI, the 16S rDNA sequences of the two strains have high similarity (98%-100%) with the sequences in the GenBank database. The phylogenetic tree (Fig. 2, Fig. 3) showed that the isolate Lb18-01 was clustered with P. vulgaris, and the isolate Lb18-02 was clustered with C. meningosepticum.

Artificial infection test results of isolates

After injection of Lb18-01 and Lb18-02 bacterial suspensions, the Chinese soft-shelled turtles appeared sluggish on the second day, showed decreased appetite, and died thereafter. The mortality rate increased with the increase of bacterial liquid concentration. On the 15th d, the mortality rate in the 1.0×109

CFU/ml Lb18-01 test group was 70%, and the dying Chinese soft-shelled turtles first appeared red spots on the back and abdomen, which then turned white and gradually became purulent. In the 1.0×109 CFU/ml Lb18-02 test group, the mortality rate reached 100% (Table 2), but there were no red spots on the backs and abdomens of Chinese soft-shelled turtles. The anatomy showed that the livers and gallbladders of Chinese soft-shelled turtles in the Lb18-01 and Lb18-02 test groups were slightly enlarged, and the intestines were red and swollen and contained no food, which were the same as the symptoms in natural disease cases. The artificially-induced soft-shelled turtles were used for pathogen isolation again, and the strains with the same morphological and physical and chemical properties as the isolated bacteria Lb18-01 and Lb18-02 were obtained. The control group had no abnormal symptoms and no deaths.

Drug susceptibility test results of isolates

The susceptibility of the isolated bacteria Lb18-01 and Lb18-02 to 20 kinds of antibiotic drugs showed that they were tolerant to the 20 test drugs, and there was no inhibition zone around the susceptibility paper, as shown in Table 3.

Guomin JIANG et al. Isolation and Identification of Bacterial Pathogens of Proteus vulgaris and Chryseobacterium meningosepticum from Peodiscus sinensis and Observation on the Histopathology

Pathological observation

The liver, spleen, kidney and intestinal tissues of the diseased soft-shelled turtles showed different pathological characteristics. In the liver the arterial wall was thickened and the inflammatory cells around the blood vessel increased (arrow 1 in Fig. 4A); the vein blood vessel ruptured (arrow 2 in Fig. 4A), the sinusoid was dilated, and a large number of red blood cells were deposited (arrow 1 in Fig. 4B); and the liver cell boundary was blurred (arrow 2 in Fig. 4B), and a large amount of hemosiderin could be seen (arrow 3 in Fig. 4B).The tunica of the spleen was thickened, the blood vessels of the tunica were enlarged, and there were a large number of red blood cells and lymphocytes (Fig. 4D); the red pulp area was enlarged, and a large number of red blood cells, inflammatory cells and hemosiderin deposits could be seen (arrows 1, 2, 3 in Fig. 4E);and a large number of lymphocytes and red blood cells were abnormal in structure, and ever ruptured and disintegrated in severe cases, the white pulp area was relatively reduced, the number of lymphocytes was significantly reduced, the capillaries were dilated, and the red blood cells were deposited (arrow 4 in Fig. 4E).

A large number of inflammatory cells infiltrated into the lateral and medial areas of the kidney, and the medial area was more severely affected than the lateral area; in renal tubular the epithelial cells and nuclei were enlarged and granular degeneration occurs, the microvilli on the free surface of the cells were not obvious, the lumen became smaller or disappeared, and the boundary between kidney tubules disappeared, resulting in the formation of a combined inclusion body (arrow 2 in Fig. 4G); the capillaries in the glomeruli were dilated, a large number of blood cells were accumulated (arrow 1 in Fig. 4G), and some glomeruli appeared necrosis and disintegration (Fig. 4H). Intestinal mucosal epithelial cells were arranged disorderly (arrow 1 in Fig. 4J), densely, goblet cells were darkened, and a large number of inflammatory cells infiltrated (arrow 3 in Fig. 4J); in lamina propria the capillaries are dilated, a lot of erythrocytes were deposited, and lymphocytes in the mucosal muscle layer increase suddenly (arrow 2 in Fig.4J); and in the submucosal and muscle layer blood vessels were swollen and many blood cells were deposited (Fig. 4K).

Discussion

In this study, two strains of bacteria Lb18-01 and Lb18-02 were isolated from Chinese soft-shelled turtles with "scabies". Inthe artificial inoculation test, the infected Chinese soft-shelled turtles showed symptoms similar to the natural disease, with a fatality rate of more than 70%. So the isolates Lb18-01 and Lb18-02 showed high pathogenicity and were the pathogenic bacteria of the disease. Through morphological characteristics, physiological and biochemical characteristics, 16S rDNA gene sequencing and alignment and phylogenetic tree cluster analysis, the isolates Lb18-01 and Lb18-02 were identified as P. vulgaris and C. meningosepticum, respectively.

P. vulgaris and C. meningosepticum are widely distributed in nature and are conditional pathogens of humans and aquatic animals［10-14］, as well as important pathogens that are common to humans, livestock and fish［11,15］. P. vulgaris can cause wound infection and scabies［15］, also cause Chinese soft-shelled turtle caverned disease and white abdominal shell disease［3］. C. meningosepticum can cause disease outbreaks with a high mortality rate, but only bullfrog infections have been reported in aquatic animals［16］. Co-infection of P. vulgaris and C. meningosepticum resulted in an outbreak of Chinese soft-shelled turtle scabies with a mortality rate of 100%, which is the first time in China.

Generally Chinese soft-shelled turtle scabies disease is highly contagious, and has a long epidemic time, resulting in a mortality rate of more than 20%. And with the development of disease, scabies will deteriorate into shot-hole［5,17］. However, in this case, the naturally-diseased Chinese soft-shelled turtles had a course of 6-10 months, scabies ulcers on the body surface were obvious, but the shot-hole phenomenon did not appear. Moreover, with the prolonged course of the disease, the mortality of Chinese soft-shelled turtles increased significantly to 100%.The reason of this phenomenon might be that the Chinese soft-shelled turtles were first infected with P. vulgaris which caused scabies, and then as the disease aggravated and the bodys resistance declined, they were infected with C. meningosepticum and the internal organs showed different degrees of inflammation, bleeding and symptoms of congestion, which in turn caused multiple organ dysfunction［18-19］, and a significant increase in mortality.

The isolates Lb18-01 and Lb18-02 were tolerant to 20 kinds of antibiotics in the drug susceptibility test, which might be due to the fact that farmers used the treatment drugs incorrectly based on only the pathological characteristics of the diseased Chinese soft-shelled turtles to lead to tolerance, or the abuse of antibiotics in the environment led to the emergence of drug tolerance of pathogenic bacteria. Therefore, with the increase of the scale of breeding and the increase of diseases, the types of pathogenic bacteria become more diverse, and the pathological characteristics become more and more complicated, in the process of aquatic animal disease diagnosis, technicians should not judge the types of diseases and prescribe based on the superficial pathological characteristics alone, they should also carry out pathogen drug susceptibility tests to ensure the scientific and reasonable selection and use of drugs to strictly prevent the occurrence of drug tolerance.

References

［1］ HE R. Research on nutrition and preservation technology of Peodiscus sinensis［D］. Shanghai: Shanghai Ocean University, 2014. (in Chinese)

［2］ YAN SF. Ultrastructural pathological studies on furunculosis disease of soft-shelled turtle (Triongx sinensis)［J］. Journal of Jimei University: Natural Science, 2001, 6(3): 197-203.(in Chinese)

［3］ SUN PF, CAI WQ, WU JN, et al. Pathology of the caverned disease of soft-shelled turtle［J］. Journal of Fisheries of China, 1996, 20(2): 120-124. (in Chinese)

［4］ MA YZ, SHU MA. Studies on the pathogens of Aeromonas sobria in Trionyx Sinensis［J］. Journal of Zhejiang University: Science Edition, 2000, 27(3): 329-333. (in Chinese)

［5］ WU HX, WANG SX, LI XX, et al. Studies on the  Aeromonas Furunculosis of Trionyx sinensis［J］. Donghai Marine Science, 1998, 16(4): 40-46. (in Chinese)

［6］ ZENG D, ZHONG L, LUO FX, et al. Isolation and identification of pathogenic bacteria from complications of Trionyx sinensis"skin-rotting" disease and furunculosis disease and drug sensitivity analysis［J］. Journal of Natural Science of Hunan Normal University, 2013, 36(4): 74-78. (in Chinese)

［7］ DONG XZ, CAI MY. Common bacterial system identification manual［M］. Beijing: Science Press, 2001. (in Chinese)

［8］ HOLT J G, KRIEG N R, PHA S, et al. Bergeys manual of determinative bacteriology［M］. Baltimore: Willians﹠Wilkins, 1994.

［9］ XU SY, BIAN RL, CHEN X. Pharmacological experiment methodology［M］. Beijing: Peoples Medical Publishing House, 2002: 192. (in Chinese)

［10］ FANG H, CHEN CZ, ZHANG XJ. Pathogenic bacteriology of aquaculture animals［M］. Beijing: China Agriculture Press, 2010: 237-245. (in Chinese)

［11］ LEE YJ, JUNG IO, OH DY. A rare case of ecthyma gangrenosum caused by Proteus vulgaris and Candida albicans in a patient with castleman disease［J］. Archives of Plastic Surgery, 2015, 42(6):805-807.

［12］ ZHAO NX, YUAN GY. Name and classification of medical bacteria［M］. Jinan: Shandong University Press, 2013: 321-322.(in Chinese)

［13］ SILVA CJD, HEO GJ, HOSSAIN S, et al. Comparison of virulence genes in Proteus species isolated from human and pet turtle［J］. Iranian Journal of Veterinary Research, 2018, 19(1):48-52.

［14］ TRIVEDI KM,  BRANTON A,  TRIVEDI D, et al. Phenotyping and genotyping characterization of Proteus vulgaris after biofield treatment［J］. International Journal of Genetics and Genomics, 2015, 6(3): 66-73.

［15］ YANG YB, YU LX, YANG QH, et al. Isolation, identification and antimicrobial susceptibility of Proteus vulgaris isolated from Ictalurus punctatus［J］. Microbiology China, 2017, 44(10): 2391-2397. (in Chinese)

［16］ CHEN XF, ZHOU CY, CHEN ML. Study on pathogen of Flavobacterium menigosepticum disease of bullfrog［J］. Journal of Jimei University: Natural Science, 1999, 4(3): 30-35. (in Chinese)

［17］ QIAN MM, SONG J, WU B, et al. The diagnosis and integrated prevention and treatment of common diseases in Pelodiscus sinensis［J］. Progress in Veterinary Medicine, 2009, 30(4): 103-107.  (in Chinese)

［18］ SHI LL, HAN YQ, REN HJ, et al. Research advance of pathology and physiology of sepsis［J］. Chinese Journal of Nosocomiology, 2016, 26(8): 1914-1916. (in Chinese)

［19］ FAN HG, LUAN L, GUAN W, et al. Pathogenesis of septic acute kidney injury and protective mechanisms of dexmedetomidine［J］. Journal of Northeast Agricultural University, 2017, 48(6): 91-96. (in Chinese)