Isolation Identification and Susceptibility Analysis of Bacillus pyocyaners from a Canine Pyometra

Kunyuan ZHU Lan WU Yanfei LIU Jiande YANG

Abstract In order to clarify the causes of the fester disease in companion animals， this study involved isolation， identification and drug susceptibility analysis of pathogenic bacteria from the uterus of an 11-year-old female dog who was admitted to the veterinary hospital of Tianjin Agricultural University. According to the results of colony morphology， smear staining microscopy， physiological and biochemical reaction and 16S rRNA gene analysis， the bacteria was identified as a Bacillus pyocyaners. The susceptibility analysis showed that the isolate is highly sensitive to aztreonam， cefdinir， cefotaxime， cefepime， ceftazidime， ofloxacin， streptomycin， kanamycin， piperacillin and tobramycin. Moreover， the isolate is moderately sensitive to ceftriaxone， cefuroxime and cefoxitin， but resistant to ampicillin， cefazolin， cefazine and medemycin. The research results provide references for controlling infection caused by B. pyocyaners.

Key words Bacillus pyocyaners; Isolation and identification; 16S rRNA gene; Susceptibility analysis

Bacillus pyocyaners， also known as Pseudomonas aeruginosa， is a gram-negative bacillus in Pseudomonas. It was first isolated from wound pus in 1882 by Gersard[1]. It is a conditional pathogen that is infectious to both humans and animals. In veterinary clinics， P. aeruginosa can cause a variety of clinical symptoms： bovine mastitis， metritis， horse abortion， suppurative pneumonia， snake necrotizing stomatitis， chicken sepsis， etc. In human medicine clinics， P. aeruginosa is a kind of common pathogenic bacteria that can cause postoperative and wound infections， and can cause human pleurisy， pneumonia and even meningitis in severe cases[2]. In recent years， reports of diseases caused by animal infection with P. aeruginosa have increased[3-7]. With the improvement of the quality of life， more and more people raise companion animals. While pets bring joy to people， they also spread zoonotic diseases， which seriously threaten the health of livestock owners. Therefore， the health of pets has become the focus of attention. Bacteria are a main cause of the onset of purulent diseases in companion animals. In this study， we investigated a case of canine pyometra caused by P. aeruginosa， aiming to provide reference for the prevention and treatment of related infections caused by P. aeruginosa.

Materials and Methods

Materials

Source of disease material

A case of pyometra of an 11-year-old female dog raised by a livestock owner in Tianjin was received by College of Animal Science and Veterinary Medicine， Tianjin Agricultural University on September 14， 2017.

Reagents

Media

The media used included ordinary nutrient agar medium， MacConkey medium， nutrient broth medium， and blood agar medium. These media were made in the laboratory of Tianjin Agricultural University， and the preparation materials were purchased from Hangzhou Institute of Biology.

（i） Enterobacteriaceae biochemical identification tubes： Sucrose， sodium citrate， VP-MR reagent， mannitol， maltose， lactose， indole， hydrogen sulfide， urea， and semi-solid agar power， biochemical identification tubes were all purchased from Hangzhou Institute of Biology.

（ii） Antimicrobial susceptibility disk： Ampicillin， aztreonam， cefazolin， cefazine， cefuroxime， cefdinir， cefotaxime， ceftriaxone， cefepime， ceftazidime， cefoxitin， ofloxacin， streptomycin， tobramycin， kanamycin， midecamycin， and piperacillin were all purchased from Hangzhou Microbial Reagent Co.， Ltd.

（iii） Ezup column bacterial genomic DNA extraction kit， Ezup column fungal genomic DNA extraction kit and Ezup column yeast genomic DNA extraction kit were purchased from Sangon Biotech （Shanghai） Co.， Ltd. DreamTaq-TM DNA Polymerase was purchased from MBI. dNTP was purchased from Sangon Biotech （Shanghai） Co.， Ltd. Agarose was purchased from BBI. SanPrep column DNAJ gel recovery kit and DNA Ladder Mix maker were purchased from Sangon Biotech （Shanghai） Co.， Ltd.

（iv） Primers： forward primer 5′-AGTTTGATCMTGGCTCAG-3′and reverse primer 5-GGTTACCTTGTTACGACTT-3′ were synthesized by Sangon Biotech （Shanghai） Co.， Ltd.

Methods

Medium preparation

Raw materials were weighed quantitatively， mixed， and diluted to 1 L. Each medium was autoclaved at 120-125 ℃ for 15 min. Common nutrient agar medium， MacConkey medium， and blood agar medium were poured into plates on an ultra-clean table， cooled and placed in a refrigerator at 4 ℃ for later use. After cooling the nutrient broth， it was placed in a refrigerator at 4 ℃ for later use.

Collection and separation of disease materials

The pus was aseptically taken from the uterus of this case of canine pyometra， streak-inoculated on ordinary nutrient agar medium， and placed in a constant temperature incubator at 37 ℃ for 24 h. The dominant colonies were picked for pure culture， streak-inoculated separately on ordinary nutrient agar medium， MacConkey medium and blood agar medium， respectively， and cultured in a constant temperature incubator at 37 ℃ for 24 h， followed by storing in a refrigerator at 4 ℃ for later use.

Smear staining microscopy

A single colony was picked from a pure culture and spread on a glass slide， and gram staining was then performed. After finding a field of view under a low power lens （4×） of the biological microscope， the morphology and staining characteristics of bacteria were observed under an oil lens.

Physiological and biochemical test

A pure culture was picked aseptically， inoculated in the nutrient broth medium， and incubated in a constant temperature incubator at 37 ℃ for 24 h to obtain a young bacterial liquid. The young bacterial liquid was inoculated into such 10 kinds as sucrose， sodium citrate， VP-MR reagent， mannitol， maltose， lactose， indigo， hydrogen sulfide， urea， and semi-solid agar dynamic biochemical identification tubes and placed in a 37 ℃ constant temperature incubator for 24 h. The results were recorded and analyzed.

Drug susceptibility test

A pure culture was picked aseptically， inoculated in the nutrient broth medium， and incubated in a constant temperature incubator at 37 ℃ for 24 h to obtain a young bacterial liquid. A certain amount of the young bacterial liquid （150 ml） was pipetted with a micropipettor and coated on the surface of common nutrient agar medium with a T-shaped rod. Then， 17 kinds of antimicrobial susceptibility disks were aseptically taken with tweezers and evenly and equidistantly put on the surface of the culture medium. After culturing in a 37 ℃ constant temperature incubator for 24 h， The results were recorded and analyzed.

PCR amplification of the 16S rRNA gene sequence of the isolate

The genomic DNA of the bacteria was extracted according to the operating instruction of the SK8255 （bacteria） kit. PCR amplification was performed using the genomic DNA as a template， and the pre-amplified band was 1 500 bp. The amplification system included template （genomic DNA 20-50 ng/μl） 0.5 μl， 10×Buffer （with Mg2+） 2.5 μl， dNTP （2.5 mmol/L each） 1 μl， enzyme 0.2 μl， F （10 μmol/L） 0.5 μl， R （10 μmol/L） 0.5 μl， and double-steamed H2O added to 25 μl. The PCR cycle conditions were pre-denaturation at 94 ℃ for 4 min， denaturation at 94 ℃ for 45 s， annealing at 55 ℃ for 45 s， and extension at 72 ℃ for 1 min， and 30 cycles was performed in total， followed by extension at 72 ℃ for 10 min， and termination at 4 ℃. The PCR product was subjected to 1% agarose electrophoresis under 150 V and 100 mA for 20 min， and the electrophoresis result was observed. The product was finally sent to Sangon Biotech （Shanghai） Co.， Ltd. for purification and sequencing. The sequencing primers were 5′-AGTTTGATCMTGGCTCAG-3′ and 5′-GGTTACCTTGTTACGACT T-3′.

Homology analysis of the 16S rRNA gene sequence from the isolate and construction of phylogenetic tree

The 16S rRNA gene sequence was subjected to Blast alignment in the NCBI database to obtain the sequences closest to the 16S rRNA sequence of the isolate. Multi-sequence homology alignment and analysis and the construction of the phylogenetic trees were performed using DNAstar software.

Results and Analysis

Morphological characteristics of cultured colonies

On the common nutrient agar medium， the colonies were medium-sized， light green， round， smooth and wet， and showed a waved edges; and the medium was also yellow-green， and after a few days， the green of the medium gradually became darker （Fig. 1）. Round colonies light gray in the middle with a transparent periphery and a large pin top were formed on the MacConkey medium. In the blood agar medium， large irregular gray round colonies with a darker center， showing with β hemolysis， were formed （Fig. 2）.

Smear staining and microscopy

Under the oil lens， gram-negative bacilli of medium size could be seen in single， paired or connected short chains （Fig. 3）

Results of physiological and biochemical test

A biochemical test was performed on the bacteria， and the results are shown in Table 1. The isolate did not decompose sucrose， maltose， lactose and mannitol; it did not produce indole; it did not use urea; both MR and VP tests were negative; it could use sodium citrate; and it did not produce hydrogen sulfide. With reference to the physiological and biochemical characteristics of the standard strain， the isolate was basically the same as P. aeruginosa， and preliminarily determined to be P. aeruginosa.

Results of drug susceptibility test

The results of the susceptibility test for the isolate are shown in Table 2. According to the CLSI susceptibility test standard， the resistance of the bacteria to various drugs could be divided into drug resistant （R）， moderately sensitive （I）， and highly sensitive （S）. The susceptibility analysis showed that the isolate is highly sensitive to aztreonam， cefdinir， cefotaxime， cefepime， ceftazidime， ofloxacin， streptomycin， kanamycin， piperacillin and tobramycin. Moreover， the isolate is moderately sensitive to ceftriaxone， cefuroxime and cefoxitin， but resistant to ampicillin， cefazolin， cefazine and medemycin.

PCR amplification of 16S rRNA gene sequence of the isolate

The PCR amplification and electrophoresis of the 16S rRNA gene sequence of the isolate showed that the length of the amplified fragment of the bacteria was about 1 480 bp. The length of the pre-amplified fragment was 1 500 bp， so the result was consistent with the expected fragment length.

Homology analysis of 16S rRNA gene sequence of the isolate

The gene sequence obtained by sequencing of the isolate was subjected to Blast on the NCBI website， obtaining 13 similar reference sequences. The 16S rRNA gene sequence of the isolate was analyzed for homology with the 13 16S rRNA gene sequences by Megalign in the DNAStar software. The results showed that the 16S rRNA gene sequence of the isolate shared extremely high homology with the 16S rRNA gene sequences of the 13 P. aeruginosa strains， and the homology values were all above 99%， as shown in Fig. 4.

Construction of phylogenetic tree of the isolate

The 16S rRNA gene sequence of the isolate and the 13 similar sequences were constructed by the Neighbor-Joining method through the Megalign in the DNAStar software. According to the phylogenetic tree analysis， the isolate was closest to KX268504.1， but relatively far from MG585077.1 and KM894176.1 in the database， as shown in Fig. 5.

Conclusions and Discussion

P. aeruginosa is an important conditional pathogen for human and animal infections， which can infect a variety of animals including humans. Companion animals share a living environment with humans， and they are either sensitive or play a role in the spread of diseases[8]. In this study， the growth morphological characteristics and gram staining microscopy results of the isolate colonies on common nutrient agar medium， MacConkey medium and blood nutrient agar medium are consistent with the morphological and culture characteristics of P. aeruginosa mentioned in research report by Han et al.[9]. The physiological and biochemical test results are consistent with the biochemical characteristics of P. aeruginosa mentioned in the research report of Niu et al.[1]. 16S rRNA gene sequencing is currently the most common molecular technique used to identify strains[10]. The 16S rRNA gene sequence of the isolate shared a homology as high as 99% with the 16S rRNA gene molecular sequence of the reference P. aeruginosa， and combined with the colony morphology observation， smear staining microscopy， physiological and biochemical test， and 16S rRNA gene sequence analysis and identification， the isolate from the uterine pus of canine pyometra was judged to be P. aeruginosa.

The results of the susceptibility test showed that the isolate is highly sensitive to aztreonam， cefdinir， cefotaxime， cefepime， ceftazidime， ofloxacin， streptomycin， kanamycin， piperacillin and tobramycin. Moreover， the isolate is moderately sensitive to ceftriaxone， cefuroxime and cefoxitin， but resistant to ampicillin， cefazolin， cefazine and medemycin. These results are consistent to the results of the susceptibility test on isolates No.1 and No.15 from cobra rotten skin disease to the 17 drugs， only the drug tolerance to ceftriaxone， ofloxacin and kanamycin changed， which might be related to the pre-diagnostic medication or test error of the dog. According to the test results， cefdinir and cefotaxime with high sensitivity can be selected as therapeutic drugs. The results of the drug susceptibility test show that the isolate has extensive drug tolerance， which is consistent with the drug resistance test results of CHINET P. aeruginosa reported by Zhang et al.， 2005-2014[11]. Many factors lead to the resistance of bacteria to antibiotics. In veterinary clinics， it is mainly because the unreasonable use or abuse of antibiotics[4]. The sensitivity of P. aeruginosa to antibiotics shows a trend of decreasing year by year[12]. Therefore， the drug resistance test of the isolate is an important reference index for clinical medication. In order to obtain rapid and effective treatment of P. aeruginosa and delay the emergence of drug resistance， veterinarians not only need to choose the most effective treatment drugs based on the results of drug resistance test， but also need to strictly control the use of antibiotics to avoid long-term use of the same antibiotic.

Companion animal purulent disease is a common disease in veterinary clinics， and the focal area contains a variety of zoonotic pathogens. Once humans come into contact with diseased animals that carry zoonotic pathogens without paying attention to disinfection and hygiene， they might be infected. It has been reported that the corpus luteum secretes a large amount of progesterone in the late stage of estrus in dogs， and the female dog's uterus is most sensitive to bacterial infection at this time[13]. Affected by the increase in the concentration of progesterone and the decrease in the concentration of estrogen， the immunity of the uterus will decrease， and the pathogenic bacteria that enter the uterus through the opened cervix in the late stage of estrus can easily proliferate in large numbers， leading to endometritis and finally causing pyometra[14]. Therefore， the disease in this case of canine pyometra was judged to be caused by the proliferation of P. aeruginosa in the uterus in a large quantity in the late stage of estrus of the dog under the pre-action of progesterone and estrogen. Clinically， the bacteria isolated from pyometra cases are mainly Escherichia coli[15]， in addition to Klebsiella pneumoniae， Streptococcus， Enterococcus faecalis， Pseudomonas， Staphylococcus， etc.[16-17]. There are few reports on pyometra caused by P. aeruginosa. This study has enriched the research data of P. aeruginosa， and also provides reference for the prevention and treatment of related infections caused by the bacteria. In order to reduce the probability of infection of animals and humans， livestock owners should do regular cleaning work， strictly vaccinate， and treat their pets in time after the illness.

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