A nosocomial Pseudomonas aeruginosa ST3495 isolated from a wild Burmese python (Python bivittatus) with suppurative pneumonia and bacteremia in Hainan, China

Abstract

Pseudomonas aeruginosa is a common infectious agent associated with respiratory diseases in boas and pythons, however, the histopathology, resistance and virulence are yet described for this species. In this study, we investigated a dying Burmese python rescued from tropical rainforest in Hainan. Clinical signs were open-mouthed breathing, abnormal shedding and anorexia. Abundant yellow mucopurulent secretions were observed in highly ectatic segmental bronchi by postmortem. Histopathological lesions included systemic pneumonia, enteritis, nephritis and carditis. P. aeruginosa was the only species isolated from heart blood, kidney, trachea and lung. The phenotype analysis demonstrated that the isolates had strong biofilm, and were sensitive to amikacin, spectinomycin, ciprofloxacin, norfloxacin and polymyxin B, moreover, the LD₅₀ of the most virulent isolate was 2.22×10⁵ cfu/mL in a zebrafish model. Molecular epidemiological analysis revealed that the isolates belonged to sequence type 3495, the common gene patterns were toxA + exoSYT + phzIM + plcHN in virulence and catB + bla_TEM + ant (3'')-I+ tetA in resistance. This study highlights that P. aeruginosa should be worth more attention in wildlife conservation and raise the public awareness for the cross infection and cross spread between animals and human.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-023-01038-7.

Highlights

• Epidermal cells of multiple organs were invaded by Pseudomonas aeruginosa in the wild Burmese python.

• Common gene patterns in the isolates were toxA + exoSYT + phzIM + plcHN for virulence and catB + blaTEM + ant (3'')-I + tetA for resistance.

• Biofilm was necessary for virulence of the isolates infection in a zebrafish model.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-023-01038-7.

Introduction

The Burmese python (Python bivittatus) is one of the longest and heaviest non-venomous pythons in the world. This reptile is native to south and southeast Asia, including the south of China (for example, Hainan province and Hong Kong city) [1]. Currently, the population is largely declining in the wild as a result of illegal capture, overexploitation, diseases and natural habitat loss, and the Burmese python has been listed in CITES Appendix II, of the IUCN red list (https://cites.org/eng/app/appendices.php or https://www.iucnredlist.org/en) as vulnerable and is nationally protected in China [2, 3].

Respiratory tract disease, such as Mycoplasma agassizii infection [4], is common in captive and wild Burmese pythons, although few data are available, According to our research, pneumonia and tracheitis are typically caused by opportunistic Gram-negative bacterial infections, such as Salmonella enterica subsp. houtenae and Pseudomonas aeruginosa [5, 6], but it is noticed only when the disease is advanced and the clinical signs and symptoms are very obvious, which leads to high mortality rates among captive pythons.

P. aeruginosa is a ubiquitous pathogen in natural settings that causes infection not only of the respiratory system, urinary tract, blood and other parts of the body in humans and animals, but also causes rotten root in plants and environmental contaminations, particularly those strains with increased antibiotic resistance [7–10]. The pathogenesis, virulence factors, antibiotic resistance and interactions with host have been intensively studied for this pathogen. P. aeruginosa displays a vast repertoire of virulence factors, antibiotic resistance determinants, regulatory circuits and signaling systems, which confer remarkable plasticity and adaptability. The main virulence factors include cell-associated lipopolysaccharide, biofilm, motility structures, the quorum sensing system, secretion systems, two-component systems, extracellular outer membrane vesicles, cytotoxins, and antioxidant enzymes and their regulation [11]. P. aeruginosa readily acquires high resistance to various antibiotics (for example aminoglycosides, quinolones and β-lactams) through a myriad of intrinsic and acquired mechanisms, including outer membrane permeability, efflux systems, antibiotic-inactivating enzyme and biofilm-mediated adaptive antibiotic resistance [12]. Histologically, severe immune cell infiltration into the bronchioles, severe alveolitis with pulmonary infiltrates and hemorrhagic necrosis are the common signs in the infected lungs of patients, a mouse model and in farmed mink, respectively [13–15]. The infection of tropical snakes (one Python molurus and two Boa constrictors) by P. aeruginosa was also described in 2013 as deposition of fibrinoid in the walls of the myocardial blood vessels, necrotic stomatitis and necrotic exudative pneumonia, hyaline droplet degeneration of the hepatocytes and the kidney epithelium, diffuse fibrinoid degeneration of the connective tissue within all viscera and focal infiltrations of pseudoeosinophile leukocytes in the spleen [16]. However, the pathogenesis of P. aeruginosa infection in the Burmese python is yet to be elucidated, but is necessary for the effective and conservation of this reptile. This study reports a natural-P. aeruginosa-infection case in a wild Burmese python and reveals the histopathology, the genotype/phenotype profiles of virulence and resistance, and the multilocus sequence types of the isolates.

Materials and methods

Burmese python

A wild Burmese python was rescued in Hainan Tropical Rainforest National Park by the Boa Rescue and Breeding Center of Hainan Province (BRBCH) in 2019. However, it was too weak to eat and died in several days later. The body was then sent to Hainan University for etiological analysis.

Necropsy and histopathology

The trachea, lungs, heart, liver and kidney were collected by routine anatomical methods [6]. Histological analysis was carried out by paraffin sectioning and observation at 100× and 400× magnification under a light microscope [17].

Bacterial examination

Heart blood and the bacterial inocula from trachea exudates and each internal organ including the spleen, lungs, heart, liver and kidney were collected during necropsy as previously described [6, 17]. The samples were then streaked onto Luria-Bertani (LB) agar and incubated at 35 ℃ for 24 h. The bacterial isolates were identified by Gram staining, 16S rRNA gene sequencing (primer set listed in Table S1) and phylogenetic analysis using GenBank BLAST and MEGA-X software [17, 18].

Antimicrobial susceptibility testing

Based on the Kirby-Bauer disk diffusion method and the standard protocol of the Clinical and Laboratory Standards Institute guidelines [19], seven classes of antibiotics including β-lactams, aminoglycosides, quinolones, chloramphenicol, macrolides, sulfonamides and peptides (Table 1) were used in a drug sensitivity test. The susceptibility was determined as sensitive, intermediate or resistant by measuring the size of the inhibitory zone of each test isolates with a quality control strain of Escherichia coli (ATCC25922).

Table 1.

Resistance pattern of the P. aeruginosa isolates

Group	Antibiotics	Standard(mm)				Isolates (100%)
		R	I	S	R
β-lactams	Amoxicillin	≤13	14-16	≥17	≤13	R
	Penicillin	≤10	11-15	≥16	≤10	R
	Cephalexin	≤14	15-17	≥18	≤14	R
	Cefalotin	≤14	15-17	≥18	≤14	R
	Ampicillin	≤13	14-16	≥17	≤13	R
	Cefoperazone	≤15	16-20	≥21	≤15	I
Aminoglycosides	Streptomycin	≤11	12-14	≥15	≤11	R
	Gentamicin	≤12	13-14	≥15	≤12	R
	Tobramycin	≤12	13-14	≥15	≤12	R
	Kanamycin	≤13	14-17	≥18	≤13	R
	Amikacin	≤14	15-16	≥17	≤14	S
	Spectinomycin	≤12	13-18	≥19	≤12	S
Quinolones	Levofloxacin	≤13	14-16	≥17	≤13	R
	Ciprofloxacin	≤15	16-20	≥21	≤15	S
	Norfloxacin	≤12	13-16	≥17	≤12	S
Tetracyclines	Tetracycline	≤14	15-18	≥19	≤14	R
	Doxycycline	≤12	13-15	≥16	≤12	R
Chloramphenicol	Chloramphenicol	≤12	13-17	≥18	≤12	R
	Florfenicol	≤12	13-17	≥18	≤12	R
Macrolides	Erythromycin	≤13	14-22	≥23	≤13	R
	Roxithromycin	≤10	11-15	≥26	≤10	R
Sulfonamides	Compound trimethoprim	≤12	13-16	≥17	≤12	R
Peptides	Polymyxin B	≤8	9-11	≥12	≤8	S

‘R’ represents resistant, ‘I’ represents moderately resistant and ‘S’ represents sensitive to antibiotic

Assessment of biofilm formation

Biofilm formation was measured using a 96-well microtiter plate and the crystal violet assay [20]. In brief, an overnight bacterial culture was adjusted to an OD₅₉₅ of 1.0 then diluted 1:40. The dilution was then added to a microtiter plate (200 μL per well), for 24 h at 37 °C to allow for biofilm formation. The wells were washed with sterile PBS three times, fixed with 100 μL of methanol solution for 15 min, stained with 125 μL of 0.1% crystal violet solution for 5 min, rewashed with sterile PBS three times, finally, the dye was resolubilized the dye with 100 μL of 95% ethanol solution for 30 min at 37 °C. The OD₅₉₅ value of each well was quantified by a microplate reader. Escherichia coli (ATCC 25922) was used as a negative control. Biofilm production was assessed as described before [21]. The average optical density value (ODc) of the negative control (LB broth) was used as the cut-off. Biofilm formation by each isolate was then determined as absent, weak, moderate or strong, as shown in Table S1.

Genotype assay of resistance and virulence

The resistance and virulence genes were screened using PCR amplification. Primers, annealing temperatures and the amplicon length of each gene are listed in Table S2 [22–27]. The size of each PCR product was detected via 1% agarose gel electrophoresis. Genotypic patterns were determined according to the detector complement of genes.

Multilocus sequence typing (MLST)

According to the schemes, loci and primers of the P. aeruginosa typing database (https://pubmlst.org/bigsdb?db=pubmlst_paeruginosa_seqdef), seven endogenous genes: acsA (acetyl coenzyme A synthetase), aroE (shikimate dehydrogenase), guaA (GMP synthase), mutL (DNA mismatch repair protein), nuoD (NADH dehydrogenase I chain C, D subunit), ppsA (phosphoenolpyruvate synthase) and trpE (antioxidant synthetase component I) were amplified and sequenced. The sequence type (ST) was assigned based on the allelic profile of the seven amplicons. The specific ST of each isolate was obtained from the above MLST website.

Zebrafish experiment

As previously described [6], three-month-old healthy zebrafish (Danio rerio) were raised in a laboratory for at least 1 week. The fish were then fasted for 24 h and infected with 10 µl of bacterial suspension by intraperitoneal injection. Table 3 shows the doses of each isolate used in this experiment. Reactions and cumulative mortalities (%) were recorded daily for 96 h. The half-maximal lethal dose (LD₅₀) was calculated via the Bliss method using SPSS software. Bacterial examination was repeated from the seroperitoneum of the diseased fish to confirm whether the infection was successful or not. The Ethical Committee for Animal Laboratories, at Hainan University permitted all protocols.

Table 3.

Genotypic profiles of resistance and virulence of P. aeruginosa isolates

Category	Function	Genes	Coding protein	Isolates(100%)
Resistance	Chloramphenicol	catB	Type B chloramphenicol O-acetyltransferase	+
	β-lactams	blaTEM	Class A extended-spectrum beta-lactamase	+
		blaPER	Class A extended-spectrum beta-lactamase	-
	Aminoglycosides	ant (3’)-I	Nucleotidyltransferase	+
		ac (6’)-II	N-acetyltransferase	-
	Tetracyclines	tetA	Tetracycline efflux MFS transporter	+
	Macrolides	ermA	23S rRNA (adenine(2058)-N(6))-methyltransferase	-
		ermB	23S rRNA (adenine(2058)-N(6))-methyltransferase	-
	Quinolone	gyrA	DNA gyrase subunit A	-
		gyrB	DNA topoisomerase (ATP-hydrolyzing) subunit B	-
Virulence	Type III secretion system	exoU	Succinoglycan biosynthesis glycosyltransferae	-
		exoS	Exoenzyme S	+
		exoY	Adenylate cyclase	+
		exoT	Exoenzyme T	+
	Type II secretion system	toxA	Exotoxin A	+
	Oxidative stress	phzM	Phenazine-specific methyltransferase	+
		phzI	Autoinducer synthase
	Phospholipase C	plcH	Hemolytic phospholipase C	+
		plcN	Non-hemolytic phospholipase C	+
	Type IV pili	pilB	ATPase	-

‘+’ indicates positive and ‘–’ indicates negative

Results

Gross examination and histopathology

The wild Burmese python was approximately 2 meters long and had dysecdysis, with patches of dry and peeling skin remaining (Fig. 1). According to the BRBCH the snake was lethargic and weak, displayed dyspnea and open-mouth breathing, and was not eating when rescued. After the death of the snake a few days later, a postmortem revealed a blocked airway with excess yellow mucus (Fig. 2). Histopathology showed that the exudate was composed of heterophilic cells, necrotic debris and tissue fluid in the lungs (Fig. 3B), hemorrhagic foci in the heart (Fig. 3E), and epithelial cell abscission in the trachea (Fig. 3C), intestine (Fig. 3A) and kidneys (Fig. 3D).

Fig. 1.

Burmese python. Arrows indicate the pieces remaining after abnormal ecdysis

Fig. 2.

Anatomical analysis of the lung of the Burmese python. The lung was blocked with excess yellow mucus

Fig. 3.

Histopathology of various tissues of the Burmese python. A Intestine. Shed intestinal columnar epithelial cells (black arrow), bar = 500 µm. B Lung. Exudates in the air chambers (black arrow), bar = 500 µm. C Trachea. The mucosal epithelium of the trachea is sloughed off (white arrow), bar = 500 µm. D. Kidney. Dropped renal tubular epithelial cells (Black arrow); steatosis (blue arrow); cell pyknosis and necrosis (red circle), bar = 50 µm. E Heart. Hemorrhagic foci (white arrow), bar = 500 µm

Bacterial examination

Rough blue-green colonies were isolated from the samples of blood, trachea, lung and kidney on LB agar. Gram staining revealed that the cells were slender rod-shaped and Gram-negative. Four isolates, designated MDM1, MDM2, MDM3 and MDM4 (per isolate per sample), were randomly selected for 16S rRNA gene sequencing and were identified as P. aeruginosa via BLAST and phylogenetic analysis (Fig. 4).

Fig. 4.

16S rRNA phylogenetic tree of the isolates. Phylogenetic analysis of P. aeruginosa isolated from the wild Burmese pythons. The evolutionary history was inferred using the neighbor-joining method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The evolutionary distances were computed using the maximum composite likelihood method. The phylogenetic tree was constructed using MEGA-X software

Antimicrobial susceptibility testing

The four isolates showed the same antimicrobial susceptibility profile (Table 1) with multiple antibiotic resistance to seven types of antibiotics, including 17 drugs in total seven β-lactams (with the exception of moderate sensitivity to cefoperazone), four aminoglycosides, one quinolone (levofloxacin), two tetracyclines, two chloramphenicol, two macrolides and one sulfonamide (compound trimethoprim). The four isolates were susceptible to two aminoglycosides (amikacin and spectinomycin), two quinolones (ciprofloxacin and norfloxacin), and polypeptide polymyxin B.

Assessment of biofilm formation

The results of the crystal violet assay showed that all four isolates formed biofilm and the values of OD₅₉₅ were seven to nine folds higher than that of the negative control (Table 2), which is more than the threshold (four folds) and revealed that all isolates were strong biofilms producers (Table S1).

Table 2.

Biofilm production capability of the P. aeruginosa isolates

Isolates	OD595	ratio	Biofilm
MDM1	0.5900±0.008718	7.2544	Strong
MDM2	0.6470±0.009539	7.9552	Strong
MDM3	0.4490±0.009000	5.5207	Strong
MDM4	0.7387±0.01007	9.0827	Strong
Negative control	0.08133±0.001528	1	0

Genotype patterns of resistance and virulence

In total, 10 resistance genes and 10 virulence genes were detected by PCR and sequencing. The results were identical for the four isolates (Table 3). The typical genotype pattern of resistance was catB (chloramphenicol resistance) + bla_TEM (β-lactam resistance) + ant (3'')-I (aminoglycoside resistance) + tetA (tetracycline resistance), while the typical genotype pattern of virulence included eight genes, namely toxA (T2SS secretion system), exoS/exoY/exoT (T3SS secretion system), phzI/phzM (oxidative stress-related genes) and plcH/plcN (phospholipase genes). Notably, the gyrA and gyrB genes encoding quinolone resistance were not detected by PCR.

MLST

According to the analysis of the sequences and the allele spectrum in the MLST network database of P. aeruginosa, all four strains belonged to ST3495, which has been registered in database.

Zebrafish infection model

As shown in Table 3, all of the isolates were virulent to zebrafish and death occurred among the groups injected with high doses of isolates, with LD₅₀ values ranging from 2.22×10⁵ cfu/mL to 4.0×10⁷ cfu/mL. Using zebrafish as an animal model, the obvious signs of P. aeruginosa infection was bleeding under the gills and abdomen (Fig 5A and B). Infection was confirmed by recovering isolates from the diseased fish and identifying them by PCR with the primers listed in Table S2 [28].

Fig. 5.

Zebrafish infected with the P. aeruginosa isolates. Hemorrhagic foci in the gills and abdomen are indicated by arrows in (A) and (B), respectively

Discussions

P. aeruginosa is an opportunistic pathogen causing acute or chronic infection in its hosts, which include humans and various domestic and wild animals [7]. In this study, we describe a case of P. aeruginosa infection in a wild Burmese python. According to the gross examination, histopathology and bacterial examination, the death was resulted from asphyxia caused by a blockage in the respiratory tract of excess yellow mucus, sepsis caused by the spread of P. aeruginosa among the organs of the heart, kidney and lung via the blood circulation.

Histopathology showed that the infection of P. aeruginosa caused the death of epithelial cells, including intestinal columnar cells, renal tubular epithelial cells and respiratory epithelial cells and detachment from the main tissues (Fig 3A-C), with some cells showing fatty degeneration and necrosis (Fig 3D). Previous studies have shown that P. aeruginosa infection involves the adherence, invasion, and remodeling of host epithelial cells and finally resulting in cell death by inducing the mitochondrial dysfunction or other mechanisms [29–31]. Hence, the invasion and destruction of epithelial cells appears to be a common and crucial mechanism of host infection by P. aeruginosa including the case of the Burmese python. The pathology of disease in the python therefore confirmed that death was caused by the P. aeruginosa isolates. Moreover, disease was caused by a single virulent strain rather than a mixture of different strains, because the four isolates from blood and from three different organs were identical in terms of their genotypic/phenotypic profiles of resistance and their sequence type (ST3495).

According to a recent molecular epidemiological study on P. aeruginosa, two isolates of ST3495 were also recovered from the feces of residents at long-term care facilities in France [32]. By comparing of the resistance and virulence profiles of these two human isolates with the four isolates from the Burmese python, all possessed the exoS gene and lacked the exoU gene, while the two human isolates were sensitive to tobramycin and had moderate resistance to ciprofloxacin, whereas the four isolates were resistant to both tobramycin and ciprofloxacin (Table 1). It is contradictory that tobramycin is recommended for the clinical management of moderate to severe bacterial infections, especially in cystic fibrosis patients with P. aeruginosa infection, but is seldomly used as a veterinary drug in domestic animals, such as dogs and cats, while ciprofloxacin is a broad-spectrum antibiotic widely used to treat infections both in animals and nosocomial infections in humans. Resistance to both tobramycin and ciprofloxacin is acquired by mutations and by adaptions involving biofilm-mediated resistance rather than being acquired via horizontal transfer of resistance genes from microbial communities or soil/water environments [12]. We speculate that such uncorrelated evolution of resistance to antibiotic exposition would be generated via the accumulation of random mutations. The four isolates in this study were surprisingly consistent with the nosocomial clinical isolates from Panama hospital [33] in terms of their sensitivity to aminoglycosides (amikacin and spectinomycin), quinolones (ciprofloxacin and norfloxacin), and polypeptide polymyxin B. This highlights the challenges facing antibiotic therapy for P. aeruginosa infection in humans and animals, and the risk of cross-transmission and cross-infection at the interface of wild environments and human communities.

Inconsistencies were observed between the resistance phenotypes to macrolides (resistance to erythromycin and roxithromycin) and quinolone (resistance to levofloxacin, but sensitivity to ciprofloxacin and norfloxacin) and the genetic profiles showing a lack of the ermAB genes and intact gyrAB genes in the isolates ; although there was a partial correlation between resistance to chloramphenicol, β-lactam, aminoglycoside and tetracycline and the presence of the catB, bla_TEM, ant (3'')-I and tetA genes. Many factors may contribute to those discrepancies such as undetected resistance genes and undiscovered resistance mechanisms. In fact, macrolide resistance can be generated via many mechanisms, including ribosomal protection protein genes (ermA, ermB and ermD), efflux pump genes (msrA, mefA and mefE), enzymatic modification genes (mphB, ereA and ereB), and integron genes (intI2 and intI3) [34, 35]. In the same way, the mechanisms of quinolone resistance include two categories of mutations and the acquisition of resistance-conferring genes. Resistance mutations generally refer to the genes encoding DNA topoisomerase IV, DNA gyrase, and efflux pumps, whereas acquired resistance mainly refer to plasmid-encoded Qnr proteins [36]. Future studies involving whole genome sequencing [37] may aid our understanding of the resistance mechanisms in P. aeruginosa isolates

Our findings showed that the isolates possessed 8 of the 10 tested virulence genes, including exoA, which encodes ExoA, an exotoxin secreted by the type II secretion system that inhibits protein synthesis in host cells [38], and exoY, which encodes cytotoxin ExoY, an effector secreted through the type III secretion system to regulate host cytoskeletal organization and signal transduction [39]. Our results also revealed that the presence of exoS and exoU differs significantly between isolates and appears to be mutually exclusive [40], because exoS was detected but not exoU in the four isolates. A hypothesis for this exclusivity is that ExoS and ExoU confer enhanced fitness in distinct ecological niches.

In addition, the pilB gene which encodes the ATPase PilB that promotes polar type IV pili assembly in P. aeruginosa, was not detected in the four isolates in this study. It has previously been reported that pilB mutants show no reduction in virulence [41], but were unable to assemble surface pili [42], showed decreased motility [43] and disrupted levels of intracellular cAMP [44]. Considering that type IV pili promote early biofilm formation [45], a lack of pili would be expected to weaken the development of biofilm, yet in fact all of the isolates produced strong biofilm. This may indicate that type IV pili are not necessary for biofilm formation, but are reportedly involved in shaping the cellular architecture of P. aeruginosa [46].

Biofilms plays an important role in the generation of antimicrobial resistance, evasion of host immune defenses, and persistent infections. Recent studies have shown that bacterial biofilms are involved in 65% of infectious diseases and more than 80% of chronic infections [47]. P. aeruginosa is the main pathogen associated with this type of medical infection [48]. Our data displayed a negative correlation between the amount of biofilm and the LD₅₀ value (Tables 3 and 4), which implied that the virulence was positively associated with the ability to form biofilm for the four isolates, as the profiles of resistance and virulence genes were consistent among the isolates.

Table 4.

The values of LD₅₀ of P. aeruginosa isolates

Isolate	Dose(cfu/mL)	Death number of /tails	Mortality rate/%	LD50(cfu/mL)
MDM1	108	4/6	66.7	1.33×107
	107	3/6	50
	106	2/6	33.3
	105	0	0
MDM2	108	6/6	100	2.98×106
	107	4/6	66.7
	106	2/6	33.3
	105	1/6	16.7
MDM3	108	4/6	66.7	4.0×107
	107	2/6	33.3
	106	1/6	16.7
	105	0	0
MDM4	108	6	100	2.22×105
	107	5	83.3
	106	4	66.7
	105	3	50

In summary, this study primarily described the case of a wild Burmese python with suppurative pneumonia and sepsis caused by P. aeruginosa infection. Histopathology highlighted that the invasion of epidermal cells was a common mechanism by which this pathogen infects the host including the Burmese python. Bacteria were isolated from the blood and multiple organs. Our findings highlight the risk of potential cross- infection between animals and humans because all isolates belonged to the originally detected nosocomial ST3495, as determined by molecular epidemiology. Our findings reconfirmed the discrepancies between phenotype and genotype regarding the intricate mechanisms of resistance in this pathogen and emphasize that susceptibility testing is irreplaceable by molecular methods and plays an important role in clinical practice. Finally, the zebrafish infection model revealed that biofilm was important for the virulence of P. aeruginosa as PCR analysis confirmed that the isolates had the same genotypic profiles of resistance and virulence. In conclusion, this study highlights that attention should be paid to P. aeruginosa as an emerging opportunistic pathogen in wild animals.

Authors’ contributions

Jiping Zheng, Jifeng Zeng conceived the experiments. Roushan Li, Bo Ling, Xin Wang, Nuo Yang, Lixia Fan performed the experiments. Guiying Guo, Xuesong Li and Fei Yan analyzed the results. Roushan Li and Jiping Zheng wrote the paper. All authors read and approved the final manuscript.

Funding

This study was supported by the Hainan Provincial Natural Science Foundation of China (Grant 321MS007 to G. Guo, 821MS029 to N. Yang, 821RC1052 to L. Fan and 2019RC084 to J. Zheng) and the Natural Science Foundation of China (32060131 to J. Zheng and 32060788 to J. Zeng).

Availability of data and material

GenBank accession numbers for the 16S rRNA gene of Pseudomonas aeruginosa isolated from Burmese python are MDM1 (trachea) (MZ618953), MDM2 (lung) (MZ618954), MDM3 (heart) (MZ618955) and MDM4 (liver) (MZ618956).

Declarations

Ethics approval

This study was conducted as per the recommendations for the care and use of laboratory animals. The Hainan University Application for Animal Welfare and Ethical Review approved the experimental protocol under approval number HNUAUCC-2020-00183.

Conflicts of interest

The authors confirm that there are no conflicts of interest regarding the publication of this article.