First Molecular Identification and Whole Genome Sequencing of Listeria monocytogenes Isolated From an African Lion

ABSTRACT

Listeria monocytogenes (LM) is a zoonotic pathogen that causes sporadic infectious listeriosis, which is a foodborne disease associated with consumption of contaminated food or feed. The internal organs of an African lion from a zoo in Shanghai were analysed to determine the cause of death. LM infection was suspected on the basis of the clinical symptoms and pathological changes and confirmed by polymerase chain reaction, whole genome sequencing and phylogenetic analysis. This is the first report of LM infection of an African lion in China.

This study describes for the first time the molecular evidence of Listeria monocytogenes infection in African lions. Through various methodologies of classical and molecular microbiology, pathological analyses and in vivo experimentation, as well as whole genome sequencing, it was possible to identify LM as the cause of disease in an African lion. This implies that LM is also a pathogenic microorganism in this feline.

1. Introduction

Listeriosis is most commonly caused by consumption of foods contaminated with the bacterium Listeria monocytogenes (LM). The main symptoms of listeriosis are fever, severe headache and nausea but can progress to meningitis (Beamonte Vela et al. 2020). As the mortality rate of listeriosis is especially high, early diagnosis and treatment are crucial for prevention and control. Sporadic outbreaks of listeriosis have been reported in humans, birds and ruminants in many countries and regions (Gu et al. 2015; Hydeskov et al. 2019) but have not been reported in African lions.

Zoonotic pathogens break down barriers between species and can be transmitted to humans through natural contact and the food chain (Markovich et al. 2024; Tola 2024). LM is a rapidly spreading and highly destructive opportunistic pathogen that can be transmitted through oral and faecal routes. Listeriosis is a zoonotic disease associated with a high mortality rate that is mainly caused by consumption of contaminated meat, poultry and seafood products, as well as unprocessed fruits, vegetables and milk (Farber and Peterkin 1991; Ragon et al. 2008). In August 2019, consumption of contaminated pork led to 222 cases of human listeriosis in Spain, demonstrating the potential for infection of pigs by virulent strains of LM, which has important implications in veterinary medicine and food safety (Gomez‐Laguna et al. 2020).

We report the isolation and identification of a virulent strain of LM isolated from the internal organs of an African lion from a zoo in Shanghai, China. These findings clarify the effects of LM infection in wildlife and will help to develop effective prevention and control strategies to protect zoo wildlife.

2. Materials and Methods

2.1. Source of Diseased Materials and Reference LM Strain

The internal organs of an African lion (heart, liver, spleen, lungs and kidneys) were obtained from a zoo in Shanghai, China. The LM standard strain C53004 was purchased from the China Institute for Biological Products and Drug Supervision.

2.2. Biochemical Testing

The VITEK2 automatic microbiological identification system and the accompanying Gram‐positive identification card were obtained from bioMérieux SA (Marcy‐l′Étoile, France). The analyses performed with the VITEK system included fermentation of glucose, mannitol and rhamnose, in addition to the Voges–Proskauer (V–P), aesculin hydrolysis, nitrate reduction and hippuric acid tests, which were all conducted in accordance with Bergey′s Manual of Determinative Bacteriology (8th edition).

2.3. Observation of Bacterial Morphology by Transmission Electron Microscopy

Bacterial cells were fixed in 0.25% glutaraldehyde solution for 24 h, washed three times by centrifugation with sterile water and then mixed with an equal amount of an aqueous solution of 2% sodium phosphotungstate and copper omentum added drop‐wise through a sterile capillary straw. After drying, the bacteria were uniformly distributed onto a copper mesh and examined under a low‐power optical microscope followed by a transmission electron microscope.

2.4. Virulence Gene Detection of Isolates

Bacterial DNA was isolated and amplified by polymerase chain reaction (PCR) for detection of virulence genes using gene‐specific primers (Table S1; Shanghai Sanyi Biotechnology Co. Ltd., Shanghai, China). Each 50‐µL PCR reaction system included 25 µL of Ex Taq enzyme (5 U/µL), 2 µL of the upstream and downstream primers (each) (10 µm mol/L), 4 µL of DNA as template and 17 µL of sterilized water. The PCR amplification conditions included a predenaturation step at 95°C for 2 min, followed by 35 cycles of denaturation at 95°C for 15 s, annealing at 60°C for 30 s and extension at 72°C for 90 s with a final extension step at 72°C for 10 min. The target bands were detected by 1% agarose gel electrophoresis.

2.5. Sequencing of the 16S rRNA Gene and Phylogenetic Tree Analysis

Using the isolated DNA as a template, 16S rRNA was amplified by PCR using the universal primers 27F (5′‐AGA GTT TGA TCC TGG CTC AG‐3′) and 1492R (5′‐CTA CGG CTA CCT TGT TAC GA‐3′). Each 50‐µL PCR reaction system included 12.5 µL of Ex Taq enzyme (5 U/µL), 1 µL of the upstream and downstream primers (each) (10 µm mol/L), 2 µL of DNA as template and 8.5 µL of sterilized water. The PCR reaction conditions included a predenaturation step at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 52°C for 30 s and extension at 72°C for 2 min, with a final extension step at 72°C for 10 min. The PCR products were sequenced by Shanghai Saiheng Biotechnology Co. Ltd. (Shanghai, China). The sequences were blasted with known nucleic acid sequences in GenBank database Analyze. The phylogenetic tree was constructed with MEGA 5.0 software.

2.6. Histopathological Analysis

The heart, liver, spleen, lung and kidney tissues of the African lion were fixed with 10% formalin, dehydrated with gradient alcohol, cleared with xylene, impregnated with wax, embedded in paraffin and cut into thin slices, which were stained with haematoxylin and eosin and then observed under an optical microscope.

2.7. Drug Susceptibility Test

The sensitivity of isolates to common drugs was determined using the paper AGAR diffusion method referring to the operating standards and determination methods recommended by the American Institute for Clinical and Laboratory Standardization (CLSI).

2.8. Determination of the Median Lethal Dose (LD₅₀) of LM in Mice

The mice were randomly assigned to the experimental group or the control group (six mice each). Mice in the experimental group were intraperitoneally inoculated with 0.1 mL of suspended LM cells (1 × 10⁸), whereas mice in the control group were intraperitoneally inoculated with 0.1 mL of normal saline. The mice were observed every 2 h after inoculation for 12 h. The deaths were recorded, calculated by the Karber method. The hearts, livers, spleens and kidneys of the dead mice were collected aseptically, ground and streaked on blood agar plates.

2.9. Whole Genome Sequencing

A library of different inserted fragments was constructed using the whole genome shotgun method. Next‐generation sequencing was conducted with the NovaSeq 6000 sequencing platform (Illumina Inc., San Diego, CA, USA), and third‐generation single molecule sequencing was performed with a sequencing platform obtained from Oxford Nanopore Technologies (Oxford, England).

Prokka v 1.10 software was used to predict gene elements, including genes, tRNA and rRNA. RepeatMasker and CRT v 1.2 were used for genome repeat and CRISPR prediction analysis. BLAST software was used to compare gene protein sequences with multiple databases to obtain functional annotation information. Virulence factor database (VFDB) and the comprehensive antibiotic research database (CARD) predicted virulence genes and resistance genes present.

3. Results

3.1. Bacterial Isolation and Culture

The African lion analysed in this study exhibited symptoms of depression and decreased appetite before death. Under sterile conditions, the heart, liver and spleen tissues of the lion were cultured on blood agar plates at 37°C for 24 h. Small transparent colonies (diameter, ∼0.5 mm) with smooth surfaces, neat edges and surrounded by very narrow ß‐haemolytic rings were observed by microscopic examination (Figure 1A). The colonies were positive for Gram staining. The bacterial cells were slightly curved, and some were arranged in a ‘V’ shape (Figure 1B). Transmission electron microscopy showed that the bacterial cells were obtuse at both ends (Figure 1C).

FIGURE 1.

Bacterial isolation and culture: (A) colony morphology on a blood agar plate, (B) Gram‐stained bacteria, (C) morphology of individual colonies by transmission electron microscopy.

3.2. Biochemical Test Results

The biochemical test results of the isolated strain were consistent with those of the standard LM strain C54001. The isolated strain was positive for fermentation of glucose and rhamnose, but not xylose and mannitol, hydrolysis of escautin, negative for nitrate reduction and positive for the hippuric acid test (Table 1).

TABLE 1.

Biochemical test results.

Biochemical test	Glucose	Xylose	Rhamnose	Mannitol	Hippuric acid	Nitrate	Esculin	V–P
Results	+	−	+	−	+	−	+	+

Abbreviation: V–P, Voges–Proskauer.

3.3. VITEK2 Identification Results

The results of 43 reaction items on the bacterial identification card with the VITEK2 system are shown in Table 2, which were consistent with the standard strain C54001. The isolate was confirmed as LM.

TABLE 2.

Bacterial identification card reaction result.

Projects	Results	Projects	Results	Projects	Results
AMY	+	NC6.5	+	D‐MNE	+
APPA	−	O129R	+	SAC	−
LeuA	−	D‐XYL	−	BGAL	−
AlaA	−	AspA	−	AMAN	+
D‐RIB	−	BGURr	−	PyrA	−
NOVO	+	D‐SOR	−	POLYB	+
D‐RAF	−	LAC	−	D‐MAL	+
OPTO	+	D‐MAN	−	MBdG	+
PIPLC	+	SAL	+	D‐TRE	+
CDEX	+	ADH1	−	AGLU	+
L‐ProA	−	BGAR	−	PHOS	−
TyrA	+	AGAL	−	BGUR	−
ILATk	−	NAG	+	D‐GAL	−
BACI	+	PUL	−	ADH2s	−
URE	−

3.4. Results of 16S rRNA Identification of Isolates

The isolated strains were amplified by PCR using bacterial 16S rRNA universal primers. BLAST was used to compare the sequenced sequences, and the results showed that the homology with LM was the highest, and the consistency reached more than 99%. The isolated strain was in the same cladism as FDA00006907 (CP022020) (Figure 2).

FIGURE 2.

Phylogenetic tree constructed by neighbour‐joining method based on 16S rRNA nucleotide sequences.

3.5. Virulence Gene Test Results of the Isolates

The PCR amplification products of the virulence genes hlyA, plcA, prfA, iap and actA were consistent with the expected sizes of 456, 1484, 1060, 131 and 839 bp, respectively (Figure 3).

FIGURE 3.

Virulence gene test results of the isolates.

3.6. Drug Susceptibility Test Results

The isolates were sensitive to aminoglycosides, β‐lactams and tetracyclines and resistant or moderately sensitive to quinolones, sulphonamides, macrocyclic lactones, rifamycin and lincomycin (Table 3).

TABLE 3.

Drug susceptibility test results.

Classification	Drugs	Diameter of inhibition zone (mm)	Judgement results	Judgement standard (mm)
				R	I	S
Aminoglycosides	Neomycin	26	S	≤12	13–16	≥17
	Gentamicin	24	S	≤12	13–14	≥15
Quinolones	Norfloxacin	14	I	≤12	13–16	≥17
β‐Lactams	Cefradine	26	S	≤14	15–17	≥18
	Ceftriaxone	30	S	≤13	14–20	≥21
Sulphonamides	Cotrimoxazole	8	R	≤10	11–15	≥16
Macrocyclic lactones	Azithromycin	16	I	≤14	15–16	≥17
Rifamycins	Rifampicin	10	R	≤14	15–16	≥17
Lincomycin class	Clindamycin	8	R	≤14	15–20	≥21
Tetracyclines	Doxycycline	28	S	≤12	13–15	≥16

3.7. Histopathological Observation

The liver was characterized by hepatocyte degeneration and focal necrosis, with cloudy and swollen hepatocytes, mononuclear macrophages scattered among the cords of hepatocytes, and a large number of mononuclear cells and small number of lymphocytes at the sites of focal necrosis (Figure 4A). The pulmonary alveolar wall was thickened with interstitial widening and infiltration by a large number of inflammatory cells. Perivascular focal monocytes and heterotrophic granulocytosis were observed (Figure 4B). Analysis of the spleen showed the lack of white pulp lymph nodules, a significantly reduced number of lymphocytes and monocytosis, along with red pulp congestion, bleeding and a marked decrease in lymphocytes, with scattered monocytes and a small amount of xenophil infiltration (Figure 4C). Renal tubular epithelial cells were enlarged and denatured, some epithelial cell membranes were broken, and a large number of protein particles were observed in the narrowed lumen. Some renal tubular epithelial cells lacked nuclei, and interstitial blood vessels were dilated and congested, with varying amounts of infiltrating monocytes and lymphocytes. Focal bleeding was observed in the medullary area (Figure 4D). These results are clear evidence of pathogenicity.

FIGURE 4.

Histopathological changes to the internal organs of African lions: (A) liver; (B) lungs; (C) spleen and (D) kidney.

3.8. Pathogenicity Test of Isolated Strains in Mice

The mice developed symptoms within 12 h after inoculation, which included loss of appetite, mental lethargy and difficulty breathing. All of the infected mice had died within 24 h. Necropsy of the dead mouse showed pleural effusion, spleen enlargement and cerebral congestion. The same bacteria were isolated from the organs of the dead mice. The LD₅₀ of mice, as determined by the Karber method, was 2.55 × 10⁸ CFU/mL.

Histopathological observations revealed swelling and degeneration of hepatocytes and local formation of necrotic lesions in the liver (Figure 5A), nerve cell degeneration, perivascular oedema and space enlargement in the parenchyma (Figure 5B), myocardial fibre swelling and mild granular degeneration in the heart (Figure 5C), coagulation necrosis of some renal tubules in the kidneys (Figure 5D), lymphocyte fragmentation and necrosis in the spleen (Figure 5E) and congestion and interstitial thickening of blood vessels in the lung (Figure 5F). These results indicate that the bacterium was pathogenic.

FIGURE 5.

Histopathological changes to the internal organs of mice: (A) liver; (B) brain; (C) heart muscle; (D) kidney; (E) spleen and (F) lungs.

3.9. Whole‐Genome Sequencing

For the isolated LM strain SH120021, the whole genome shotgun method was used to construct a library of different inserted fragments, and next‐generation sequencing was conducted. The final assembly consisted of 133 overlapping groups with an N50 length of 12,227 bp, which produced a genome size of approximately 2974,038 bp with a GC content of 37.96%. The predicted number of coding genes was 2959, the total length of all coding genes was 2646,093 bp, and the average length of each coding gene was 894.25 bp, including 67 tRNA and 18 rRNA genes. A circular genome map was generated with CGView software to visualize the annotation results (Figure 6).

FIGURE 6.

Genomic circle of SH120021.

Reference to the Comprehensive Antibiotic Resistance Database identified 14 genes associated with resistance to glycopeptides (vanRB, vanRG), quinolones (gyrA, gyrB, norB, parC), rifampicin (rpoB, rpoC), lincosamides (lmrC, lmrD), streptomycin (strA), macrolides (bcrA), sulphonamides (floP) and cyclo‐lipopeptides (pgsA) (Table 4).

TABLE 4.

Analysis result of drug resistance genes.

Categories of drug resistance genes	Drug resistance genes
Glycopeptides	vanRB, vanRG
Quinolones	gyrA, gyrB, norB, parC
Rifampicin	rpoB, rpoC
Lincosamides	lmrC, lmrD
Streptomycin	strA
Macrolides	bcrA
Sulphonamides	floP
Cyclo‐lipopeptides	pgsA

Reference to the VFDB identified 62 virulence genes of LM strain SH120021. Of these, the four most common types of virulence genes are adherence, invasion, exotoxin and motility, respectively. The classification of virulence genes involving more than 10 genes is shown in the Figure 7.

FIGURE 7.

Classification of virulence genes.

Reference to the Kyoto Encyclopaedia of Genes and Genomes identified 3088 genes enriched in 45 metabolic pathways (Figure 8). Most of the genes were associated with pathways involved in metabolism, genetic information processing and environmental information processing.

FIGURE 8.

Kyoto Encyclopaedia of Genes and Genomes annotations of predicted genes.

4. Discussion

The results of pathological and biochemical analyses, bacterial identification and virulence gene detection confirmed that the cause of death of the African lion was LM infection. This study is the first to describe LM infection of an African lion.

The gram‐positive bacterium LM is a ubiquitous, intracellular pathogen which has been implicated within the past decade as the causative organism in several outbreaks of foodborne disease (Farber and Peterkin 1991). Most cases of LM infection of humans and animals are related to contamination of food and feed, respectively, which jeopardizes food safety and causes huge economic losses to the livestock industry (Ayaz and Cufaoglu 2016). As LM infection can be fatal, early diagnosis and treatment are essential for effective management.

A prior study of 1275 batches of fish products imported from 29 countries found that 36 (2.8%) batches from 8 countries were contaminated with Listeria, with LM accounting for 2.6% (33/1275) (Chen et al. 2010). Consumption of contaminated food has been linked to the spread of the LM. In this study, a strain of Listeria was identified by sequencing of the 16S rRNA gene, which demonstrated homology of >99% with LM strain FDA00006905 isolated in the USA. These results suggest that the death of the African lion may have been caused by consumption of meat products contaminated with Listeria. Histopathological analysis showed varying degrees of damage to the liver, lungs, spleen and kidney of the African lion. Notably, damage to the liver was particularly severe, as demonstrated by local necrosis. Due to the lack of tissues, it was not possible to assess damage to the brain.

LM is an intracellular facultative parasite that infects humans and animals in a process that includes four steps: adhesion, invasion, intracellular proliferation and intercellular migration. Each of these steps involves regulation of virulence genes (Skowron et al. 2018; Siderakou et al. 2022). The virulence genes of LM are mainly distributed on the first and second virulence islands (LIPI‐1 and LIPI‐2, respectively). The virulence genes of LIPI‐1 (hlyA, prfA, mpl, plcA and actA) are mainly involved in intracellular infection (Dramsi et al. 1997; Chen et al. 2022; Kayode and Okoh 2022), whereas those of LIPI‐2 (inlA, inlB, inlC and inlJ) are mainly involved in adhesion and invasion (Vazquez‐Boland et al. 2001; Cossart, Pizarro‐Cerda, and Lecuit 2003; Ragon et al. 2008; Panera‐Martinez et al. 2023). Virulence genes play crucial roles in the pathogenesis of LM. In this study, various virulence genes of LM were detected by PCR, whole genome sequencing and comparative analysis with the VFDB. Regression infection experiments showed that mice infected with LM exhibited typical clinical symptoms, such as pleural effusion, spleen enlargement and brain congestion, which confirmed the strong pathogenicity of LM in mice, suggesting that measures should be strengthened to prevent and control listeriosis.

Drug‐resistant strains of Listeria have recently become more common in livestock, poultry and food for human consumption. For example, Gianluca et al. detected 98 strains of LM isolated from two pig farms and found that 57% of the strains was resistant to clindamycin, and 20%–50% was resistant to ciprofloxacin, oxacillin, levofloxacin and daptomycin. Krzysztof et al. reported that 47.1% of 237 LM isolates from fish plants was sensitive to only erythromycin and sulfamethoxazole using the disk diffusion method, indicating that LM was resistant to a variety of antibiotics. In the present study, LM strain SH120021 was sensitive to aminoglycosides, β‐lactams and tetracyclines but resistant or moderately sensitive to quinolones, sulphonamides, macrocyclic lactones, rifamycin and lincomycin. Reference to the Comprehensive Antibiotic Resistance Database identified 14 genes associated with resistance to sulphonamides and lincomycin, consistent with the drug resistance phenotype of LM strain SH120021, which also harboured the quinolone resistance genes gyrA, gyrB, norB and parC, and the macrolide resistance gene bcrA. However, LM strain SH120021 was moderately sensitive to quinolones and macrocyclic lactones, which was presumably related to the drug concentration used in the drug susceptibility test or external environmental factors that cause resistance genes not to be expressed.

LM is known to infect more than 40 kinds of domestic and wild animals, including reptiles, birds and mammals. Arumugaswamy and Gibson detected LM in 18.6% of faecal samples of animals in the New South Wales Zoo, Australia, whereas Bauwens et al. found that 7.5% of wild animals in zoos was positive for pathogenic LM. The reason for the high percentage of wild animals in zoos infected with LM is usually related to the transmission of foodborne bacteria (Hale et al. 2012; Heiderich et al. 2024). Animal feed, including raw meat, milk and dairy products, seafood and silage, is easily contaminated with LM, demonstrating an important pathway of LM infections of carnivores (van Bree et al. 2018; Silva et al. 2024). Therefore, feed, such as broilers, raw meat and fodder, in addition to the zoo environment, must be disinfected to prevent further infection. In addition, continuous monitoring of drug resistance of LM should be conducted to control the emergence of drug‐resistant strains.

5. Conclusions

This study describes for the first time the molecular evidence of LM infection in African lions. Through various methodologies of classical and molecular microbiology, pathological analyses and in vivo experimentation, as well as whole genome sequencing, it was possible to identify LM as the cause of disease in an African lion. This implies that LM is also a pathogenic microorganism in this feline. It further enriched the related research of LM and also provided a reference for the prevention and treatment of LM.

Author Contributions

P.X. and X.Q. conceived and designed the study. X.W., F.X., and H.Z. executed the experiment and finalized the data. L.S. and Y.Z. helped in sampling. S.H. and J.W. assisted in experimentation. P.X. wrote the manuscript. All authors read and approved the final manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

Peer Review

The peer review history for this article is available at https://publons.com/publon/10.1002/vms3.70110.

Supporting information

Supporting Information

Data Availability Statement

Data will be made available on request.