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Co-infections of Klebsiella pneumoniae and Elizabethkingia miricola in black-spotted frogs (Pelophylax nigromaculatus).
Li S
,
Wang X
,
Lu Y
,
Wang J
,
Yu D
,
Zhou Z
,
Wei J
,
Liu L
,
Liu J
,
Liu F
,
Su Y
,
Xu H
.
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Pelophylax nigromaculatus is a common commercial specie of frogs that generally cultured throughout China. With the application of high-density culture, P. nigromaculatus can be co-infected by two or more pathogens, which thereby induce synergistic influence on the virulence of the infection. In this study, two bacterial strains were simultaneously isolated from diseased frogs by incubating on Luria-Bertani (LB) agar. Isolates were identified as Klebsiella pneumoniae and Elizabethkingia miricola by morphological, physiological and biochemical features, as well as 16S rRNA sequencing and phylogenetic analysis. The whole genome of K. pneumoniae and E. miricola isolates consist single circular chromosome of 5,419,557 bp and 4,215,349 bp, respectively. The genomic sequence analysis further indicated that K. pneumoniae isolate conserved 172 virulent and 349 antibiotic-resistance genes, whereas E. miricola contained 24 virulent and 168 antibiotic resistance genes. In LB broth, both isolates could grow well at 0%-1% NaCl concentration and pH 5-7. Antibiotic susceptibility testing revealed that both K. pneumoniae and E. miricola were resistant to kanamycin, neomycin, ampicillin, piperacillin, carbenicillin, enrofloxacin, norfloxacin and sulfisoxazole. Histopathological studies showed that co-infection caused considerable lesions in the tissues of brain, eye, muscle, spleen, kidney and liver, including cell degeneration, necrosis, hemorrhage and inflammatory cell infiltration. The LD50 of K. pneumoniae and E. miricola isolates were 6.31 × 105 CFU/g and 3.98 × 105 CFU/g frog weight, respectively. Moreover, experimentally infected frogs exhibited quick and higher mortality under coinfection with K. pneumoniae and E. miricola than those single challenge of each bacterium. To date, no natural co-infection by these two bacteria has been reported from frogs and even amphibians. The results will not only shed light on the feature and pathogenesis of K. pneumoniae and E. miricola, but also highlight that co-infection of these two pathogen is a potential threat to black-spotted frog farming.
Fig. 1. Clinical signs in Pelophylax nigromaculatus naturally infected with Klebsiella pneumoniae and Elizabethkingia miricola. A. Head biased to one side (green arrow). B. Unilateral blindness/opacity of the eye (red arrow). C. Abdomen swelling (red arrow), ulcer on frog mouth skin (green arrow). D. Swollen of liver (green arrow) and pneumorrhagia (red arrow). E. Redness on frog thigh (red arrow). F. Swollen on frog thigh (green arrow). G. Double staining technique with alcian blue and alizarin red showing skeletal deformities (green arrow).
Fig. 2. The morphological characteristics and identification of isolated Klebsiella pneumoniae and Elizabethkingia miricola. A. Phylogenetic tree of isolated K. pneumoniae and E. miricola as determined by 16S rRNA gene sequences. The evolutionary history was inferred using the Neighbor-Joining method by the MEGA X software. Bootstrap values out of 1000 repetitions were indicated above each branch. The isolate 2022KP07 and 2022EM07 identified in this study were indicated by the shaded square and triangle, respectively. B. Bacteria K. pneumoniae colonies on LB agar (left) and 5% sheep blood agar (right) after incubation at 28 °C for 18 h. C. K. pneumoniae was Gram-negative, short, plump and straight rods. D. Bacteria E. miricola colonies on LB agar (left) and 5% sheep blood agar (right) after incubation at 28 °C for 18 h. E. E. miricola was Gram-negative and indicated in red, straight rod-shaped, and non-endospore. Scale bars = 20 μm.
Fig. 3. The analysis of whole genome of Klebsiella pneumoniae 2022KP07 and Elizabethkingia miricola 2022EM07. A and B. Circular complete genome map of isolate 2022KP07 and 2022EM07. From outside to inside, the first and fourth circles showed forward and reverse CDSs with different colors representing different COG functional classifications (right-top of map). The second and third circles showed the CDS, tRNA and rRNA genes on positive and negative strands, respectively. The fifth and sixth circles showed the GC content plot and the GC-skew. COG, Clusters of orthologous groups; CDS, coding sequence; tRNA, transfer RNA; rRNA, ribosomal RNA; GC, guanine-cytosine. C and D. Statistics of functional classification of Kyoto Encyclopedia of Genes and Genomes (KEGG) protein of 2022KP07 and 2022EM07.
Fig. 4. Growing characteristics of Klebsiella pneumoniae 2022KP07 and Elizabethkingia miricola 2022EM07 strains at different pH and NaCl concentration. A. Growth of K. pneumoniae 2022KP07 at different pH. B. Growth of K. pneumoniae 2022KP07 at different NaCl concentration. C. Growth of E. miricola 2022EM07 at different pH. D. Growth of E. miricola 2022EM07 at different NaCl concentration.
Fig. 5. Histopathologic lesions in black-spotted frogs naturally co-infected with the Klebsiella pneumoniae and Elizabethkingia miricola. A, B. Brain: hemorrhage (black arrow), hyaline degeneration (yellow arrow) and necrosis (green arrow). C. Eye: necrotic neurons (green arrow) and neuronophagia (red arrow). D. Lung: hyperemia of the pulmonary interstitium (black arrow). E, F. Spleen: hyaline degeneration (green arrow). G. Liver: hemorrhage (black arrow), hyaline degeneration (yellow arrow), necrosis (green arrow), congestion and inflammatory cells infiltration (blue arrow). H, I. Kidney: hyaline degeneration (yellow arrow), cell necrosis (green arrow), congestion and inflammatory cells infiltration (blue arrow). J. Muscle: hemorrhage (black arrow) and lymphocyte infiltration (blue arrow). K. Heart: hemorrhage (black arrow) and inflammatory cell infiltration (blue arrow). L. Intestine: necrosis (green arrow) and inflammatory cells infiltration (blue arrow). Scale bars, = 100 μm.
Fig. 7. Prediction and classification of antibiotic resistance genes of Klebsiella pneumoniae 2022KP07 (A) and Elizabethkingia miricola 2022EM07 (B) isolated from black-spotted frogs. Note: Different colors represented different drug class classifications, and their areas indicated the relative proportion of genes in the classification.
Fig. 8. Cumulative mortalities of black-spotted frogs following intramuscular challenge experiments with Klebsiella pneumoniae, Elizabethkingia miricola and combination of K. pneumoniae and E. miricola. A. The mortalities of frogs infected with K. pneumonia. B. The mortalities of frogs infected with E. miricola. C. The mortalities of frogs co-infected with K. pneumoniae and E. miricola.
