Delineating virulence of Vibrio campbellii: a predominant luminescent bacterial pathogen in Indian shrimp hatcheries

Abstract

Luminescent vibriosis is a major bacterial disease in shrimp hatcheries and causes up to 100% mortality in larval stages of penaeid shrimps. We investigated the virulence factors and genetic identity of 29 luminescent Vibrio isolates from Indian shrimp hatcheries and farms, which were earlier presumed as Vibrio harveyi. Haemolysin gene-based species-specific multiplex PCR and phylogenetic analysis of rpoD and toxR identified all the isolates as V. campbellii. The gene-specific PCR revealed the presence of virulence markers involved in quorum sensing (luxM, luxS, cqsA), motility (flaA, lafA), toxin (hly, chiA, serine protease, metalloprotease), and virulence regulators (toxR, luxR) in all the isolates. The deduced amino acid sequence analysis of virulence regulator ToxR suggested four variants, namely A123Q150 (AQ; 18.9%), P123Q150 (PQ; 54.1%), A123P150 (AP; 21.6%), and P123P150 (PP; 5.4% isolates) based on amino acid at 123rd (proline or alanine) and 150th (glutamine or proline) positions. A significantly higher level of the quorum-sensing signal, autoinducer-2 (AI-2, p = 2.2e-12), and significantly reduced protease activity (p = 1.6e-07) were recorded in AP variant, whereas an inverse trend was noticed in the Q150 variants AQ and PQ. The pathogenicity study in Penaeus (Litopenaeus) vannamei juveniles revealed that all the isolates of AQ were highly pathogenic with Cox proportional hazard ratio 15.1 to 32.4 compared to P150 variants; PP (5.4 to 6.3) or AP (7.3 to 14). The correlation matrix suggested that protease, a metalloprotease, was positively correlated with pathogenicity (p > 0.05) and negatively correlated (p < 0.05) with AI-2 and AI-1. The syntenic organization of toxS-toxR-htpG operon in V. campbellii was found to be similar to pathogenic V. cholerae suggesting a similar regulatory role. The present study emphasizes that V. campbellii is a predominant pathogen in Indian shrimp hatcheries, and ToxR plays a significant role as a virulence regulator in the quorum sensing-protease pathway. Further, the study suggests that the presence of glutamine at 150th position (Q150) in ToxR is crucial for the pathogenicity of V. campbellii.

Figure 1

Maximum likelihood phylogenetic tree of luminescent bacterial isolates and other species under Harveyi clade was constructed using (A) 16S rRNA (B) rpoD (C) toxR and (D) concatenated sequence of 16S rRNA, rpoD and toxR. The tree was constructed at 1000 bootstraps replication and the length of each branch is proportional to the estimated number of substitutions per position. The tree is rooted using V. cholerae as outgroup. The 29 isolates of present study are represented by LB1 to LB516.

Figure 2

Amino acid sequence alignment of the variable region of ToxR of V. campbellii.

Figure 3

Syntenic gene organization of toxR gene loci in V. campbellii, V. cholerae and V. parahaemolyticus. HTPG: High Temperature Protein G (heat shock protein G); ADK: Adenylate kinase; FC: Ferrochelatase; DTPA: Dipeptide/tripeptide permease; RFAH: Transcription antitermination protein.

Figure 4

Distribution of virulence markers in Vibrio campbellii isolates based on gene-specific PCR. The phylogenetic tree was constructed based on toxR gene using maximum likelihood algorithm. The presence and absence of virulence markers was depicted by applying an additional panel using iTOL v4 (Interactive Tree of Life). The blue, red, green, and black shaded mark represents presence, while the white square box represents the absence of the gene.

Figure 5

Box plot analysis of virulence markers and pathogenicity in Vibrio campbellii isolates among the variants of ToxR. (A) Luminescence, (B) Autoinducer 1 (AI-1), (C) Autoinducer 2 (AI-2), (D) Protease, (E) Biofilm, (F) Chitinase, (G) Motility (H) Mortality. One way ANOVA followed by Tukey post hoc comparison was carried out using rstatix package. The graphics were prepared using ggpubr package in R4.0.2. The AQ (A123Q150), PQ (P123Q150), AP (A123P150) and PP (P123P150) represents four variants of ToxR based upon amino acid at 123rd and 150th position.

Figure 6

Principal component and cluster analysis of virulence factors and pathogenicity. (A) Principal component analysis. Dim1, First principal component; Dim2: second principal component; Lum: Luminescence; AI-1: Autoinducer-1; AI-2: autoinducer-2. (B) Scree plot showing the first three dimension for most of the cumulative variability in data represented by the eigenvalues (C) Hierarchical clustering analysis with the heatmap. The red to blue transition indicates a higher to a lower value.

Figure 7

Correlation matrix (Pearson correlation coefficients) among virulence factors and pathogenicity. The highlighted block indicates a significant relationship (p < 0.05). Blue shade represents a significant positive correlation, while red indicates a significant negative correlation.

Figure 8

Pathogenicity of Vibrio campbellii in Penaeus vannamei juveniles. (A) Kaplan–Meier survival estimates of four variants of ToxR. (B) Relative pathogenicity of V. campbellii isolates against reference strain V. campbellii BAA-1116. The result was analysed by Cox proportional hazard analysis. The significance level is indicated at * (p < 0.05), ** (0.01) and *** (p < 0.001). The isolates within a ToxR variant is represented with a similar pattern. The AQ (A123Q150), PQ (P123Q150), AP (A123P150) and PP (P123P150) represents four variants of ToxR based upon amino acid at 123rd and 150th position.