Development of a multi-locus sequence typing scheme for Laribacter hongkongensis, a novel bacterium associated with freshwater fish-borne gastroenteritis and traveler's diarrhea

Abstract

Background: Laribacter hongkongensis is a newly discovered, facultative anaerobic, Gram-negative, motile, sea gull-shaped rod associated with freshwater fish borne gastroenteritis and traveler's diarrhea. A highly reproducible and discriminative typing system is essential for better understanding of the epidemiology of L. hongkongensis. In this study, a multilocus sequence typing (MLST) system was developed for L. hongkongensis. The system was used to characterize 146 L. hongkongensis isolates, including 39 from humans and 107 from fish.

Results: Fragments (362 to 504 bp) of seven housekeeping genes were amplified and sequenced. Among the 3068 bp of the seven loci, 332 polymorphic sites were observed. The median number of alleles at each locus was 34 [range 22 (ilvC) to 45 (thiC)]. All seven genes showed very low d(n)/d(s) ratios of < 0.04, indicating that no strong positive selective pressure is present. A total of 97 different sequence types (STs) were assigned to the 146 isolates, with 80 STs identified only once. The overall discriminatory power was 0.9861. eBURST grouped the isolates into 12 lineages, with six groups containing only isolates from fish and three groups only isolates from humans. Standardized index of association (I(S)(A)) measurement showed significant linkage disequilibrium in isolates from both humans and fish. The I(S)(A) for the isolates from humans and fish were 0.270 and 0.636, indicating the isolates from fish were more clonal than the isolates from humans. Only one interconnected network (acnB) was detected in the split graphs. The P-value (P = 0) of sum of the squares of condensed fragments in Sawyer's test showed evidence of intragenic recombination in the rho, acnB and thiC loci, but the P-value (P = 1) of maximum condensed fragment in these gene loci did not show evidence of intragenic recombination. Congruence analysis showed that all the pairwise comparisons of the 7 MLST loci were incongruent, indicating that recombination played a substantial role in the evolution of L. hongkongensis. A website for L. hongkongensis MLST was set up and can be accessed at http://mlstdb.hku.hk:14206/MLST_index.html.

Conclusion: A highly reproducible and discriminative MLST system was developed for L. hongkongensis.

Figure 1

Phylogenetic tree showing the relationships of the 97 STs of L. hongkongensis in this study. The genetic relatedness among the 97 STs was assessed by ClonalFrame algorithm based on the pair-wise differences in the allelic profiles of the seven housekeeping genes. Numbers immediately to the right of the dendrogram show the eBURST clonal clusters to which the STs belong.

Figure 2

BURST analysis of L. hongkongensis isolates in this study. Each number represents a MLST sequence type (ST) and each line connects STs that differ in only one of the seven housekeeping genes. Boxed numbers represent STs found in both human and fish, shaded numbers represent STs found only in human, and un-boxed and un-shaded numbers represent STs found only in fish. Hollow circles and squares represent predicted group and subgroup founders respectively. The sizes of the circles and squares are proportional to the number of isolates within each ST.

Figure 3

Split decomposition analysis of MLST data of L. hongkongensis isolates in this study. Split decomposition network was constructed using the individual (rho, acnB, ftsH, trpE, ilvC, thiC and eno) gene sequences. The scale bar represents the number of substitutions per site.