RESUMEN
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.
Asunto(s)
Proteínas Bacterianas/genética , Diarrea/microbiología , Enfermedades de los Peces/microbiología , Gastroenteritis/microbiología , Neisseriaceae/clasificación , Análisis de Secuencia de ADN , Animales , Técnicas de Tipificación Bacteriana , Peces/microbiología , Agua Dulce , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Neisseriaceae/genética , Neisseriaceae/aislamiento & purificación , Filogenia , Análisis de Secuencia de ADN/métodos , ViajeRESUMEN
We report the complete nucleotide sequence and characterization of a cryptic plasmid, pHLHK26, recovered from a strain of Laribacter hongkongensis isolated from a patient with community acquired gastroenteritis. pHLHK26 consists of 8700 bp, with G + C content 51.3%. The copy number (mean +/- SD) is 0.57 +/- 0.07 and it is stable after four passages (about 240 generations) in the absence of selection. There is a predicted origin of replication that consists of a DnaA box and five 22-bp direct repeats. pHLHK26 has four ORFs with two genes encoded in the sense direction and the other two in antisense direction. These four ORFs encode a putative plasmid partitioning protein of the ParA family, a putative protein that contains putative ADP-ribose 1"-phosphatase activity belonging to the Appr-1-p processing enzyme family, a putative recombinase (TniR) of the resolvase/invertase family, and a putative replication protein, respectively. We speculate that pHLHK26 is a theta, possibly Class A, replicative plasmid, as it contains an origin of replication with AT-rich region, a number of iterons and a DnaA box and a gene that encodes a replicative protein most homologous to those of other theta replicative plasmids and it shares eight of the nine positions of the consensus sequence TTAT(C/A)CA(C/A)A (TTTTCCACA in pHLHK26) in the DnaA boxes observed in other classical examples of Class A plasmids of this group.
Asunto(s)
Neisseriaceae/genética , Plásmidos/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Composición de Base , Secuencia de Bases , Sitios de Unión/genética , Infecciones Comunitarias Adquiridas/microbiología , Secuencia de Consenso , ADN Helicasas/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Gastroenteritis/microbiología , Humanos , Datos de Secuencia Molecular , Neisseriaceae/aislamiento & purificación , Sistemas de Lectura Abierta , Monoéster Fosfórico Hidrolasas/genética , Filogenia , Recombinasas/genética , Origen de Réplica/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Transactivadores/genéticaRESUMEN
An Escherichia coli-Laribacter hongkongensis shuttle vector (pPW380) was constructed by ligating the 4701-bp EcoRI digested fragment of pHLHK8 to EcoRI digested pBK-CMV. An E. coli-L. hongkongensis inducible expression shuttle vector was further constructed by ligating a 2105-bp fragment that contains the tetracycline repressor and tetracycline-inducible promoter region of pALC2084 to the 8897-bp fragment of pPW380, deletion of the green fluorescent protein gene, and insertion of a multiple cloning site. This inducible expression system was able to express two commonly used reporter genes, the green fluorescent protein gene and the glutathione S-transferase gene, efficiently in E. coli and L. hongkongensis.
Asunto(s)
Escherichia coli/genética , Gastroenteritis/microbiología , Regulación Bacteriana de la Expresión Génica , Vectores Genéticos , Neisseriaceae/genética , Secuencia de Bases , Escherichia coli/metabolismo , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Datos de Secuencia Molecular , Neisseriaceae/aislamiento & purificación , Neisseriaceae/metabolismo , Infecciones por Neisseriaceae/microbiología , Plásmidos , Análisis de Secuencia de ADN , Transformación GenéticaRESUMEN
Among 21 human strains of Laribacter hongkongensis, small plasmids were observed in four strains, and large ones in six strains. The smallest, 3264-bp plasmid, pHLHK19, has only one ORF that encodes a putative replication initiator protein and a predicted origin of replication (ori) with a DnaA box, three 18-bp direct repeats and five pairs of inverted repeats. An Escherichia coli-L. hongkongensis shuttle vector was constructed by ligating the HindIII-digested pHLHK19, containing the replication initiator protein and ori of pHLHK19, to HindIII-digested pBK-CMV. This shuttle vector can propagate in E. coli and L. hongkongensis with good transformation efficiencies.
Asunto(s)
Vectores Genéticos/genética , Neisseriaceae/genética , Plásmidos/genética , Secuencia de Bases , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Humanos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Origen de Réplica/genética , Transactivadores/genética , Transformación BacterianaRESUMEN
In a territory-wide surveillance study, Laribacter hongkongensis was isolated solely from freshwater fish (60% of grass carps, 53% of bighead carps, and 25% of mud carps). Comparing the pulsed-field gel electrophoresis patterns of fish and patient isolates revealed that most patient isolates were clustered together, suggesting that some clones could be more virulent.