Qingpi Yan

Portal of entry for pathogenic Vibrio alginolyticus into large yellow croaker Pseudosciaena crocea, and characteristics of bacterial adhesion to mucus

The portal of entry for pathogenic Vibrio alginolyticus into large yellow croaker Pseudosciaena crocea is via the intestinal tract rather than gill or skin according to the kinetics of the bacterial adhesion to different mucus. The different effects on adhesion caused by proteolytic enzymes and heat treatment might be due to the different chemical compositions of mucus. Adhesion of V. alginolyticus to mucus depends on concerted action of bacterial surface structures such as cell-surface proteins, somatic antigens, flagella, etc. In addition, starvation and monosaccharides, especially fructose, inhibit the bacterial adhesion to the mucus. Knowledge of these adhesive characteristics should be very useful for designing more efficacious prophylactic strategies.

Isolation and characterization of a hepcidin peptide from the head kidney of large yellow croaker, Pseudosciaena crocea

Large yellow croaker (Pseudosciaena crocea) is one of the most important marine cultured fish in China. Acidic extracts of five tissues of large yellow croaker showed strong anti-Vibrio alginolyticus activity. Acidic extract of head kidney tissue was subjected to heat-treatment in boiling water, and solid-phase extraction on Sep-Pak C(18) cartridge. It was found that the antibacterial substances were heat stable, and 20% acetonitrile effluent exhibited strong antibacterial activity. Active extract was further applied to Sephadex G-25 gel permeation chromatography and StableBond C(18) RP-HPLC. An antibacterial peptide with a single peak was obtained. The results of amino acid sequencing and MALDI-TOF MS suggested that the peptide was RCRFCCRCCPRMRGCGICCRF with an observed molecular mass of 2523.2 Da. BLAST searching suggested that the purified antibacterial peptide was the mature peptide section of the hepcidin preproprotein presumed from cDNA of large yellow croaker, thus designated hepcidin-Pl. Hepcidin-P1 exhibited strong antibacterial activity against four marine vibrios.

Flagellar motility contributes to the invasion and survival of Aeromonas hydrophila in Anguilla japonica macrophages.

The interaction between pathogenic bacteria and the host phagocytes is complicated. It is generally believed that only obligate intracellular pathogens can invade and survive in host phagocytes. In this study, we revealed that the pathogenic Aeromonas hydrophila B11 can also invade and survive in the macrophages of its host Anguilla japonica in vitro. To further investigate the mechanisms of A. hydrophila invasion and survival in host macrophages, a mini-Tn10 transposon mutagenesis system was used to generate an insertion mutant library by cell conjugation between the donor Escherichia coli Sm10 (pLOFKm) and the recipient A. hydrophila B11. Out of 465 individual colonies, 13 mutants impaired in survival within macrophages were selected, and the mutant BM116 was the most seriously impaired strain. Molecular analysis showed that an ORF of approximately 1335 bp (GenBank accession numbers JQ974982) of the mutant BM116 was inserted by mini-Tn10. This ORF putatively encodes a deduced 445 amino acids protein that displays the highest identity (99.6%) with the flagellar hook protein FlgE of A. hydrophila subsp. hydrophila ATCC 7966. The biological characteristics of the wild-type B11, the mutant B116 and the complemented strain were investigated. The results reveal that the flagella of the mutant BM116 was absent and that these mutant bacteria exhibited defective motility, adhesion, and invasion and survival in host macrophages when compared with the wild type and the complemented strain. These findings indicate that flgE is required for flagellum biogenesis in A. hydrophila and that flagellar motility is required for A. hydrophila invasion and survival in the macrophages of its host. Our findings provide an important new understanding of the nonintracellular pathogenic bacteria invasion and survival in host phagocytes and the interactions between the pathogens and their host.

Identification and characterization of three Vibrio alginolyticus non-coding RNAs involved in adhesion, chemotaxis, and motility processes

The capability of Vibrio alginolyticus to adhere to fish mucus is a key virulence factor of the bacteria. Our previous research showed that stress conditions, such as Cu2+, Pb2+, Hg2+, and low pH, can reduce this adhesion ability. Non-coding (nc) RNAs play a crucial role in regulating bacterial gene expression, affecting the bacterias pathogenicity. To investigate the mechanism(s) underlying the decline in adhesion ability caused by stressors, we combined high-throughput sequencing with computational techniques to detect stressed ncRNA dynamics. These approaches yielded three commonly altered ncRNAs that are predicted to regulate the bacterial chemotaxis pathway, which plays a key role in the adhesion process of bacteria. We hypothesized they play a key role in the adhesion process of V. alginolyticus. In this study, we validated the effects of these three ncRNAs on their predicted target genes and their role in the V. alginolyticus adhesion process with RNA interference (i), quantitative real-time polymerase chain reaction (qPCR), northern blot, capillary assay, and in vitro adhesion assays. The expression of these ncRNAs and their predicted target genes were confirmed by qPCR and northern blot, which reinforced the reliability of the sequencing data and the target prediction. Overexpression of these ncRNAs was capable of reducing the chemotactic and adhesion ability of V. alginolyticus, and the expression levels of their target genes were also significantly reduced. Our results indicated that these three ncRNAs: (1) are able to regulate the bacterial chemotaxis pathway, and (2) play a key role in the adhesion process of V. alginolyticus.

Incidence of antimicrobial-resistance genes and integrons in antibiotic-resistant bacteria isolated from eels and aquaculture ponds

The overuse of antimicrobials in aquaculture has promoted the selection of antimicrobial-resistant bacteria. Here we investigated the abundance of antimicrobial-resistance genes and integrons in 108 strains of antibiotic-resistant bacteria isolated from eels and aquaculture ponds in China. Conventional PCR was implemented to examine common antibiotic-resistance genes, integrons, and their gene cassette arrays. The results showed that the antibiotic-resistance genes blaTEM, tetC, sulI, aadA, floR, and qnrB were detected at high percentages, as were a number of other resistance genes. Class I integrons were present in 79.63% of the strains, and 10 out of 108 isolates carried class II integrons. Class III integrons were not detected. Three strains carried both class I and class II integrons, and 73.26% of the class I integron-positive isolates contained the qacEΔ1/sul1 gene. Fourteen types of integron cassette arrays were found among class I integron-positive isolates. A new array, dfrB4-catB3-blaOXA-10-aadA1, was discovered in this study. The gene cassette array dfrA12-orfF-aadA2 was the most widely distributed. In summary, 23 different gene cassettes encoding resistance to 8 classes of antibiotics were identified in the class I integrons, and the main cassettes contained genes encoding resistance to aminoglycosides (aad) and trimethoprim (dfr). All class II integron-positive strains had only a single gene cassette array, viz. dfrA1-catB2-sat2-aadA1. High levels of antimicrobial-resistance genes and integrons in eels and auqauculture ponds suggest that the overuse of antimicrobials should be strictly controlled and that the levels of bacterial antimicrobial-resistance genes in aquaculture should be monitored.

Disruption of chemotaxis-related genes affects multiple cellular processes and the virulence of pathogenic Vibrio harveyi

Chemotactic motility is involved in the virulence of many pathogenic bacteria. In order to understand the role of chemotactic motility of Vibrio harveyi in cellular processes and virulence, mini-Tn10/Kan transposon-induced mutants with deficient chemotactic motility were constructed, screened, and identified. Sequence analysis revealed that the 465-bp fragment (Gen Bank accession number HM630274) flanking the transposon insertion site in mutant TS-CM1 had the highest identity (96.9%) with a hypothetical protein gene of V. harveyi ATCC BAA-1116 and the second-highest identity (91.8%) with the pgk gene of V. parahaemolyticus RIMD 2210633. In another mutant, TS-CM2, 356 bp of transposon-flanking sequence (Gen Bank accession number HM630275) also showed the highest identity (94.6%) with a hypothetical protein gene of V. harveyi ATCC BAA-1116 and the second-highest identity (92.4%) with the flaB gene of V. alginolyticus HY9901. Studies on virulence-related biological characteristics such as growth, motility, adhesion, and infectivity of themutants showed that disruption of either the flagellin gene or energy metabolism gene led to subsequent loss of chemotactic motility and changes in growth, motility, adhesion, and virulence of the pathogenic V. harveyi. Hence, the flagellin gene and crucial energy metabolism gene played an important role in the chemotactic motility of V. harveyi.

Purification and characterization of an adhesion protein from the skin mucus of Paralichthys olivaceus

Higher adhesion to mucus and subsequent growth is associated with the pathogenicity of Vibrio. Vibrio fluvialis adherence to and growth in skin mucus of Paralichthys olivaceus were examined, and a adhesion-specific protein was purified from the skin mucus of P. olivaceus. The results indicate that V. fluvialis adhered readily to and grew efficiently in skin mucus of P. olivaceus. By Sephadex G-100 column, fractionation of skin mucus resolved 2 major peaks and 1 small peak, and the fractions with higher adhesion of V. fluvialis were focused on the first peak, 5 fractions especially. By the high performance liquid chromatography (HPLC), the fractions 5 of Sephadex G-100 resolved into 6 major peaks at OD280, and V. fluvialis adhered strongly to fractions of peak 1, 3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the fractions 5 of skin mucus on Sephadex G-100 columns yielded two bands, the fractions of peak 3 on HPLC yielded one band with a molecular mass of 66.2 kDa. The results suggest that pathogenic V. fluvialis can adhere to and grow in P. olivaceus skin mucus, and the V. fluvialis -specific receptor present in P. olivaceus skin mucus is a 66.2 kDa glycoprotein.