The cabABC Operon Essential for Biofilm and Rugose Colony Development in Vibrio vulnificus.

A transcriptome analysis identified Vibrio vulnificus cabABC genes which were preferentially expressed in biofilms. The cabABC genes were transcribed as a single operon. The cabA gene was induced by elevated 3',5'-cyclic diguanylic acid (c-di-GMP) and encoded a calcium-binding protein CabA. Comparison of the biofilms produced by the cabA mutant and its parent strain JN111 in microtiter plates using crystal-violet staining demonstrated that CabA contributed to biofilm formation in a calcium-dependent manner under elevated c-di-GMP conditions. Genetic and biochemical analyses revealed that CabA was secreted to the cell exterior through functional CabB and CabC, distributed throughout the biofilm matrix, and produced as the biofilm matured. These results, together with the observation that CabA also contributes to the development of rugose colony morphology, indicated that CabA is a matrix-associated protein required for maturation, rather than adhesion involved in the initial attachment, of biofilms. Microscopic comparison of the structure of biofilms produced by JN111 and the cabA mutant demonstrated that CabA is an extracellular matrix component essential for the development of the mature biofilm structures in flow cells and on oyster shells. Exogenously providing purified CabA restored the biofilm- and rugose colony-forming abilities of the cabA mutant when calcium was available. Circular dichroism and size exclusion analyses revealed that calcium binding induces CabA conformational changes which may lead to multimerization. Extracellular complementation experiments revealed that CabA can assemble a functional matrix only when exopolysaccharides coexist. Consequently, the combined results suggested that CabA is a structural protein of the extracellular matrix and multimerizes to a conformation functional in building robust biofilms, which may render V. vulnificus to survive in hostile environments and reach a concentrated infective dose.

Correction: The cabABC Operon Essential for Biofilm and Rugose Colony Development in Vibrio vulnificus.

[This corrects the article DOI: 10.1371/journal.ppat.1005192.].

Enrichment Broth for the Detection of Campylobacter jejuni and Campylobacter coli in Fresh Produce and Poultry.

Although campylobacteriosis caused by Campylobacter jejuni and Campylobacter coli has been increasingly reported worldwide owing to the consumption of contaminated poultry and fresh produce, the current detection protocols are not selective enough to inhibit unspecific microbes other than these pathogens. Five antibiotics were separately added to Bolton broth, and the survival rates of 18 Campylobacter spp. and 79 non-Campylobacter spp. were evaluated. The survival rate of the non-Campylobacter spp. was the lowest in Bolton broth with rifampin (6.3%), followed by cefsulodin (12.7%), novobiocin (16.5%), and potassium tellurite and sulfamethozaxole (both 17.7%). Also the most effective concentration of rifampin was found to be 12.5 mg/L, which markedly inhibited non-Campylobacter strains while not affecting the survival of Campylobacter strains. After the Campylobacter spp. were enriched in Bolton broth supplemented with 12.5 mg/L rifampin (R-Bolton broth), CampyFood Agar (CFA) was found to be better in selectively isolating the pathogens in the enrichment broth than the International Organization for Standardization method of using modified charcoal cefoperazone deoxycholate agar (mCCDA) for this step. When applied to natural food samples-here, romaine lettuce, pepper, cherry tomato, Korean leek, and chicken-the R-Bolton broth-CFA combination decreased the number of false-positive results by 50.0, 4.2, 20.8, 50.0, and 94.4%, respectively, compared with the International Organization for Standardization method (Bolton broth-mCCDA combination). These results demonstrate that the combination of R-Bolton broth and CFA is more efficient in detecting C. jejuni and C. coli in poultry and fresh produce and thus should replace the Bolton broth-mCCDA combination.

Role of extracellular matrix protein CabA in resistance of Vibrio vulnificus biofilms to decontamination strategies.

Biofilms are recalcitrant and raise safety problems in the food industry. In this study, the role of CabA, an extracellular matrix protein, in the resistance of the biofilms of Vibrio vulnificus, a foodborne pathogen, to decontamination strategies was investigated. Biofilms of the cabA mutant revealed reduced resistance to detachment by vibration and disinfection by sodium hypochlorite compared to the biofilms of the parental wild type in vitro. The reduced resistance of the cabA mutant biofilms was complemented by introducing a recombinant cabA, indicating that the reduced resistance of the cabA mutant biofilms is caused by the inactivation of cabA. The expression of cabA was induced in cells bound to oyster, the primary vehicle of the pathogen. The cabA mutant biofilms on oyster are defective in biomass and resistance to detachment and disinfection. The bacterial cells in the wild-type biofilms are clustered by filaments which are not apparent in the cabA mutant biofilms. The combined results indicated that CabA contributes to the structural integrity of V. vulnificus biofilms possibly by forming filaments in the matrix and thus rendering the biofilms robust, suggesting that CabA could be a target to control V. vulnificus biofilms on oyster.