GcpA (stm1987) is critical for cellulose production and biofilm formation on polystyrene surface by Salmonella enterica serovar Weltevreden in both high and low nutrient medium

Abstract

Biofilm formation by Salmonella is a serious concern in the food-processing industry and the persistence of the organism in biofilms becomes a constant source of contamination. Since there is zero tolerance for Salmonella in foods, it is important to understand the mechanism of biofilm formation and to prevent the formation. Therefore, this study aimed at investigating the biofilm-forming ability of seafood isolates of Salmonella enterica serovar Weltevreden (S. Weltevreden) under two different nutrient conditions (normal strength trypticase soy broth (TSB) and 1:100 diluted TSB). The role of cellulose production in biofilm formation and in the expression of multicellular behavior (rough, dark, red morphotype: rdar) was investigated. Fourteen isolates of seafood associated S. Weltevreden were studied for biofilm production in polystyrene microtitre plates. Only one (SW49) of 14 was a strong biofilm former on polystyrene template and was able to produce biofilm in both undiluted TSB and 1:100 diluted TSB at 24h. All others produced moderate or weak biofilms which was higher in 1:100 diluted TSB compared to undiluted medium. All the isolates except one were positive by PCR for the three genes, gcpA (stm1987), adrA (yaiC) and csgD. Gene expression of gcpA, adrA and csgD was studied by real-time PCR with the one strong (SW49) and one representative weak (SW30) biofilm former. In SW49 at 24h of incubation, the expression of gcpA from biofilm cells was 33 and 36 times higher than from planktonic cells grown in TSB and diluted TSB respectively and at 72h the expression from biofilm cells was 57 and 61 times higher than that from planktonic cells. Quantification of gene expression did not reveal any significant difference in the expression of csgD and adrA gene. Deletion of gcpA in SW49 resulted in its inability to produce cellulose and consequent inability to bind calcoflour, inability to form rdar colony on Congo Red-agar plates and failure to produce biofilm on polystyrene substrate. The data indicated that, in case of S. Weltevreden, gcpA is critical for activating cellulose synthesis and biofilm formation both in undiluted and diluted TSB. The results of this study suggest the existence of an alternative biofilm regulatory pathway in S. Weltevreden. Role of adrA in cellulose production in nutrient rich medium is known but role of gepA in the above phenomenon is proved in this study. An understanding of the genes involved would help in looking at strategies of repression of the gene to control formation of biofilm.