The role of the general stress response regulator RpoS in Cronobacter sakazakii biofilm formation
Graphical abstract
Introduction
Cronobacter sakazakii is an opportunistic pathogen capable of causing rare but life-threatening cases of meningitis, necrotizing enterocolitis and sepsis in neonates, infants and immunocompromised individuals. It is a Gram-negative, non-spore forming, facultatively anaerobic and motile rod-shaped bacterium from the Enterobacteriaceae family that multiplies at temperatures ranging from 6 to 45 °C, with an optimum temperature of 37–43 °C, and that can survive at water activity levels as low as 0.3 for up to 12 months (Almajed and Forsythe, 2016, Ray and Bhunia, 2014).
Although it is found in a number of low-moisture foods, the most common vehicles implicated in C. sakazakii infections are powdered infant formula and powdered milk (Henry & Fouladkhah, 2019). Similar to other foodborne pathogens, C. sakazakii is able to survive under numerous stressful conditions, both in the food chain (Du et al., 2018, Sánchez-Maldonado et al., 2018) and during host infection (Feeneyy, Kropp, O’Connor, & Sleator, 2015). Therefore, a better understanding of the mechanisms involved in C. sakazakii survival in these environments will help identify new means to control this microorganism and shed light on its behaviour under harsh environmental conditions.
In Gram-negative bacteria, the general stress response of stationary-phase cells is modulated by the alternative sigma factor RpoS, encoded by the rpoS gene. Starvation, as well as a wide range of stress conditions, induce the interaction of RpoS with the core RNA polymerase activating the expression of a specific but large set of genes that govern the general stress response (Battesti et al., 2010, Weber et al., 2005, Wong et al., 2017). The rpoS gene is located at a highly polymorphic region of the chromosome and it has been described as a highly mutable gene in Escherichia coli and Salmonella spp. (Ferenci, 2003, Larsen et al., 2014, Robbe-Saule et al., 2003, Saxer et al., 2014). Loss-of-function mutations in the rpoS gene have been related to an increased sensibility to food-related stresses in verocytotoxigenic E. coli strains (A. Álvarez-Ordóñez et al., 2013) and the expression level of this gene in environmental E. coli isolates has been reported to contribute to the overall intraspecies variability in stress resistance (Chiang, Dong, Edge, & Schellhorn, 2011).
Bacterial persistence in natural and industrial settings may emerge through the development of surface-associated biological structures, so-called biofilms. These sessile communities, embedded in a self-produced matrix of biopolymers, are of concern in the food industry due to their tolerance to environmental stresses, including cleaning and disinfection agents, which can result in the persistence of bacterial pathogens in equipment and processing environments as well as in the recurrent cross-contamination of food products (Alvarez-Ordóñez et al., 2019, Bridier et al., 2015).
C. sakazakii ability to form biofilms on both biotic and abiotic environments has been widely described (Du et al., 2018, Endersen et al., 2017, Ye et al., 2015). Of special concern is its ability to colonize enteral feeding tubes of neonates being fed with reconstituted powdered infant formula (Kim, Ryu, & Beuchat, 2006). Moreover, within the food industry, biofilm formation by C. sakazakii on equipment surfaces can be enhanced by the presence of nutrients from the food product (Henry & Fouladkhah, 2019).
Gene expression studies in E. coli biofilms have shown a major relevance of the rpoS gene in the establishment of mature biofilms in this species through shifts in global gene expression, affecting functions related to energy metabolism, motility and response to stresses (Ito et al., 2008, Ren et al., 2004). Similar to E. coli, a significant phenotypic diversity has been observed among field isolates of C. sakazakii in relation to their ability to cope with various (sub)lethal stress conditions and rpoS has been also shown to be a highly polymorphic gene whose functionality has been related to increased tolerance to stress conditions (Álvarez-Ordóñez, Begley, & Hill, 2012). However, the role of RpoS in C. sakazakii biofilm formation remains still unknown.
The current study was undertaken to evaluate the influence of the alternative sigma factor RpoS in the ability to form biofilms of a number of field isolates of C. sakazakii, with known status in relation to their RpoS functionality. Biofilm formation was evaluated on different abiotic surfaces, including polystyrene and stainless steel. Furthermore, the relationship between biofilm formation and RpoS status was examined by comparing the biofilms formed by a strain with an already known loss-of-function mutation in the rpoS gene, before and after its complementation with a functional rpoS gene.
Section snippets
Bacterial strains, media and culture conditions
The bacterial isolates used in this study are shown in Table 1. The master stocks of all strains were maintained at −20 °C in cryovials with 40% of glycerol as cryoprotectant. The strains were recovered by streaking them on Brain Heart Infusion (BHI, Merck, Germany) agar plates, which in the case of the complemented strain were supplemented with 10 µg/mL of chloramphenicol. After incubation at 37 °C for 24 h the plates were stored at 4 °C until further use.
Biofilm formation assays on polystyrene 96-well plates
The biofilm formation of the C.
Results
The ability of nine C. sakazakii field isolates to form biofilms was evaluated in 16 different culture conditions, including two different growth media (BHI and MM), two pH levels (non-buffered and buffered to pH 7.0), and supplementation with three distinct amino acids (arginine, lysine and glutamic acid). The results obtained on polystyrene 96-well plates show that biofilm formation was influenced by the culture media, the pH and the amino acid used to supplement the growth media (Fig. S1).
In
Discussion
The increased public health concern about the emergence of C. sakazakii as a foodborne pathogen has boosted the interest in understanding the mechanisms involved in its persistence in low-moisture food products and environments. The general stress response of stationary–phase cells of C. sakazakii, as happens for other Gram-negative bacteria, is controlled by the alternative sigma factor RpoS (Álvarez-Ordóñez et al., 2012, Jameelah et al., 2018). In E. coli, an optimal functionality of the rpoS
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
This study was funded by the Ministry of Science, Innovation and Universities of the Spanish Government, under grant number AGL2017-82779-C2-2-R. PFG is grateful to Junta de Castilla y León and the European Social Fund (ESF) for awarding her a pre-doctoral grant (BOCYL-D-15122017-4).
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