The role of the general stress response regulator RpoS in Cronobacter sakazakii biofilm formation

Highlights

• Heterogeneity existed in biofilm formation ability among C. sakazakii field isolates.

• Biofilm formation was higher in buffered minimum media and on stainless steel.

• A strain impaired in the rpoS gene showed lower biofilm formation ability.

• Loss of RpoS caused a delay in the development of mature biofilms in C. sakazakii.

Abstract

The relationship between biofilm formation and RpoS status was assessed in nine field isolates of C. sakazakii. Their ability to form biofilms was studied in BHI and minimum media with different pH values and supplemented or not with the amino acids arginine, lysine and glutamic acid. Biofilm formation, both on polystyrene and stainless steel, was measured by spectrometric determination of the fixed crystal violet and the biofilms were visualized by confocal laser scanning microscopy and scanning electron microscopy. Despite the existing heterogeneity among the different strains, biofilm formation was generally higher in buffered minimum media (pH 7.0) supplemented with lysine than in other culture media and on stainless steel plates than on polystyrene. The results showed a lower ability to form biofilms for a strain with a loss-of-function mutation in the rpoS gene, the general stress response regulator of Gram-negative bacteria, when compared to the rest of the strains, which harboured a functional rpoS. The complementation of this strain with a functional rpoS gene resulted in an increase in its biofilm formation ability up to levels comparable to those observed for strains with a functional rpoS. However, the differences were markedly reduced when the incubation time was increased from 24 to 48 h, indicating that the loss of RpoS caused a delay in the development of mature biofilms, rather than a complete inhibition of biofilm production in C. sakazakii.

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.