Assessment of the of acid shock effect on viability of Bacillus cereus and Bacillus weihenstephanensis using flow cytometry

Highlights

• Flow cytometry successfully assessed on acid shock effect on Bacillus cells.

• Acid pH caused membrane disruption and bacterial cell death in the first 24 h.

• B. weihenstephanensis KBAB4 was more resistant to low pH than B. cereus ATCC 14579.

Abstract

The present work studies the effect of acid shock on cell viability of Bacillus cereus and Bacillus weihenstephanensis. Experiments were performed by means of flow cytometry (FCM) combined with fluorescent labelling. Propidium iodide, for membrane evaluation, and carboxyfluorescein diacetate, for esterase activity were used for differential staining of both strains. pH values of 3.4, 3.8 and 4.2 were selected on the basis of previous screening tests. FCM analysis showed to clearly discriminate between different populations, viable and damage cells, leading to successful assessment of acid shock effect on B. cereus and B. weihenstephanensis vegetative cell viability. Cellular viability decreased at lower pH values and longer exposure times, being B. weihenstephanensis more acid resistant than B. cereus. Reliability of FCM analysis for detection of B. cereus and B. weihenstephanensis vegetative cells was compared with classical viable plate count techniques (VPC). Although FCM and VPC data were not directly correlated and classical analytical methods show higher reliability, FCM analysis provides high-speed information at real time on damage at single cell level, whereas VPC only gives an indication of cells able to grow at a certain time. These data could help to establish more accurately the potential risk associated to foods or processing conditions where pH is involved, taking damage into consideration.

Introduction

Recent changes on eating habits have enhanced consumption of refrigerated processed food which are a favourable cold niche for B. cereus and B. weihenstephanensis (Stenfors Arnesen, Fagerlund, & Granum, 2008). Both species are able to survive mild heat treatments and then grow at low temperature increasing the probability of spoilage incidents and outbreaks caused by these microorganisms. In this context, a thorough understanding of Bacillus pathogenicity and the identification of suitable food preservatives will be essential to prevent food spoilage and toxin production.

It is well known that pH is a key parameter to control bacterial growth. Low-pH foods are widely used to ensure microbiological quality and stability by adding organic acids. Besides, adaptation and survival in low pH environments are factors of particular interest in food safety and to engineer probiotic Bacilli (Ter Beek & Brul, 2010). The risk of B. cereus food poisoning is strongly determined by their resistance to low pH since the diarrhoeal syndrome is caused by spores and vegetative cells ingested with food. These cells subsequently produce enterotoxins in the intestine after surviving to the acid environment of human stomach (Kotiranta, Lounatmaa, & Haapasalo, 2000).

The efficacy of low pH has been generally assessed using classical plate count methods which are supposed to be accurate under optimal conditions, but may underestimate the numbers of viable bacteria due to biases introduced by selected incubation conditions, as well as by different environmental factors (Paparella et al., 2008). Besides, development of new detection methods is needed for resolution at single cell level to make the data applicable for predictive modelling and risk assessment approaches capable of being implemented in the food industry (Ter Beek & Brul, 2010). In this regard, flow cytometry (FCM) offers many potential advantages over conventional techniques. It has been proved to be faster than growth based methods, allowing both the measurement of heterogeneity within a population and the analysis of individual microorganisms, and providing real-time information on physiological status of the cell. Indeed, FCM has been used as a powerful, reliable and fast tool to assess the resistance and survival of B. cereus vegetative cells to acid and osmotic stress, antibiotics, food processing treatments and process equipment sanitation regimes (Cronin & Wilkinson, 2010). Moreover, previous studies reveal this technique as an effective method of verifying the viability of microorganisms present in milk, wine and water (Gunasekera et al., 2000, Malacrinò et al., 2001, Porter et al., 1995) or the antimicrobial activity of essential oils (Muñoz et al., 2009, Paparella et al., 2008).

The present research aims to characterize the physiological behaviour of B. weihenstephanensis and B. cereus vegetative cell populations after exposure to acid shock by using flow cytometric analysis (FCM) in combination with fluorescent techniques. To discriminate cells in different physiological states, two specific labels, propidium iodide (PI) and carboxyfluorecein diacetate (cFDA), were successfully used as previously reported by Biesta-Peters, Mols, Reij, and Abee (2011). Both of them selectively stain different structures allowing differentiation of viable and damaged cells. PI is a membrane impermeant dye which binds to DNA by intercalating between the bases when membrane integrity is lost (Ananta, Heinz, & Knorr, 2004). cFDA is a lipophilic dye able to diffuse inside the cells where it is cleaved by unspecific esterases converting in a membrane-impermeant fluorescent compound, carboxyfluorescein (cF), which is retained in viable cells with intact cytoplasmic membrane (Petit, Denis-Gay, & Ratinaud, 1993). Viable cells stained with cFDA emitted green fluorescence, whereas DNA of membrane-damaged cells binds PI emitting red fluorescence. Likewise, reliability of FCM analysis for detection and quantification of B. cereus and B. weihenstephanensis vegetative cells was assessed in comparison with classical techniques (VPC).