Modeling the effect of pH, water activity, and ethanol concentration on biofilm formation of Staphylococcus aureus

doi:10.1016/j.fm.2018.06.006

Food Microbiology

Volume 76, December 2018, Pages 287-295

https://doi.org/10.1016/j.fm.2018.06.006 Get rights and content

Highlights

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Biofilm formation was significantly stronger at pH and aw close S. aureus growth optimal conditions.
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Biofilm formation was significantly high at ethanol concentration around 2.5–3.5%.
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CPM showed a better quality of fit compare to CPMI whatever the environmental factors.
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High extracellular matrix production depend on stress induced by ethanol at non-lethal concentrations.

Abstract

In this work, the effect of environmental factors on Staphylococcus aureus (ATCC 13150) biofilm formation in tryptic soy broth was investigated under different ranges of pH (3.0–9.5), ethanol concentration (EtOH 0.0–20.0%), and a_w (NaCl, 0.866–0.992). Biofilm formation was quantified using the crystal violet staining method and optical density (OD: 590 nm) measurements. Biofilm formation was significantly stronger at pH and a_w close to S. aureus optimal growth conditions, while it was high at EtOH around 2.5–3.5%. Data sets from the difference between the OD measurements of the test and control (ΔOD) were fitted to the cardinal parameter model (CPM) and cardinal parameter model with inflection (CPMI) to describe the effect of the environmental factors. The models showed good quality of fit for the experimental data in terms of calculated RMSE, with the latter ranging from 0.276 to 0.455. CPM gave a good quality of fit compared to CPMI for the environmental factors tested. Optimal pH was close to neutral (6.76–6.81) and biofilm formation was possible till pH = 3.81–3.78 for CPM and CPMI, respectively. Optimum EtOH and a_w conditions for biofilm formation were in the range of 1.99–2.75 and 0.98–0.97, respectively. Predicted OD values observed using strain 13150 were very closely correlated to the OD values predicted with strain 12600 with R² of 0.978, 0.991, and 0.947 for pH, EtOH, and a_w, respectively. The cultivable bacterial cells within the biofilm were enumerated using standard plate counting and a linear model was applied to correlate the attached biofilm cells to ΔOD of biofilm formation. It was found that the biofilm formation correlated with S. aureus population growth. At 2.5–3.5% of EtOH the maximum population density was lower than that observed at 0.0% of EtOH. As 2.5–3.5% of EtOH initiated a stronger biofilm formation, biofilm formation seems to be induced by ethanol stress. The development of cardinal parameter models to describe the effect environmental factors of importance to biofilm formation, offers a promising predictive microbiology approach to decrypting the S. aureus population growth and survival ability on food processing surfaces.

Introduction

In natural and human-made ecosystems some bacteria have the ability to attach to surfaces and form organized communities called biofilm. Its formation on food processing equipment leads to the contamination of food products, which causes foodborne illnesses and significant economic losses (Rode et al., 2007; Sharma and Anand, 2002). The microbial biofilm is a complex three-dimensional structure composed of microbial cells and an extracellular matrix principally consisting of polysaccharides, proteins, nucleic acids and lipids of microbial origin (Flemming and Wingender, 2010). These polysaccharides provide the structural scaffold of the biofilm and act as a shield against various types of antimicrobials treatments (Arciola et al., 2012; Bridier et al., 2015). It has been reported that contaminated surfaces play a pivotal role in spreading foodborne pathogens to food by contact with food processing equipment and it is one of the main contributing factors to foodborne outbreaks (Gormley et al., 2011; Perez-Rodriguez et al., 2013). It seems that biofilm formation is essential for the bacterial survival on the food processing surface and poses a potential risk of post-processing food contamination (Møretrø et al., 2003).

Staphylococcus aureus has the potential to colonize the surfaces of food processing equipment and form biofilms. S. aureus has often been isolated from biofilms that developed in food processing plant such as dairy, egg, seafood, and meat processing industries (Bridier et al., 2015; Gutiérrez et al., 2012; Rohde et al., 2007; Shi and Zhu, 2009; Tango et al., 2015). The survival of the S. aureus in hostile environments such as food processing equipment may be due to the biofilm formation, which enhances the recurrence of planktonic bacterial populations when foods are processed. Biofilms defy most antimicrobial agents and represent a potential source of bacterial contamination in the food industry. It has been reported that S. aureus produces polysaccharide intercellular adhesion (PIA) surrounding the cell, which can form a capsule and protects the bacterial cell against host immune response (phagocytes) (Fox et al., 2005). Previous research has shown the existence of a strong causal connection between staphylococcal biofilm and staphylococcal food poisoning (SFP) through the consumption of staphylococcal enterotoxin (SE) produced in food (da Silva Meira et al., 2012; Planchon et al., 2006). It is therefore essential for the food industry to understand the conditions, under which S. aureus is able to survive, grow, and contaminate food products with respect to biofilm formation. This knowledge is critical for a successful risk assessment program.

Biofilm formation is a multistep process, involving a large number of physiological changes in bacteria and takes place in response to environmental and biochemical factors (Arrizubieta et al., 2004). The mechanism of staphylococcal biofilm formation in food industry and on medical materials has been evaluated in detail and it is reported that S. aureus cells form biofilms though various means. This ability to form biofilm can be thwarted by the suboptimal growth temperature and the presence of nitrite (Gustafson et al., 2014; Rode et al., 2007). However, sodium chloride induces biofilm formation of S. aureus (Planchon et al., 2006). It has been reported that alcohol can induce haemolytic properties on otherwise non-haemolytic microorganisms, phenomenon referred to as “microbial alcohol-conferred haemolysis” (MACH) (Korem et al., 2010; Knobloch et al., 2001). Korem et al. (2010) demonstrated that alcohols selectively increased the hemolytic properties of certain staphylococci strains and resulted in an increased biofilm formation. Ethanol is commonly used as plant disinfectant in food industry, medical applications, and household products, and hence may induce MACH on certain strains of S. aureus, thereby increasing also biofilm production when used at inappropriate concentration.

The transition from a planktonic to a complex three-dimensional structure is a dynamic process that involves environmental and biochemical phenomena, thus is possibly implemented through a developmental model (Hermanowicz, 2001; Monds and O'Toole, 2009). Considerable effort has been employed during recent decades to develop mathematical models for describing substrate use and microbial population dynamics during biofilm formation. Developed models, such as individual-based models, successfully predict biofilm structure dynamics and clarify the processes which govern biofilm formation and development (Bridier et al., 2015). The development of new approaches of predictive microbiology may contribute to understanding the role of different environmental conditions on biofilm development (Hermanowicz, 2001; Xavier et al., 2004). The experimental methods will ultimately quantify the biofilm formation and mathematical models can be useful tools for investigating the effects of different environmental factors on biofilm formation by assumptions in which the enhancement or inhibitory effect of these factor is multiplicative (Ross and Dalgaard, 2003). Regarding these aspects, this study was performed with the objective of evaluating the S. aureus response to pH, ethanol concentration (EtOH), and water activity (a_w) during switching between planktonic and biofilm modes and to develop a predictive microbiology model to describe the effects of these environment factors on the biofilm formation ability of S. aureus.

Section snippets

Bacterial strains for biofilm testing

The modeling of biofilm development was performed using S. aureus strain ATCC 13150, which was provided by the Department of Food Science and Biotechnology, Kangwon National University, South Korea. The bacterial stock was maintained by cryopreservation at −80 °C in tryptic soy broth (TSB, Difco, Sparks, MD, USA) supplemented with 15% glycerol (Sigma-Aldrich, Co, Saint-Louis, USA). Two days before each biofilm experiments, bacterial stocks were thawed and recovered from deep freezing by

Modeling S. aureus biofilm development

Initially, 25 strains isolated from different food products and plants were evaluated to select the highest biofilm producer. The evaluation was performed in TSB at 37 °C for 48 h in polystyrene microtiter plates. Among these strains, ATCC 13150 produced the strongest biofilm (data no shown). S. aureus 13150 was studied in more detail as a target strain while investigating the effect of pH, EtOH, and a_w on the biofilm formation in TSB at 37 °C after 48 h of incubation in 96-well microtiter

Discussion

S. aureus has been recognized as one of the greater biofilm producer bacteria and the connection between staphylococcal biofilm and staphylococcal food poisoning has been previously established (da Silva Meira et al., 2012; Planchon et al., 2006). In the present study, the effect of environmental parameters on S. aureus biofilm formation was modeled using CPM and CPMI for pH, EtOH, and a_w. The cardinal parameters are a family of models which define cardinal values (minimum, optimum and maximum)

Conclusion

The present study developed and validated a predictive model to quantitatively assess the effects of pH, EtOH, and a_w on the biofilm formation ability of S. aureus. The developed cardinal models allow defining the range of environmental factors for which the biofilm formation is probable. These models were able to estimate the rate of S. aureus biofilm formation. Subsequently these models can play a great role in risk assessment. The models are based on simplified growth media and refer to

Acknowledgments

This research was supported by a research grant of Kangwon National University (2015), Central laboratory of Kangwon National University, project PFV/10/002 (Center of Excellence OPTEC-Optimization in Engineering) of the KU Leuven Research Council, projects G093013N of the Fund for Scientific Research-Flanders, and the Belgian Program on Interuniversity Poles of Attraction, initiated by the Belgian Federal Science Policy Office.

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Modeling the effect of pH, water activity, and ethanol concentration on biofilm formation of Staphylococcus aureus

Highlights

Abstract

Introduction

Section snippets

Bacterial strains for biofilm testing

Modeling S. aureus biofilm development

Discussion

Conclusion

Acknowledgments

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