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The Scientific World Journal
Volume 2014, Article ID 153956, 5 pages
http://dx.doi.org/10.1155/2014/153956
Research Article

Effect of Simulated Gastrointestinal Conditions on Biofilm Formation by Salmonella 1,4,[5],12:i:-

1Interdisciplinary Centre of Research in Animal Health (CIISA), Faculdade de Medicina Veterinária da Universidade de Lisboa, Avenida da Universidade Técnica, 1300-477 Lisboa, Portugal
2ISPA - Instituto Universitário das Ciências Psicológicas, Sociais e da Vida, Rua Jardim do Tabaco 34, 1149-041 Lisboa, Portugal
3National Reference Laboratory of Gastrointestinal Infections, Centro Nacional de Salmonella, National Health Institute Doutor Ricardo Jorge, Avenida Padre Cruz, 1649-016 Lisboa, Portugal

Received 12 March 2014; Revised 25 May 2014; Accepted 6 June 2014; Published 30 June 2014

Academic Editor: Paul Cos

Copyright © 2014 R. Seixas et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Salmonella Typhimurium 1,4,[5],12:i:- is a major serovar responsible for human salmonellosis whose biofilm-forming ability, influenced by environmental conditions like those found in the gastrointestinal tract, is one of the main contributing factors to its ability to persist in the host and thus one of the main causes of chronic relapsing infections. Most studies to evaluate biofilm formation are performed in microtiter assays using standard media. However, no reports are available on the ability of this serovar to produce biofilm under in vitro simulated gastrointestinal conditions which better correlate with the environment found in the gastrointestinal tract. To address this, a modified biofilm assay simulating intestinal fluid was conceived to assess the biofilm formation of 133 Salmonella Typhimurium 1,4,[5],12:i:- isolates with and without agitation and at three different time points (24 h, 48 h, and 72 h). The results were then compared to the existing microtiter method using conventional biofilm growth medium (Mueller Hinton Broth). Statistical analysis revealed significant differences in the results obtained between the three protocols used. The simulated human intestinal environment impaired biofilm production demonstrating that conditions like pH, agitation or the presence of enzymes can influence biofilm production. Therefore, results from in vitro simulation of in vivo conditions may contribute to unravelling factors relating to biofilm formation and persistence in the context of the human host.

Dedicated to Professor Cristina Lobo Vilela, 1958–2013, Interdisciplinary Centre of Research in Animal Health (CIISA), Faculty of Veterinary Medicine from the University of Lisbon

1. Introduction

The emergence of a pandemic monophasic variant of Salmonella Typhimurium, S. enterica subsp. enterica serovar 1,4,[5],12:i:-, was first reported in Europe in the mid-1990s and is presently considered to be one of the major serovars responsible for human salmonellosis worldwide [1].

Many studies have demonstrated that Salmonella bacteria are capable of forming biofilms on a wide variety of abiotic and biotic surfaces [2, 3]. These highly organized multicellular bacterial structures, responsible for chronic or persistent infections, decrease antimicrobial therapy efficacy and improve resistance to environmental stresses such as desiccation, high temperatures, and antiseptics [4, 5].

Since its conception by Christensen and collaborators in 1985, the 96-well microtiter plate test has been the most frequently used assay for high throughput quantitative evaluation of biofilm-forming ability by bacteria [6, 7]. Over the years, modifications have been made to improve its accuracy [8, 9]. It is generally performed under static conditions using different media, such as Mueller Hinton Broth (MHB) or Tryptic Soy Broth (TSB), and enables quantitative biofilm determination through the application of different dyes such as crystal violet, resazurin, or dimethyl methylene blue [7, 8].

However these in vitro conditions differ greatly from the human intestinal environment, in terms of organic composition (enzymes), pH, or dynamics (peristalsis), which is the preferential location for Salmonella infection.

Several factors, including pH, temperature, and media composition [10, 11], affect biofilm formation. We aimed to evaluate the influence of conditions mimicking the intestinal human tract environment on biofilm formation by Salmonella Typhimurium 1,4,[5],12:i:- in vitro. With these modifications, which better simulate real conditions, we aim to provide a better insight into the influence the gastrointestinal environment has upon the biofilm-forming ability of this serovar and ultimately provide more reliable laboratory and clinically relevant results.

2. Materials and Methods

2.1. Bacterial Isolates and Identification

In this study, 133 Salmonella Typhimurium 1,4,[5],12:i:- isolates, collected in Portugal from 2006 to 2011 from different origins, were used. Isolates were obtained from clinical (), environmental (), and animal () samples. All Salmonella isolates were serotyped and identification was confirmed by multiplex PCR as recommended by EFSA (EFSA Panel on Biological Hazards 2010).

2.2. Evaluation of Biofilm Formation by a Standard Microtiter Biofilm Assay

Alamar Blue (AB) (Thermo Fisher Scientific, Oxford, UK) biofilm assay was performed according to the protocol described by Pettit et al. (2005) [8], with minor modifications. Overnight cultures were used to prepare bacterial suspensions with  CFU/mL in MHB (Liofilchem, Roseto degli Abruzzi, Italy). Suspensions were placed in flat-bottom, polystyrene, tissue-culture-treated 96-well microtiter plates (Orange Scientific, Braine-l’Alleud, Belgium). Three microtiter wells were used per isolate. Plates were incubated in a humidity chamber at 37°C without agitation for 24 h, 48 h, and 72 h. After each time point, plates were removed from the incubator and 5 μL of AB was added to the wells, gently shaken, and incubated for 1 h at 37°C, in order to stain the adherent and viable bacteria. Absorbances at 570 nm were determined using a Spectra MAX 340PC microplate reader (Molecular Devices, Sintra, Portugal). All microtiter assays were carried out in triplicate and repeated on three different occasions and the results were averaged.

2.3. Biofilm Formation under In Vitro Simulated Intestinal Conditions by a Microtiter Biofilm Assay
2.3.1. In Vitro Passage of Salmonella Typhimurium 1,4,[5],12:i:- under Simulated Gastric Conditions

Microtiter biofilm assay was also performed using simulated gastrointestinal conditions as described by de Angelis et al. (2006) [12]. Briefly, stationary-phase bacteria grown in 5 mL of TSB were harvested at 6000 g (Hermle Labortechnik, Wehingen, Germany) for 10 min and suspended in 5 mL of simulated gastric fluid which contained NaCl (125 mM/L), KCl (7 mM/L), NaHCO (45 mM/L), and pepsin (3 g/L) (Sigma-Aldrich, St. Louis, USA), pH 3. Bacterial suspensions were submitted to agitation conditions for 180 min with a minishaker apparatus (VWR, Lisboa, Portugal) at 175 rpm, in order to simulate the passage through the stomach. Aliquots were taken in order to determine the number of colony forming units per mL by measuring optical density (O.D.) values, based on standard curves previously determined (data not shown).

2.3.2. In Vitro Biofilm Formation under Simulated Intestinal Conditions

After gastric digestion, bacteria cells were harvested using the same conditions, washed with 0.9% sterile sodium chloride solution, and suspended in simulated intestinal fluid (SIF), containing 0.1% (w/v) pancreatin (AppliChem, Darmstadt, Germany) and 0.15% (w/v) bile bovine (Sigma-Aldrich, St. Louis, USA), pH 8.0 [12].

Then, 100 μL of bacterial suspensions in SIF was incubated in flat-bottom, polystyrene, and tissue-culture-treated 96-well microtiter plates (Orange Scientific, Braine-l’Alleud, Belgium). For each isolate, three microtiter wells were used. Plates were incubated in a humidity chamber at 37°C under stationary and agitation conditions with a minishaker apparatus (VWR, Lisboa, Portugal) at 100 rpm for 24, 48, and 72 h, and, after each time point, plates were removed from the incubator and 5 μL of AB was added to the wells. The plates were then incubated for a further 1 h at 37°C. Absorbances at 570 nm were determined using a Spectra MAX 340PC microplate reader (Molecular Devices, Sintra, Portugal). All microtiter assays were carried out in triplicate and repeated on three different occasions and the results were averaged.

2.3.3. Classification of Biofilm-Forming Ability by Microtiter Plates

Based on the O.D. and O.D. cut-off (O.D.c) values, isolates were classified into different categories according to their biofilm-forming ability, as previously described by Stepanovic et al. (2000) [9]. The O.D. cut-off was defined as three standard deviations above the mean O.D. of the negative control and isolates were classified as follows: if O.D. O.D.c, isolates were considered to be nonbiofilm producers; if , isolates were considered weak biofilm producers; if , isolates were considered moderate biofilm producers; and if ., isolates were considered strong biofilm producers [13]. AB assays were performed in triplicate and repeated on different occasions, and results were averaged. Results are presented as mean value ± standard deviation (SD). Statistical analyses were performed using the SPSS 20.0 software (IBM Corporation, NY, USA). Differences between time points and techniques were evaluated by repeated measures ANOVA and one-way ANOVA, respectively. Tukey post hoc tests were used to compare biofilms O.D. mean values. Correlation between CFU/mL after gastric passage and biofilm production at 24 h was determined by Pearson coefficient. values 0.05 were considered statistically significant.

3. Results and Discussion

Standard microtiter biofilm assay staining with resazurin (Alamar Blue), a metabolic activity indicator frequently used for quantitative biofilm determination, revealed that Salmonella Typhimurium 1,4,[5],12:i:- isolates possess a high ability for biofilm formation on plastic surfaces, which is in accordance with previous studies [7, 10, 14]. O.D. mean values in MHB increased over time; it was observed that biofilms with the highest O.D. mean values are produced at 72 h (Figure 1). This increase was statistically significant (repeated measures ANOVA, ).

153956.fig.001
Figure 1: Time course of biofilm production by 133 Salmonella Typhimurium 1,4,[5],12:i:- isolates using an Alamar Blue microtiter assay applied in different incubation conditions. Mean and standard deviation for MHB were at 24 h 0,856 ± 0,095, at 48 h 0,977 ± 0,105, and at 72 h 1,044 ± 0,118. SIF under static conditions were at 24 h 0,531 ± 0,217, at 48 h 0,443 ± 0,222, and at 72 h 0,409 ± 0,146. SIF under agitation conditions were at 24 h 0,377 ± 0,136, at 48 h 0,355 ± 0,142, and at 72 h 0,297 ± 0,108. MHB: Mueller Hinton Broth; SIF: simulated intestinal fluid.

Following the simulated gastric passage using the modified microtiter biofilm assay, CFU/mL values have a significant positive correlation, although weak, with biofilm production at 24 h in SIF under static conditions (Pearson ,183, ) and in SIF under dynamic conditions (Pearson ,158, ). Higher numbers of CFU/mL can lead to a higher biofilm formation, even though the effects of gastric stress conditions on biofilm formation may be strain specific, as demonstrated by other authors [15].

The largest number of isolates forming weak biofilms was found in SIF under dynamic conditions (83.5% at 24 h, 51.1% at 48 h, and 57.9% at 72 h), while the largest number of isolates able to form moderate and strong biofilms was found in MHB at 48 h and at 72 h (66.2% and 99.2%, resp.) (Table 1). However, 21% of the isolates showed strong biofilm-forming ability at 24 h in SIF under static conditions, and this percentage decreased with time (9% at 48 h and 3% at 72 h). In MHB, more than one-third (37.6%) of the Salmonella Typhimurium 1,4,[5],12:i:- isolatestested were only able to produce strong biofilms at 72 h.

tab1
Table 1: Characterization of biofilm-forming ability of 133 Salmonella Typhimurium 1,4,5,12:i:- isolates using an Alamar Blue microtiter assay applied in different incubation conditions.

Human gastrointestinal conditions may decrease bacteria’s ability to adhere to a substratum, the first step required for biofilm formation and which impaired the ability to form strong biofilm [16]. O.D. mean values of biofilm production in SIF under dynamic conditions decreased significantly with incubation time (repeated measures ANOVA, ) and are significantly lower in comparison with static conditions (ANOVA, ), at all the time points studied. This can be explained by the fact that the dynamic conditions applied may have impaired bacterial adhesion and are in accordance with other reports that used dynamic methodologies [11, 16].

Biofilm O.D. mean values obtained in SIF with static conditions, although lower than the ones from MHB, are higher than the ones obtained in SIF with dynamic conditions; these differences are statistically significant (ANOVA, ) showing that conditions, like agitation, have a significant influence on biofilm formation. Dynamics of intestinal peristalsis may strongly influence bacteria’s ability to adhere to a surface and should be included as a parameter during biofilm evaluation, as already stated in previous studies [16].

The decrease in biofilm OD mean values between 48 h and 72 h at SIF with dynamic condition was significantly higher than in SIF under static conditions (Tukey, ), which can be due to a decrease in the number of viable bacteria. The higher number of dead bacteria cells may be due to a decrease in nutrients together with an accumulation of toxic compounds originating from bacterial metabolism that were disseminated by the agitation conditions during this assay [17].

There were significant differences between results obtained by the three protocols at the three time points evaluated (ANOVA, ), which indicates that intestinal conditions can influence biofilm production by Salmonella. White et al. 2008 [18] previously showed that expression of biofilm related genes like curli genes is turned off during in vivo infection but turned on again once the bacteria is shed into the environment. This may explain why biofilm production is lower in SIF than in MHB, especially if considering the dynamic conditions present in the intestinal tract due to peristalsis.

4. Conclusions

The simulated gastrointestinal environment impaired biofilm production by Salmonella, demonstrating that conditions simulating those encountered in vivo like pH, agitation, or the presence of enzymes can influence in vitro biofilm formation results, emphasizing the importance of experimental conditions in the results obtained. In conclusion, the provision of dynamic and environmental conditions that better simulate the in vivo gastrointestinal stress that Salmonella is subjected to should be included as one of the parameters in the evaluation of biofilm producing strains, enabling a more accurate correlation between in vitro biofilm formation and what happens in the gastrointestinal tract. By approximating experimental conditions to those that bacteria encounter in the human host it may be possible to obtain more insight into the real ability and importance of biofilm production when compared with MHB used in standard biofilm assays.

Conflict of Interests

The authors declare that there is no conflict of interests regarding the publication of this paper.

Acknowledgments

The authors would like to thank Dr. Sinclair Owen for the revision of the paper. This study was conducted with the financial support of “Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária da Universidade de Lisboa” (CIISA/FMV/ULisboa). Rui Seixas holds a Ph.D. fellowship (SFRH/BD/75836/2011) from FCT, Portugal.

References

  1. A. I. M. Switt, Y. Soyer, L. D. Warnick, and M. Wiedmann, “Emergence, distribution, and molecular and phenotypic characteristics of Salmonella enterica serotype 4,5,12:i:-,” Foodborne Pathogens and Disease, vol. 6, no. 4, pp. 407–415, 2009. View at Publisher · View at Google Scholar · View at Scopus
  2. T. Møretrø, L. K. Vestby, L. L. Nesse, S. E. Storheim, K. Kotlarz, and S. Langsrud, “Evaluation of efficacy of disinfectants against Salmonella from the feed industry,” Journal of Applied Microbiology, vol. 106, no. 3, pp. 1005–1012, 2009. View at Publisher · View at Google Scholar · View at Scopus
  3. N. A. Ledeboer and B. D. Jones, “Exopolysaccharide sugars contribute to biofilm formation by Salmonella enterica serovar typhimurium on HEp-2 cells and chicken intestinal epithelium,” Journal of Bacteriology, vol. 187, no. 9, pp. 3214–3226, 2005. View at Publisher · View at Google Scholar · View at Scopus
  4. K. Scher, U. Romling, and S. Yaron, “Effect of heat, acidification, and chlorination on Salmonella enterica serovar typhimurium cells in a biofilm formed at the air-liquid interface,” Applied and Environmental Microbiology, vol. 71, no. 3, pp. 1163–1168, 2005. View at Publisher · View at Google Scholar · View at Scopus
  5. C. Marin, A. Hernandiz, and M. Lainez, “Biofilm development capacity of Salmonella strains isolated in poultry risk factors and their resistance against disinfectants,” Poultry Science, vol. 88, no. 2, pp. 424–431, 2009. View at Publisher · View at Google Scholar · View at Scopus
  6. E. Peeters, H. J. Nelis, and T. Coenye, “Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates,” Journal of Microbiological Methods, vol. 72, no. 2, pp. 157–165, 2008. View at Publisher · View at Google Scholar · View at Scopus
  7. S. Stepanović, I. Cirković, L. Ranin, and M. Svabić-Vlahović, “Biofilm formation by Salmonella spp. and Listeria monocytogenes on plastic surface,” Letters in Applied Microbiology, vol. 38, no. 5, pp. 428–432, 2004. View at Publisher · View at Google Scholar
  8. R. K. Pettit, C. A. Weber, M. J. Kean et al., “Microplate alamar blue assay for Staphylococcus epidermidis biofilm susceptibility testing,” Antimicrobial Agents and Chemotherapy, vol. 49, no. 7, pp. 2612–2617, 2005. View at Publisher · View at Google Scholar · View at Scopus
  9. S. Stepanovic, D. Vukovic, I. Dakic, and B. Savic, “Svabic-Vlahovic M: a modified microtiter-plate test for quantification of staphylococcal biofilm formation,” Journal of Microbiological Methods, vol. 40, no. 2, pp. 175–179, 2000. View at Publisher · View at Google Scholar
  10. E. B. Solomon, B. A. Niemira, G. M. Sapers, and B. A. Annous, “Biofilm formation, cellulose production, and curli biosynthesis by Salmonella originating from produce, animal, and clinical sources,” Journal of Food Protection, vol. 68, no. 5, pp. 906–912, 2005. View at Google Scholar · View at Scopus
  11. S. Stepanović, I. Cirkovic, V. Mijac, and M. Svabic-Vlahovic, “Influence of the incubation temperature, atmosphere and dynamic conditions on biofilm formation by Salmonella spp,” Food Microbiology, vol. 20, no. 3, pp. 339–343, 2003. View at Publisher · View at Google Scholar
  12. M. de Angelis, S. Siragusa, M. Berloco et al., “Selection of potential probiotic lactobacilli from pig feces to be used as additives in pelleted feeding,” Research in Microbiology, vol. 157, no. 8, pp. 792–801, 2006. View at Publisher · View at Google Scholar · View at Scopus
  13. S. Stepanović, I. Cirković, L. Ranin, and M. Svabić-Vlahović, “Biofilm formation by Salmonella spp. and Listeria monocytogenes on plastic surface,” Letters in Applied Microbiology, vol. 38, no. 5, pp. 428–432, 2004. View at Google Scholar
  14. L. K. Vestby, T. Møretrø, S. Langsrud, E. Heir, and L. L. Nesse, “Biofilm forming abilities of Salmonella are correlated with persistence in fish meal- and feed factories,” BMC Veterinary Research, vol. 5, article 20, 2009. View at Publisher · View at Google Scholar · View at Scopus
  15. A. Lianou and K. P. Koutsoumanis, “Strain variability of the biofilm-forming ability of Salmonella enterica under various environmental conditions,” International Journal of Food Microbiology, vol. 160, no. 2, pp. 171–178, 2012. View at Publisher · View at Google Scholar · View at Scopus
  16. S. Stepanović, D. Vuković, P. Jezek, M. Pavlović, and M. Svabic-Vlahović, “Influence of dynamic conditions on biofilm formation by staphylococci,” European Journal of Clinical Microbiology & Infectious Diseases, vol. 20, no. 7, pp. 502–504, 2001. View at Google Scholar
  17. S. Sillankorva, P. Neubauer, and J. Azeredo, “Pseudomonas fluorescens biofilms subjected to phage phiIBB-PF7A,” BMC Biotechnology, vol. 8, article 79, 2008. View at Publisher · View at Google Scholar · View at Scopus
  18. A. P. White, D. L. Gibson, G. A. Grassl et al., “Aggregation via the red, dry, and rough morphotype is not a virulence adaptation in Salmonella enterica serovar typhimurium,” Infection and Immunity, vol. 76, no. 3, pp. 1048–1058, 2008. View at Publisher · View at Google Scholar · View at Scopus