Transfer of bacteria between stainless steel and chicken meat: A CLSM and DGGE study of biofilms

Afraa Said Al-Adawi; Christine C. Gaylarde; Jan Sunner; Iwona B. Beech; Afraa Said Al-Adawi; Christine C. Gaylarde; Jan Sunner; Iwona B. Beech

doi:10.3934/microbiol.2016.3.340

AIMS Microbiology

2016, Volume 2, Issue 3: 340-358. doi: 10.3934/microbiol.2016.3.340

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Transfer of bacteria between stainless steel and chicken meat: A CLSM and DGGE study of biofilms

1.
Microbiology Research Laboratory, School of Pharmacy and Biomedical Sciences, University of Portsmouth, St. Michael’s Building, White Swan Rd, Portsmouth PO1 2DT, UK
2.
Department of Microbiology and Plant Biology, Oklahoma University, 770 Van Vleet Oval, Norman, OK 73019, USA
3.
Department of Applied Sciences, Higher College of Technology, Muscat, Oman

Received: 14 May 2016 Accepted: 14 August 2016 Published: 18 August 2016

This study aimed to assess the interaction between bacteria and food processing surfaces using novel methods. Microbial cross contamination between stainless steel, a common food processing material, and raw chicken was studied using microbiological culture, specialized microscope and molecular techniques. Confocal laser scanning microscopy (CLSM) allowed the visualization of biofilms containing single or dual species of Escherichia coli O157:H7, Salmonella typhimurium, Bacillus cereus, Staphylococcus aureus and Pseudomonas aeruginosa, formed after 6 days’ incubation on stainless steel or 4h on raw chicken. The results provided information on intra-biofilm location and stratification of species within dual species biofilms. Top-to-bottom Z-stack images revealed that, on both materials, S. typhimurium and E. coli attached concurrently, the former in greater numbers. E. coli and B. cereus segregated on steel, E. coli more frequent near the metal surface, B. cereus almost the only species in outer layers. Few cells of S. aureus, found at all depths, were seen in the 2.9 µm thick biofilm on steel with E. coli. Greatest attachment was shown by P. aeruginosa, followed by S. typhimurium, E. coli and finally Gram positive species. Large amounts of EPS in P. aeruginosa biofilms made visualization difficult on both materials, but especially on chicken meat, a limitation of this technique. Nevertheless, CLSM was useful for determining time sequence of adhesion and species makeup of thin biofilms. The technique showed that five min contact between bacterially-contaminated chicken and sterile steel resulted in greatest transfer of P. aeruginosa, followed by S. typhimurium. This was confirmed using DGGE. Gram positive bacteria transferred poorly. A biofilm containing 2.3 × 10⁵ cfu·cm⁻²B. cereus on steel transferred an undetectable number of cells to chicken after 5 min contact. This species was unable to form biofilm on chicken when incubated for 4 h in growth medium. S. typhimurium and P.aeruginosas were most efficiently transferred from contaminated steel to raw chicken within 5 min contact, with 20–30% transfer from single species biofilms. All other species, and all cells in dual species biofilms, showed less than 2% transfer. CLSM and DGGE were shown to be useful techniques for the study of bacterial adhesion to stainless steel.
- biofilms,
- CLSM,
- cross-contamination,
- food-processing surfaces,
- stainless steel
Citation: Afraa Said Al-Adawi, Christine C. Gaylarde, Jan Sunner, Iwona B. Beech. Transfer of bacteria between stainless steel and chicken meat: A CLSM and DGGE study of biofilms[J]. AIMS Microbiology, 2016, 2(3): 340-358. doi: 10.3934/microbiol.2016.3.340

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Abstract

This study aimed to assess the interaction between bacteria and food processing surfaces using novel methods. Microbial cross contamination between stainless steel, a common food processing material, and raw chicken was studied using microbiological culture, specialized microscope and molecular techniques. Confocal laser scanning microscopy (CLSM) allowed the visualization of biofilms containing single or dual species of Escherichia coli O157:H7, Salmonella typhimurium, Bacillus cereus, Staphylococcus aureus and Pseudomonas aeruginosa, formed after 6 days’ incubation on stainless steel or 4h on raw chicken. The results provided information on intra-biofilm location and stratification of species within dual species biofilms. Top-to-bottom Z-stack images revealed that, on both materials, S. typhimurium and E. coli attached concurrently, the former in greater numbers. E. coli and B. cereus segregated on steel, E. coli more frequent near the metal surface, B. cereus almost the only species in outer layers. Few cells of S. aureus, found at all depths, were seen in the 2.9 µm thick biofilm on steel with E. coli. Greatest attachment was shown by P. aeruginosa, followed by S. typhimurium, E. coli and finally Gram positive species. Large amounts of EPS in P. aeruginosa biofilms made visualization difficult on both materials, but especially on chicken meat, a limitation of this technique. Nevertheless, CLSM was useful for determining time sequence of adhesion and species makeup of thin biofilms. The technique showed that five min contact between bacterially-contaminated chicken and sterile steel resulted in greatest transfer of P. aeruginosa, followed by S. typhimurium. This was confirmed using DGGE. Gram positive bacteria transferred poorly. A biofilm containing 2.3 × 10⁵ cfu·cm⁻²B. cereus on steel transferred an undetectable number of cells to chicken after 5 min contact. This species was unable to form biofilm on chicken when incubated for 4 h in growth medium. S. typhimurium and P.aeruginosas were most efficiently transferred from contaminated steel to raw chicken within 5 min contact, with 20–30% transfer from single species biofilms. All other species, and all cells in dual species biofilms, showed less than 2% transfer. CLSM and DGGE were shown to be useful techniques for the study of bacterial adhesion to stainless steel.

References

[1]	Trachoo N (2003) Biofilms and the food industry. Songklanakarin J. Sci Technol 25: 807–815.
[2]	Vestby LK, Møretrø T, Langsrud S, et al. (2009) Biofilm forming abilities of Salmonella are correlated with persistence in fish meal- and feed factories. BMC Vet Res 5: 20. doi: 10.1186/1746-6148-5-20
[3]	Holah J, Gibson H (2000) Food Industry Biofilms. In: Evans, L.V. Editor Biofilm Recent Advances in their Study and Control. Amsterdam: Harwood Academic Publishers, 211–235.
[4]	Stepanović S, Ćirković I, Mijač V, et al. (2002) Influence of the incubation temperature, atmosphere and dynamic conditions on biofilm formation by Salmonella spp. Food Microbiol 20: 339–34.
[5]	Almohamad S, Somarajan SR, Singh KV, et al. (2014) Influence of isolate origin and presence of various genes on biofilm formation by Enterococcus faecium. FEMS Microbiol Lett 353: 151–156. doi: 10.1111/1574-6968.12418
[6]	Papenfort K, Förstner KU, Cong J-P, et al. (2015) Differential RNA-seq of Vibrio cholera identifies the VqmR small RNA as a regulator of biofilm formation. PNAS 112: E766–E775. doi: 10.1073/pnas.1500203112
[7]	Stoodley P, Sauer K, Davies DG, et al. (2002) Biofilms as complex differentiated communities. Ann Rev Microbiol 56: 187–209.
[8]	Alavi HE, Hansen LT (2013) Kinetics of biofilm formation and desiccation survival of Listeria monocytogenes in single and dual species biofilms with Pseudomonas fluorescens, Serratia proteamaculans or Shewanella baltica on food-grade stainless steel surfaces. Biofouling 29: 1253–1268. doi: 10.1080/08927014.2013.835805
[9]	Bremer PJ, Monk I, Osborne CM (2001) Survival of Listeria monocytogenes attached to stainless steel surfaces in the presence or absence of Flavobacterium spp. J Food Protect 64: 1369–1376.
[10]	Pompermayer DMC, Gaylarde CC (2000) The influence of temperature on the adhesion of dual species cultures of Staphylococcus aureus and Escherichia coli to polypropylene. Food Microbiol 17: 361–365.
[11]	Schwab U, Hu Y, Wiedmann M, et al. (2005) Alternative sigma factor sigmaB is not essential for Listeria monocytogenes surface attachment. J Food Protect 68: 311–317.
[12]	Rodríguez A, McLandsborough LA (2007) Evaluation of the transfer of Listeria monocytogenes from stainless steel and high density polyethylene to Bologna and American cheese. J Food Protect 70: 600–606.
[13]	Moore G, Blair I, McDowell DA (2007) Recovery and transfer of Salmonella typhimurium from four different domestic food contact surfaces. J Food Protect 70: 2273–2280.
[14]	Malheiros PS, Passos CT, Casarin LS, et al. (2010) Evaluation of growth and transfer of Staphylococcus aureus from poultry meat to surfaces of stainless steel and polyethylene and their disinfection. Food Control 21: 298–301. doi: 10.1016/j.foodcont.2009.06.008
[15]	Midelet G, Carpentier B (2004) Impact of cleaning and disinfection agents on biofilm structure and on microbial transfer to a solid food model. J Appl Microbiol 97: 262–270. doi: 10.1111/j.1365-2672.2004.02296.x
[16]	Jensen DA, Friedrich LM, Harris LJ, et al. (2013) Quantifying transfer rates of Salmonella and Escherichia coli O157:H7 between fresh-cut produce and common kitchen surfaces. J Food Protect, Number 9, September 2013, 1530–1538.
[17]	Niemira BA, Solomon EB (2005) Sensitivity of planktonic and biofilm-associated Salmonella spp. to ionizing radiation. Appl Env Microbiol 71: 2732–2736. doi: 10.1128/AEM.71.5.2732-2736.2005
[18]	Marques SC, Rezende GCOS, Alves LAF, et al. (2007) Formation of biofilms by Staphylococcus aureus on stainless steel and glass surfaces and its resistance to some selected chemical sanitizers. Braz J Microbiol 38: 538–543. doi: 10.1590/S1517-83822007000300029
[19]	Kives J, Orgaz B, SanJosé C (2006) Polysaccharide differences between planktonic and biofilm-associated EPS from Pseudomonas fluorescens B52. Colloid Surface B 52: 123–127.
[20]	Filoche SK, Zhu M, Wu CD (2004) In situ biofilm formation by multi-species oral bacteria under flowing and anaerobic conditions. J Dent Res 83: 802–806. doi: 10.1177/154405910408301013
[21]	Rickard AH, McBain AJ, Ledder RG, et al. (2003) Coaggregation between freshwater bacteria within biofilm and planktonic communities. FEMS Microbiol Lett 220: 133–140. doi: 10.1016/S0378-1097(03)00094-6
[22]	Rao D, Webb JS, Kjelleberg S (2006) Microbial colonization and competition on the marine algae Ulva australis. Appl Env Microbiol 72: 3916–3923. doi: 10.1128/AEM.03022-05
[23]	Rossoni EMM, Gaylarde CC (2000) Comparison of sodium hypochlorite and peracetic acid as sanitising agents for stainless steel food processing surfaces using epifluorescence microscopy. Int J Food Microbiol 61:81–85. doi: 10.1016/S0168-1605(00)00369-X
[24]	Kumari S, Sarkar PK (2014) In vitro model study for biofilm formation by Bacillus cereus in dairy chilling tanks and optimization of clean-in-place (CIP) regimes using response surface methodology. Food Control 36: 153–158. doi: 10.1016/j.foodcont.2013.08.014
[25]	Harimawan A, Rajasekar A, Ting Y-P (2011) Bacteria attachment to surfaces—AFM force spectroscopy and physicochemical analyses. J Colloid Interf Sci 364: 213–218. doi: 10.1016/j.jcis.2011.08.021
[26]	Montville R, Schaffner DW (2003) Inoculum size influences bacterial cross contamination between surfaces. Appl Env Microbiol 69: 7188–7193. doi: 10.1128/AEM.69.12.7188-7193.2003
[27]	Sheen S, Hwang, C-A (2010) Mathematical modeling the cross-contamination of Escherichia coli O157:H7 on the surface of ready-to-eat meat product while slicing. Food Microbiol 27: 37–43. doi: 10.1016/j.fm.2009.07.016
[28]	Mertz AW, Koo OK, O'Bryan CA, et al. (2014) Microbial ecology of meat slicers as determined by denaturing gradient gel electrophoresis. Food Control 42: 242–247. doi: 10.1016/j.foodcont.2014.02.027

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