The objective of this study was to standardize a PVC microtiter plate assay to compare the ability of 31 coded L. monocytogenes strains to form biofilms. Cellular growth rates and final cell density did not correlate with biofilm formation after 40 h at 32°C, indicating that differences in biofilm formation were due to factors other than the ability to grow in MWB (Fig. ). The majority of lineage I strains fell within the upper end of biofilm formation, and overall this lineage had significantly greater biofilm formation than lineage II and lineage III strains. In addition, L. monocytogenes biofilm formation on PVC and stainless steel was analyzed by quantitative epifluorescence microscopy and results were compared to the PVC microtiter plate biofilm assay. Similar trends in biofilm formation were observed using the microscopic and the microtiter plate assays, indicating that the PVC microtiter plate assay may be used as a rapid, simple method to assess biofilm production.
strains were observed to differ in the amount of biofilm produced at 40 h (Fig. ), and the microtiter plate assay appeared to detect differences in biofilm-forming ability among L. monocytogenes
strains. It has been suggested that motility and flagella formation can play a role in initial phases of biofilm formation (21
). In this study, initial cell attachment was not studied and biofilm formation was examined over a longer time period (40 h), so that possible flagella formation and its influence in the early stages of cell attachment (30
) could be excluded. However, motility was observed for all strains after 40 h of incubation at 32°C in MWA.
It is important to note that the assay presented here measured biofilm production under a minimal nutrient environment. L. monocytogenes
growing in a food-processing environment may be exposed to fluctuating levels of nutrients, depending upon location in the plant. For example, populations existing in floor drains will be exposed to high levels of nutrients during prerinsing of equipment prior to cleaning and sanitizing. On the other hand, if living on walls, ceilings, or within condensate (on the outside of cooling equipment or pipes), it is likely that these bacteria are surviving under reduced nutrient conditions. Although this research has focused upon biofilms of pure L. monocytogenes
, in the food-processing environment they most likely exist as a member of a complex bacterial community consisting of many different types of bacteria. This is supported by the research of Sasahara and Zottola in which higher numbers of L. monocytogenes
ScottA were observed in biofilms when cocultured with Pseudomonas fragi
). If growing under low nutrient conditions in the processing plant, L. monocytogenes
could possibly use other environmental bacteria as a source for the seven essential amino acids needed for growth.
After all testing, L. monocytogenes
strains were decoded into three lineages (Table ) (19
) and arranged from the highest to the lowest biofilm producers (Fig. ). It was observed that most of the lineage I strains (Table ) were at the upper end of the continuum, with the exception of strains LM24 and LM25 (Fig. ). In general, lineage II and III strains had lower destained biofilm values (Fig. ). Using a one-way analysis of variance for statistical analysis of the mean, biofilm production by lineage I strains was found to be higher and statistically different from biofilm formation of lineage II and lineage III strains (Table ). According to Wiedmann et al. (31
), lineage I contains all food-borne epidemic strains and isolates from sporadic cases among humans and animals (Table ). Lineage II contains both human and animal isolates, and lineage III contains strains isolated from animals only (19
). We thus hypothesize that the ability of lineage I strains to form biofilms may contribute to their prevalence among humans. Lineage I strains may represent an evolutionarily distinct group of L. monocytogenes
, and the increased biofilm formation of isolates in this lineage could be due to the presence or absence of specific genes or by lineage-specific allelic variations. The presence or absence of specific genes within subtypes of the same species has been observed within group A streptococci (26
Biofilm formation assessed by microtiter plate assay was compared to biofilm formation assessed by quantitative epifluorescence microscopy. The microtiter plate biofilm assay revealed greater differences in biofilm production than did the microscopy biofilm assay. In general, though, trends observed with the microtiter plate assay were also observed with the microscopic analysis on both PVC and stainless steel, although differences were not always statistically significant in the microscopic analysis.
Microscopic analysis indicated that biofilm coverage was higher on PVC slides for all strains tested than on stainless steel slides (Fig. ). This may be due to differences in surface hydrophobicity compared to cellular hydrophobicity when grown under low nutrient environments; however, others have found L. monocytogenes
LO28 to be negatively charged and hydrophilic in nature when grown in a defined medium at various temperatures (8
). Beresford et al. (3
) observed that L. monocytogenes
attachment was higher on PVC than on stainless steel after a short contact time and after 2 h of incubation, but listerial attachment was not significantly different between these two materials (3
). Helke et al. (11
) and Sommer et al. (28
) proposed that the interactions between bacterial cells and an inorganic surface are different for adhesion onto hydrophobic or hydrophilic surfaces. Besides material properties, it has been observed that bacterial characteristics such as charge, hydrophobicity, and surface appendages can enhance the adhesion process as well (6
). It has been reported that factors other than hydrophobic interactions, such as electrostatic and exopolymer interactions, account for the attachment of L. monocytogenes
to various materials (4
). Moreover, Sommer et al. (28
) showed that cell wall hydrophobicity may be closely related to adhesion on inert substrata, either to hydrophobic or hydrophilic surfaces. For hydrophobic surfaces such as PVC, hydrophobic interactions are considered the main forces and, thus, cell wall hydrophobicity of strains plays a major role (28
). For hydrophilic surfaces such as stainless steel, electrostatic interactions are considered dominant and the charge of the bacterial cell wall is more important than other factors (28
). Since PVC and stainless steel differ in hydrophobicity, we believe that adhesion on PVC may be higher due to its hydrophobic nature compared to stainless steel.
When comparing the microtiter plate assay with the quantitative microscopic biofilm assay, it is important to consider the inherent advantages and disadvantages of each assay. The major advantage of microscopic analysis is the ability to observe the biofilm directly. The microscopic method, on the other hand, is relatively time-consuming compared to the microtiter plate assay. Blackman and Frank (4
) also reported that epifluorescence microscopy may overestimate the area covered by cells, since some extracellular polymer is stained as well (4
). A possibility for bias in selecting fields for analysis and usage of the program for microscopy data analysis (NIH Image software was used for calculation of the percentage of area covered by cells) could result in a human error by over- or underestimation of the percentage of area covered. In addition, the magnification for determining surface coverage should be considered. We used high magnification (×1,000) since fluorescence was brightest but, ideally, surface coverage should be observed at lower magnification to obtain a larger field. In addition, the surface areas examined by each method are different: in the microtiter plate assay the whole individual well area covered by cells (approximately 130 mm2
) is evaluated, while the area of the 50 fields examined by microscopy represents only about 0.135 mm2
The microtiter plate has the advantage of enabling researchers to rapidly analyze adhesion of multiple bacterial strains or growth conditions within each experiment. The major disadvantage is that the microtiter plate method is an indirect indication of the level of biofilm produced; the adsorption of the crystal violet in the destaining solution was used as an indication of the amount of biofilm. In order to assure consistency between experiments, the manufacturer's lot numbers of the PVC plates were monitored, since researchers have reported dramatic differences in adhesion between different lots of PVC (1
). In addition, care was taken to thoroughly dry films before staining and to adhere to consistent timing of staining and destaining steps. Despite the shortfall as an indirect method, we found more consistent results (lower standard deviations between replicates within an experiment and between experiments) with the microtiter plate assay than with direct microscopy.
The results of the microtiter plate assay had a strong correlation to microscopic coverage on PVC. Although the microscopic assay showed greater coverage on PVC than on stainless steel, a significant correlation (P < 0.05) was observed between both materials. Direct observation will always be very important to studying adhesive cells and biofilms, but we believe that the microtiter plate assay can be used in conjugation with microscopy as a rapid and reproducible screening method. This assay will allow researchers to rapidly screen for differences between strains or growth conditions prior to performing labor-intensive microscopic quantification.