Biofilm and Microbial Food Safety

Biofilm and Microbial Food Safety: History

Please note this is an old version of this entry, which may differ significantly from the current revision.

Subjects: Agriculture, Dairy & Animal Science

Contributor: Sapna Chitlapilly Dass

Food-processing facilities harbor a wide diversity of microorganisms that persist and interact in multispecies biofilms, which could provide an ecological niche for pathogens to better colonize and gain tolerance against sanitization. Biofilm formation by foodborne pathogens is a serious threat to food safety and public health. Biofilms are formed in an environment through synergistic interactions within the microbial community through mutual adaptive response to their long-term coexistence. Mixed-species biofilms are more tolerant to sanitizers than single-species biofilms or their planktonic equivalents. Hence, there is a need to explore how multispecies biofilms help in protecting the foodborne pathogen from common sanitizers and disseminate biofilm cells from hotspots and contaminate food products. This knowledge will help in designing microbial interventions to mitigate foodborne pathogens in the processing environment. As the global need for safe, high-quality, and nutritious food increases, it is vital to study foodborne pathogen behavior and engineer new interventions that safeguard food from contamination with pathogens.

biofilm
microbial ecology
black queen hypothesis

. Introduction

Our understanding of the microbial world has evolved from single-species existence to highly complex and diverse microbial communities [1,2]. Previously, microbiological studies have involved studying microorganisms in axenic stetting, overlooking the fact that in many environments, microorganisms coexist [3]. With the recent shift in studying microorganisms as a mixed community, there has been a surge of research focusing on biofilms and cell–cell communication, thus sparking the importance of examining multispecies systems and their combined metabolic properties [4,5,6,7,8]. Biofilms are complex communities that are anchored to a substratum and are enveloped in an extracellular polymeric substance (EPS) matrix [9,10]. The EPS layer in the biofilm provides higher resilience to various environmental stress and resistance to antimicrobial, chemical, or sanitizer treatments [11,12]. Biofilm formation is highly variable among different microorganisms, thus adding complexity in understanding the mechanism of biofilm formation [10,13,14]. Biofilms are of high medical and economical significance, as they have been associated with chronic illness, food contamination, antibiotic tolerance, plant health, bioremediation, natural product discovery, and waste-water treatment [1,3,10,11,15,16].

In food-processing environments, biofilms have been a major cause for food spoilage associated economic losses and food safety issues leading to number of outbreaks [16,17,18,19]. An estimated 9.4 million foodborne diseases due to known pathogens are reported each year in the United States [20,21,22]. In the food industry, owing to high risk of food safety and spoilage involving the persistence of biofilm, research has been directed towards better understating of biofilm formation, interventions, and approaches to mitigate them.

4. Interactions of Microorganism in a Biofilm Community

Survival of an organism in the environment depends on its ability to sense and respond to the changes in its surroundings. Cell-to-cell communication is key for social construct of biofilm formed by different groups of microorganisms and to escape adverse environmental conditions [1,23,88]. These communications are essential for cell attachment, maturation, and detachment from biofilms [90]. The signals produced by microorganisms vary among different groups [1]. In a multispecies biofilm, how these communication signals are expressed and interpreted by other species are important in establishing a successful biofilm.

4.1. Signaling in Interspecies, Intraspecies, and Cross-Kingdom Communication

Signaling molecules are often referred to as autoinducers; these are small, diffusible molecules [88]. A number of microorganisms use diffusible signal molecules to monitor the population density and to modulate their behavior in response to their environment [90]. At low concentrations, some antibiotics also act as signaling molecules, and this may contribute to the antibiotic tolerance [90]. Cell–cell communication allows groups of microorganisms to behave in a coordinated fashion to regulate biofilm formation [91,92].

Gram-positive and -negative bacteria produce and sense small diffusible compounds called autoinducers in cell-to-cell communication. The mechanisms for producing and detecting autoinducers is referred to as quorum sensing [91]. As the name suggests, quorum sensing provides demographic information about the population level to other microbial groups nearby that senses the autoinducer. Other than population sensing, these diffusing autoinducers also provide information on spatial distribution of cells and conditions of the local environment [91]. The production of autoinducers is directly proportional to cell density, when the cell population increases, which in turn produces more of the autoinducer molecules [13,14,88,91].

In gram-positive bacteria, the cell signaling system is composed of two component systems: a membrane bound sensor kinase and cytoplasmic transcription factors [11]. Gram-negative bacteria are composed on LUX IR circuit [93]. Species–species communication allows recognition of self in a mixed population and also a mechanism to sense other bacteria [11]. Gram-positive and gram-negative bacteria communicate with by producing a signal molecule AI-2, which is common in both [16]. The signal molecule facilitates interspecies communication to sense each other’s presence and enable the biofilm formation.

Cross-kingdom cell signaling occurs between both prokaryotes and eukaryotes. The signaling involves small molecules, such as hormones by eukaryotes and hormones similar to chemicals produced by bacteria [13,94]. The cross-kingdom communication helps in establishing biofilms in animal and plant tissues. In food-processing facilities, the food matrix can be of both plant and animal origin and introduction of biofilms through their tissues can be a high food safety risk [12,13,93,95].

4.2. Metabolic Interactions

Microbial communities in the biofilm possess a combined metabolic activity or obligatory mutualistic metabolism shared among all the species [37]. Metabolic interactions among microorganisms leads to the success of the biofilm [96]. These interactions contribute to division of labor among the different groups and lead to an increased virulence [11]. Combined metabolic activity of microorganisms enable a microbial community to survive with minimal energy resources [11]. Successful establishment of multispecies biofilm can result from an association between metabolically cooperative organisms that facilitates interspecies substrate exchange and the removal or distribution of metabolic products [91,93]. Biofilms provide an ideal environment for the establishment of obligatory mutualistic metabolism or syntrophic relationships [10]. Syntrophism is a special case of symbiosis in which two metabolically distinct types of bacteria depend on each other to utilize certain substrates, typically for energy production [10,93].

4.3. Enhanced Biofilm Formation through Microbial Interactions

Synergistic interactions can enhance the biofilm formation. In a study carried out by Bharathi et al., 2011 [93], when four poor biofilm formers were cocultured, it enhanced the potential to form strong multispecies biofilms [97]. Metabolic interactions that enhanced coaggregations and organized spatial distribution could be responsible for the shift from weak biofilm formation to strong biofilm formers [2,98,99]. Species that do not form biofilms as single strains may benefit from the advantages associated with biofilm formation, including enhanced protection from external stress and expanded niche availability, through engagement with multispecies communities [78,99].

Enhanced biofilm formation and protection to less tolerant species with low ability to form biofilms has been observed in multispecies biofilms [78,91,100]. This is achieved by cooperative behavior between bacterial communities rather than competition among them. With the higher the number of species, the complexity of interactions increases. The cooperation in a multispecies biofilm is explained in the “Black Queen Hypothesis” [101]. This hypothesis considers cooperation in complex bacterial communities as being a consequence of species adapting to the presence of each other [11,78]. Some species in the complex community delete vital function or pathways that are provided by the surrounding bacteria, leading to communal dependency [2,88] and thus leading to irreversible commitment to coexistence [102].

2. Biofilm Community and Genetic Element Exchange

Bacterial coexistence in natural environment could lead to the development of mixed biofilms that constitute a reservoir to facility exchanges of genetic materials within the biofilm community via physical contact. Higher levels of horizontal gene transfer promoted within the biofilm community have been demonstrated in laboratories [115,116], and enhanced biofilm formation as a result of genetic material transfer has been observed as well. For instance, conjugative transfer of the F-like plasmid R1drd19 in E. coli strains [117] and transfer of plasmid RP4 between Pseudomonas species [118] were both shown to occur at significantly higher frequencies in biofilms. Studies [119,120] using E. coli systems also demonstrated horizontal transfer of non-conjugative plasmids and genetic elements. On the other hand, transmission of conjugative plasmids R1drd19 [121] and pMAS2027/pOLA52 [122] could induce greater biofilm development in E. coli cultures through pili and type 3 fimbriae synthesis, respectively. More importantly, it has been shown that the E. coli plasmid pOLA52 could retain its ability to induce biofilm development after it was transferred to other bacterial species, including Salmonella Typhimurium strains [123]. Transformation of non-conjugative plasmids pET28 and pUC8 could also increase biofilm cell growth in E. coli cultures [124].

In addition to facilitate plasmid transfer, biofilm development could also affect plasmid copy-number control, which has been investigated with plasmids pBR322 and pCF10. The copy-numbers of the two plasmids were both increased in biofilms compared to the planktonic cells in E. coli [125] and E. faecalis [126] cultures, respectively. The higher copy number of the plasmid pBR322, which encoded antibiotic resistance genes against ampicillin and tetracycline, was also correlated with stronger antibiotic resistance phenotype, as the presence of sub-lethal concentrations of the antibiotic increased the copy number of the plasmid [125]. A recent report [107] showed that compared to the diversity control panel strains, HEP E. coli O157:H7 strains overall retained significantly higher copy number of the pO157 plasmid, and a positive correlation was observed among the high plasmid copy number, strong biofilm forming ability, low sanitizer susceptibility, and high survival/recovery capability of the biofilm cells after sanitization. Since the highly conserved pO157 plasmid has been associated with biofilm formation and bacterial optimal survival/persistence in the environment [108,109,110], the high copy number of the pO157 plasmid might therefore constitute the genetic basis for the strong biofilm forming ability and high sanitizer tolerance of these HEP E. coli O157:H7 strains that pose stronger survival capability and higher potential of causing contamination at the meat plants.

Biofilm promoted horizontal gene transfer that might potentially lead to dissemination of antibiotic resistance determinants is another concern to food safety and public health. Increased transfer of multidrug resistance plasmids has been shown in E. coli [127,128] and Staphylococcus aureus biofilms [115]. Resistance against oxyimino-cephalosporins encoded by the bla CTX-M genes was investigated in E. coli, K. pneumoniae, and E. cloacae, and results also showed that the transfer frequency of the bla CTX-M genes was higher in strains at their biofilm stage than those at planktonic state [128]. In addition, it was reported that the natural blaNDM-1 plasmids could be successfully transferred from E. coli trans-conjugants to strong biofilm formers of P. aeruginosa and A. baumannii in dual-species biofilms, demonstrating the potential spread of the blaNDM-1 carbapenemase gene conferring resistance to carbapenem antibiotics within the multispecies biofilm community [129].

It is worthy to note that experiments performed under laboratory settings and longitudinal studies conducted at real commercial/industry environment involving animal population movements and antimicrobial usage over time may provide different observations. For instance, transfer of bla CMY-2-carrying plasmids conferring cephalosporins resistance was observed from E. coli donor strains to Serratia marcescens strains in biofilms, and the recipient strains with the resistant phenotype as a result of bla CMY-2 acquisition further acted as secondary plasmid donors [127]. However recently, a 24-month longitudinal study [130] reported that the presence of third-generation cephalosporins (3GC)-resistant Salmonella in commercial cattle feed yards was driven by the persistent pathogen subtypes instead of actively acquiring and maintaining the bla genes from the more frequently isolated 3GC-resistant E. coli, which has been suggested as the reservoir of 3GC resistance. Such observation contradicts the traditional reservoir theory, which was based on the isolation of Salmonella and E. coli harboring the IncA/C2 plasmids with similar genetic structures, including the bla CMY-2 gene, that may facilitate horizontal gene transfer between the two bacterial species. These various findings brought additional dimension to biofilm research concerning genetic material exchange under real life conditions because critical information, such as bacterial species, plasmid replicon profile, presence/location of antibiotic resistant genes, as well as evolutionary relationships, should be all taken into consideration when investigating biofilm related dissemination and persistence of genetic elements.

3. Conclusions

In the food-processing environment, the coexistence of multiple bacterial species profoundly affects biofilm structure, composition, and, more importantly, the tolerance levels of the biofilm cells to sanitizers and other antimicrobial interventions. Understanding how foodborne pathogens are protected and released into the food-processing environment from biofilms will lead to new knowledge on sanitizer tolerance and recurrent contamination. Studies related to foodborne pathogen or microbial interventions on biofilms are mostly designed on single-species biofilms or planktonic cells in axenic settings, overlooking the fact that in most environments, microorganisms coexist. Mixed-species biofilms are more tolerant to sanitizers than single-species biofilms or their planktonic equivalents. Hence, there is a need to explore how multispecies biofilms help in protecting the foodborne pathogen from common sanitizers and disseminate biofilm cells from hotspots and contaminated food products. This knowledge will help in designing microbial interventions to mitigate foodborne pathogens in the processing environment. As the global need for safe, high-quality, and nutritious food increases, it is vital to study foodborne pathogen behavior in a multi-partite interaction and engineer new interventions that safeguard food from contamination with pathogens.

This entry is adapted from the peer-reviewed paper 10.3390/pathogens11030346

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