Article

Formation and resistance to cleaning of biofilms at air-liquid-wall interface. Influence of bacterial strain and material

Authors:
  • French National Institute for Agricultural Research, France, Lille
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Abstract

Interfaces between air, liquid and walls (ALW interfaces) are known to be conducive to the formation of biofilms, at least in some bacteria, yet little information is available on the influence of material properties on the amount of biofilms formed and their resistance to a cleaning procedure. In this study, we investigated the ability of four bacterial strains (Pseudomonas fluorescens [Pf1], Escherichia coli [Ec-SS2], Bacillus cereus [Bc-98/4] and B. subtilis [Bs-PY79]) to form biofilms in static conditions at the ALW interface on four materials with very different topographic and hydrophilic/hydrophobic properties (stainless steels with 2R or 2B finishes, polypropylene and glass). Biofilms were observed after staining with orange acridine visually, by epifluorescence microscopy and by confocal scanner laser microscopy. The number of culturable cells within biofilms was also estimated after growth on agar. After one-day of incubation in a bacterial suspension, three strains (except Bc-PY79) were found to form large amounts of biofilm, easily observable to the naked eye. However, great differences were observed between strains in the number of CFU (between 4.7 and 7.4 log CFU cm⁻²), as well in the biofilm structure. Furthermore, the material also affected the amount and/or structure of biofilms, and a 3D-biofilm organisation was only observed for two of the four tested strains (Bc-98/4 and Pf1) on PP, a hydrophobic material. After a standard cleaning-in-place treatment involving NaOH 0.5% at 60 °C, cultivable cells were only detected from Bc-98/4 biofilms (growth on agar), while biofilms were also still visible on coupons contaminated with Pf1. Furthermore, most residual biofilms after cleaning appeared orange by epifluorescence microscopy after staining with orange acridine suggesting the presence of many viable but non-culturable cells within the residual biofilms. In Bc-98/4 biofilms, spores were also clearly observed by epifluorescence microscopy. Knowing their ability to survive the conditions encountered during cleaning procedures, this could account for the high level of CFU enumerated after cleaning. Lastly, Bc-98/4 biofilms formed on stainless steel 2R were more resistant to cleaning than on PP and glass. All of these results highlighted the importance of biofilms at the ALW interfaces in the control of surface hygiene, particularly in the food industry. We then investigated whether the shape of the menisci at the interfaces (convex vs concave, kinetics over time) could at least partly explain the shape or even the resistance to detachment of the ALW biofilms.

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... their structure or their resistance to rinsing and/or cleaning procedures. These include bacteria (Faille et al., 2014), flow arrangements (Bouvier et al., 2021), material's surface properties , or position of the surface within the processing line such as in fully submerged areas vs. at an air-liquid-wall interface (Jha et al., 2020). ...
... The other three materials are found in food environments: AISI 316 stainless steel with pickled (2B) and bright annealed (2R) finishes (kindly provided by APERAM, Isbergues, France), and polypropylene (PP) (API Plastiques, Brenelles, France). Prior to each experiment, the coupons were cleaned and disinfected according to a previously described procedure (Jha et al., 2020). Briefly, coupons were cleaned using an alkaline detergent RBS T105 (Traitements Chimiques des Surfaces, France). ...
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In the food industry, the control of surface hygiene is a major issue. It is therefore essential to identify those parameters that can affect the bacterial contamination of surfaces and the effectiveness of hygiene procedures. Numerous studies have focused on the role of bacteria, flow arrangements or materials, but almost nothing has been reported on the possible impact of vertical or horizontal positioning of surfaces on bacterial contamination. The aim of the current study was firstly to determine the ability of bacterial species usually found in food processing lines to form biofilms on surfaces positioned vertically or horizontally and then to assess the resistance of these biofilms to detachment. The experiments were carried out using three bacterial strains (Escherichia coli SS2, Bacillus cereus 98/4, and Pseudomonas fluorescens Pf1) that produced biofilms on glass, polypropylene and stainless steel (surface finishes 2B and 2R). We first observed that not only did the bacterial strain type impacts its ability to form biofilms (Ec-SS2 > Pf1 > Bc-98/4), but that the vertical vs horizontal position of the surface would also affect biofilm formation, probably due to the accumulation through sedimentation of bacteria on horizontal surfaces. However, the horizontally formed Pf1 biofilms were very fragile and could be partially removed by a gentle rinsing step. Lastly, no significant differences could be found in the ability to form biofilms on the different materials. The resistance to detachment to a standard rinsing process in a pilot rig was also investigated. While both strains and materials significantly affected the amount of biofilm detached, only Bc-98/4 biofilms were impacted by the surface position, with horizontal biofilms showing extreme resistance to shear forces. In conclusion, this study shows that horizontal surfaces in food environments probably represent an increased risk of contamination by bacteria frequently isolated from these environments and should be subjected to increased monitoring.
... Indeed, it is largely observed that thick biofilms often preferentially develop at the air-liquid-substratum interface, rather than in wholly submerged areas. Recently, the same P. fluorescens strain (Jha et al., 2020) was used and compared to three other strains E. coli, Bacillus cereus, and Bacillus subtilis to form biofilms in static conditions at the ALW interface on four materials with very different topographic and hydrophilic/hydrophobic properties (stainless steels with 2R or 2B finishes, polypropylene and glass). After one day of incubation in a bacterial suspension, all but B. cereus was found to form large amounts of biofilm, easily observable to the naked eye with a 3D structure at the interface corresponding to the meniscus area. ...
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... of antifouling materials are more commonly focused on the fouling of fully submerged substrates. [9] Accordingly, less is known about specific design principles that operate well at the triple interface beyond what is already known about the solid-liquid interface. Despite their exceptional antifouling performance in submerged applications ranging from medical device coatings to reverse osmosis membranes, [10,11] zwitterionic polymers incur considerable growth of P. aeruginosa biofilm at the three-phase contact line. ...
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Biofilms are known to play important roles in bacterial survival and persistence in food-processing environments. This study aimed to determine the ability of the top 7 STEC serotypes to form biofilms on polystyrene (POL) and stainless steel (SS) plates and to quantify their survival and transfer from dry-surface biofilms to lettuce pieces. The ability of 14 STEC strains to form biofilms on these two materials at different exposure times and temperatures was assessed using crystal violet, Congo red and SEM. At 10 °C all serotypes were weak biofilm producers on both surfaces. In contrast, serotypes O45-040, O45-445, O103-102, O103-670 and O157-R508 were strong biofilm producers at 25 °C. Strains O103-102, O103-670, O111-CFS, O111-053 and O157:H7-R508 were expressers of curli. Under scanning electron microscopy, strains O103-670, O111-CFS, O157-R508, and O121-083 formed more discernible multilayer, mature biofilms on SS coupons. Regardless of the surface (POL/SS), all STEC strains were able to transfer viable cells onto fresh lettuce within a short contact time (2 min) to varying degrees (up to 6.35 log cfu/g). On POL, viable cell of almost all serotypes exhibited decreased detachment (p = 0.001) over 6 days; while after 30 days on SS, serotypes O45-040, O103-102, O103-670, O111-053, O111-CFS, O121-083, O145-231 O157:H7-R508 and O157:H7-122 were transferred to lettuce. After enrichment, all 14 STEC strains were recovered from dry-surface biofilms on POL and SS plates after 30 days. Results demonstrated that the top 7 STEC remained viable within dry-surface biofilms for at least 30 days, transferring to lettuce within 2 min of exposure and acting as a source of adulteration.
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Public authorities, chain stakeholders and consumers are all concerned by microbial food safety. Microbiological hazards are one of the most common causes of food poisoning that has been considered for many years but still on the track nowadays considering the recent foodborne disease outbreaks largely reported by the media. Microbial contamination origins are diverse from the field to the plate e.g. soil, air, equipment surfaces, packaging material and staff. Firstly, this article highlights the ways in which the choice of materials plays a major role in surface hygiene. Hydrodynamic conditions directly linked to the equipment geometry are focused on, as is the role played by surfaces in contact with air in surface drying. Surface environmental conditions during processing or cleaning are discussed and new proposals described. Better knowledge of surface contamination and cleaning mechanisms would positively impact hygienic design principles, thereby mitigating any environmental impact of the cleaning operations in the food and beverage industries: new strategies are therefore proposed. Industrial relevance Hygienic design of food processing equipment is nowadays considered to be mandatory in the reduction of the risk microbial food contamination. The presentation of potential roles of materials on the remaining bacterial soil, after soiling and cleaning, provide new insights when envisaging any hygienic improvements. Equipment design plays a major role in contamination trapping or in the ease of removal via the flow arrangements, notably during cleaning and rinsing operations. More detailed knowledge of the flow mechanical actions combined with the unavoidable chemical action of the detergents would allow industries to go further in enhancing certain “good” phenomena, such as pulsed flow conditions. Last but not least are those possibilities offered by a hygienically designed processing line, in the quest for environmental impact mitigation of cleaning operations i.e. drinking water and energy reductions, or the use of new “green” mild detergents.
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The objective of this study was to investigate the roles of mechanical and chemical actions on the detachment kinetics during CIP of biofilms of Pseudomonas fluorescens at a pilot-plant scale.Biofilms were grown 48 h at 20 °C in diluted milk. Shear stress conditions ranged from 0 (static) to 20 Pa and NaOH concentrations from 0.1 to 0.5% w/w. A simple two-phase model was applied to model the removal kinetics and compare detachment parameters.A quick removal of the biofilm during the first phase (4-5 log decrease in three minutes) was followed by a second phase of slow removal of the cells and small aggregates still present at the surface (0-2 log decrease). Hydrodynamics was responsible for removal of both biofilm and single cells while chemicals mainly disrupted biofilm clusters during the first phase. No complete biofilm removal was observed, suggesting a significant role of the interaction forces between bacteria and substrata in the CIP efficiency.
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Escherichia coli typically colonizes food contact surfaces in the presence of other bacterial strains. The aim of this work was to evaluate the influence of a resident strain isolated from a fresh-cut salad industry (Pseudomonas grimontii 13A10) on the development of a model pathogen (E. coli) on bare stainless steel (SST) and stainless steel coated with diamond-like carbon (DLC) films, a-C:H:Si:O designated by SICON® and a-C:H:Si designated by SICAN. The bacterial composition and spatial organization of single- and dual-species biofilms were analyzed by confocal laser scanning microscopy (CLSM). Biofilms were developed for 1 and 3 days at 10 °C and it was observed that the biovolume of E. coli biofilms in the presence of P. grimontii was lower than in axenic conditions, suggesting that the isolate can protect food contact surfaces from pathogen colonization. After 3 days, the dual-species biofilms contained essentially P. grimontii cells and no preferential vertical distribution of bacterial strains was observed. The use of a-C:H:Si:O coated surfaces reduced the short-term colonization of the model pathogen in single- and dual-species biofilms, whereas decreased colonization by the non-pathogenic strain was only observed after 3 days. Free access to the final version: https://authors.elsevier.com/a/1VWiV5O41LDNy
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The importance and challenges presented by Bacillus spores in the food industry are briefly outlined with a focus on Bacillus cereus. The structure and the mechanism of resistance exhibited by Bacillus spores are described, and the steps involved in their germination are included. Novel technologies, using no or only mild heat treatments, to inactivate Bacillus spores are covered, including ultraviolet radiation, pulsed electric field, and high-pressure processing, both as stand-alone techniques or techniques as part of a hurdle approach.
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In various environments, including that of food processing, adherent bacteria are often subjected to drying conditions. These conditions have been shown to result in changes in the ability of biofilms to cross-contaminate food in contact with them. In this study, we investigated the consequences of a drying step on the further ability of adherent bacterial spores to resist detachment. An initial series of experiment was set up with latex microspheres as a model. A microsphere suspension was deposited on a glass slide and incubated at 25, 35 and 50 °C for times ranging from 1 h to 48 h. By subjecting the dried slides to increasing water flow rates, we showed that both time and temperature affected the ease of microsphere detachment. Similar observations were made for three Bacillus spores despite differences in their surface properties, especially regarding their surface physicochemistry. The differences in ease of adherent spore detachment could not be clearly linked to the minor changes in spore morphology, observed after drying in various environmental conditions. In order to explain the increased interaction between spheres or spores and glass slides, the authors made several assumptions regarding the possible underlying mechanisms: the shape of the liquid bridge between the sphere and the substratum, which is greatly influenced by the hydrophilic/hydrophobic characters of both surfaces; the accumulation of soil at the liquid/air interface; the presence of trapped nano-bubbles around and/or under the sphere.
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This study investigated the effect of material types (polystyrene, polypropylene, glass, and stainless steel) and glucose addition on Staphylococcus aureus biofilm formation, and the relationship between biofilm formation measured by crystal violet (CV) staining and the number of biofilm cells determined by cell counts was studied. We also evaluated the efficacy of chlorine sanitizer on inhibiting various different types of S. aureus biofilms on the surface of stainless steel. Levels of biofilm formation of S. aureus were higher on hydrophilic surfaces (glass and stainless steel) than on hydrophobic surfaces (polypropylene and polystyrene). With the exception of biofilm formed on glass, the addition of glucose in broth significantly increased the biofilm formation of S. aureus on all surfaces and for all tested strains (P ≤ 0.05). The number of biofilm cells was not correlated with the biomass of the biofilms determined using the CV staining method. The efficacy of chlorine sanitizer against biofilm of S. aureus was not significantly different depending on types of biofilm (P > 0.05). Therefore, further studies are needed in order to determine an accurate method quantifying levels of bacterial biofilm and to evaluate the resistance of bacterial biofilm on the material surface. Portuguese Biofilm formation of Staphylococcus aureus on the surface was different depending on the surface characteristics and S. aureus strains. There was low correlation between crystal violet staining method and viable counts technique for measuring levels of biofilm formation of S. aureus on the surfaces. These results could provide helpful information for finding and understanding the quantification method and resistance of bacterial biofilm on the surface.
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Environmental conditions and growth history can affect the sporulation process as well as subsequent properties of formed spores. The sporulation dynamics was studied in wet and air-dried biofilms formed on stainless steel (SS) and polystyrene (PS) for Bacillus cereus ATCC 10987 and the undomesticated food isolate B. cereus NIZO 4080. After harvesting and maturation, the wet heat resistance of spores obtained from these biofilms was tested and compared to planktonic and agar plate-derived spores. Drying/air exposure of the preformed 24 h old biofilms accelerated spore formation for both strains and resulted in higher final spore percentages. Prolonged dry incubation of more than three days triggered germination of spores in the biofilms of ATCC 10987. Spores harvested from wet biofilms on SS displayed the highest heat resistance compared to liquid, agar plate and PS biofilm derived spores. The D95 °C values for these spores were 17 and 22 min for NIZO 4080 and ATCC 10987, respectively, which was 2 and 1.2 fold higher compared to planktonic spores of these strains. Spores obtained from dried biofilms of ATCC 10987 displayed reduced heat resistance compared to wet biofilm spores. The results indicate that environmental conditions encountered by biofilms affect sporulation dynamics and spore heat resistance, thus affecting subsequent quality issues and safety risks related to these biofilms.
Article
Biofilm formation of Bacillus cereus reference strains ATCC 14579 and ATCC 10987 and 21 undomesticated food isolates was studied on polystyrene and stainless steel as contact surfaces. For all strains, the biofilm forming capacity was significantly enhanced when in contact with stainless steel (SS) as a surface as compared to polystyrene (PS). For a selection of strains, the total CFU and spore counts in biofilms were determined and showed a good correlation between CFU counts and total biomass of these biofilms. Sporulation was favoured in the biofilm over the planktonic state. To substantiate whether iron availability could affect B. cereus biofilm formation, the free iron availability was varied in BHI by either the addition of FeCl3 or by depletion of iron with the scavenger 2,2-Bipyridine. Addition of iron resulted in increased air-liquid interface biofilm on polystyrene but not on SS for strain ATCC 10987, while the presence of Bipyridine reduced biofilm formation for both materials. Biofilm formation was restored when excess FeCl3 was added in combination with the scavenger. Further validation of the iron effect for all 23 strains in microtiter plate showed that fourteen strains (including ATCC10987) formed a biofilm on PS. For eight of these strains biofilm formation was enhanced in the presence of added iron and for eleven strains it was reduced when free iron was scavenged. Our results show that stainless steel as a contact material provides more favourable conditions for B. cereus biofilm formation and maturation compared to polystyrene. This effect could possibly be linked to iron availability as we show that free iron availability affects B. cereus biofilm formation. Copyright © 2015 Elsevier B.V. All rights reserved.
Article
The Hydrodynamic conditions as well as design and surface properties within fresh-cut food processing equipment create a complex environment for biofilms. A new experimental approach was thus proposed to identify those physical parameters impacting biofilm development in such conditions. A set-up comprising original mock-ups mimicking generic features of washing tanks (e.g. welds, folds, flat surfaces, air/liquid/wall interface) was designed. The flow pattern therein was characterized using two computational fluid dynamic calculation approaches. Full trials were run for 48 h at 10 °C with a Pseudomonas fluorescens strain to identify the preferential biofilm formation areas. As in current industrial systems, the pilot rig had recirculation areas and low wall shear stress rates (τw < 0.1 Pa) in corners and angles. These were identified as critical areas with Surface Microbial Loads (SML) over 5 Log10/cm2. However, τw alone failed to explain why SML in areas under unidirectional flow was higher than in the mock-ups. Lastly, air/liquid/wall interface conditions were more critical than immersed surfaces. This study validated the possibility of using CFD methods to understand the way in which flow pattern influences biofilm formation. The methodology proposed would be helpful in quantifying equipment components criticality based on biofilm growth parameters.
Article
Bacillus strains are often isolated from biofilms in the food industries. Previous works have demonstrated that sporulation could occur in biofilms, suggesting that biofilms would be a significant source of food contamination with spores. In this study, we investigated the properties of mono-species and mixed Bacillus biofilms and the ability of Bacillus strains to sporulate inside biofilms. Bacillus strains were able to form mono-species biofilms on stainless steel coupons, with up to 90% spores after a 48 h-incubation. These spores were highly resistant to cleaning but were easily transferred to agar, mimicking the cross-contamination of food, thereby suggesting that biofilms would be of particular concern due to a potential for Bacillus spore food contamination. This hypothesis was strengthened by the fact that Bacillus strains were able to form mixed biofilms with resident strains and that sporulation still occurred easily in these complex structures.
Article
We investigated the effects of cleaning-in-place (CIP) cleaning agents, food additives and other compounds (EDTA, Tween20 and SDS) on the removal of Staphylococcus aureus and Escherichia coli biofilm from stainless steel plates. S. aureus formed thick biofilm and was more resistant to cleaning than E. coli biofilm. Strong acidic and strong alkaline CIP cleaning agents showed a significant cleaning effect. Strong alkaline CIP cleaning agents were especially effective for the removal of S. aureus biofilm.
Article
There is increasing evidence for a viable but non-culturable (VBNC) state in microbes, particularly in the stressing environment presented by modern foods with their varied pre-treatment and packaging strategies. This is a cause for concern because of evidence that microbial pathogens in such a state may retain their capacity to cause infections after ingestion by the consumer despite their inability to grow under the conditions employed in procedures for determining their presence in foods. Heavily stressed pathogenic species of bacteria in a VBNC or not immediately culturable state are potentially dangerous public health problems, particularly as stressed cells may be more virulent than well-fed bacteria. In this viewpoint article, I wish to focus on possible procedures for detecting such organisms and assessing their physiological state.
Article
This study was designed to evaluate how conditions encountered by spores during cleaning-in-place (CIP) procedures affected their surface properties, their viability and ability to contaminate materials. Spores from five Bacillus cereus strains were treated with NaOH at high temperature. Results revealed that high temperatures (exceeding 60 degrees C) and NaOH concentrations (over 0.5%) were required to significantly decrease spore viability (3-5log decrease). In these conditions, modifications were also clearly observed by microscopy to various surface structures of spores (appendages, exosporium, and especially to the hair-like nap) but also to their coat. Therefore, the ability of culturable spores to adhere decreased for the majority of strains tested. We then demonstrated that spores in suspension in NaOH could adhere to surfaces of a CIP rig and that the contamination level was controlled by flow pattern. Consequently, re-adhesion along the processing line might occur during CIP procedures and this phenomenon must be taken into account when defining cleaning strategies.
Article
Enteropathogenic Escherichia coli (EPEC) is an important causal agent of diarrheal illness throughout the world. Nevertheless, researchers have only recently begun to explore its capacity to form biofilms. Strain O55:H7 (DMS9) is a clinical isolate belonging to the atypical EPEC (aEPEC) group, which displays a high degree of genetic relatedness to enterohemorrhagic E. coli. Strain DMS9 formed a robust biofilm on an abiotic surface at 26 degrees C, but not at 37 degrees C. It also formed a dense pellicle at the air-liquid interface and developed a red, rough, and dry (RDAR) morphotype on Congo red agar. Unlike a previously described E. coli O157:H7 strain, the aEPEC strain seems to express cellulose. Transposon mutagenesis was used to identify biofilm-deficient mutants. One of the mutants was inactivated in the csgFG genes, required for assembly and secretion of curli fimbriae, while a second mutant had a mutation in crl, a thermosensitive global regulator that modulates sigma(S) activity and downstream expression of curli and cellulose. The two mutants were deficient in their biofilm formation capabilities and did not form a pellicle at the air-liquid interface. Unlike in Salmonella, the csgFG mutant in aEPEC completely lost the RDAR phenotype, while the crl mutant displayed a unique RDAR "pizza"-like morphotype. Genetic complementation of the two mutants resulted in restoration of the wild-type phenotype. This report is the first to describe and analyze a multicellular behavior in aEPEC and support a major role for curli and the crl regulator in biofilm development at low temperatures corresponding to the nonmammalian host environment.
Article
The ability of Campylobacter jejuni strains to attach to stainless steel as they became nonculturable during storage in distilled water at 4 degrees C for 30 days was investigated. From an initial count of approximately 7 log colony-forming units/mL all strains completely lost culturability by day 20, but the numbers of cells attaching to stainless steel remained constant at approximately 3.5 log cells/cm(2). These findings suggest that viable but nonculturable Campylobacter in a liquid matrix can still attach to stainless steel.
Article
Biofilm formation is a long-standing problem in ultrapure water and bioprocess fluid transport lines. The standard materials used in these applications (316L stainless steel, polypropylene and glass) have long been known to be good surfaces for the attachment of bacteria and other biological materials. To compare the relative tenacity of biofilms grown on materials used in manufacturing processes, a model system for biofilm attachment was constructed that approximates the conditions in industrial process systems. New fluorinated polymers were compared to the above materials by evaluating the surface area coverage of bacterial populations on materials before and after mild chemical treatment. In addition, contact angle studies compared the relative hydrophobicity of surfaces to suspensions of bacteria in growth media, and scanning electron microscopy and atomic force microscopy studies were used to characterize surface smoothness and surface defects. Biofilm adherence to polymer-based substrata was determined to be a function of both surface finish and surface chemistry. Specifically, materials that are less chemically reactive, as indicated by higher contact angle, can have rougher surface finishes and still be amenable to biofilm removal.
Article
The regulatory programme of multicellular behaviour in Salmonella typhimurium is determined by mutations in the agfD promoter. AgfD has already been identified to regulate the extracellular matrix associated with the multicellular morphotype composed of thin aggregative fimbriae (agf). To detect additional components contributing to the multicellular morphotype in S. typhimurium, we constructed a mutant in agfD, the positive transcriptional regulator of the agfBA(C) operon encoding for fimbrial subunit proteins. The agfD mutant lacked any form of multicellular behaviour as shown by analysis at the macroscopic and microscopic level. In contrast, the agfBA mutant unable to form thin aggregative fimbriae still maintained long-range intercellular adhesion. Promoter and expression analysis revealed that the genes downstream of agfD agfEFG most likely did not contribute to the remaining aggregative behaviour. Screening of transcriptional fusions for agfD dependency uncovered adrA, a homologue of yaiC in Escherichia coli. Environmental factors regulating adrA correspond to the regulation of thin aggregative fimbriae. AdrA is a putative transmembrane protein with a C-terminal GGDEF domain of unknown function although it is present in over 50 bacterial proteins. AdrA mutant cells, which still formed thin aggregative fimbriae with all binding characteristics, exhibited community behaviour but, unlike the wild type, lacked long-range intercellular adhesion. An agfBA adrA double mutant behaved like the agfD mutant. Therefore, it was concluded that agfD regulates at least two independent pathways contributing to the multicellular morphotype in S. typhimurium.