Microbial biofilms in food environments: study approaches and intervention strategies

The main objective of this study was to examine in-depth the problems related to the for-mation of microbial biofilms in food plants. Although it has long been known that the ability to form biofilms is a common trend in natural environments including food processing environments, most of the studies in the literature concern the medical context, where microbial biofilms are often the cause of serious hospital infections. In the food field, however, the interest in biofilms has only recently arisen, even though the formation of microbial biofilms appears to be the major cause of cross-contamination in food products. Currently, data on microbial communities in the different food processing areas are only limited, and the influence of environmental parameters on the char-acteristics of biofilms have been studied for only a few microbial species. This knowledge is, how-ever, necessary for the development of intervention strategies for the prevention and removal of biofilms, which allows at the same time to obtain a high degree of hygienic-sanitary safety of the surfaces, as well as to reduce the impact conventional strategies have on the environment and on the safety of operators. The study discussed in this thesis was carried out on specific groups of organisms known to be pathogenic or food spoilers, including Listeria monocytogenes, Staphylococcus aureus and Pseudomonas spp. The need for the availability of appropriate study models that allow to obtain, in as little time as possible, a high number of biofilm samples with homogeneous characteristics was addressed in Chapter 2, where the use of a microtiter plate system and a reactor able to grow mi-crobial biofilms was evaluated on materials widely used in the food industry. In addition, to facili-tate the microbiological laboratory activities, two techniques of plate counts were compared in or-der to highlight problems and benefits in the context of the study of biofilms for both. The micro-titer plate assay and the CDC biofilm reactor assay showed to be sufficiently reliable and repeatable tools to produce a number of samples large enough to provide sufficient information on the ability of food microorganisms to produce biofilms under different operating conditions. Moreover, the drop plate method has proved particularly suitable, sufficiently accurate and reliable, as well as ad-vantageous from the economic point of view, for the quantification of viable cells present in the biofilm. The influence of environmental parameters on biofilm formation was studied in Chapter 3 and Chapter 4. In particular, in Chapter 3 the effect of the synergy of multiple parameters on the formation of biofilms in a static model system was studied, both in terms of quantification of total the biomass (dead and live cells and EPS matrix) and of the only evaluation of the cell count. The use of a Central Composite Design allowed to mimic the real environmental conditions in the food industry and to obtain the greatest amount of information limiting the number of experiments to be carried out. Therefore, useful data were obtained to increase the information in the literature about the synergistic effects of the environmental parameters on biofilm formation regarding the food sector. In Chapter 4 the effect of temperature on the adhesion and on the biofilm structure, as well as on resistance to disinfectants commonly used in the food industry sanitation plans was studied. The study showed that temperature significantly affects the kinetics of adhesion, but also the cell density and the amount of EPS produced, and consequently the resistance to biocides. The use of CLSM technique for microscopic observation allowed the study of biofilms in undisturbed condi-tions, and thus it is well suited to a possible use during the biofilm growth. The evaluation of dif-ferent strategies for the removal of biofilms was the subject of Chapter 5 and Chapter 6, in which conventional and non-conventional approaches were considered. A comparison between chemical, physical and biological treatments shows that a hurdle-approach, in which different strategies are used in sequence, could help in limiting the health and hygiene problems related to microbial bio-films in the production of foodstuffs. Finally, in Chapter 7 the gained knowledge was used to study the problem of biofilms in specific food contexts.openDottorato di ricerca in Scienze degli alimentiembargoed_20150912Frigo, Francesc

Characterising filamentous fungal biofilm in drinking water distribution systems using microscopic and molecular techniques

Tese de doutoramento em Engenharia Química e BiológicaA qualidade microbiológica de água de consumo tem sido uma das grandes preocupações de fornecedores governamentais e privados. Sistemas de distribuição de água são complexos ambientes onde algas, bactérias, fungos e protozoários cohabitam. Muitos destes microrganismos são patogenicos ao homem. Uma vez no sistema de distribuição, microorganimos podem aderir às superfícies das tubulações e formar biofilmes. Biofilmes são comunidades microbianas nas quais seus constituintes apresentem características diferenciadas, por exemplo, maior resistência a fatores adversos como desidratação, altas temperaturas, condições oligotroficas e ação de biocidas. Fungos são conhecidos por habitarem diversos ambientes aquáticos. Sua presença em sistemas de distribuição de água está associada a problemas de bloqueio de tubulações, produção de metabólitos e mudanças em propriedades organolépticas como odor e turbidez, e consequentemente, os fungos estão ligados à perda da qualidade da água. Além disso, por conta da sua habilidade em aderir a superfícies, os fungos são capazes de crescer como biofilmes em diferentes materiais, inclindo as paredes das tubulações. O presente trabalho visou o estudo de biofilmes de fungos filamentosos em sistemas de distribuição de água. Para isto, técnicas de microscopia de fluorescência e fluorescent in situ hybridization (FISH) foram utilizadas pretendendo-se detectar, monitorar e caracterizar biofilmes de fungos filamentosos em condições reais e laboratoriais. Corantes e sondas fluorescentes específicos foram aplicados, i.e. Calcofluor White R2R, DAPI, FUN-1, e as sondas EUK516 FUN1429. Microspheres Adhesion Assay, coloração com Cristal Violeta e MTT foram também utilizados. Adicionalmente, foi projetado um amostrador para formação de biofilmes in situ. Como resultados, foi observado que biofilmes de fungos filamentosos, quando comparados com sua forma planquitonica, possuem um comportamento diferenciado com relação à resistência contra agentes desinfectantes e a nívels de hidrophobicidade celular. Biofilmes de fungos filamentosos foram detectados em tubulações, amostradores e sistemas laboratoriais. Fungus filamentosos isolados de sistemas de água são capazes de formar biofilmes, apresentando caracteristicas morfológicas e fisiológicas diferenciadas. Em conclusão, fungos filamentosos são capazes de formar biofilmes sob diversas condições reais e laboratoriais. É provável que os fungos desempenhem um papel importante nas interações nos biofilmes em sistemas de água e, conseqüentemente, também na qualidade microbiológica da água. Assim, os fungos devem ser incluídos como membros consistentes dos biofilmes. Os resultados relatados neste trabalho representam novos conceitos na pesquisa micológica, e mais estudos na área podem levar a novas descobertas sobre a biologia dos fungos.Microbial quality of drinking water has been one of the greatest concerns of governmental and privative water suppliers. Water distribution systems are complex environments where algae, bacterial, fungi and protozoa cohabit. Many of these microorganisms are pathogenic to human and are associated with loss of water quality. Once within a water distribution system, microorganisms can attach to pipe surface and form biofilms. Biofilms are microbial communities wherein theirs constituents present differentiate features when compared with their planktonic form, e.g. increased resistance against adverse factors such as dryness, high temperatures, oligotrophic conditions and biocides. Fungi are known to habit diverse aquatic environments. Their presence in water systems is associated with pipe blockage, produce of metabolites involved in organoleptic changes such as odour and turbidity, and consequently are linked with loss of water quality. Moreover, because of their ability to adhere to surfaces, fungi are able to grown as biofilms on different surfaces, including pipe walls. The present work aimed to study filamentous fungal biofilms in water distribution systems. For this, epifluorescence microscopy and fluorescent in situ hybridization (FISH) were applied intending to detect, monitor and characterise filamentous fungal biofilms in real and laboratorial water systems. Specific fluorescent dyes and probes were used, i.e. Calcofluor White R2R, DAPI, FUN-1, EUK516 and FUN1429 probes. Microspheres Adhesion Assay, Cristal Violet staining and MTT assay were also applied. Moreover, a device was designed for sampling fungal biofilms in situ. As results, was observed that filamentous fungal biofilms have differentiated behaviour concerning resistance against disinfectant and levels of cell hydrophobicity when compared with their planktonic form. Filamentous fungal biofilms were detected in replaced pipes, sampler devices and laboratorial water system. Moreover, filamentous fungi recovered from water systems were capable to form biofilms with specific morphological and physiological features. In conclusion, filamentous fungi are able to form biofilms under diverse real and laboratorial conditions. Fungi are likely to play an important role in microbial interactions within water biofilms and consequently in microbial water quality. Thus, fungi may be included as a consistent member of biofilms in drinking water systems. The results reported in this work represent new concepts in mycological research, and further studies in the area may lead to new insights in fungal biology