Listeria monocytogenes and salmonella enterica adhesion, biofilm formation and control

Food contamination leads to wide economic loss and has a strong impact on public health worldwide. Listeria monocytogenes and Salmonella enterica Enteritidis are two of the most sight threatening and frequent foodborne pathogens, being responsible for listeriosis and salmonellosis foodborne outbreaks, respectively. The work presented in this thesis aimed at investigating adhesion and biofilm formation ability of these two bacteria regarding yet unexplored growth conditions and exposure to antimicrobials, as well as study possible repercussions of chemical disinfection on the genetic expression of virulence factors and stress response by surviving biofilm cells. L. monocytogenes has been a polemic bacterium as far as its biofilm formation capability is concerned, with different, and sometimes controversial, conclusions being stated by several authors. After testing this biological process under batch and fed-batch growth modes, both previously used by several authors but never compared simultaneously before, the results herein presented showed that the different growth modes influenced biofilm formation by L. monocytogenes on polystyrene, both in terms of biofilms’ total biomass and cellular viability. Temperature also played an important role on L. monocytogenes biofilm formation since refrigeration temperatures led to biofilms with less biomass but highly metabolic active, while at 37ºC biofilms had higher amount of biomass but were metabolically weaker. Surface coatings and antimicrobial incorporated materials have been two of the most promising attempts to produce new and improve already existing materials to be applied in food processing environments, in order to prevent microbial contaminations. A nitrogen-doped titanium dioxide coating on glass and on stainless steel was tested and showed to have bactericidal effect upon L. monocytogenes after only 30 minutes irradiation with visible light (fluorescent and/or incandescent light), when compared to non-coated surfaces. This fact indicated that such coated materials are likely to be applied on food contact surfaces as a means to reduce the risk of bacterial colonization and, thus, to improve food safety. The action of incorporated triclosan was assessed through S. enterica adhesion and biofilm formation on yet poorly studied food contact materials - stones. In this way, silestones (artificial stones mainly made of quartz, with triclosan incorporated) were tested and their performance compared with regular bench cover stones (granite and marble, without any antimicrobial compound) and stainless steel (one of the most commonly found surfaces in food processing environments). Similar levels of bacterial colonization and biofilm formation were observed on all materials, and lower numbers of S. enterica viable-culturable cells were found within biofilms formed on silestones. This indicates that, despite having shown some bactericidal effect upon biofilm cells, triclosan incorporated in silestones did not prevent bacterial colonization or biofilm formation. Once means to prevent contamination have failed and biofilms had already colonized the food contact surfaces, or in those cases where it is practically impossible to avoid microbial colonization during food processing, the greater concern becomes the surface cleaning through disinfection. In this work, susceptibility of L. monocytogenes and S. enterica monoculture-biofilms to disinfection was evaluated by determining the minimum biofilm eradication concentration (MBEC) of four distinct disinfectants commonly used in food industry – sodium hypochlorite, benzalkonium chloride, hydrogen peroxide and triclosan. Biofilm from both bacterial species were more susceptible to sodium hypochlorite than to any other disinfectant, whereas S. enterica biofilms were found to resist to triclosan’s action. Moreover, these assays revealed L. monocytogenes biofilms to be more susceptible to disinfection than S. enterica biofilms, which MBEC mean values concerning each disinfectant were higher than those found by the former bacterium. In order to investigate if disinfection had genetic repercussions on these biofilms, more specifically regarding stress-response and virulence genes expression by the surviving cells, quantitative real-time polymerase chain reaction was performed. Significant up-regulations were observed for L. monocytogenes and S. enterica stress-response genes cplC and ropS, respectively, as well as for S. enterica virulence gene avrA. These findings bring to discussion the fact that, even at concentrations that are able to significantly reduce biofilms biomass, chemical disinfectants seem to induce genetic alterations on the surviving cells that might not only lead to a stress response but, and even more worrying, may also increase their virulence.A contaminação de alimentos não só leva a grandes perdas a nível económico como tem também um forte impacto negativo na saúde pública em todo o mundo. Listeria monocytogenes e Salmonella enterica Enteritidis são dois dos patogénicos alimentares mais perigosos e frequentes, sendo responsáveis por surtos de listeriose e salmonelose alimentar, respectivamente. O trabalho apresentado nesta tese teve como objectivo estudar a capacidade de adesão e de formação de biofilme por parte de ambas as espécies mencionadas tendo em consideração condições de crescimento e exposição a agentes antimicrobianos, até então não investigados, assim como analisar possíveis repercussões que a desinfecção química possa ter a nível de expressão de genes de resposta ao stresse e de virulência por parte de células de biofilme sobreviventes. Tem havido alguma controvérsia no que respeita à capacidade de formação de biofilme da espécie L. monocytogenes, com vários autores a apresentar conclusões diferentes, e por vezes contraditórias, sobre esta matéria. Após testar o efeito de dois modos de crescimento – em sistema fechado e com alimentação escalonada (ambos usados previamente por vários autores mas que nunca tinham sido comparados simultaneamente) -, os resultados aqui apresentados mostraram que os diferentes modos de crescimento influenciaram a formação de biofilme de L. monocytogenes em poliestireno, quer em termos de biomassa total como também a nível da viabilidade celular dos biofilmes. A temperatura também desempenhou um papel importante na formação de biofilmes de L. monocytogenes, dado que à temperatura de refrigeração formou-se biofilmes com menos biomassa mas metabolicamente muito activos, enquanto que a 37ºC formou-se biofilmes com mais biomassa mas metabolicamente mais fracos. O revestimento de superfícies e a incorporação de antimicrobianos em materiais têm sido duas das tentativas mais promissoras para produção de novos materiais, e melhoria dos já existentes, para aplicação em meios de processamento de alimentos. Neste contexto, foi testado um revestimento de dióxido de titânio com azoto em vidro e em aço inoxidável, o qual mostrou ter efeito bactericida sobre a L. monocytogenes após apenas 30 minutos de irradiação com luz visível (fluorescente e/ou incandescente) quando comparado com superfícies não-revestidas Este facto indica que tais materiais são passíveis de serem aplicados em superfícies de contacto com os alimentos como forma de reduzir o risco de colonização bacteriana e, assim, melhorar a segurança alimentar. A acção do triclosano incorporado foi avaliada através da capacidade de adesão e de formação de biofilme de S. enterica em materiais de contacto com alimentos ainda pouco estudados – as pedras. Para tal, testou-se o desempenho de silestones (pedras artificiais constituídas maioritariamente por quartzo, com triclosan incorporado) comparando-o com pedras comuns usadas em bancadas de cozinha (granito e mármore, sem qualquer composto antimicrobiano) e aço inoxidável (uma das superficies mais frequentemente encontradas em meios de processamento de alimentos). Verificaram-se níveis semelhantes de colonização bacteriana e formação de biofilme em todos os materiais e que o número de células viáveiscultiváveis de S. enterica foi mais baixo nos biofilmes formados nos silestones. Isto indica que, embora tendo algum efeito bactericida sobre as células do biofilme, o triclosan incorporado nos silestones não preveniu a colonização bacteriana nem a formação de biofilme. Uma vez falhadas as medidas de prevenção de contaminação e colonizadas por biofilmes as superfícies de contacto com alimentos, ou nos casos em que é praticamente impossível evitar a colonização microbiana durante o processamento dos alimentos, a maior preocupação torna-se a limpeza de superfícies através da desinfecção. Neste trabalho, avaliou-se a susceptibilidade à desinfecção por parte de biofilmes simples de L. monocytogenes e S. enterica por meio da determinação da concentração mínima de erradicação de biofilme (CMEB) de quatro desinfectantes diferentes frequentemente usados na indústria alimentar – hipoclorito de sódio, cloreto de benzalcónio, peróxido de hidrogénio e triclosano. Os biofilmes de ambas as espécies bacterianas foram mais susceptíveis ao hipoclorito de sódio do que a qualquer outro desinfectante, tendo-se ainda verificado alguma resistência por parte dos biofilmes de S. enterica à acção do triclosano. Além disso, estes ensaios revelaram uma maior susceptibilidade à desinfecção por parte dos biofilmes de L. monocytogenes comparativamente com os biofilmes de S. enterica, cujos valores médios de CMEB de cada desinfectante foram maiores do que os registados para a primeira bactéria. De modo a investigar-se se a desinfecção teve repercussões genéticas nestes biofilmes, mais especificamente no que respeita à expressão de genes de resposta ao stress e de virulência por parte das células sobreviventes, realizaram-se reacções quantitativas em cadeia da polimerase em tempo-real. Verificou-se a sobre-expressão significativa dos genes de resposta ao stress cplC e rpoS de L. monocytogenes e S. enterica, respectivamente, assim como do gene de virulência avrA de S. enterica. Estas descobertas levantam a questão de que, mesmo submetidas a concentrações de desinfectante capazes de reduzir significativamente a biomassa dos biofilmes, as células sobreviventes parecem sofrer alterações genéticas relacionadas não só com a uma reposta ao stresse mas também, e mais preocupante ainda, com um possível aumento da sua virulência

Effect of sub-lethal chemical disinfection on the biofilm forming ability, resistance to antibiotics and expression of virulence genes of Salmonella Enteritidis biofilm-surviving cells

Although disinfection procedures are widely implemented in food environments, bacteria can survive and present increased virulence/resistance. Since little is known about these phenomena regarding biofilms, this study aimed to investigate the effect of chemical disinfection on biofilm-derived cells of Salmonella Enteritidis. Using a reference strain (NCTC 13349) and a food isolate (350), biofilm susceptibility to benzalkonium chloride (BAC), sodium hypochlorite (SH) and hydrogen peroxide (HP) was evaluated and biofilms were exposed to sub-lethal concentrations of each disinfectant. Biofilm-derived cells were characterized for their biofilm forming ability, antibiotic resistance and expression of virulence-associated genes. Except for a few instances, disinfectant exposure did not alter antibiotic susceptibility. However, SH and HP exposure enhanced the biofilm forming ability of Salmonella Enteritidis NCTC 13349. After BAC and HP exposure, biofilm-derived cells presented a down-regulation of rpoS. Exposure to BAC also revealed an up-regulation of invA, avrA and csgD on Salmonella Enteritidis NCTC 13349. The results obtained suggest that biofilm-derived cells that survive disinfection may represent an increased health risk.This study was supported by the Portuguese Foundation forScience and Technology (FCT) under the scope of the stra-tegic funding of unit UIDB/04469/2020 and BioTecNorteoperation (NORTE-01-0145-FEDER-000004) funded by theEuropean Regional Development Fund under the scope ofNorte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Adhesion and biofilm formation by a Salmonella enterica enteritidis isolate on kitchen bench stones: evaluation of the antibacterial effect of MICROBAN®

Contamination of food processing materials by microorganisms is the basis of the cross-contamination phenomenon and, therefore, is directly associated with the occurrence of severe foodborne diseases. Some approaches have already been made in order to control microbes’ attachment and development on food-contact surfaces, being the incorporation of antimicrobial compounds one of the most promising. In this context, the aim of this work was to assess bacterial adhesion and biofilm formation by Salmonella Enteritidis on kitchen bench stones (materials yet poorly studied but largely used in Mediterranean kitchens) and to compare the results between regular stones (without any antimicrobial added) and stones with Microban® incorporated (with triclosan as active agent). The assays were performed with a clinical isolate of Salmonella Enteritidis and four kitchen bench stones - granite, marble and two kinds of silestone (a material mainly made from quartz and with Microban® incorporated) – cut into squares of 2.0 x 2.0 cm2. Both adhesion and biofilm formation processes were assessed in 6-weel plates, at room temperature (25ºC), with shaking at 120 rpm and using LB as culture medium. Total cell counts of adhered bacteria were accomplished after 2 hours of incubation through epifluorescence microscopy enumeration of cells stained with DAPI, while biofilms biomass was evaluated by cell scraping and CFUs enumeration after a 48 hours growth period. The results revealed that all stones tested are prone to bacterial adhesion and no considerable effect of triclosan was observed in both silestones, indicating that Microban® does not act upon the interactions between the bacterial cells and stones surface. On the other hand, biofilm outcomes point out a possible bacteriostatic activity of this compound since, even though bacterial load in silestones remained high (≈ 7x106 cfu/cm2), significantly higher numbers of bacterial cells were found in granite and marble. In conclusion, given their propensity to bacterial colonization and biofilm growth, all these kitchen bench stones are not suitable for food processing. Moreover, Microban® incorporated in silestones had a poor performance in controlling microbial spread and, therefore, seems to be insufficient to prevent cross-contamination

Cross-contamination in food-contacting surfaces: novel approaches to control food-borne pathogens

Este resumo faz parte de: Book of abstracts of the Meeting of the Institute for Biotechnology and Bioengineering, 2, Braga, Portugal, 2010. A versão completa do livro de atas está disponível em: http://hdl.handle.net/1822/1096

Characterization of biofilms of isolates from meat retail facilities

According to EFSA cross contamination is implicated in about 40% of reported foodborne outbreaks, as pathogens can be transferred from several sources including raw foods during food processing to hands, cloths and food contact surfaces. Specifically, during slaughtering meat can be contaminated with bacteria directly by fecal contamination and indirectly by inadequately disinfected surfaces. This is aggravated by the persistence of foodborne pathogens due to their ability to adhere and form biofilms in surfaces despite of disinfection procedures. Besides, there is also a concern that the use of biocides may contribute to the development of antibiotic-resistant strains. Accordingly, isolates were collected from meat processing surfaces of retail facilities, isolated by selective media, identified by 16S sequencing and phenotypically characterized in terms of biofilm formation ability, susceptibility to disinfectants and antibiotics, viability and acquisition of cross-resistance. Planktonic cells showed low susceptibility to the disinfectants tested (hydrogen peroxide and sodium hypochlorite) and to antibiotics (rifampicin and linezolid). Moreover, isolates presented good biofilm formation ability and, as expected, a lower susceptibility to disinfectants and to antibiotics compared to planktonic cells. After exposure of biofilms to hydrogen peroxide at concentrations higher than the recommended ones, biofilms were still able to survive to antibiotics at a 10 x MIC. These results showed that bacteria may be exposed to only sublethal concentrations and survive which may contribute to bacterial resistance to these compounds, as well as cross-resistance to antibiotics

Monoculture and mixed biofilms of listeria monocytogenes and pseudomonas fluorescens: effect of different culture media and temperatures

Like most microorganisms, Listeria monocytogenes and Pseudomonas fluorescens are able to form biofilms and are rarely found as monoculture biofilms in natural environments. Previous works showed that associations between bacteria from different genus commonly found in food-processing environments may affect their growth, attachment and biofilm formation. This work studied L. monocytogenes and P. fluorescens monoculture and multispecies biofilm formation, and investigated how different culture media and temperatures may influence such bacterial interactions. L. monocytogenes strains assayed were CECT 4031T, 747 and 994 (food isolates), 1559 (environmental isolate) and 1562 (clinical isolate). P. fluorescens strains used were ATCC 27663 and PF7A (food isolate). Each strain was tested for monoculture and mixed culture biofilm formation with each one of the other bacterium’s strains. Assays were performed during three days in 96-weel microtitre plates, at 4ºC, 22ºC and 37ºC. Brain Heart Infusion (BHI) and Skim Milk (SM) were the culture media and biofilm formation was assessed by Crystal Violet staining. Overall results showed that both media and temperature affect biofilm formation, as monoculture and as multispecies biofilms, and confirmed that the influence of different bacterial genus on biofilm formation is dependent on strains. Although a decrease of biomass was observed on multispecies biofilms formed at 22ºC in SM and at 37ºC in BHI, significantly higher OD values were found at 4ºC in both media, and at 22ºC in SM, indicating that the combination of these two bacteria on meat and dairy food processing environments may seriously compromise food safety potentiating higher contamination levels

Control of bacterial cells growths by magnetic hyperthermia

In this work, we report the effectiveness of magnetic hyperthermia as a potential disinfection method against food spoilage microorganisms. High structural-magnetic quality magnetite nanoparticles have been found to be effective against bacterial microorganisms in solution under an oscillating magnetic field. Samples containing both magnetite nanoparticles and Pseudomonas fluorescens cells in aqueous solution have been subjected to an alternating magnetic field of chosen amplitude 100 Oe with frequency of 873 kHz for different times, achieving different maximum temperatures ranging from 35°C to 55°C. The subsequent colony forming units count evidenced an important decreasing of the cell survival with temperature in comparison to a conventional direct heating, ending in the total eradication of the microorganisms in relatively short times (~8 min). This ability of magnetic hyperthermia to control bacteria cells constitutes a novel contribution to the finding of new useful applications of hyperthermia different from biomedicine.This work was supported in part by the European Community’s under the FP7-Cooperation Programme through the MAGISTER project “Magnetic Scaffolds for in vivo Tissue Engineering” Large Collaborative Project FP7 and from European Regional Development Fund (ERDF) under the Northern Regional Operational Programme ON.2-O Novo Norte- for the acquisition of the main equipment used in this research. D. R. also acknowledges the financial support of Portuguese Foundation for Science and Technology (FCT) through the grant SFRH/BPD/72632/2010

Control of planktonic bacterial cells and biofilms through magnetic hyperthermia

Disinfection of surfaces is a challenging task aggravated by bacteria's capacity to form biofilms, which enables them to survive and resist a wide variety of antimicrobial agents and hostile conditions. Potential application of magnetic hyperthermia (MH) as a new disinfection method against biofilms has been recently proposed however, studies comparing its performance and effectiveness on planktonic and biofilm cells from the same bacterial species remain unexplored. This work evaluated the effect of MH generated by iron oxide magnetic nanoparticles (MNP) against planktonic and biofilm cells Pseudomonas fluorescens, a major food spoilage microorganism. A P. fluorescens collection strain (ATCC 27663) was used and its biofilms allowed to form on silicone coupons during three days incubation in tryptic soy broth culture medium, at room temperature (20 ± 2ºC) and constant agitation of 120 rpm. Hyperthermia experiments were performed by applying an oscillating magnetic field of 873kHz and 100 Oe to several identical solutions of bacteria and MNP. To study cell viability as a function of temperature, magnetic heatings were performed at the same heating rate and up to different maximum temperatures. Bacterial survival was assessed through colony forming units count, while confocal laser scanning microscopy (CLS) was used to evaluate cellular membrane integrity of both bacterial life forms, as well as eventual effects of MH in biofilms' structure. Results showed a significant reduction (3 log) of viable planktonic cells when a maximum temperature of 40QC was reached, corresponding to only about 3 minutes of exposure to alternate magnetic field. A complete cellular eradication was achieved after only 8 minutes, when the maximum temperature was increased up to 55ºC. ln contrast, a significantly lower reduction of cellular viability was accomplished for biofilm s at the same temperatures, and no eradication was achieved even after 17 minutes of magnetic field exposure, reaching a maximum temperature of 60ºC. CLS images showed that MH inflicted cellular membrane damages both in planktonic and biofilm s cells, and also suggested that the outer cell layers of biofilms were more damaged than inner ones, as denoted by the higher amount of injured cells observed in the external layers. Summarizing, this work confirms the potential of MH as a disinfection method and shows for the first time its efficacy against a food spoilage microorganism. More importantly, it presents the first insights about how different bacterial life forms are affected by MH, showing a significantly different effectiveness against planktonic cells and biofilms

Effect of batch and fed-batch growth modes on biofilm formation by Listeria monocytogenes at different temperatures

The influence of Listeria monocytogenes (L. monocytogenes) biofilm formation feeding conditions (batch and fed-batch) at different temperatures on biofilm biomass and activity was determined. Biofilm biomass and cellular metabolic activity were assessed by Crystal Violet (CV) staining and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT) colorimetric method, respectively. Live/Dead staining was also performed in order to get microscopic visualization of the different biofilms. Results revealed that at refrigeration temperature (4°C) a higher amount of biofilm was produced when batch conditions were applied, while at higher temperatures the fed-batch feeding condition was the most effective on biofilm formation. Moreover, independently of the temperature used, biofilms formed under fed-batch conditions were metabolically more active than those formed in batch mode. In conclusion, this work shows that different growth modes significantly influence L. monocytogenes biofilm formation on abiotic surfaces as well as the metabolic activity of cells within biofilms.Fundação para a Ciência e a Tecnologia (FCT) - PPCDT/AGR/59358/2004, SFRH/ BD/28887/2006, SFRH/BPD/26803/200