Resistance of Listeria monocytogenes biofilms to sanitizing agents in a simulated food processing environment

The objective of this study was to evaluate the resistance of biofilms of Listeria monocytogenes to sanitizing agents under laboratory conditions simulating a food processing environment. Biofilms were initially formed on stainless steel and Teflon coupons using a five-strain mixture of L. monocytogenes. The coupons were then subjected to repeated 24-h daily cycles. Each cycle consisted of three sequential steps: (i) a brief (60 s) exposure of the coupons to a sanitizing agent (a mixture of peroxides) or saline as a control treatment, (ii) storage of the coupons in sterile plastic tubes without any nutrients or water for 15 h, (iii) and incubation of the coupons in diluted growth medium for 8 h. This regimen was repeated daily for up to 3 weeks and was designed to represent stresses encountered by bacteria in a food processing environment. The bacteria on the coupons were reduced in number during the first week of the simulated food processing (SFP) regimen, but then adapted to the stressful conditions and increased in number. Biofilms repeatedly exposed the peroxide sanitizer in the SFP regimen developed resistance to the peroxide sanitizer as well as other sanitizers (quaternary ammonium compounds and chlorine). Interestingly, cells that were removed from the biofilms on peroxide-treated and control coupons were not significantly different in their resistance to sanitizing agents. These data suggest that the resistance of the treated biofilms to sanitizing agents may be due to attributes of extracellular polymeric substances and is not an intrinsic attribute of the cells in the biofilm.     

Lactic Acid Bacteria (LAB) and Their Bacteriocins as Alternative Biotechnological Tools to Control <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> Biofilms in Food Processing Facilities

Bacteriocins are antimicrobial peptides produced by bacteria Gram-negative and Gram-positive, including lactic acid bacteria (LAB), organisms that are traditionally used in food preservation practices. Bacteriocins have been shown to have an aptitude as biofilm controlling agents in Listeria monocytogenes biofilms, a major risk for consumers and the food industry. Biofilms protect pathogens from sanitization procedures, allowing them to survive and persist in processing facilities, resulting in the cross-contamination of the end products. Studies have been undertaken on bacteriocinogenic LAB, their bacteriocins, and bioengineered bacteriocin derivatives for controlling L. monocytogenes biofilms on different surfaces through inhibition, competition, exclusion, and displacement. These alternative strategies can be considered promising in preventing the development of resistance to conventional sanitizers and disinfectants. Bacteriocins are “friendly” antimicrobial agents, and with high prevalence in nature, they do not have any known associated public health risk. Most trials have been carried out in vitro, on food contact materials such as polystyrene and stainless steel, while there have been few studies performed in situ to consolidate the results observed in vitro. There are strategies that can be employed for prevention and eradication of L. monocytogenes biofilms (such as the establishment of standard cleaning procedures using the available agents at proper concentrations). However, commercial cocktails using alternatives compounds recognized as safe and environmental friendly can be an alternative approach to be applied by the industries in the future.     

Listeria monocytogenes Can Form Biofilms in Tap Water and Enter Into the Viable but Non-Cultivable State

Listeria monocytogenes is a foodborne pathogen that can be transmitted through contaminated raw food or by ready-to-eat products that have been in contact with contaminated surfaces. Tap water (TW) is used to wash produce, as a processed food constituent and to wash processing surfaces and floors. The main aim of this work was to investigate the formation and survival of L. monocytogenes biofilms on stainless steel (SS) coupons in TW at 4, 22, 30 and 37 °C. For that, coupons with biofilm were visualised in situ while other coupons were scraped to quantify total cells by SYTO 9, cultivable numbers by plating onto brain heart infusion agar and viable numbers by the direct viable count method. Results showed that L. monocytogenes can form biofilms on SS surfaces in TW at any temperature, including at 4 °C. The number of total cells was similar for all the conditions tested while cultivable numbers varied between the level of detection (<8.3 CFU cm^?2) and 3.5?×?10⁵ CFU cm^?2, meaning between 7.0?×?10⁴ and 1.1?×?10⁷ cells cm^?2 have entered the viable but non-cultivable (VBNC) state. This work clearly demonstrates that L. monocytogenes can form biofilms in TW and that sessile cells can remain viable and cultivable in some conditions for at least the 48 h investigated. On the other hand, VBNC adaptation suggests that the pathogen can remain undetectable using traditional culture recovery techniques, which may give a false indication of processing surface hygiene status, leading to potential cross-contamination of food products.     

Competition of Listeria monocytogenes Serotype 1/2a and 4b Strains in Mixed-Culture Biofilms

The majority of Listeria monocytogenes isolates recovered from foods and the environment are strains of serogroup 1/2, especially serotypes 1/2a and 1/2b. However, serotype 4b strains cause the majority of human listeriosis outbreaks. Our investigation of L. monocytogenes biofilms used a simulated food-processing system that consisted of repeated cycles of growth, sanitation treatment, and starvation to determine the competitive fitness of strains of serotypes 1/2a and 4b in pure and mixed-culture biofilms. Selective enumeration of strains of a certain serotype in mixed-culture biofilms on stainless steel coupons was accomplished by using serotype-specific quantitative PCR and propidium monoazide treatment to prevent amplification of extracellular DNA or DNA from dead cells. The results showed that the serotype 1/2a strains tested were generally more efficient at forming biofilms and predominated in the mixed-culture biofilms. The growth and survival of strains of one serotype were not inhibited by strains of the other serotype in mixed-culture biofilms. However, we found that a cocktail of serotype 4b strains survived and grew significantly better in mixed-culture biofilms containing a specific strain of serotype 1/2a (strain SK1387), with final cell densities averaging 0.5 log₁₀ CFU/cm² higher than without the serotype 1/2a strain. The methodology used in this study contributed to our understanding of how environmental stresses and microbial competition influence the survival and growth of L. monocytogenes in pure and mixed-culture biofilms.A prominent food-borne pathogen, Listeria monocytogenes can cause severe infections in humans, primarily in high-risk populations, though the disease (listeriosis) is relatively rare (11, 30, 43). Outbreaks of listeriosis have resulted from the contamination of a variety of foods by L. monocytogenes, especially meat and dairy products (27). L. monocytogenes is ubiquitous in the environment, able to grow at refrigeration temperature, and tolerant of the low pHs (3 to 4) typical of acidified foods (28, 32, 44). The capacity to produce biofilms confers protection against stresses common in the food-processing environment (13, 33).Biofilms are characterized by dense clusters of bacterial cells embedded in extracellular polymeric substances which are secreted by cells to aid in adhesion to surfaces and to other cells (4, 5). Strains of L. monocytogenes have been known to persist for years in food-processing environments, presumably in biofilms. Of the 13 known serotypes of L. monocytogenes, three (1/2a, 1/2b, and 4b) account for >95% of the isolates from human illness (21). Serotype 1/2a accounts for >50% of the L. monocytogenes isolates recovered from foods and the environment, while most major outbreaks of human listeriosis have been caused by serotype 4b strains (1, 3, 14, 15, 17, 22, 29, 31, 41, 47, 49,). No correlation between L. monocytogenes strain fitness and serotype has been identified (16, 19). Some studies have reported that strains repeatedly isolated from food and environmental samples (defined as persistent strains) had a higher adherence capacity than strains that were sporadically isolated (2, 36), while this phenomenon was not observed by others (7). Serotype 4b strains exhibited a higher capacity for biofilm formation than did serotype 1/2a strains (36), whereas this was not observed by Di Bonaventura and colleagues (6). It has been suggested that serotype 1/2a strains could be more robust than serotype 4b strains in biofilm formation under a variety of environmental conditions. Furthermore, strains of these serotypes differ in terms of the medium that promotes biofilm formation. Biofilm formation by serotype 4b strains was higher in full-strength tryptic soy broth than in diluted medium, whereas the opposite was observed with serotype 1/2a strains, which produced more biofilm in diluted medium (12).There is limited information on microbial competition between strains of different serotypes in biofilms or on how the environmental stresses present in food-processing environments may affect the biofilm formation and survival of L. monocytogenes of different serotypes. In food-processing plants, the environmental stresses encountered by bacteria are more complex and variable than most laboratory systems used for microbial ecology and biofilm studies. A simulated food-processing (SFP) system has been developed to address this issue (38). The SFP system incorporates several stresses that may affect bacteria in biofilms in the food-processing environment, including exposure to sanitizing agents, dehydration, and starvation. When biofilms were subjected to the SFP regimen over a period of several weeks, the cell numbers of L. monocytogenes strains in the biofilms initially were reduced and then increased as the culture adapted (38). The development of resistance to sanitizing agents was specific to the biofilm-associated cells and was not apparent in the detached cells (38). This suggested that extracellular polymeric substances present in the biofilm matrix were responsible for the resistance to sanitizing agents. It was subsequently found that real-time PCR, in combination with propidium monoazide (PMA) treatment of samples prior to DNA isolation, was an effective method for enumerating viable cells in biofilms (37).The objective of this study was to determine if strains of serotype 1/2a or 4b have a selective advantage under stress conditions. We investigated and compared the initial attachment and biofilm formation capabilities of L. monocytogenes strains of these two serotypes and analyzed the survival and growth of bacteria of each serotype in mixed-serotype biofilms in the SFP system by using PMA with quantitative PCR.     

Antimicrobial activity and effectiveness of a combination of sodium hypochlorite and hydrogen peroxide in killing and removing Pseudomonas aeruginosa biofilms from surfaces

AIMS: To evaluate both the antimicrobial activity and the effectiveness of a combination of sodium hypochlorite and hydrogen peroxide (Ox-B) for killing Pseudomonas aeruginosa ATCC 19142 cells and removing P. aeruginosa biofilms on aluminum or stainless steel surfaces. METHODS AND RESULTS: Pseudomonas aeruginosa biofilms were developed in tryptic soy broth containing vertically suspended aluminium or stainless steel plates. Biofilms were exposed to a mixed sodium hypochlorite and hydrogen peroxide solution as a sanitizer for 1, 5 and 20 min. The sanitizer was then neutralized, the cells dislodged from the test surfaces, and viable cells enumerated. Cell morphologies were determined using scanning (SEM) and transmission electron microscopy (TEM). Cell viability was determined by confocal scanning laser microscopy (CSLM). Biofilm removal was monitored by Fourier transform infrared (FTIR) spectrophotometry. Cell numbers were reduced by 5-log to 6-log after 1 min exposure and by 7-log after 5 min exposure to Ox-B. No viable cells were detected after a 20 min exposure. Treatment with equivalent concentrations of sodium hypochlorite reduced viable numbers by 3-log to 4-log after 1 min exposure and by 4-log to 6-log after 5 min, respectively. A 20 min exposure achieved a 7-log reduction. Hydrogen peroxide at test concentration treatments showed no effect. FTIR analysis of treated pseudomonad biofilms on aluminium or stainless steel plates showed either a significant reduction or complete removal of biofilm material after a 5 min exposure to the mixed sodium hypochlorite and hydrogen peroxide solution. SEM and TEM images revealed damage to cell wall and cell membranes. CONCLUSIONS: A combination of sodium hypochlorite and hydrogen peroxide effectively killed P. aeruginosa cells and removed biofilms from both stainless steel and aluminium surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of sodium hypochlorite and hydrogen peroxide can be used as an alternative disinfectant and/or biofilm remover of contaminated food processing equipment.     

Enhanced inactivation of salmonella and pseudomonas biofilms on stainless steel by use of T-128, a fresh-produce washing aid, in chlorinated wash solutions

The effect of the washing aid T-128 (generally recognized as safe [GRAS] formulation, composed mainly of phosphoric acid and propylene glycol) on inactivation of Salmonella and Pseudomonas populations in biofilms on stainless steel was evaluated under conditions of increasing organic matter loads in chlorinated wash solutions dominated by hypochlorous acid. Biofilms were formed statically on stainless steel coupons suspended in 2% lettuce extract after inoculation with Salmonella enterica serovar Thompson or Newport or with Pseudomonas fluorescens. Coupons with biofilms were washed in chlorine solutions (0, 0.5, 1, 2, 5, 10, or 20 mg/liter at pH 6.5, 5.0 and 2.9), with or without T-128, and with increasing loads of organic matter (0, 0.25, 0.5, 0.75, or 1.0% lettuce extract). Cell populations on coupons were dispersed using intermittent, pulsed ultrasonication and vortexing and enumerated by colony counts on XLT-4 or Pseudomonas agars. Cell responses to fluorescent viability staining of biofilm treatment washing solutions were examined using confocal laser scanning microscopy. Results showed that 0.1% T-128 (without chlorine) reduced P. fluorescens biofilm populations by 2.5 log(10) units but did not reduce Salmonella populations. For both Salmonella and Pseudomonas, the sanitizing effect of free chlorine (1.0 to 5.0 mg/liter) was enhanced (P < 0.05) when it was combined with T-128. Application of T-128 decreased the free chlorine depletion rate caused by increasing organic matter in wash waters and significantly (P < 0.05) augmented inactivation of bacteria in biofilms compared to treatments without T-128. Image analysis of surfaces stained with SYTO and propidium iodide corroborate the cultural assay results showing that T-128 can aid in reducing pathogen viability in biofilms and thus can aid in sanitizing stainless steel contact surfaces during processing of fresh-cut produce.     

Colorimetric assay for biofilms in wet processing conditions

Controlling bacterial biofilms is necessary for food safety and industrial processing in clean room environments. Our goal was to develop a method to quantitatively measure biofilm produced by pathogens under wet poultry production and processing conditions. Stainless steel and glass coupons were incubated in aqueous media containing reduced nutrients and exposed to Listeria monocytogenes under static temperature and humidity conditions. Samples were measured separately by biofilm assay and viable cell density, and then confirmed by spectrophotometry and microscopy. The biofilm assay resulted in different t groupings from the cell density. The mean from the biofilm assay was 0.50, and the error% was 0.595. The mean of the log₁₀ density (cfu/cm²) was 5.90, and the standard deviation ranged from 0.127 to 0.438 on 24 coupons. The typical sequence of biofilm development, followed by microscopy of biofilm grown on glass coupons, exhibited a change from dispersed single cells to an all-over pattern of clumps with few dispersed cells. L. monocytogenes formed biofilms on all of the substrata tested. Bacterial counts from planktonic cultures at 24, 48, 72, and 144 h confirmed that L. monocytogenes remained viable throughout the experiment and reached equilibrium between 6 and 24 h. The cell density log₁₀/ml was 8.01, 8.03, 7.69, and 6.66, respectively; and the standard deviation ranged from 0.156 to 0.394. The data will be used to grow stable biofilms of Listeria spp. collected from the food processing environment for further study. This is the first use of the crystal violet assay for measurement of bacterial biofilms on stainless steel under these conditions. The methods tested are applicable to other bacteria and substrata.     

Effect of Octenidine Hydrochloride on Planktonic Cells and Biofilms of Listeria monocytogenes

Listeria monocytogenes is a food-borne pathogen capable of forming biofilms and persisting in food processing environments for extended periods of time, thereby potentially contaminating foods. The efficacy of octenidine hydrochloride (OH) for inactivating planktonic cells and preformed biofilms of L. monocytogenes was investigated at 37, 21, 8, and 4°C in the presence and absence of organic matter (rehydrated nonfat dry milk). OH rapidly killed planktonic cells and biofilms of L. monocytogenes at all four temperatures. Moreover, OH was equally effective in killing L. monocytogenes biofilms on polystyrene and stainless steel matrices in the presence and absence of organic matter. The results underscore OH''s ability to prevent establishment of L. monocytogenes biofilms by rapidly killing planktonic cells and to eliminate preformed biofilms, thus suggesting that it could be used as a disinfectant to prevent L. monocytogenes from persisting in food processing environments.Listeria monocytogenes is a major bacterial pathogen (2), accounting for approximately 28% of the deaths resulting from food-borne illnesses in the United States (22). It is widespread in nature and occurs in soil, vegetation, fecal matter, sewage, water, and animal feed (14). Because it is ubiquitous, L. monocytogenes is frequently isolated from foods and food processing environments (13, 23), thereby presenting a significant challenge to the food industry. Several studies have shown that L. monocytogenes is capable of adhering to food contact surfaces, such as glass, stainless steel, rubber, and polystyrene (6, 11, 28). Upon attachment to such surfaces, L. monocytogenes establishes biofilms and persists for long periods of time in the food processing environment (18, 30). This potentially poses a food safety hazard since biofilms are an important source of contamination of food products that come into contact with them. In addition, biofilms also protect the underlying bacteria from desiccation, antimicrobials, and sanitizing agents (7, 16). Thus, eradication of L. monocytogenes biofilms in processing plants is critical for improving food safety.When problems with persistent L. monocytogenes are encountered in food processing facilities, plant hygiene and sanitation are emphasized (31). This involves preventing the establishment of L. monocytogenes biofilms in the food processing environment and reducing contamination of product contact surfaces. A variety of cleaners and disinfectants, including quaternary ammonium compounds and hypochlorite, have been evaluated for this purpose (20). Although these compounds are approved by the Food and Drug Administration for use as disinfectants in processing plants, they are not effective in killing L. monocytogenes (24, 25), especially in the presence of soil or organic matter and at low temperatures. Therefore, there is a need for an effective disinfectant that can eliminate listerial biofilms in the presence of organic matter at a wide range of temperatures. Octenidine hydrochloride (OH) is a positively charged bispyridinamine that exhibits antimicrobial activity against plaque-producing organisms, such as Streptococcus mutans and Streptococcus sanguis (5). Toxicity studies with a variety of species have shown that OH is not absorbed through mucous membranes and the gastrointestinal tract, and there have been no reports of carcinogenicity, genotoxicity, or mutagenicity of this compound (17, 19, 29).The objective of this study was to investigate the efficacy of OH for inactivating planktonic cells and preformed biofilms of L. monocytogenes at 37, 21, 8, and 4°C in the presence and absence of organic matter on two matrices, polystyrene and stainless steel.     

Control of Listeria monocytogenes in a Biofilm by Competitive-Exclusion Microorganisms

Biofilms from drains in food processing facilities with a recent history of no detectable Listeria monocytogenes in floor drains were cultured for microorganisms producing antilisterial metabolites. A total of 413 microbial isolates were obtained from 12 drain biofilm samples and were assayed at 15 and 37°C for activities that were bactericidal or inhibitory to L. monocytogenes, by two agar plate assays. Twenty-one of 257 bacterial isolates and 3 of 156 yeast isolates had antilisterial activity. All 24 isolates which produced metabolites inhibitory to L. monocytogenes were assayed for antilisterial activity in coinoculated broth cultures containing tryptic soy broth with yeast extract (TSB-YE). A five-strain mixture of 10³ CFU of L. monocytogenes/ml and 10⁵ CFU of the candidate competitive-exclusion microorganism/ml was combined in TSB-YE and incubated at 37°C for 24 h, 15°C for 14 days, 8°C for 21 days, and 4°C for 28 days. Substantial inhibition of L. monocytogenes growth (4 to 5 log CFU/ml) was observed for nine bacterial isolates at 37°C, two at 15 and 8°C, and three at 4°C. The inhibitory isolates were identified as Enterococcus durans (six isolates), Lactococcus lactis subsp. lactis (two isolates), and Lactobacillus plantarum (one isolate). The anti-L. monocytogenes activity of these isolates was evaluated in biofilms of L. monocytogenes on stainless steel coupons at 37, 15, 8, and 4°C. Results revealed that two isolates (E. durans strain 152 and L. lactis subsp. lactis strain C-1-92) were highly inhibitory to L. monocytogenes (growth inhibition of >5 log₁₀ CFU of L. monocytogenes/cm²). These two bacterial isolates appear to be excellent competitive-exclusion candidates to control L. monocytogenes in biofilms at environmental temperatures of 4 to 37°C.     

Co-Culture with Listeria monocytogenes within a Dual-Species Biofilm Community Strongly Increases Resistance of Pseudomonas putida to Benzalkonium Chloride

Biofilm formation is a phenomenon occurring almost wherever microorganisms and surfaces exist in close proximity. This study aimed to evaluate the possible influence of bacterial interactions on the ability of Listeria monocytogenes and Pseudomonas putida to develop a dual-species biofilm community on stainless steel (SS), as well as on the subsequent resistance of their sessile cells to benzalkonium chloride (BC) used in inadequate (sub-lethal) concentration (50 ppm). The possible progressive adaptability of mixed-culture biofilms to BC was also investigated. To accomplish these, 3 strains per species were left to develop mixed-culture biofilms on SS coupons, incubated in daily renewable growth medium for a total period of 10 days, under either mono- or dual-species conditions. Each day, biofilm cells were exposed to disinfection treatment. Results revealed that the simultaneous presence of L. monocytogenes strongly increased the resistance of P. putida biofilm cells to BC, while culture conditions (mono-/dual-species) did not seem to significantly influence the resistance of L. monocytogenes biofilm cells. BC mainly killed L. monocytogenes cells when this was applied against the dual-species sessile community during the whole incubation period, despite the fact that from the 2nd day this community was mainly composed (>90%) of P. putida cells. No obvious adaptation to BC was observed in either L. monocytogenes or P. putida biofilm cells. Pulsed field gel electrophoresis (PFGE) analysis showed that the different strains behaved differently with regard to biofilm formation and antimicrobial resistance. Such knowledge on the physiological behavior of mixed-culture biofilms could provide the information necessary to control their formation.     

Current knowledge and perspectives on biofilm formation: the case of Listeria monocytogenes

Listeriosis is a rare, serious, and mainly food-borne infection caused by the bacterium Listeria monocytogenes. This food-borne infection primarily affects pregnant women and immunologically compromised individuals. L. monocytogenes is recognized as a problem for the food industry, mainly due to its environmental persistence, attributed in part to its ability to form biofilms. Biofilms are microbial communities adhered to biotic or abiotic surfaces coated by self-produced extracellular polymers. These structures confer protection to bacterial cells and decrease the efficiency of cleaning and disinfection procedures. This article presents a brief review of current perspectives on the formation of biofilms, with emphasis on L. monocytogenes, highlighting the importance of cell-to-cell communication and structural composition of the microbial communities. The techniques currently used to study biofilms and the need to develop new strategies for the prevention and control of biofilm-forming pathogens are also discussed.     

Identification of Listeria monocytogenes Determinants Required for Biofilm Formation

Listeria monocytogenes is a Gram-positive, food-borne pathogen of humans and animals. L. monocytogenes is considered to be a potential public health risk by the U.S. Food and Drug Administration (FDA), as this bacterium can easily contaminate ready-to-eat (RTE) foods and cause an invasive, life-threatening disease (listeriosis). Bacteria can adhere and grow on multiple surfaces and persist within biofilms in food processing plants, providing resistance to sanitizers and other antimicrobial agents. While whole genome sequencing has led to the identification of biofilm synthesis gene clusters in many bacterial species, bioinformatics has not identified the biofilm synthesis genes within the L. monocytogenes genome. To identify genes necessary for L. monocytogenes biofilm formation, we performed a transposon mutagenesis library screen using a recently constructed Himar1 mariner transposon. Approximately 10,000 transposon mutants within L. monocytogenes strain 10403S were screened for biofilm formation in 96-well polyvinyl chloride (PVC) microtiter plates with 70 Himar1 insertion mutants identified that produced significantly less biofilms. DNA sequencing of the transposon insertion sites within the isolated mutants revealed transposon insertions within 38 distinct genetic loci. The identification of mutants bearing insertions within several flagellar motility genes previously known to be required for the initial stages of biofilm formation validated the ability of the mutagenesis screen to identify L. monocytogenes biofilm-defective mutants. Two newly identified genetic loci, dltABCD and phoPR, were selected for deletion analysis and both ΔdltABCD and ΔphoPR bacterial strains displayed biofilm formation defects in the PVC microtiter plate assay, confirming these loci contribute to biofilm formation by L. monocytogenes.     

Role of RpoS and AlgT in Pseudomonas aeruginosa biofilm resistance to hydrogen peroxide and monochloramine

The role of two sigma factors, AlgT and RpoS, in mediating Pseudomonas aeruginosa biofilm resistance to hydrogen peroxide and monochloramine was investigated. Two knock out mutant strains, SS24 (rpoS-) and PAO6852 (algT-), were compared with a wild type, PAO1, in their susceptibility to monochloramine and hydrogen peroxide. When grown as biofilms on alginate gel beads (mean untreated areal cell density 3.7 +/- 0.27 log cfu cm-2) or on glass slides (mean untreated areal cell density 7.6 +/- 0.9 log cfu cm-2), wild type bacteria exhibited reduced susceptibility to both antimicrobial agents in comparison with suspended cells. On alginate gel beads, all strains were equally resistant to monochloramine. rpoS- and algT- gel bead biofilms of 24-hour-old were more susceptible to hydrogen peroxide disinfection than were biofilms formed by PAO1. Biofilm disinfection rate coefficients for the two mutant strains were statistically indistinguishable from planktonic disinfection rate coefficients, indicating complete loss of biofilm resistance. While 48-hour-old algT- biofilm cells became resistant to hydrogen peroxide, 48-hour-old rpoS- biofilm cells remained highly susceptible. With the thicker biofilms formed on glass coupons, all strains were equally resistant to both hydrogen peroxide and monochloramine. It is concluded that while RpoS and AlgT may play a transient role in protecting thin biofilms from hydrogen peroxide, these sigma factors do not mediate resistance to monochloramine and do not contribute significantly to the hydrogen peroxide resistance of thick biofilms.     

Failure of foodborne pathogens to develop resistance to sanitizers following repeated exposure to common sanitizers

The increased focus on food safety hygiene in the home and the food industry has resulted in an increasing use of chemical sanitizers. Whether the increased use of sanitizers will result in emergence of sanitizer-resistant foodborne pathogens has been speculated. Sanitizers that may be used in the home for sanitizing of surfaces or in hand sanitizers were evaluated and included sodium hypochlorite, chlorhexidine digluconate, and benzalkonium chloride. The adaptation of surface-associated and planktonic Escherichia coli O157:H7, Salmonella enterica subsp. enterica serovar Enteritidis, Staphylococcus aureus, Listeria monocytogenes and Pseudomonas aeruginosa to those sanitizers was investigated. In the suspension test, all sanitizers effectively reduced microbial number >5 log units after an exposure of 5 min. In the surface test, generally <3 log unit reduction was achieved when sanitizers were tested at 256 ppm. Repeated exposure of surface-associated bacteria resulted in a significant decrease in log number of cells after the first and second exposure to the sanitizer. Serial subculture of S. Enteritidis in sublethal concentrations of chlorexidine digluconate resulted in a stable decreased susceptibility to the agent, but not resistance. Results of this study suggest that foodborne bacterial pathogens remain susceptible to the sanitizers tested under conditions evaluated in this study.     

The elimination effects of lavender essential oil on Listeria monocytogenes biofilms developed at different temperatures and the induction of VBNC state

Listeria monocytogenes is a typical foodborne pathogen that causes hard-to-treat bacterial infections, mainly due to its ability to form biofilm and enter into a viable but non-culturable state (VBNC). In this study, we investigated the removal effects of four antimicrobial agents on L. monocytogenes biofilms formed at 32°C and 10°C, analysed the resistances of the mature biofilms to antimicrobial agents, and explored the VBNC state of cells in mature biofilms induced by lavender essential oil (LEO). The results showed that the growth of L. monocytogenes was completely inhibited when 1·6% (v/v) of the LEO was added. Meanwhile, the results of the crystal violet staining and XTT reduction method indicated that different concentrations of LEO significantly reduced L. monocytogenes biofilms biomass and metabolic activities, followed by sodium hypochlorite, lactic acid, and hydrogen peroxide. Moreover, the confocal laser scanning microscopy (CLSM) images confirmed that the treated biofilms became thinner, the structure was sparse, and the appearance was blurry. More interestingly, L. monocytogenes biofilms developed at 10°C were less susceptible to the sanitizers than those formed at 32°C. In addition, LEO presented a more significant dispersing effect on the biofilm cells, and 1/2 MIC to 4 MIC of LEO could induce fewer VBNC state cells in biofilm and plankton compared with sodium hypochlorite. This study indicated that the LEO could be considered as an ideal antibiofilm agent for controlling L. monocytogenes. But we should pay attention to the resistance of the biofilms developed at low temperatures.     

Incidence of Staphylococcus aureus and Analysis of Associated Bacterial Communities on Food Industry Surfaces

Biofilms are a common cause of food contamination with undesirable bacteria, such as pathogenic bacteria. Staphylococcus aureus is one of the major bacteria causing food-borne diseases in humans. A study designed to determine the presence of S. aureus on food contact surfaces in dairy, meat, and seafood environments and to identify coexisting microbiota has therefore been carried out. A total of 442 samples were collected, and the presence of S. aureus was confirmed in 6.1% of samples. Sixty-three S. aureus isolates were recovered and typed by random amplification of polymorphic DNA (RAPD). Profiles were clustered into four groups which were related to specific food environments. All isolates harbored some potential virulence factors such as enterotoxin production genes, biofilm formation-associated genes, antibiotic resistance, or lysogeny. PCR-denaturing gradient gel electrophoresis (PCR-DGGE) fingerprints of bacterial communities coexisting with S. aureus revealed the presence of bacteria either involved in food spoilage or of concern for food safety in all food environments. Food industry surfaces could thus be a reservoir for S. aureus forming complex communities with undesirable bacteria in multispecies biofilms. Uneven microbiological conditions were found in each food sector, which indicates the need to improve hygienic conditions in food processing facilities, particularly the removal of bacterial biofilms, to enhance the safety of food products.     

A green biocide enhancer for the treatment of sulfate-reducing bacteria (SRB) biofilms on carbon steel surfaces using glutaraldehyde

Generally speaking, a much higher concentration of biocide is needed to treat biofilms compared to the dosage used to for planktonic bacteria. With increasing restrictions of environmental regulations and safety concerns on large-scale biocide uses such as oil field applications, it is highly desirable to make more effective use of biocides. In this paper a green biocide enhancer ethylenediaminedisuccinate (EDDS) that is a biodegradable chelator, was found to enhance the efficacy of glutaraldehyde in its treatment of sulfate-reducing bacteria (SRB) biofilms. Experiments were carried out in 100 ml anaerobic vials with carbon steel coupons. The ATCC 14563 strain of Desulfovibrio desulfuricans was used. Biofilms on coupon surfaces were visualized using scanning electron microscopy (SEM). Experimental results showed that EDDS reduced the glutaraldehyde dosages considerably in the inhibition of SRB biofilm establishment and the treatment of established biofilms on carbon steel coupon surfaces.     

Biofilm Formation by Escherichia coli O157:H7 on Stainless Steel: Effect of Exopolysaccharide and Curli Production on Its Resistance to Chlorine

The resistance of Escherichia coli O157:H7 strains ATCC 43895-, 43895-EPS (an exopolysaccharide [EPS]-overproducing mutant), and ATCC 43895+ (a curli-producing mutant) to chlorine, a sanitizer commonly used in the food industry, was studied. Planktonic cells of strains 43895-EPS and/or ATCC 43895+ grown under conditions supporting EPS and curli production, respectively, showed the highest resistance to chlorine, indicating that EPS and curli afford protection. Planktonic cells (ca. 9 log₁₀ CFU/ml) of all strains, however, were killed within 10 min by treatment with 50 μg of chlorine/ml. Significantly lower numbers of strain 43895-EPS, compared to those of strain ATCC 43895-, attached to stainless steel coupons, but the growth rate of strain 43895-EPS on coupons was not significantly different from that of strain ATCC 43895-, indicating that EPS production did not affect cell growth during biofilm formation. Curli production did not affect the initial attachment of cells to coupons but did enhance biofilm production. The resistance of E. coli O157:H7 to chlorine increased significantly as cells formed biofilm on coupons; strain ATCC 43895+ was the most resistant. Population sizes of strains ATCC 43895+ and ATCC 43895- in biofilm formed at 12°C were not significantly different, but cells of strain ATCC 43895+ showed significantly higher resistance than did cells of strain ATCC 43895-. These observations support the hypothesis that the production of EPS and curli increase the resistance of E. coli O157:H7 to chlorine.     

Genome Analysis of Listeria monocytogenes Sequence Type 8 Strains Persisting in Salmon and Poultry Processing Environments and Comparison with Related Strains

Listeria monocytogenes is an important foodborne pathogen responsible for the disease listeriosis, and can be found throughout the environment, in many foods and in food processing facilities. The main cause of listeriosis is consumption of food contaminated from sources in food processing environments. Persistence in food processing facilities has previously been shown for the L. monocytogenes sequence type (ST) 8 subtype. In the current study, five ST8 strains were subjected to whole-genome sequencing and compared with five additionally available ST8 genomes, allowing comparison of strains from salmon, poultry and cheese industry, in addition to a human clinical isolate. Genome-wide analysis of single-nucleotide polymorphisms (SNPs) confirmed that almost identical strains were detected in a Danish salmon processing plant in 1996 and in a Norwegian salmon processing plant in 2001 and 2011. Furthermore, we show that L. monocytogenes ST8 was likely to have been transferred between two poultry processing plants as a result of relocation of processing equipment. The SNP data were used to infer the phylogeny of the ST8 strains, separating them into two main genetic groups. Within each group, the plasmid and prophage content was almost entirely conserved, but between groups, these sequences showed strong divergence. The accessory genome of the ST8 strains harbored genetic elements which could be involved in rendering the ST8 strains resilient to incoming mobile genetic elements. These included two restriction-modification loci, one of which was predicted to show phase variable recognition sequence specificity through site-specific domain shuffling. Analysis indicated that the ST8 strains harbor all important known L. monocytogenes virulence factors, and ST8 strains are commonly identified as the causative agents of invasive listeriosis. Therefore, the persistence of this L. monocytogenes subtype in food processing facilities poses a significant concern for food safety.