Biofilm formation by heat-resistant dairy bacteria: multispecies biofilm model under static and dynamic conditions.

In the food industry, especially dairy, biofilms can be formed by heat-resistant spoilage and pathogenic bacteria from the farm. Such biofilms may persist throughout the processing chain and contaminate milk and dairy products continuously, increasing equipment cleaning, maintenance costs, and product recalls. Most biofilms are multispecies, yet most studies focus on single-species models. A multispecies model of dairy biofilm was developed under static and dynamic conditions using heat-resistant Bacillus licheniformis, Pseudomonas aeruginosa, Clostridium tyrobutyricum, Enterococcus faecalis, Streptococcus thermophilus, and Rothia kristinae isolated from dairies. C. tyrobutiricum and R. kristinae were weak producers of biofilm, whereas the other four were moderate to strong producers. Based on cross-streaking on agar, P. aeruginosa was found to inhibit B. licheniformis and E. faecalis. In multispecies biofilm formed on stainless steel in a CDC reactor fed microfiltered milk, the strong biofilm producers were dominant while the weak producers were barely detectable. All biofilm matrices were dispersed easily by proteinase K treatment but were less sensitive to DNase or carbohydrases. Further studies are needed to deepen our understanding of multispecies biofilms and interactions within to develop improved preventive strategies to control the proliferation of spoilage and pathogenic bacteria in dairies and other food processing environments. IMPORTANCE A model of multispecies biofilm was created to study biofilm formation by heat-resistant bacteria in the dairy industry. The biofilm formation potential was evaluated under static conditions. A continuous flow version was then developed to study multispecies biofilm formed on stainless steel in microfiltered milk under dynamic conditions encountered in dairy processing equipment. The study of biofilm composition and bacterial interactions therein will lead to more effective means of suppressing bacterial growth on food processing equipment and contamination of products with spoilage and pathogenic bacteria, which represent considerable economic loss.

Inactivation of foodborne viruses by novel organic peroxyacid-based disinfectants.

Viruses are responsible for most enteric foodborne illnesses worldwide. The foods most frequently involved are fresh fruits and vegetables since they undergo little or no processing. Washing with a chemical disinfectant is a convenient way of inactivating viruses on foods. Peracetic acid, widely used as a disinfectant in the food industry, has the drawback of leaving a strong odor and is ineffective alone against some foodborne viruses. In this study, four disinfectants, namely per levulinic acid with or without sodium dodecyl sulfate, peracetic acid and a commercial peracetic acid-based disinfectant were tested on murine norovirus 1 (MNV-1), hepatitis A virus (HAV), and hepatitis E virus (HEV). Disinfectant concentrations were 50, 80, 250, 500, and 1000 mg l-1 and contact times were 0.5, 1, 5, and 10 min. Under these conditions, per levulinic acid supplemented with 1% SDS reduced MNV-1 infectious titer by 3 log cycles vs. 2.24 log cycles by peracetic acid within 0.5 min. On stainless steel at 80 ppm, only peracetic acid produced 3-log reductions within 0.5 min. None of these peroxyacids was able to reduce infectious titers of HAV or HEV by even 2 log cycles at any concentration or time-tested. This study will guide the development of new chemical formulas that will be more effective against major foodborne viruses and will have less impact on food quality and the environment.

Protective Effect of Select Bacterial Species Representative of Fresh Produce on Human Norovirus Surrogates Exposed to Disinfecting Pulsed Light.

Contamination of berries and leafy greens with human norovirus (HuNoV) is a major cause of outbreaks of epidemic gastroenteritis worldwide. Using murine norovirus type 1 (MNV-1) and Tulane virus, we studied the possible extension of HuNoV persistence by biofilm-producing epiphytic bacteria on fresh produce. Nine bacterial species frequently found on the surface of berries and leafy greens (Bacillus cereus, Enterobacter cloacae, Escherichia coli, Kocuria kristinae, Lactobacillus plantarum, Pantoea agglomerans, Pseudomonas fluorescens, Raoultella terrigena, and Xanthomonas campestris) were evaluated for the ability to form biofilms in the MBEC Assay Biofilm Inoculator and in 96-well microplates. The biofilm-forming bacteria were further tested for binding MNV-1 and Tulane virus and the ability to protect them against loss of capsid integrity upon exposure to disinfecting pulsed light at a fluence of 11.52 J/cm2. Based on viral reductions, MNV-1 did not benefit from attachment to biofilm whereas Tulane virus was significantly more resistant than the control when attached to biofilms of E. cloacae (P ≤ 0.01), E. coli (P ≤ 0.01), K. kristinae (P ≤ 0.01), P. agglomerans (P ≤ 0.05), or P. fluorescens (P ≤ 0.0001). Enzymatic dispersion of biofilm and microscopic observations suggest that the biofilm matrix composition may contribute to the virus resistance. Our results indicate that direct virus-biofilm interaction protects Tulane virus against disinfecting pulsed light, and that HuNoV on fresh produce therefore might resist such treatment more than suggested by laboratory tests so far. IMPORTANCE Recent studies have shown that bacteria may be involved in the attachment of HuNoV to the surface of fresh produce. Because these foods are difficult to disinfect by conventional methods without compromising product quality, nonthermal nonchemical disinfectants such as pulsed light are being investigated. We seek to understand how HuNoV interacts with epiphytic bacteria, particularly with biofilms formed by bacterial epiphytes, with cells and extracellular polymeric substances, and to determine if it thus escapes inactivation by pulsed light. The results of this study should advance understanding of the effects of epiphytic biofilms on the persistence of HuNoV particle integrity after pulsed light treatment and thus guide the design of novel pathogen control strategies in the food industry.

Evaluation of the Efficacy of Organic Peroxyacids for Eradicating Dairy Biofilms Using an Approach Combining Static and Dynamic Methods.

The presence of biofilms in the dairy industry is of major concern, as they may lead to the production of unsafe and altered dairy products due to their high resistance to most clean-in-place (CIP) procedures frequently used in processing plants. Therefore, it is imperative to develop new biofilm control strategies for the dairy industry. This protocol is aimed at evaluating the efficacy of organic peroxyacids (peracetic, perpropionic, and perlactic acids and a commercial peracetic acid-based disinfectant) for eradicating dairy biofilms using a combination of static and dynamic methods. All the disinfectants were tested on the strongest biofilm-producing bacteria in either a single or a mixed biofilm using the minimum biofilm eradication concentration (MBEC) assay, a static high-throughput screening method. A contact time of 5 min with the disinfectants at the recommended concentrations successfully eradicated both the single and mixed biofilms. Studies are currently ongoing to confirm these observations using the Center for Disease Control (CDC) biofilm reactor, a dynamic method to mimic in situ conditions. This type of bioreactor enables the use of a stainless-steel surface, which constitutes most industrial equipment and surfaces. The preliminary results from the reactor appear to confirm the efficacy of organic peroxyacids against biofilms. The combined approach described in this study may be used to develop and test new biological or chemical formulations for controlling biofilms and eradicating microorganisms.

Comparative Study of Different Sampling Methods of Biofilm Formed on Stainless-Steel Surfaces in a CDC Biofilm Reactor.

The formation of biofilms in dairy processing plants can reduce equipment efficiency, contribute to surface deterioration, and contaminate dairy products by releasing the microorganisms they contain, which may cause spoilage or disease. However, a more representative identification of microbial communities and physico-chemical characterization requires to detach and recover adequately the entire biofilm from the surface. The aim of this study is to develop an efficient technique for in-plant biofilm sampling by growing a strain of Pseudomonas azotoformans PFl1A on stainless-steel surface in a dynamic CDC biofilm reactor system using tryptic soy broth (TSB) and milk as growth media. Different techniques, namely, swabbing, scraping, sonic brushing, synthetic sponge, and sonicating synthetic sponge were used and the results were compared to a standard ASTM International method using ultrasonication. Their efficiencies were evaluated by cells enumeration and scanning electron microscopy. The maximum total viable counts of 8.65 ± 0.06, 8.75 ± 0.08, and 8.71 ± 0.09 log CFU/cm2 were obtained in TSB medium using scraping, synthetic sponge, and sonicating synthetic sponge, respectively, which showed no statistically significant differences with the standard method, ultrasonication (8.74 ± 0.02 log CFU/cm2). However, a significantly (p < 0.05) lower cell recovery of 8.57 ± 0.10 and 8.60 ± 0.00 log CFU/cm2 compared to ultrasonication were achieved for swabbing and sonic brushing, respectively. Furthermore, scanning electron microscopy showed an effective removal of biofilms by sonic brushing, synthetic sponge, and sonicating synthetic sponge; However, only the latter two methods guaranteed a superior release of bacterial biofilm into suspension. Nevertheless, a combination of sonication and synthetic sponge ensured dislodging of sessile cells from surface crevices. The results suggest that a sonicating synthetic sponge could be a promising method for biofilm recovery in processing plants, which can be practically used in the dairy industries as an alternative to ultrasonication.

Secondary Staphylococcus aureus intramammary colonization is reduced by non-aureus staphylococci exoproducts.

Non-aureus staphylococci (NAS) and Staphylococcus aureus are pathogens that cause bovine mastitis, a costly disease for dairy farmers, however; many NAS are considered part of the normal udder microbiota. It has been suggested that through a mechanism that remains to be elucidated, NAS intramammary colonization can prevent subsequent infection with other bacterial pathogens. This study shows that in a murine mastitis model, secondary Staph. aureus intramammary colonization is reduced by exoproducts from Staph. chromogenes and Staph. simulans, both NAS, while Streptococcus spp. exoproducts have much less ability to affect the course of the infection caused by S. aureus.

Efficacy of Organic Peroxyacids for Eliminating Biofilm Preformed by Microorganisms Isolated from Dairy Processing Plants.

The aim of this study was to evaluate the ability of microorganisms isolated from the dairy industry to form biofilms and to investigate the efficacy of organic peroxyacids (peracetic, perpropionic, and perlactic acids and BioDestroy) to eradicate those biofilms. Eighteen microorganisms were isolated from Quebec dairy processing plants that have issues associated with biofilm formation and were presumptively identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The single-species biofilm-producing ability of the isolates was then evaluated using 96-well microplates. Eight out of 18 of these isolates were identified as moderate or strong biofilm producers, and 10 out of 18 were negative or weak biofilm producers. The efficacy of the above-mentioned disinfectants was tested on the stronger biofilm-producing bacteria using the MBEC (minimum biofilm eradication concentration) assay. After 5 min, all disinfectants tested successfully eradicated both the single and mixed biofilms when applied following the recommended concentration. However, the efficacy of organic peroxyacids was significantly variable at lower concentrations. For example, 25 ppm of BioDestroy was sufficient to eradicate all the biofilms, except for Pseudomonas azotoformans PFl1A. Unfortunately, microscopic observations highlighted those dead cells were still attached to the surfaces. In conclusion, our results suggest that some microorganisms found in dairy plants can produce tenacious biofilms that are still susceptible to disinfectants, including organic peroxyacids. Further studies would be needed to confirm these observations using a dynamic method to mimic in vivo conditions. IMPORTANCE Biofilm-forming microorganisms are a major issue in the food industry, including the dairy industry, because of their negative impact on product quality. Biofilms are difficult to remove by clean-in-place (CIP) procedures commonly used in processing plants and may be less sensitive to sanitizers. Therefore, it is important to identify these microorganisms to develop biofilm control strategies. The results gathered in the present study could contribute to this aim, even though it was carried out using only static methods.

Physicochemical Parameters Affecting Norovirus Adhesion to Ready-To-Eat Foods.

The adhesion of noroviruses to strawberry, turkey slices, ham, and cheddar cheese was studied using murine norovirus 1 (MNV-1) as a surrogate for human norovirus (NoV). Based on plaque assay, the recovery and adhesion of MNV-1 depended on the food type (turkey versus strawberry), pH of the initial suspension buffer (pH 4 versus pH 7), and food fat composition (C8 versus C18). Recovery of infectious particles from turkey was 68% compared to 9.4% from strawberry. On turkey, adhesion of MNV-1 was lower at pH 7 (pH of fecal matter), and virus particles adhered to this pH were recovered more easily (33,875 PFU) than at pH 4 (pH of vomitus). The presence of fat and the composition of fatty acids seemed to increase MNV-1 recovery and adherent viral particles recovered but did not affect adhesion (68% on fat-free turkey and regular turkey). Adherent MNV-1 particles recovered from stainless steel coated with saturated fatty acid (C8, C14, C18) increased significantly with chain length (P < 0.05), but adhesion did not seem to change. Using liquid droplet contact angle to measure surface energy, it was deduced that hydrophobic interactions contribute considerably to the adhesion of MNV-1 to stainless steel, polyvinyl chloride, and high-density polyethylene. IMPORTANCE Ready-to-eat (RTE) foods are major vehicles of transmission of foodborne viral pathogens, including NoV. The high incidence of gastroenteritis caused by viruses is due largely to their persistence in the environment and adhesion to different kinds of surfaces in the food industry, including the foods themselves. Compared with bacteria, adhesion of viruses to surfaces is poorly understood. Better knowledge of the physicochemical parameters involved in the adhesion of NoV to ready-to-eat foods is essential to devising effective strategies for reducing the persistence and, thus, the transmission of this virus.

Counteracting Bacterial Motility: A Promising Strategy to Narrow Listeria monocytogenes Biofilm in Food Processing Industry.

Listeria monocytogenes (L. monocytogenes) is often associated with processed food as it can form biofilms that represent a source of contamination at all stages of the manufacturing chain. The control and prevention of biofilms in food-processing plants are of utmost importance. This study explores the efficacy of prospect molecules for counteracting bacterial mechanisms leading to biofilm formation. The compounds included the phytomolecule tomatidine, zinc chloride (ZnCl2), ethylenediaminetetraacetic acid (EDTA), and a more complexed mixture of bacterial compounds from coagulase-negative staphylococci (CNS exoproducts). Significant inhibition of L. monocytogenes biofilm formation was evidenced using a microfluidic system and confocal microscopic analyses (p < 0.001). Active molecules were effective at an early stage of biofilm development (≥50% of inhibition) but failed to disperse mature biofilms of L. monocytogenes. According to our findings, prevention of surface attachment was associated with a disruption of bacterial motility. Indeed, agar cell motility assays demonstrated the effectiveness of these molecules. Overall, results highlighted the critical role of motility in biofilm formation and allow to consider flagellum-mediated motility as a promising molecular target in control strategies against L. monocytogenes in food processing environments.

The New Face of Berries: A Review of Their Antiviral Proprieties.

Due to rising consumer preference for natural remedies, the search for natural antiviral agents has accelerated considerably in recent years. Among the natural sources of compounds with potential antiviral proprieties, berries are interesting candidates, due to their association with health-promoting properties, including antioxidant, antimutagenic, anticancer, antimicrobial, anti-inflammatory, and neuroprotective properties. The past two decades have witnessed a flurry of new findings. Studies suggest promising antiviral proprieties against enveloped and non-enveloped viruses, particularly of cranberries, blueberries, blackcurrants, black raspberries, and pomegranates. The aim of this review is to assemble these findings, to list the implied mechanisms of action, and thereby point out promising subjects for research in this field, in the hope that compounds obtainable from natural sources such as berries may be used someday to treat, or even prevent, viral infections.

Coagulase-negative staphylococci species affect biofilm formation of other coagulase-negative and coagulase-positive staphylococci.

Coagulase-negative staphylococci (CNS) are considered to be commensal bacteria in humans and animals, but are now also recognized as etiological agents in several infections, including bovine mastitis. Biofilm formation appears to be an important factor in CNS pathogenicity. Furthermore, some researchers have proposed that CNS colonization of the intramammary environment has a protective effect against other pathogens. The mechanisms behind the protective effect of CNS have yet to be characterized. The aim of this study was to evaluate the effect of CNS isolates with a weak-biofilm phenotype on the biofilm formation of other staphylococcal isolates. We selected 10 CNS with a weak-biofilm phenotype and 30 staphylococcal isolates with a strong-biofilm phenotype for this study. We measured biofilm production by individual isolates using a standard polystyrene microtiter plate assay and compared the findings with biofilm produced in mixed cultures. We confirmed the results using confocal microscopy and a microfluidic system with low shear force. Four of the CNS isolates with a weak-biofilm phenotype (Staphylococcus chromogenes C and E and Staphylococcus simulans F and H) significantly reduced biofilm formation in approximately 80% of the staphylococcal species tested, including coagulase-positive Staphylococcus aureus. The 4 Staph. chromogenes and Staph. simulans isolates were also able to disperse pre-established biofilms, but to a lesser extent. We also performed a deferred antagonism assay and recorded the number of colony-forming units in the mixed-biofilm assays on differential or selective agar plates. Overall, CNS with a weak-biofilm phenotype did not inhibit the growth of isolates with a strong-biofilm phenotype. These results suggest that some CNS isolates can negatively affect the ability of other staphylococcal isolates and species to form biofilms via a mechanism that does not involve growth inhibition.