Category Archives: Biofilm

Research – Interaction and inactivation of Listeria and Lactobacillus cells in single and mixed species biofilms exposed to different disinfectants

Wiley Online

Abstract

Listeria spp. are ubiquitously found in both the natural and the food processing environment, of which Listeria monocytogenes is of an important health risk. Here, we report on the formation of single and mixed species biofilms of L. monocytogenes/Listeria innocua and Lactobacillus plantarum strains in 24‐well polystyrene microtiter plates and on the inactivation of 24‐hr and 72‐hr biofilms using quaternary ammonium compound‐, tertiary alkyl amine‐, and chlorine‐based disinfectants. Fluorescent in situ hybridization (FISH) and LIVE/DEAD BacLight staining were applied for 72‐hr L. innocuaL. plantarum mixed biofilms in the LabTek system for the species identification and the reaction of biofilm cells to disinfectants, respectively. L. monocytogenes/L. innocua were more resistant to disinfectants in 72‐hr than in 24‐hr biofilms, whereas L. plantarum strains did not show any significant differences between 72‐hr and 24‐hr biofilms. Furthermore, L. innocua when grown with L. plantarum was more resistant to all disinfection treatments, indicating a protective effect from lactobacilli in the mixed species biofilm. The biofilm formation and reaction to disinfectants, microscopically verified using fluorescence in situ hybridization and LIVE/DEAD staining, showed that L. innocua and L. plantarum form a dense mixed biofilm and also suggested the shielding effect of L. plantarum on L. innocua in the mixed species biofilm.

Research – Modulation of flagellar rotation in surface-attached bacteria: A pathway for rapid surface-sensing after flagellar attachment

PLOS

Abstract

Attachment is a necessary first step in bacterial commitment to surface-associated behaviors that include colonization, biofilm formation, and host-directed virulence. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa can initially attach to surfaces via its single polar flagellum. Although many bacteria quickly detach, some become irreversibly attached and express surface-associated structures, such as Type IV pili, and behaviors, including twitching motility and biofilm initiation. Paeruginosa that lack the GTPase FlhF assemble a randomly placed flagellum that is motile; however, we observed that these mutant bacteria show defects in biofilm formation comparable to those seen for non-motile, aflagellate bacteria. This phenotype was associated with altered behavior of ΔflhF bacteria immediately following surface-attachment. Forward and reverse genetic screens led to the discovery that FlhF interacts with FimV to control flagellar rotation at a surface, and implicated cAMP signaling in this pathway. Although cAMP controls many transcriptional programs in Paeruginosa, known targets of this second messenger were not required to modulate flagellar rotation in surface-attached bacteria. Instead, alterations in switching behavior of the motor appeared to result from direct or indirect effects of cAMP on switch complex proteins and/or the stators associated with them.

Author summary

Attachment to a surface often triggers programs of gene expression that alter the behavior, virulence and fitness of bacteria. Initial contact is usually mediated by surface exposed adhesins, such as flagella or pili/fimbriae, and there is much interest in how these structures might sense and respond to surface attachment. The human bacterial pathogen Pseudomonas aeruginosa can initially contact surfaces via its polar flagellum, the structure that also powers bacterial swimming. We observed that wild-type bacteria quickly stopped rotating their flagellum after surface attachment, but that a mutant lacking the flagellar-associated protein FlhF did not. Using a combination of genetic approaches, we demonstrated that FlhF interacts with a component of the flagellar rotor (FliG) and with a polar scaffolding protein that positively regulates cAMP production (FimV) to stop flagellar rotation and thereby favor bacterial persistence at a surface. We provide evidence that the second messenger cAMP is the likely signal generated by flagellar-mediated surface attachment and show that cAMP is sufficient to alter the behavior of the flagellar motor.

Research – Interaction and inactivation of Listeria and Lactobacillus cells in single and mixed species biofilms exposed to different disinfectants

Wiley Online

Abstract

Listeria spp. are ubiquitously found in both the natural and the food processing environment, of which Listeria monocytogenes is of an important health risk. Here, we report on the formation of single and mixed species biofilms of L. monocytogenes/Listeria innocua and Lactobacillus plantarum strains in 24‐well polystyrene microtiter plates and on the inactivation of 24‐hr and 72‐hr biofilms using quaternary ammonium compound‐, tertiary alkyl amine‐, and chlorine‐based disinfectants. Fluorescent in situ hybridization (FISH) and LIVE/DEAD BacLight staining were applied for 72‐hr L. innocuaL. plantarum mixed biofilms in the LabTek system for the species identification and the reaction of biofilm cells to disinfectants, respectively. L. monocytogenes/L. innocua were more resistant to disinfectants in 72‐hr than in 24‐hr biofilms, whereas L. plantarum strains did not show any significant differences between 72‐hr and 24‐hr biofilms. Furthermore, L. innocua when grown with L. plantarum was more resistant to all disinfection treatments, indicating a protective effect from lactobacilli in the mixed species biofilm. The biofilm formation and reaction to disinfectants, microscopically verified using fluorescence in situ hybridization and LIVE/DEAD staining, showed that L. innocua and L. plantarum form a dense mixed biofilm and also suggested the shielding effect of L. plantarum on L. innocua in the mixed species biofilm.

Research – Antibiofilm Efficacy of Peptide 1018 against Listeria monocytogenes and Shiga Toxigenic Escherichia coli on Equipment Surfaces

Journal of Food Protection

ABSTRACT

Listeria monocytogenes and Shiga toxigenic Escherichia coli (STEC) are important foodborne bacterial pathogens that can form biofilms on equipment surfaces at food processing facilities. Pathogens in biofilms are resistant to conventional antimicrobials and require higher antimicrobial concentrations to be inactivated. In this study, the efficacy of a synthetic innate defense regulator peptide 1018 (peptide 1018) for inactivating L. monocytogenes and STEC (O26, O111, O145, O157) biofilms on stainless steel and polycarbonate surfaces was investigated. Stainless steel and polycarbonate coupons (12 mm in diameter) were used in a Centers for Disease Control and Prevention biofilm reactor containing 400 mL of 10% tryptic soy broth (TSB) that had been inoculated with an individual strain of L. monocytogenes or STEC to obtain 6 log CFU/mL populations. The reactor was set with a constant flow rate at 50 mL/h of 10% TSB for 48 h. After 48 h, coupons were treated with peptide 1018 at 0, 10, 20, or 50 μg/mL in phosphate buffer saline (PBS) for 24 h. Surviving bacterial populations were determined by scraping off the coupons and spiral plating on selective media. Significantly higher levels of pathogens in biofilms formed by certain bacterial strains, including L. monocytogenes F6854, E. coli O157:H7 RM4407 and NADC5713, and non-O157 E. coli NADC3629, were recovered on polycarbonate surfaces than on stainless steel. Antibiofilm efficacy of peptide 1018 against pathogens was concentration-dependent and varied with the type of pathogen and material surfaces. Peptide 1018 at 50 μg/mL significantly inactivated all tested bacterial biofilms on both surfaces compared with the PBS control (P < 0.05). L. monocytogenes was the bacterium most sensitive to peptide 1018; on stainless steel surfaces treated with 50 μg/mL peptide 1018, there was a 3.7- to 4.6-log CFU/cm2 reduction in Listeria populations compared with a 1.0- to 3.5-log CFU/cm2 reduction of STEC. Results suggest that peptide 1018 may be used to inactivate L. monocytogenes and STEC biofilms on equipment surfaces.

HIGHLIGHTS
  • Bacteria attach at higher levels on polycarbonate surfaces than on stainless steel.

  • L. monocytogenes is more sensitive than STEC to peptide 1018.

  • Peptide 1018 can be used to inactivate biofilms on equipment surfaces.

Research – Probiotics Lactobacillus strains: A promising alternative therapy against to biofilm-forming enteropathogenic bacteria?

Academic Journals

Biofilms formation stands out in context of persistent intestinal infections caused by Enterobacteriaceae, which are associated with a high resistance to antimicrobial agents’ and phagocytosis by host defense cells. Hence, understanding the mechanisms involved in this process becomes major for the development of new preventive and therapeutic strategies. Lactic acid bacteria, including species of the genus Lactobacillus, have been associated with the prevention or dispersion of biofilms formed by pathogenic microorganisms. This effect is often associated with the antimicrobial substances production, among them organic acids, bacteriocins, hydrogen peroxide and biorsurfactants. However, the antibiofilm action of Lactobacillus seems to be strain-specific and may not be demonstrated by strains of the same genus. Thus, diet supplementation with beneficial microorganisms represents a possible strategy for prevention and treatment of intestinal infectious diseases, such as persistent or acute diarrhea caused by enteropathogenic bacteria. However, in vitro and in vivo further studies are needed to clarify the efficacy of different probiotic candidates, including commercially available products.

 

Research – Inactivation of Staphylococcus aureus Biofilms on Food Contact Surfaces by Superheated Steam Treatment

Journal of Food Protection Staph

ABSTRACT

The objective of this study was to compare the inactivation efficacy of saturated steam (SS) and superheated steam (SHS) on Staphylococcus aureus biofilms on food contact surfaces, including type 304 stainless steel coupons with No. 4 finish (STS No. 4), type 304 stainless steel coupons with 2B finish (STS 2B), high-density polyethylene (HDPE), and polypropylene (PP). In addition, the effects of the surface characteristics on the inactivation efficacy were evaluated. Biofilms were formed on each food contact coupon surface using a three-strain cocktail of S. aureus. Five-day-old biofilms on STS No. 4, STS 2B, HDPE, and PP coupons were treated with SS at 100°C and SHS at 125 and 150°C for 2, 4, 7, 10, 15, and 20 s. Among all coupon types, SHS was more effective than SS in inactivating the S. aureus biofilms. S. aureus biofilms on steel coupons were more susceptible to most SS and SHS treatments than the biofilms on plastic coupons. S. aureus biofilms on HDPE and PP coupons were reduced by 4.00 and 5.22 log CFU per coupon, respectively, after SS treatment (100°C) for 20 s. SS treatment for 20 s reduced the amount of S. aureus biofilm on STS No. 4 and STS 2B coupons to below the detection limit. With SHS treatment (150°C), S. aureus biofilms on HDPE and PP needed 15 s to be inactivated to below the detection limit, while steel coupons only needed 10 s. The results of this study suggest that SHS treatment has potential as a biofilm control intervention for the food industry.

HIGHLIGHTS
  • SHS was more effective than SS for inactivating biofilm cells of S. aureus.

  • Biofilms on steel coupons were more susceptible than those on plastic coupons.

  • The thermal conductivity of the coupon was an important factor in SHS treatment.

  • Biofilm; Saturated steam; Staphylococcus aureus; Superheated steam

Research – Preliminary Assessment of Visible, Near-Infrared, and Short-Wavelength–Infrared Spectroscopy with a Portable Instrument for the Detection of Staphylococcus aureus Biofilms on Surfaces

Journal of Food Protection

ABSTRACT

Bacterial biofilms constitute a major source of sanitary problems and economic losses in the food industry. Indeed, biofilm removal may require intense mechanical cleaning procedures or very high concentrations of disinfectants or both, which can be damaging to the environment and human health. This study assessed the efficacy of a technique based on spectroscopy in the visible, near-infrared, and short-wavelength infrared range for the quick detection of biofilms formed on polystyrene by the pathogenic bacterium Staphylococcus aureus. To do that, biofilms corresponding to three S. aureus strains, which differed in biofilm-forming ability and composition of the extracellular matrix, were allowed to develop for 5 or 24 h, representing an active formation stage and mature biofilms, respectively. Spectral analysis of the samples, corresponding to three biological replicates of each condition, was then performed by using a portable device. The results of these experiments showed that partial least-squares discriminant analysis of the spectral profile could discriminate between surfaces containing attached bacterial biomass and noninoculated ones. In this model, the two first principal components accounted for 39 and 19% of the variance and the estimated error rate stabilized after four components. Cross-validation accuracy of this assessment was 100%. This work lays the foundation for subsequent development of a spectroscopy-based protocol that allows biofilm detection on food industrial surfaces.

HIGHLIGHTS
  • A biofilm detection spectroscopy–based technique with a portable device was tested.

  • Staphylococcus aureus biofilms of different strengths were scanned with the device.

  • Spectral data showed correlation with crystal violet staining quantification results.

  • Data from spectral analysis was suitable for prediction of biofilm contamination.

Research – Effect of Sodium Hypochlorite on Biofilm-Forming Ability of Histamine-Producing Bacteria Isolated from Fish

Journal of Food Protection

ABSTRACT

Histamine poisoning occurs when temperature-abused marine fish containing elevated levels of histamine are consumed. Histamine-producing bacteria found in fish can colonize processing surfaces and form biofilms. In this study, the biofilm-forming abilities of histamine-producing bacteria from Indian mackerel (Rastrelliger kanagurta) and the effect of hypochlorite treatment on biofilm formation were studied. The isolates of this study produced histamine in the range of 471 to 2,126 ppm. The histidine decarboxylase gene hdc was detected in all isolates producing histamine except in one strain each of Psychrobacter pulmonis and Proteus vulgaris. All isolates tested in this study produced moderate biofilms under control conditions, whereas exposure to 1 and 3 ppm of sodium hypochlorite significantly enhanced biofilm formation. However, exposure to 5 ppm of sodium hypochlorite showed an inhibitory effect on biofilm formation by all the isolates except Klebsiella variicola. The results of this study suggest that histamine-producing bacteria can form stable biofilms and that this activity may be enhanced by the application of low levels of sodium hypochlorite, a phenomenon that might influence the persistence of histamine-producing bacteria in fish processing areas.

HIGHLIGHTS
  • Bacteria isolated from Indian mackerel produced histamine in the range of 471 to 2,126 ppm.

  • Histamine-producing bacteria isolated from the same fish can vary in the levels of histamine produced.

  • The hdc gene was not detected in one strain each of Psychrobacter pulmonis and Proteus vulgaris.

  • All histamine-producing bacteria formed moderate biofilms under control conditions.

  • Exposure to 1 and 3 ppm of sodium hypochlorite increased biofilm formation by histamine-producing bacteria.

Research -Insights into Psychrotrophic Bacteria in Raw Milk: A Review

Journal of Food Protection

ABSTRACT

Supply of high-quality dairy products is the goal of every dairy producer throughout the world. However, psychrotrophic bacteria in raw milk, generally comprising bacterial species of the genera Pseudomonas, Acinetobacter, Aeromonas, Serratia, Bacillus, Lactococcus, Microbacterium, and Staphylococcus, are of special concern to the dairy industry because they can produce heat-stable enzymes. These enzymes may withstand various heat treatments during dairy processing, causing quality defects over the product storage period. The levels and diversity of psychrotrophic bacteria in raw milk are tightly linked to natural habitats, milking practices, and hygiene practices of farms in different countries. Most psychrotrophic bacteria can form biofilms on various milk storage and processing equipment, which serve as persistent sources of microbial contamination due to their biotransfer potential. A comprehensive review of the diversity of psychrotrophic bacteria in raw milk, the spoilage potential of these bacteria, the specific technological problems caused by biofilms and heat-resistant enzymes, and potential strategies for controlling dairy spoilage starting at the farm level is needed for improving our understanding of these spoilage bacteria.

HIGHLIGHTS
  • Levels of psychrotrophic bacteria in raw milk are affected by to habitats and farm hygiene.

  • Biofilms formed by psychrotrophic bacteria are persistent sources of contamination.

  • Heat-stable enzymes produced by psychrotrophic bacteria compromise product quality.

  • Various strategies are available for controlling dairy spoilage caused by psychrotrophic bacteria.

Research – How flow shapes bacterial biofilms

Science Daily

Although we tend to think of them as solitary sojourners of the world, bacteria are actually very social organisms. In fact, the vast majority of bacteria live on surfaces by forming “biofilms”: three-dimensional communities hosting thousands to millions of bacteria of such bustling activity that scientists describe them as “bacterial cities.”

Bacteria form biofilms by attaching to each other on a wide variety of surfaces: the bottom of oceans, lakes or rivers, medical equipment and even internal organs, like the intestine, lungs, and teeth — the latter is the familiar dental plaque, a large source of income for dentists.

In short, biofilms are the preferred lifestyle of bacteria. They grow wide and thick, forming a new, social dynamic among their member microorganisms, while also defending them: biofilms can be notoriously inaccessible to antibiotics, which is why they have drawn a lot of medical research.

But looking at biofilms can also give us clues about broader social dynamics that have shaped the evolution of species across the entire planet, like cooperation, competition etc. And it is such questions that drive the work of Alexandre Persat, director of EPFL’s Microbial Mechanics Lab.