Campylobacter Jejuni Biofilm Formation Research Articles

Increase in temperature facilitates Campylobacter jejuni biofilm formation under both aerobic and microaerobic incubation

The formation of Campylobacter jeuni biofilms on processing surfaces is a significant concern in poultry processing, contributing to food safety risks. This study focused on assessing the biofilm forming capabilities of 12 field isolates of C. jejuni of different aerotolerance categories on stainless steel surfaces, a prevalent material in poultry processing environments. Working cultures of each isolate were prepared to approximately 6 log CFU/mL and incubated on stainless steel coupons under microaerobic or aerobic conditions at room temperature or 42°C for 72 h. Biofilm attached cells were enumerated using direct plating and biofilm density was measured using a crystal violet assay by measuring the optical density (OD600) a. Data analysis was conducted using the PROC GLIMMIX procedure in SAS 9.4 with a significance level of 0.05. The study revealed a notable interaction between aerotolerance categories and temperature (P < 0.039) impacting the number of biofilms attached C. jejuni cells on stainless steel coupons. All isolates had significantly higher counts when incubated at 42°C compared to room temperature, regardless of oxygen level (P < 0.001). Furthermore, stronger biofilm density was observed at 42°C compared to room temperature, regardless of oxygen level. These findings underscore the influence of temperature on the biofilm forming ability of C. jejuni. The ability of these field isolates to form biofilms under various environmental conditions suggests a heightened potential for surface colonization and increased infection risk in poultry processing facilities.

A significant antibiofilm and antimicrobial activity of chitosan-polyacrylic acid nanoparticles against pathogenic bacteria

Chitosan is known to exert antimicrobial activity without the need for any chemical modification; however, new derivatives of chitosan can be created to target multi-drug resistant bacteria. In this study, chitosan (CS) was cross-linked with sodium tripolyphosphate to form nanoparticles, which were then coated with polyacrylic acid (PAA). The SEM images revealed that the CS-PAA nanoparticles had spherical shapes with smooth surfaces and the size of the dried nanoparticles was approximately 222 nm. Biofilm formation was significantly inhibited by 0.5 mg/mL of CS-PAA. In-situ optical microscopy showed that CS-PAA nanoparticles inhibited the bacterial biofilm formation in Campylobacter jejuni, Pseudomonas aeruginosa, and Escherichia coli after a single treatment with 40 μg. Additionally, 20 µg of CS-PAA nanoparticles demonstrated antibacterial activity against the growth of C. jejuni, P. aeruginosa, and E. coli with notable inhibitory zones of 9, 12, and 13 mm, respectively (P < 0.01). The development of a novel and ecofriendly method for the preparation of chitosan nanoparticles through an interaction of chitosan with PAA shows promise tool to combat bacterial infections and validates effective antibacterial and antibiofilm properties against antibiotic resistant pathogens.

Inhibition of AI-2 Quorum Sensing and Biofilm Formation in Campylobacter jejuni by Decanoic and Lauric Acids.

Campylobacter jejuni is a major bacterial cause of human diarrheal diseases worldwide. Despite its sensitivity to environmental stresses, C. jejuni ubiquitously distributes throughout poultry production chains. Biofilm formation mediated by quorum sensing is suggested to be critical to the survival of C. jejuni in agroecosystem. C. jejuni possesses LuxS, the enzyme involved in the production of autoinducer-2 (AI-2) signaling molecules. In this study, two fatty acids, namely decanoic acid and lauric acid, were identified to be effective in inhibiting AI-2 activity of C. jejuni. Both decanoic acid and lauric acid at 100 ppm inhibited ∼90% AI-2 activity (P < 0.05) of C. jejuni without bacterial inactivation. The biofilm biomass of two C. jejuni strains was reduced by 10–50% (P < 0.05) after treatment by both fatty acids, while increased biofilm formation was observed for one C. jejuni strain. In addition, both fatty acids effectively reduced the motility of all tested C. jejuni strains. These findings can aid in developing alternative C. jejuni control strategies in agri-food and clinical settings.

Mutagenic strategies against luxS gene affect the early stage of biofilm formation of Campylobacter jejuni.

Currently, it is clear that the luxS gene has an impact on the process of biofilm formation in Campylobacter jejuni. However, even within the species, naturally occurring strains of Campylobacter lacking the luxS gene exist, which can form biofilms. In order to better understand the genetic determinants and the role of quorum sensing through the LuxS/AI-2 pathway in biofilm formation, a set of mutant/complemented strains of C. jejuni 81-176 were prepared. Additionally, the impact of the mutagenic strategy used against the luxS gene was investigated. Biofilm formation was affected by both the presence and absence of the luxS gene, and by the mutagenic strategy used. Analysis by CLSM showed that all mutant strains formed significantly less biofilm mass when compared to the wild-type. Interestingly, the deletion mutant (∆luxS) showed a larger decrease in biofilm mass than the substitution (∙luxS) and insertional inactivated ([Formula: see text]luxS) mutants, even though all the mutant strains lost the ability to produce autoinducer-2 molecules. Moreover, the biofilm of the ∆luxS mutant lacked the characteristic microcolonies observed in all other strains. The complementation of all mutant strains resulted in restored ability to produce AI-2, to form a complex biofilm, and to develop microcolonies at the level of the wild-type.

Inhibition of Campylobacter jejuni Biofilm Formation by D-Amino Acids.

The ability of bacterial pathogens to form biofilms is an important virulence mechanism in relation to their pathogenesis and transmission. Biofilms play a crucial role in survival in unfavorable environmental conditions, acting as reservoirs of microbial contamination and antibiotic resistance. For intestinal pathogen Campylobacter jejuni, biofilms are considered to be a contributing factor in transmission through the food chain and currently, there are no known methods for intervention. Here, we present an unconventional approach to reducing biofilm formation by C. jejuni by the application of D-amino acids (DAs), and L-amino acids (LAs). We found that DAs and not LAs, except L-alanine, reduced biofilm formation by up to 70%. The treatment of C. jejuni cells with DAs changed the biofilm architecture and reduced the appearance of amyloid-like fibrils. In addition, a mixture of DAs enhanced antimicrobial efficacy of D-Cycloserine (DCS) up to 32% as compared with DCS treatment alone. Unexpectedly, D-alanine was able to reverse the inhibitory effect of other DAs as well as that of DCS. Furthermore, L-alanine and D-tryptophan decreased transcript levels of peptidoglycan biosynthesis enzymes alanine racemase (alr) and D-alanine-D-alanine ligase (ddlA) while D-serine was only able to decrease the transcript levels of alr. Our findings suggest that a combination of DAs could reduce biofilm formation, viability and persistence of C. jejuni through dysregulation of alr and ddlA.

Campylobacter jejuni Biofilm Formation Under Aerobic Conditions and Inhibition by ZnO Nanoparticles.

Campylobacter jejuni is a major foodborne pathogen worldwide. As it forms biofilms, it can become a persistent contaminant in the food and pharmaceutical industries. In this study, it was demonstrated that C. jejuni could make more biofilm in aerobic conditions than in microaerobic conditions, and only 13.9% C. jejuni entered coccus (a VBNC state) under microaerobic conditions; however, the rate increased to 95.5% under aerobic conditions. C. jejuni could form more biofilm in mixed culture with Escherichia coli or Pseudomonas aeruginosa than in pure culture. Scanning electron microscope results showed that C. jejuni retained its normal spiral shape under aerobic conditions for 48 h by forming crosslinks with the aerobic and facultative anaerobic bacteria. Additionally, culture medium containing 0.5 mg/ml ZnO nanoparticles inhibited biofilm formation. Our results provide information on a new approach to controlling contamination via C. jejuni.

RNA Sequencing Data Sets Identifying Differentially Expressed Transcripts during Campylobacter jejuni Biofilm Formation.

Campylobacter jejuni is a foodborne pathogen and an important contributor to gastroenteritis in humans. C. jejuni readily forms biofilms which may play a role in the transmission of the pathogen from animals to humans. Herein, we present RNA sequencing data investigating differential gene expression in biofilm and planktonic C. jejuni These data provide insight into pathways which may be important to biofilm formation in this organism.

Enhanced Biofilm Formation by Ferrous and Ferric Iron Through Oxidative Stress in Campylobacter jejuni.

Campylobacter is a leading foodborne pathogen worldwide. Biofilm formation is an important survival mechanism that sustains the viability of Campylobacter under harsh stress conditions. Iron affects biofilm formation in some other bacteria; however, the effect of iron on biofilm formation has not been investigated in Campylobacter. In this study, we discovered that ferrous (Fe2+) and ferric (Fe3+) iron stimulated biofilm formation in Campylobacter jejuni. The sequestration of iron with an iron chelator prevented the iron-mediated biofilm stimulation. The level of total reactive oxygen species (ROS) in biofilms was increased by iron. However, the supplementation with an antioxidant prevented the total ROS level from being increased in biofilms by iron and also inhibited iron-mediated biofilm stimulation in C. jejuni. This suggests that iron promotes biofilm formation through oxidative stress. Based on the results of fluorescence microscopic analysis, Fe2+ and Fe3+ enhanced both microcolony formation and biofilm maturation. The levels of extracellular DNA and polysaccharides in biofilms were increased by iron supplementation. The effect of iron on biofilm formation was also investigated with 70 C. jejuni isolates from raw chicken. Regardless of the inherent levels of biofilm formation, iron stimulated biofilm formation in all tested strains; however, there were strain variations in iron concentrations affecting biofilm formation. The biofilm formation of 92.9% (65 of 70) strains was enhanced by either 40 μM Fe2+ or 20 μM Fe3+ or both (the iron concentrations that enhanced biofilm formation in C. jejuni NCTC 11168), whereas different iron concentrations were required to promote biofilms in the rest of the strains. The findings in this study showed that Fe2+ and Fe3+ contributed to the stimulation of biofilm formation in C. jejuni through oxidative stress.

TYPLEX® Chelate, a novel feed additive, inhibits Campylobacter jejuni biofilm formation and cecal colonization in broiler chickens

Reducing Campylobacter spp. carriage in poultry is challenging, but essential to control this major cause of human bacterial gastroenteritis worldwide. Although much is known about the mechanisms and route of Campylobacter spp. colonization in poultry, the literature is scarce on antibiotic-free solutions to combat Campylobacter spp. colonization in poultry. In vitro and in vivo studies were conducted to investigate the role of TYPLEX® Chelate (ferric tyrosine), a novel feed additive, in inhibiting Campylobacter jejuni (C. jejuni) biofilm formation and reducing C. jejuni and Escherichia coli (E. coli) colonization in broiler chickens at market age. In an in vitro study, the inhibitory effect on C. jejuni biofilm formation using a plastic bead assay was investigated. The results demonstrated that TYPLEX® Chelate significantly reduces biofilm formation. In an in vivo study, 800 broilers (one d old) were randomly allocated to 4 dietary treatments in a randomized block design, each having 10 replicate pens with 20 birds per pen. At d 21, all birds were challenged with C. jejuni via seeded litter. At d 42, cecal samples were collected and tested for volatile fatty acid (VFA) concentrations and C. jejuni and E. coli counts. The results showed that TYPLEX® Chelate reduced the carriage of C. jejuni and E. coli in poultry by 2 and 1 log10 per gram cecal sample, respectively, and increased cecal VFA concentrations. These findings support TYPLEX® Chelate as a novel non-antibiotic feed additive that may help produce poultry with a lower public health risk of Campylobacteriosis.

Environmental Stress-Induced Bacterial Lysis and Extracellular DNA Release Contribute to Campylobacter jejuni Biofilm Formation.

Campylobacter jejuni is a microaerophilic bacterium and is believed to persist in a biofilm to antagonize environmental stress. This study investigated the influence of environmental conditions on the formation of C. jejuni biofilm. We report an extracellular DNA (eDNA)-mediated mechanism of biofilm formation in response to aerobic and starvation stress. The eDNA was determined to represent a major form of constitutional material of C. jejuni biofilms and to be closely associated with bacterial lysis. Deletion mutation of the stress response genes spoT and recA enhanced the aerobic influence by stimulating lysis and increasing eDNA release. Flagella were also involved in biofilm formation but mainly contributed to attachment rather than induction of lysis. The addition of genomic DNA from either Campylobacter or Salmonella resulted in a concentration-dependent stimulation effect on biofilm formation, but the effect was not due to forming a precoating DNA layer. Enzymatic degradation of DNA by DNase I disrupted C. jejuni biofilm. In a dual-species biofilm, eDNA allocated Campylobacter and Salmonella at distinct spatial locations that protect Campylobacter from oxygen stress. Our findings demonstrated an essential role and multiple functions of eDNA in biofilm formation of C. jejuni, including facilitating initial attachment, establishing and maintaining biofilm, and allocating bacterial cells.IMPORTANCECampylobacter jejuni is a major cause of foodborne illness worldwide. In the natural environment, the growth of C. jejuni is greatly inhibited by various forms of environmental stress, such as aerobic stress and starvation stress. Biofilm formation can facilitate the distribution of C. jejuni by enabling the survival of this fragile microorganism under unfavorable conditions. However, the mechanism of C. jejuni biofilm formation in response to environmental stress has been investigated only partially. The significance of our research is in identifying extracellular DNA released by bacterial lysis as a major form of constitution material that mediates the formation of C. jejuni biofilm in response to environmental stress, which enhances our understanding of the formation mechanism of C. jejuni biofilm. This knowledge can aid the development of intervention strategies to limit the distribution of C. jejuni.