Comparative Assessment of the Antibacterial and Antibiofilm Actions of Benzalkonium Chloride, Erythromycin, and L(+)-Lactic Acid against Raw Chicken Meat Campylobacter spp. Isolates.

Campylobacter spp. are significant zoonotic agents, which cause annually millions of human cases of foodborne gastroenteritis worldwide. Their inclusion in biofilms on abiotic surfaces seems to play a pivotal role in their survival outside of the host, growth, and spread. To successfully mitigate the risks that arise with these bacteria, it is crucial to decrease their prevalence within the food production chain (from farm to the table), alongside the successful treatment of the resulting illness, known as campylobacteriosis. For this, the use of various antimicrobial agents remains actively in the foreground. A general-purpose biocide and cationic surfactant (benzalkonium chloride; BAC), a widely used macrolide antibiotic (erythromycin; ERY), and a naturally occurring organic acid (L(+)-lactic acid; LA) were comparatively evaluated in this work for their potential to inhibit both the planktonic and biofilm growth of 12 selected Campylobacter spp. (of which, seven were C. jejuni and five were C. coli) raw chicken meat isolates, all grown in vitro as monocultures. The inhibitory action of LA was also studied against four mixed-culture Campylobacter biofilms (each composed of three different isolates). The results showed that the individual effectiveness of the agents varied significantly depending on the isolate, growth mode (planktonic, biofilm), intercellular interactions (monocultures, mixed cultures), and the growth medium used (with special focus on blood presence). Thus, BAC exhibited minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), and minimum biofilm inhibitory concentrations (MBICs) that ranged from 0.5 to 16 µg/mL. Interestingly enough, these values varied widely from 0.25 to 1024 µg/mL for ERY. Concerning LA, the MICs, MBCs, and MBICs varied from 1024 to 4096 µg/mL, with mixed-culture biofilm formation always being more difficult to suppress when compared to biofilm monocultures. In addition, it was evident that intercellular interactions encountered within mixed-culture Campylobacter biofilms significantly influenced both the population dynamics and the tolerance of each consortium member to acid exposure. Overall, the findings of this study provide useful information on the comparative effectiveness of three well-known antimicrobial agents for the control of Campylobacter spp. under various growth modes (i.e., planktonic, biofilm, monocultures, mixed cultures) that could potentially be encountered in food production and clinical settings.

Prevalence of Campylobacter spp., Salmonella spp., and Listeria monocytogenes, and Population Levels of Food Safety Indicator Microorganisms in Retail Raw Chicken Meat and Ready-To-Eat Fresh Leafy Greens Salads Sold in Greece.

The presence of microbial pathogens in foods compromises their safety resulting in foodborne illnesses, public health disorders, product recalls, and economic losses. In this work, 60 samples of chilled raw chicken meat and 40 samples of packaged ready-to-eat (RTE) fresh leafy greens salads, sold in Greek retail stores (butchers and supermarkets), were analyzed for the presence of three important foodborne pathogenic bacteria, i.e., Campylobacter spp., Salmonella spp., and Listeria monocytogenes, following the detection protocols of the International Organization for Standardization (ISO). In parallel, the total aerobic plate count (APC), Enterobacteriaceae, total coliforms, Escherichia coli, and staphylococci were also enumerated as hygiene (safety) indicator organisms. When present, representative typical colonies for each pathogen were biochemically verified, following the ISO guidelines. At the same time, all the Campylobacter isolates from chicken (n = 120) were identified to the species level and further phylogenetically discriminated through multiplex and repetitive sequence-based (rep) polymerase chain reaction (PCR) methods, respectively. Concerning raw chicken, Campylobacter spp. were recovered from 54 samples (90.0%) and Salmonella spp. were recovered from 9 samples (15.0%), while L. monocytogenes was present in 35 samples (58.3%). No Campylobacter was recovered from salads, and Salmonella was present in only one sample (2.5%), while three salads were found to be contaminated with L. monocytogenes (7.5%). The 65% of the Campylobacter chicken isolates belonged to C. jejuni, whereas the rest, 35%, belonged to C. coli. Alarmingly, APC was equal to or above 106 CFU/g in 53.3% and 95.0% of chicken and salad samples, respectively, while the populations of some of the other safety indicators were in some cases also high. In sum, this study unravels high occurrence percentages for some pathogenic and food safety indicator microorganisms in raw chicken meat and RTE fresh leafy greens salads sold in Greek retail, highlighting the need for more extensive microbiological control throughout the food production chain (from the farm/field to the market).

Chemical Composition, Antibacterial and Antibiofilm Actions of Oregano (Origanum vulgare subsp. hirtum) Essential Oil against Salmonella Typhimurium and Listeria monocytogenes.

Essential oils (EOs) are plant mixtures that are known to present strong bioactivities, including a wide antimicrobial action. Biofilms are microbial sessile structures that represent the default mode of growth of microorganisms in most environments. This study focused on the antimicrobial action of the EO extracted from one of the most representative oregano species, that is, Origanum vulgare (subsp. hirtum), against two important foodborne pathogens, Salmonella enterica (serovar Typhimurium) and Listeria monocytogenes. For this, the minimum inhibitory concentrations of the EO against the planktonic and biofilm growth of each bacterium were determined (MICs, MBICs), together with the minimum bactericidal and biofilm eradication concentrations (MBCs, MBECs). The EO was also analyzed for its chemical composition by gas chromatography-mass spectrometry analysis (GC-MS). The influence of EO exposure on the expression of some important virulence genes (hly, inlA, inlB and prfA) was also studied in L. monocytogenes. Results revealed a strong antibacterial and antibiofilm action with MICs and MBICs ranging from 0.03% to 0.06% (v/v) and from 0.06% to 0.13% (v/v), respectively. The application of the EO at 6.25% (v/v) for 15 min resulted in the total eradication of the biofilm cells of both pathogens. The EO was mainly composed of thymol, p-cymene, γ-terpinene and carvacrol. The 3 h exposure of L. monocytogenes planktonic cells to the EO at its MBIC (0.06% v/v) resulted in the significant downregulation of all the studied genes (p < 0.05). To sum, the results obtained advocate for the further exploitation of the antimicrobial action of oregano EO in food and health applications.

Chemical Composition, Antioxidant, and Antibiofilm Properties of Essential Oil from Thymus capitatus Plants Organically Cultured on the Greek Island of Lemnos.

Essential oils (EOs) are mixtures of volatile plant secondary metabolites and have been exploited by humans for thousands of years for various purposes because of their many bioactivities. In this study, the EO from Thymus capitatus, a thyme species organically cultured on the Greek Island of Lemnos, was analyzed for its chemical composition (through GC-FID and GC-MS), antioxidant activity (AA), and total phenolic content (TPC), as well as its antimicrobial and antibiofilm actions against three important foodborne bacterial pathogens (Salmonella enterica ser. Typhimurium, Listeria monocytogenes, and Yersinia enterocolitica). For the latter investigations, the minimum inhibitory concentrations (MICs) and minimum biofilm inhibitory concentrations (MBICs) of the EO against the planktonic and biofilm growth of each pathogen were determined, together with the minimum biofilm eradication concentrations (MBECs). Results revealed that T. capitatus EO was rich in thymol, p-cymene, and carvacrol, presenting high AA and TPC (144.66 µmol TroloxTM equivalents and 231.32 mg gallic acid equivalents per g of EO, respectively), while its MICs and MBICs ranged from 0.03% to 0.06% v/v and 0.03% to 0.13% v/v, respectively, depending on the target pathogen. The EO was able to fully destroy preformed (mature) biofilms of all three pathogenic species upon application for 15 min, with MBECs ranging from 2.00 to 6.25% v/v. Overall, the results demonstrate that the EO of organically cultured T. capitatus presents strong antioxidant, antibacterial, and antibiofilm properties and could, therefore, be further exploited as a functional and antimicrobial natural formulation for food and health applications.

A Multicomponent Home-Based Intervention for Neuropsychiatric Symptoms in People With Dementia and Caregivers' Burden and Depression: A 6-Month Longitudinal Study.

OBJECTIVES: Many people with dementia live in their home and require ongoing care, which is often provided by informal family caregivers. Thus, we examined the effectiveness of a multicomponent home-based intervention by evaluating its impact on a) neuropsychiatric symptoms of people with dementia and b) burden and depression of their caregivers. METHODS: During the first 6 months of this prospective single-center study, we applied a home-based multicomponent intervention in 205 dyads of care-recipients and caregivers. In further analyzes, we included only dyads of caregivers and care-recipients with available data both at baseline and 6-month follow-up (N = 144). All assessments were conducted at home and included sociodemographic features, care-recipients' clinical data, cognitive status (Mini-Mental State Examination), activities of daily living (Instrumental Activities of Daily Living; Katz Index of Independence in Activities of Daily Living), neuropsychiatric symptoms (Neuropsychiatric Inventory), and caregivers' burden (Zarit Burden Inventory) and depression (Center for Epidemiological Studies-Depression). RESULTS: We found significant decreases in the severity (pFDR = 0.002) and associated distress (pFDR = 0.001) of neuropsychiatric symptoms, as well as caregivers' burden (pFDR = 0.004) and depressive symptoms (pFDR = 0.001). As expected, there was significant deterioration in care-recipients' cognitive status (pFDR = 0.005) and measures of activities of daily living (pFDR < 0.005). CONCLUSION: Despite the progressive course of dementia, the home-based multicomponent intervention was effective in decreasing caregivers' burden and depression and minimizing care-recipients' neuropsychiatric symptoms' severity and associated distress after 6 months. Our study highlights the establishment of home-based care units as an advantageous solution, specifically for family members seen to have a "taken-for-granted" role in dementia caring.

Advanced Killing Potential of Thymol against a Time and Temperature Optimized Attached Listeria monocytogenes Population in Lettuce Broth.

Fresh vegetables and salads are increasingly implicated in outbreaks of foodborne infections, such as those caused by Listeria monocytogenes, a dangerous pathogen that can attach to the surfaces of the equipment creating robust biofilms withstanding the killing action of disinfectants. In this study, the antimicrobial efficiency of a natural plant terpenoid (thymol) was evaluated against a sessile population of a multi-strain L. monocytogenes cocktail developed on stainless steel surfaces incubated in lettuce broth, under optimized time and temperature conditions (54 h at 30.6 °C) as those were determined following response surface modeling, and in comparison, to that of an industrial disinfectant (benzalkonium chloride). Prior to disinfection, the minimum bactericidal concentrations (MBCs) of each compound were determined against the planktonic cells of each strain. The results revealed the advanced killing potential of thymol, with a concentration of 625 ppm (= 4 × MBC) leading to almost undetectable viable bacteria (more than 4 logs reduction following a 15-min exposure). For the same degree of killing, benzalkonium chloride needed to be used at a concentration of at least 20 times more than its MBC (70 ppm). Discriminative repetitive sequence-based polymerase chain reaction (rep-PCR) also highlighted the strain variability in both biofilm formation and resistance. In sum, thymol was found to present an effective anti-listeria action under environmental conditions mimicking those encountered in the salad industry and deserves to be further explored to improve the safety of fresh produce.

Common Plant-Derived Terpenoids Present Increased Anti-Biofilm Potential against Staphylococcus Bacteria Compared to a Quaternary Ammonium Biocide.

The antimicrobial actions of three common plant-derived terpenoids (i.e., carvacrol, thymol and eugenol) were compared to those of a typical quaternary ammonium biocide (i.e., benzalkonium chloride; BAC), against both planktonic and biofilm cells of two widespread Staphylococcus species (i.e., S. aureus and S. epidermidis). The minimum inhibitory and bactericidal concentrations (MICs, MBCs) of each compound against the planktonic cells of each species were initially determined, together with their minimum biofilm eradication concentrations (MBECs). Various concentrations of each compound were subsequently applied, for 6 min, against each type of cell, and survivors were enumerated by agar plating to calculate log reductions and determine the resistance coefficients (Rc) for each compound, as anti-biofilm effectiveness indicators. Sessile communities were always more resistant than planktonic ones, depending on the biocide and species. Although lower BAC concentrations were always needed to kill a specified population of either cell type compared to the terpenoids, for the latter, the required increases in their concentrations, to be equally effective against the biofilm cells with respect to the planktonic ones, were not as intense as those observed in the case of BAC, presenting thus significantly lower Rc. This indicates their significant anti-biofilm potential and advocate for their further promising use as anti-biofilm agents.

Exploitation of plant extracts and phytochemicals against resistant Salmonella spp. in biofilms.

Salmonella is one of the most frequent causes of foodborne outbreaks throughout the world. In the last years, the resistance of this and other pathogenic bacteria to antimicrobials has become a prime concern towards their successful control. In addition, the tolerance and virulence of pathogenic bacteria, such as Salmonella, are commonly related to their ability to form biofilms, which are sessile structures encountered on various surfaces and whose development is considered as a universal stress response mechanism. Indeed, the ability of Salmonella to form a biofilm seems to significantly contribute to its persistence in food production areas and clinical settings. Plant extracts and phytochemicals appear as promising sources of novel antimicrobials due to their cost-effectiveness, eco-friendliness, great structural diversity, and lower possibility of antimicrobial resistance development in comparison to synthetic chemicals. Research on these agents mainly attributes their antimicrobial activity to a diverse array of secondary metabolites. Bacterial cells are usually killed by the rupture of their cell envelope and in parallel the disruption of their energy metabolism when treated with such molecules, while their use at sub-inhibitory concentrations may also disrupt intracellular communication. The purpose of this article is to review the current available knowledge related to antimicrobial resistance of Salmonella in biofilms, together with the antibiofilm properties of plant extracts and phytochemicals against these detrimental bacteria towards their future application to control these in food production and clinical environments.