Sonja Smole Možina

Evaluation of diffusion and dilution methods to determine the antibacterial activity of plant extracts

The aim of this study was to evaluate diffusion and dilution methods for determining the antibacterial activity of plant extracts and their mixtures. Several methods for measurement of the minimal inhibitory concentration (MIC) of a plant extract are available, but there is no standard procedure as there is for antibiotics. We tested different plant extracts, their mixtures and phenolic acids on selected gram-positive (Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes) and gram-negative bacteria (Escherichia coli O157:H7, Salmonella Infantis, Campylobacter jejuni, Campylobacter coli) with the disk diffusion, agar dilution, broth microdilution and macrodilution methods. The disk diffusion method was appropriate only as a preliminary screening test prior to quantitative MIC determination with dilution methods. A comparison of the results for MIC obtained by agar dilution and broth microdilution was possible only for gram-positive bacteria, and indicated the latter as the most accurate way of assessing the antimicrobial effect. The microdilution method with TTC (2,3,5-triphenyl tetrazolium chloride) or INT (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) to indicate the viability of aerobic bacteria was found to be the best alternative approach, while only ATP determination was appropriate for microaerophilic Campylobacter spp. Using survival curves the kinetics of bacterial inactivation on plant extract exposure was followed for 24h and in this way the MIC values determined by the microdilution method were confirmed as the concentrations of extracts that inhibited bacterial growth. We suggest evaluation of the antibacterial activity of plant extracts using the broth microdilution method as a fast screening method for MIC determination and the macrodilution method at selected MIC values to confirm bacterial inactivation. Campylobacter spp. showed a similar sensitivity to plant extracts as the tested gram-positive bacteria, but S. Infantis and E. coli O157:H7 were more resistant.

Stress response and pathogenic potential of Campylobacter jejuni cells exposed to starvation

Campylobacter jejuni is a Gram-negative, fragile, spiral bacterium, known worldwide to be a major cause of acute human enteritis. Like many other food-borne bacteria, campylobacters must be able to survive under diverse conditions both inside the host and in the environment. Understanding stress response mechanisms provides information necessary for improving food processing and strategies that enhance food safety as well as clarifying the pathogenesis of campylobacteriosis. We investigated the relation between stress response to starvation and pathogenic potential in C. jejuni. Starvation changed the morphology and physiology of C. jejuni cells. However, the lower metabolic activity of 5-h-starved culture was not a dormant state, but probably a viable but non-culturable (VBNC) form of the cells, since starved C. jejuni induced heat stress resistance. The health hazard potential of starved cells is still unclear. We showed that, in spite of starvation, C. jejuni survived in vitro within Caco-2 enterocites up to 4 days and caused systemic campylobacteriosis in vivo in a mouse model. However, bacterial numbers in investigated organs were significantly lower and the infection was resolved sooner. Our results show that nutrient insufficiency is responsible for C. jejuni transformation, influencing but not abolishing its survival and virulence properties while in the VBNC state.

Anti-Campylobacter Activities and Resistance Mechanisms of Natural Phenolic Compounds in Campylobacter

Background Campylobacter is a major foodborne pathogen and alternative antimicrobials are needed to prevent or decrease Campylobacter contamination in foods or food producing animals. The objectives of this study are to define the anti-Campylobacter activities of natural phenolic compounds of plant origin and to determine the roles of bacterial drug efflux systems in the resistance to these natural phenolics in Campylobacter jejuni. Methodology/Principal Findings Anti-Campylobacter activities were evaluated by an MIC assay using microdilution coupled with ATP measurement. Mutants of the cmeB and cmeF efflux genes and the cmeR transcriptional repressor gene were compared with the wild-type strain for their susceptibilities to phenolics in the absence and presence of efflux-pump inhibitors (EPIs). The phenolic compounds produced significant, but variable activities against both antibiotic-susceptible and antibiotic resistant Campylobacter. The highest anti-Campylobacter activity was seen with carnosic and rosmarinic acids in their pure forms or in enriched plant extracts. Inactivation of cmeB rendered C. jejuni significantly more susceptible to the phenolic compounds, while mutation of cmeF or cmeR only produced a moderate effect on the MICs. Consistent with the results from the efflux pump mutants, EPIs, especially phenylalanine-arginine β-naphthylamide and NMP, significantly reduced the MICs of the tested phenolic compounds. Further reduction of MICs by the EPIs was also observed in the cmeB and cmeF mutants, suggesting that other efflux systems are also involved in Campylobacter resistance to phenolic compounds. Conclusion/Significance Natural phenolic compounds of plant origin have good anti-Campylobacter activities and can be further developed for potential use in controlling Campylobacter. The drug efflux systems in Campylobacter contribute significantly to its resistance to the phenolics and EPIs potentiate the anti-Campylobacter activities of plant phenolic compounds.

Seasonal Variations of Phenolic Compounds and Biological Properties in Sage (Salvia officinalis L.)

The aim was to investigate the phenolic content, antioxidant capacity, and antibacterial activity of Dalmatian sage (Salvia officinalis L.) leaves collected during different vegetation periods. Separation and quantification of the individual phenols were performed by reversed‐phase (RP)‐HPLC coupled with a PDA (photodiode array) detector and using an internal standard, while the contents of total phenols, flavonoids, flavones, and flavonols were determined spectrophotometrically. The antioxidant properties of the sage leaf extracts were evaluated using five different antioxidant assays (FRAP, DPPH, ABTS, BriggsRauscher reaction, and β‐carotene bleaching). The antimicrobial activity of the extracts was tested against two Gram‐positive (Bacillus cereus and Staphylococcus aureus) and two Gram‐negative (Salmonella Infantis and Escherichia coli) bacterial reference strains. All extracts were extremely rich in phenolic compounds, and provided good antioxidant and antibacterial properties, but the phenophase in which the leaves were collected affected the phenolic composition of the sage extracts and consequently their biological activity. The May Extract, the richest in total flavonoids, showed the best antioxidant properties and the highest antimicrobial activity. Thus, collection of the plants during May seems the best choice for further use of them in the pharmaceutical and food industry.

Attachment, invasion, and translocation of Campylobacter jejuni in pig small-intestinal epithelial cells.

Campylobacters are susceptible to environmental conditions such as starvation, temperature, and oxidative stress. Species such as Campylobacter jejuni have developed a number of mechanisms for responding to these conditions. We conducted a study to investigate whether survival of C. jejuni and pathogen-host cell interactions such as adherence, invasiveness, and intraepithelial survival in pig small-intestinal (PSI) epithelial cells are altered in response to starvation, changes in temperature, and atmospheric oxygen concentration. We assessed the ability of C. jejuni to translocate across polarized intestinal epithelial cell monolayers by measuring transepithelial electrical resistance (TER). Following heat stress, we observed loss of C. jejuni culturability but not viability. Heat-stressed C. jejuni adhered efficiently to pig intestinal epithelial cells, but their invasiveness was significantly impaired when compared with unstressed C. jejuni. Prolonged exposure to atmospheric oxygen reduced the ability of C. jejuni to adhere to intestinal epithelial cells, whereas brief exposure increased invasiveness and intraepithelial survival. By comparison, nutrient limitation reduced adherence, invasiveness, and intracellular survival of C. jejuni. Adherence of C. jejuni strongly affected the pig intestinal epithelium, as reflected by a significant decrease in TER of polarized intestinal epithelial cells. No correlation between TER and the translocation capacity of C. jejuni was observed. Additionally, campylobacters were detected in the basal chamber of a functional small-intestinal epithelial cell model at 3 hours post infection, without a significant reduction in the TER value, suggesting transcellular transport of C. jejuni into the body.

Survival of stress exposed Campylobacter jejuni in the murine macrophage J774 cell line

Although campylobacters are relatively fragile and sensitive to environmental stresses, Campylobacter jejuni has evolved mechanisms for survival in diverse environments, both inside and outside the host. Their survival properties and pathogenic potential were assessed after subjecting food and clinical C. jejuni isolates to different stress conditions. After exposure to starvation (5 h and 15 h of nutrient depletion), a temperature shock (3 min at 55 degrees C) or oxidative stress (5 h and 15 h of atmospheric oxygen) we studied the culturability, viability and capability of adhesion, internalization and survival within the in vitro cell culture model using J774 murine macrophages. Starvation severely impaired C. jejuni culturability, particularly after 15 h of nutrient depletion. The number of viable cells decreased by 30-40%. Starved bacterial cells also showed a lower capability of adhesion, internalization and survival within macrophages. Despite the reduced culturability and viability of the heat treated cells, C. jejuni efficiently adhered to, and entered murine macrophages. However, the number of heat treated cells started to decrease more quickly than non-stressed cells. Within 24 h post infection all the cells were killed. The bacterial mechanisms involved in inactivating toxic oxygen products may enhance bacterial persistence through increased binding, entry and survival of both oxidatively stressed C. jejuni isolates inside the macrophages. Oxygen exposure increased the internalization and intracellular survival, although the cells cannot remain viable for extended periods within murine macrophages. However, any prolongation of survival in macrophages may increase the probability of transmission of bacteria in the host organism and have further implications in the pathogenesis of campylobacteriosis. This indicates that environmental stress conditions may be involved.

Development of antimicrobial resistance in Campylobacter jejuni and Campylobacter coli adapted to biocides.

The potential for adaptive resistance of Campylobacter jejuni and Campylobacter coli after step-wise exposure to increasing sub-inhibitory concentrations of five biocides as triclosan, benzalkonium chloride, cetylpyridinium chloride, chlorhexidine diacetate and trisodium phosphate, was investigated, to identify the mechanisms underlying resistance. The biocide resistance and cross-resistance to the antimicrobials erythromycin and ciprofloxacin, and to sodium dodecyl sulphate, were examined according to the broth microdilution method. The presence of active efflux was studied on the basis of restored sensitivity in the presence of the efflux pump inhibitors phenylalanine-arginine beta-naphthylamide, 1-(1-naphthylmethyl)-piperazine, cyanide 3-chlorophenylhydrazone, verapamil and reserpine. Changes in the outer membrane protein profiles and morphological changes in adapted strains were studied, as compared with the parent strains. Repeated exposure of C. jejuni and C. coli to biocides resulted in partial increases in tolerance to biocides itself, to other biocides and antimicrobial compounds. The developed resistance was stable for up to 10 passages in biocide-free medium. More than one type of active efflux was identified in adapted strains. These adapted strains showed different alterations to their outer membrane protein profiles, along with morphological changes. The data presented here suggest that different mechanisms are involved in adaptation to biocides and that this adaptation is unique to each strain of Campylobacter and does not result from a single species-specific mechanism.

Effects of Efflux Pump Inhibitors on Erythromycin, Ciprofloxacin, and Tetracycline Resistance in Campylobacter spp. Isolates

The aim was to assess the potency of the efflux pump inhibitors (EPIs) phenylalanine-arginine ß-naphthylamide (PAßN) and 1-(1-naphthylmethyl)-piperazine (NMP) and the putative natural EPI phenolic (-)-epigallocatechin gallate (EGCG) for the reversal of erythromycin, ciprofloxacin, and tetracycline resistance in Campylobacter jejuni and Campylobacter coli isolates. We investigated target mutations and resistant genes involved in erythromycin and tetracycline resistance and determined the roles of the bacterial drug efflux systems (cmeB, cmeF, and cmeR) in antimicrobial resistance. Our data show that most of the high-level erythromycin resistance and all of the tetracycline resistance can be explained through mutations in 23S rRNA and the presence of the tetO gene, respectively. The EPIs show the ability to partly reverse drug resistance in these Campylobacter isolates. Based on a fourfold or greater reduction in the erythromycin minimal inhibitory concentration (MIC), PAßN and NMP had clear effects in almost of all of the isolates tested. PAßN had a highly selective action on the ciprofloxacin and tetracycline MICs. Inactivation of cmeB increased susceptibility to all of the antimicrobials tested, whereas inactivation of cmeF and cmeR had no effects. A notable decrease in resistance to erythromycin and ciprofloxacin in the presence of subinhibitory concentrations of EGCG demonstrates the resistance-modifying activities of this natural EPI, and indicates its potential use in the control of Campylobacter spp. in the food chain.

Antibiotic Resistance Modulation and Modes of Action of (-)-α-Pinene in Campylobacter jejuni

The aim of the study was to investigate the mode of action of (-)-α-pinene in terms of its modulation of antibiotic resistance in Campylobacter jejuni. Broth microdilution and ethidium bromide accumulation assays were used to evaluate the (-)-α-pinene antimicrobial activity, modulation of antimicrobial resistance, and inhibition of antimicrobial efflux. The target antimicrobial efflux systems were identified using an insertion mutagenesis approach, and C. jejuni adaptation to (-)-α-pinene was evaluated using DNA microarrays. Knock-out mutants of the key up-regulated transcriptional regulators hspR and hrcA were constructed to investigate their roles in C. jejuni adaptation to several stress factors, including osmolytes, and pH, using Biolog phenotypical microarrays. Our data demonstrate that (-)-α-pinene efficiently modulates antibiotic resistance in C. jejuni by decreasing the minimum inhibitory concentrations of ciprofloxacin, erythromycin and triclosan by up to 512-fold. Furthermore, (-)-α-pinene promotes increased expression of cmeABC and another putative antimicrobial efflux gene, Cj1687. The ethidium bromide accumulation was greater in the wild-type strain than in the antimicrobial efflux mutant strains, which indicates that these antimicrobial efflux systems are a target of action of (-)-α-pinene. Additionally, (-)-α-pinene decreases membrane integrity, which suggests that enhanced microbial influx is a secondary mode of action of (-)-α-pinene. Transcriptomic analysis indicated that (-)-α-pinene disrupts multiple metabolic pathways, and particularly those involved in heat-shock responses. Thus, (-)-α-pinene has significant activity in the modulation of antibiotic resistance in C. jejuni, which appears to be mediated by multiple mechanisms that include inhibition of microbial efflux, decreased membrane integrity, and metabolic disruption. These data warrant further studies on (-)-α-pinene to develop its use in the control of antibiotic resistance in Campylobacter.

Epigallocatechin gallate as a modulator of Campylobacter resistance to macrolide antibiotics.

Comprehensive therapeutic use of macrolides in humans and animals is important in the selection of macrolide-resistant Campylobacter isolates. This study shows high co-resistance to erythromycin, azithromycin, clarithromycin, dirithromycin and tylosin, with contributions from the 23S rRNA gene and drug efflux systems. The CmeABC efflux pump plays an important role in reduced macrolide susceptibility, accompanied by contributions from the CmeDEF efflux pump and potentially a third efflux pump. To improve clinical performance of licensed antibiotics and chemotherapeutic agents, it is important to understand the factors in Campylobacter that affect susceptibility to macrolide antibiotics. Using mutants that lack the functional genes coding for the CmeB and CmeF efflux pump proteins and the CmeR transcriptional repressor, we show that these efflux pumps are potential targets for the development of therapeutic strategies that use a combination of a macrolide with an efflux pump inhibitor (EPI) to restore macrolide efficacy. The natural phenolic compound epigallocatechin gallate (EGCG) has good modulatory activity over the extrusion across the outer membrane of the macrolides tested, both in sensitive and resistant Campylobacter isolates. Comparing EGCG with known chemical EPIs, correlations in the effects on the particular macrolide antibiotics were seen. EGCG modifies Campylobacter multidrug efflux systems and thus could have an impact on restoring macrolide efficacy in resistant strains.