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 24
h 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.
The aim of the study was to investigate the essential oil (EO) of
x
cv. Bila, which has not been studied before. The EOs were distilled from plants collected in two consecutive years on the island of ...Hvar (Croatia) and in the Karst (Slovenia) and analysed for chemical composition and antimicrobial activity. The main component of EOs was linalool, but the EOs from Hvar had higher contents of Z-β-ocimene and borneol + lavandulol than the EOs from Karst, in which camphor, linalyl acetate and 1,8-cineole predominated. The antimicrobial activity was evaluated using the minimum inhibitory concentration and proved that the EOs were effective against
spp. Studies have shown that the composition of
x
EO depends on the variety and the climatic and geographical characteristics of the plant growth. The antimicrobial activity of EO is also influenced by the type and strain of microorganisms involved in the research.
The abandoned Sitarjevec mine represents a specific environment (complete darkness; 10 °C) with waters of pH 3 and it contains various ores (e.g. lead, mercury, zinc, iron, copper) and many different ...minerals (e.g. anglesite, baryte, cinnabar, pyrite, cerussite, quartz, siderite). The aim of this study was to characterize the iron oxide/hydroxide speleothems that show rapid growth of up to 5 cm/year. Their morphological and chemical characterization showed that they have a layered structure with different ferrihydrite minerals (i.e. goethite, feroxyhyte, lepidocrocite). Scanning electron microscopy-energy dispersive X-ray spectrometry confirmed that the surface layers were very porous, while for the inner parts, the bulk was compact. Bacterial populations in these speleothems and their environment were analyzed using 16S rRNA sequencing. These data revealed a great diversity of bacteria, which included 19-34 phyla across different samples. Proteobacteria were dominant in all of the samples (60-95%), although the highest bacterial diversity was seen for a water sample from the base of the speleothem, with 670 different genera and 100 different species detected. Among these there were typical iron-oxidizing bacteria, like Gallionella capsiferriformans, Sideroxydans lithotrophicus, Sphingomonas echinoides, Candidatus Nitrotoga Nitrotoga, Leptospirillum ferrooxidans, and Ferrovum myxofaciens. These iron-oxidizing bacteria were not present in other samples from the mine. Therefore, these bacteria might be involved in the structural growth of these speleothems. The specific bacterial interactions with the minerals under the environmental conditions in the mine need to be further analyzed and evaluated.
Bacterial adhesion can be dictated by different surface characteristics. In this study we concentrate on the surface roughness of a stainless steel material. We prepared the stainless steel surfaces ...by 3D polishing, brushing, grinding and electropolishing. Untreated stainless steel surfaces were also considered. The corresponding surface roughness was assessed by profilometry and atomic force microscopy. In experiments we have used different types of bacteria. The rate of adhered bacteria on metal surfaces was determined spectrophotometrically. The results showed that the rate of adhered bacteria increases with increasing surface roughness. Scanning electron microscopy was used to image surfaces in order to determine locations of adhered bacteria. The increased adhesion of bacteria on more rough surfaces results from an increased interplay between the increasing effective surface area and increasing numbers of cracks, voids and gaps.
Using two-dimensional (2-D) electrophoresis it was shown that bovine kappa-casein could be an appropriate biomarker of adulteration of goat’s
milk with cow’s milk not only in raw milk, but also for ...milk thermally processed by pasteurization or treated with ultra-high-temperature. The presence of cow’s milk in goat’s milk was detected at level of 2 %. Furthermore, position of bovine kappa-casein spots on 2-D gels remained unchanged even with samples from two different geographical origins, Belgium and Slovenia. These results show that neither thermal processing
nor different geographical area seem to affect the position of bovine kappa-casein spots on 2-D gels.
Korištenjem dvodimenzionalne elektroforeze dokazano je da kravlji kapa-kazein može biti odgovarajući biomarker za utvrđivanje patvorenja kozjeg mlijeka kravljim mlijekom, ne samo u sirovom mlijeku, već i za mlijeko koje se termički obrađuje pasterizacijom ili ultra-visokom temperaturom. Prisutnost kravljeg mlijeka u kozjem mlijeku moguće je detektirati na razini od 2 %. Nadalje, položaj proteinskih mrlja kapa-kazeina na 2-D gelovima ostao je nepromijenjen uspoređujući uzorke mlijeka različitog geografskog podrijetla, Belgije i Slovenije. Ovi rezultati pokazuju da ni toplinska obrada niti različito geografsko podrijetlo ne utječu na položaj kravljih proteinskih mrlja kapa-kazeina na 2-D gelovima.
The purpose of this study was to develop a PCR-based method for quantification of Listeria monocytogenes adhesion in microtitre plates. We optimized isolation of DNA in the microtitre plates using ...cell lysis, ultrasound treatment, heating, and centrifugation of the lysate. Digital PCR was applied for quantification of L. monocytogenes DNA that was used for construction of the standard curve, and real-time PCR was used for quantification of the attached L. monocytogenes cells. This PCR-based method was applied to quantify different strains of L. monocytogenes at different times of biofilm formation, and to study the anti-adhesive actions of natural bioactive substances (epigallocatechin gallate, (−)-α-pinene). The results show that the PCR-based method developed here can be widely used as a novel approach for adhesion assays and biofilm research.
•Evaluation of Listeria monocytogenes adhesion with novel PCR-based method•DNA isolation in microtitre plate•Digital PCR was used to quantify the DNA used for the standard curve.•Real-time PCR was applied for quantification of L. monocytogenes biofilm cells.
is a commensal inhabitant of human skin and mucosa, and a common nosocomial pathogen in immunocompromised patients.
strains were isolated from places with precisely defined and controlled air quality ...and regular cleaning and disinfection regimes-cleanrooms. These strains were adapted to increasing concentrations of the quaternary ammonium disinfectant didecyldimethylammonium chloride (DDAC). Compared to nonadapted strains, these strains became adapted to up to 180-fold higher concentrations of DDAC, as seen by their increased minimal inhibitory concentrations. Examination of the stability of adaptation showed that three strains became permanently adapted to DDAC and named as resistant strains, and four strains were temporarily adapted to DDAC and named as strains with higher tolerance to DDAC. Some adapted strains showed cross-resistance to benzalkonium chloride and/or antibiotics. The adaptation response mechanisms of these DDAC-adapted strains were also investigated. The majority of adapted strains showed modifications to cell size and fatty acid composition. Some of the adapted strains showed changes in biofilm formation and overexpression of efflux pumps. Three adapted strains also showed altered growth rates. In this first report of adaptation of
strains to DDAC, the fatty acid profiling showed that the majority of strains had reduced ratio of saturated to unsaturated fatty acids and decreased content of straight-chain fatty acids, at the expense of the
-branched fatty acids. We can conclude that
strains can adapt or become resistant to DDAC. We have revealed several adaptive response mechanisms that can be targeted for control and inhibition of
in cleanrooms and other clean processing environments.
The food‐borne pathogen Campylobacter jejuni can cause bacterial gastrointestinal infections. Biofilm formation amplifies the risk of human infection by improving survival and persistence of ...C. jejuni in food processing environments and its transmission through the food chain. We aimed to control C. jejuni using an alternative strategy of low doses of Juniperus communis fruit preparations to target bacterial adhesion properties in the first step of biofilm formation. First, we defined the anti‐Campylobacter activity of a juniper fruit crude extract and its fractionated biflavonoids, flavone glycosides, and purified amentoflavone, of juniper fruit essential oil and of juniper fruit postdistillation waste material extract. For accurate quantification of adherent C. jejuni, we optimised digital Polymerase Chain Reaction (PCR) and quantitative real‐time PCR for construction of standard curves and quantification. We show for the first time that juniper fruit formulations can effectively inhibit adhesion of C. jejuni to polystyrene. Furthermore, ≥94% of the antiadhesion activity of juniper fruit crude extract and juniper fruit essential oil remained under food‐related conditions: modified culture medium with glucose, or a stainless steel surface, or mixed co‐cultures of C. jejuni and Listeria monocytogenes. This study indicates that addition of juniper fruit formulations can control growth and adhesion of C. jejuni and thus limit food chain transmission of campylobacters.
In this study, we initially investigated the antimicrobial activities of two commercial rosemary extracts (V20, V40) against Alicyclobacillus strains, with the ultimate purpose to determine whether ...these can be used in apple juice for the control of growth of alicyclobacilli. Minimum inhibitory concentrations (MICs) were first determined, and put through sensory analysis. Addition of the rosemary extracts to apple juice at their MICs did not change the colour, odour, taste or opacity of the apple juice. Growth kinetics studies with these rosemary extracts indicated a reduction in vegetative cells for Alicyclobacillus acidoterrestris, Alicyclobacillus hesperidum and Alicyclobacillus cycloheptanicus in Bacillus acidoterrestris broth and in apple juice. Further studies with A. acidoterrestris spores showed that the MICs of these rosemary extracts had relatively low effects on spore numbers in B. acidoterrestris broth, but had a spore number inhibition index >15% in apple juice. A four-fold increase in the rosemary extract concentrations showed the opposite effects: greater reduction in spores in B. acidoterrestris broth (inhibition index, >60%) than in apple juice (inhibition index, <10%). These data indicate that at their MICs, the V20 and V40 rosemary extracts allow outgrowth of spores but reduce vegetative cells, acting together with the low pH or other particular constituents of the apple juice. Rosemary extracts applied at their MICs thus represent an alternative method for the control of A. acidoterrestris in apple juice.
•V20/V40 rosemary extracts have anti-alicyclobacilli activity.•MICs of these rosemary extracts do not change the sensory properties of apple juice.•MICs of these rosemary extracts reduce growth of alicyclobacilli vegetative cells.•MICs of these rosemary extracts do not show sporicidal effects on alicyclobacilli.•V20/V40 rosemary extracts can provide control of alicyclobacilli in apple juice.
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