Hydrolates obtained via the hydrodistillation and steam distillation of Lavandulaangustifolia Mill., Syzygiumaromaticum L., Foeniculumvulgare Mill., and Laurusnobilis L. were analyzed by gas chromatography with flame ionization detector (GC-FID) and gas chromatography coupled to mass spectrometry (GC-MS). Additionally, the hydrolates were evaluated for antimicrobial activity (disk-diffusion and microdilution method), influence on biofilm formation (Christensen method) and cytotoxicity of concentrated hydrolates against human cell lines (A549) by xCELLigence system. Using chemical analysis, 48, 9, 13 and 33 different components were detected in lavender, clove, fennel and laurel hydrolates, respectively. Lavender hydrolate contained the largest proportion of 1,8-cineol, linalool furanoxide, and linalool. The main components of laurel hydrolate were 1,8-cineol, 4-terpineol and α-terpineol. Fenchone and estragole were the most abundant in fennel hydrolate, and eugenol and eugenyl acetate in clove hydrolate. Concentrated hydrolates showed significant antimicrobial activity. Clove hydrolate was among the most antimicrobially active agents, most preferably against C. albicans, with an inhibition zone up to 23.5 mm. Moreover, concentrated hydrolates did not show any cytotoxic effect again8 st human A549 cells. In the presence of the non-concentrated hydrolates, significantly reduced biofilm formation was observed; however, with concentrated clove hydrolate, there was an increase in biofilm formation, e.g., of A. thereius, A. lanthieri, and A. butzleri. Research shows new findings about hydrolates that may be important in natural medicine or for preservation purposes.

• MeSH
• Anti-Infective Agents pharmacology   MeSH
• Arcobacter drug effects   MeSH
• A549 Cells MeSH
• Candida albicans drug effects   MeSH
• Distillation MeSH
• Lavandula chemistry   MeSH
• Humans MeSH
• Lung Neoplasms drug therapy   MeSH
• Oils, Volatile pharmacology   MeSH
• Plant Oils pharmacology   MeSH
• Cell Proliferation MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Bacteria of the Arcobacter (A.) genus, originating mainly from food and water, are dreaded germs for humans as well as animals. However, the virulence of these bacteria has not been fully elucidated yet. This study looked at the occurrence of eight virulence-associated factors (ciaB, cj1349, pldA, irgA, hecA, tlyA, mviN, hecB) in a total of 80 isolates of Arcobacter butzleri and 22 isolates of A. cryaerophilus. The isolates were derived from food, water, and clinical samples. A polymerase chain reaction using specific primers was used to detect these virulence-associated genes. The presence of all genes in the isolates of A. butzleri (98.8% ciaB, 95.0% cj1349, 98.8% pldA, 22.5% irgA, 31.3% hecA, 95.0% tlyA, 97.5% mviN, 38.8% hecB) and A. cryaerophilus (95.5% ciaB, 0.0% cj1349, 9.1% pldA, 0.0% irgA, 0.0% hecA, 31.8% tlyA, 90.9% mviN, 0.0% hecB) was monitored. Among the tested isolates, there were 13 isolates (12.7%) of A. butzleri, in which the presence of all eight virulence-associated genes was recorded in the genome. In contrast, in one A. cryaerophilus strain, none of the observed genes were detected. The presence of ciaB and mviN genes was significantly more frequent in A. cryaerophilus isolates than other genes (P < 0.05). In general, more virulence-associated genes have been detected in A. butzleri isolates compared to A. cryaerophilus. The most common gene combination (ciaB, cj1349, pldA, tlyA, mviN) was detected in case of 39 isolates. In 50.0% of A. butzleri isolates derived from clinical samples, all eight virulence-associated genes were significantly more frequently detected (P < 0.05). The tlyA gene occurred significantly more frequent in A. butzleri isolates from meat and water samples and irgA and hecB genes in clinical samples. Therefore, our study provides information about occurrence of virulence-associated genes in genome of Arcobacter isolates. These findings could be hazardous to human health, because the presence of virulence-associated genes is the assumption for potential dangerousness of these bacteria. Our results indicate high incidence of virulence-associated genes in Arcobacter genomes and hence potentially pathogenic properties of the studied strains.

• MeSH
• Arcobacter genetics   isolation & purification   pathogenicity   MeSH
• Genes, Bacterial genetics   MeSH
• Virulence Factors genetics   MeSH
• Genetic Variation MeSH
• Gram-Negative Bacterial Infections microbiology   MeSH
• Water Microbiology * MeSH
• Food Microbiology * MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Czech Republic MeSH

Arcobacter butzleri is an emerging human and animal pathogen for which an increased prevalence of resistance to antibiotics has been observed, and so alternative compounds to modulate resistance of A. butzleri are required. This work aims to study the potential use of several polyphenols as efflux pump inhibitors (EPIs) and to evaluate their interaction with antibiotics, in order to enhance antibiotic activity against A. butzleri. The minimum inhibitory concentration (MIC) of (-)-epicatechin, (+)-catechin, rutin, gallic acid, caffeic acid, chlorogenic acid, resveratrol, pterostilbene, and pinosylvin was determined, in absence and presence of four known EPIs. Subsequently, ethidium bromide accumulation in presence of subinhibitory concentrations of polyphenols was evaluated, and the synergistic potential of the compounds with antibiotics was assessed by checkerboard dilution test. Only stilbenes presented activity against A. butzleri, with MIC values ranging between 64 and 512 μg/mL. The MIC determination of the polyphenols in the presence of subinhibitory concentrations of known EPIs showed that efflux pumps play a role in the resistance to these compounds. Stilbenes also induced a higher intracellular accumulation of ethidium bromide, indicating that they may inhibit the activity of efflux pumps. Checkerboard assays showed that several combinations of polyphenol/antibiotic had an additive effect against A. butzleri. Overall, the results indicate that some polyphenols reduce A. butzleri resistance to antibiotics, suggesting the potential of stilbenes as EPIs. The potential of resveratrol and pinosylvin as resistance modulators was evidenced, insofar as these compounds can even revert antibiotic resistance. Therefore, the use of polyphenols as resistance modulators could be an alternative to overcome the decreasing susceptibility of A. butzleri to antibiotics.

• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Arcobacter drug effects   genetics   metabolism   MeSH
• Drug Resistance, Bacterial MeSH
• Bacterial Proteins antagonists & inhibitors   genetics   metabolism   MeSH
• Gram-Negative Bacterial Infections microbiology   MeSH
• Humans MeSH
• Microbial Sensitivity Tests MeSH
• Polyphenols pharmacology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

• MeSH
• Arcobacter * MeSH
• Biofilms * MeSH

OBJECTIVES: Arcobacter spp. are considered to be potential foodborne pathogens, and consumption of contaminated food containing these bacteria could endanger human and animal health. Arcobacter butzleri and Arcobacter cryaerophilus are the species most frequently isolated from food of animal origin and from other samples. The aim of this study was to evaluate the susceptibility of arcobacters isolated in the Czech Republic. No information about antibiotic susceptibility and multidrug resistance of arcobacters isolated in the Czech Republic is available in the literature before now. METHODS: The antimicrobial resistance of A. butzleri (n=80) and A. cryaerophilus (n=20) isolated from meat of animal origin, water sources and clinical samples was examined by the disk diffusion method. RESULTS: Arcobacters were resistant to one or more antimicrobial agents in 99% (99/100) of tested isolates. Most of the Arcobacter isolates were resistant to β-lactam antibiotics, i.e. ampicillin (81.0%), amoxicillin/clavulanic acid (28.0%), cefalotin (73.0%) and aztreonam (93.0%). Arcobacters were also frequently resistant to lincosamides, i.e. clindamycin (98.0%). Of the aminoglycosides, amikacin, gentamicin and tobramycin were evaluated to be the most effective antibiotics among those tested against arcobacters. CONCLUSIONS: These results demonstrate substantial resistance in Arcobacter isolates to 18 antimicrobial agents commonly used in medical and veterinary medicine. Multidrug resistance was found in 93.8% (75/80) of A. butzleri isolates and 70.0% (14/20) of A. cryaerophilus isolates.

• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Arcobacter drug effects   isolation & purification   MeSH
• Disk Diffusion Antimicrobial Tests MeSH
• Gram-Negative Bacterial Infections microbiology   MeSH
• Humans MeSH
• Water Microbiology * MeSH
• Drug Resistance, Multiple, Bacterial * MeSH
• Food Microbiology * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic MeSH

Water plays an important role in the transmission of Arcobacter spp. to animals and humans. The aim of this study was to isolate and characterize Arcobacter spp. from 115 different water samples (66 sewage, 25 rivers, 16 spring water, and 8 drinking water) in Izmir, Turkey. In total, 41 samples (35.7 %) were found positive for Arcobacter spp. by the genus-specific PCR. Arcobacter butzleri was detected in 39 out of 115 samples (33.9 %) including 24 sewage, 13 rivers, and 2 spring water. The remaining Arcobacter spp. (n = 2) isolates could not be identified by m-PCR and 16S rRNA gene sequencing. Based on the phenotypic characterization, most of the Arcobacter species (87.8 %) indicated weak catalase activity. In addition, there were differences in phenotypic patterns among isolated species during growth at 37 °C under microaerobic and aerobic conditions, in the presence of 2 % (39/41) and 3.5 % (32/41) NaCl and 0.04 % TTC (39/41) and on MacConkey agar (38/41). The results of this study indicated that environmental water samples are common sources for Arcobacter spp. Therefore, effective control measures should be taken to protect human health.

• MeSH
• Aerobiosis MeSH
• Arcobacter classification   genetics   growth & development   isolation & purification   MeSH
• Sodium Chloride metabolism   MeSH
• DNA, Bacterial chemistry   genetics   MeSH
• Culture Media chemistry   MeSH
• Water Microbiology * MeSH
• DNA, Ribosomal chemistry   genetics   MeSH
• RNA, Ribosomal, 16S genetics   MeSH
• Sequence Analysis, DNA MeSH
• Bacterial Typing Techniques MeSH
• Temperature MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Turkey MeSH

Cíl práce: Účelem této studie bylo detekování biofilmu patogenních mikroorganismů vyskytujících se v potravinářském průmyslu a porovnání jeho tvorby při různých kultivačních podmínkách. Materiál a metody: Ke studii byly zvoleny následující mikroorganismy – Staphylococcus aureus, Listeria innocua, Listeria ivanovii, Cronobacter sakazakii, Cronobacter muyt-jensii, Arcobacter butzleri, Arcobacter cryaerophilus, Campylobacter jejuni a Campylobacter coli. Pro detekci biofilmu vybraných mikroorganismů byla použita Christensenova metoda v mikrotitračních destičkách a metoda kultivace na kuponech z nerezové oceli. Výsledky: U všech sledovaných mikroorganismů byla potvrzena schopnost tvorby biofilmu, a to jak v mikrotitračních destičkách, tak na kuponech z nerezové oceli. Tvorba biofilmu byla ovlivněna jak kultivačním médiem, použitým materiálem a dobou kultivace, tak samotným mikroorganismem. Bylo prokázáno, že různé druhy a kmeny téhož rodu tvoří biofilm rozdílně. Rozdíl byl zjištěn i při porovnání sbírkových kultur a izolátů z prostředí. Některé bakterie tvořily biofilm ve větší míře na povrchu polyetylenových mikrotitračních destiček a na nerezových kuponech pak méně, nebo tomu bylo naopak. Některé patogeny byly schopny zvýšit denzitu planktonních buněk původní suspenze během 72 hodin až o 3 řády a zároveň vytvořit velké množství biofilmu. Závěry: Sledování tvorby biofilmu obávaných patogenů je velice důležité, a to nejen v potravinářském průmyslu. Podle získaných výsledků je zřejmé, že se bakteriální biofilmy formují již po relativně krátkém čase (v našem případě 24 hodin). Vzhledem ke struktuře těchto biofilmů je jejich likvidace velice náročná, je tedy žádoucí předcházet samotnému vzniku biofilmu. Klíčová slova: nerezová ocel – biofilm – patogeny – planktonní buňky – mikrotitrační destička

Objective: Detection of biofilm formation by microbial pathogens relevant to the food industry and comparison of biofilm formation under different conditions of culture. Material and methods: The following microorganisms were selected for the study: Staphylococcus aureus, Listeria innocua, Listeria ivanovii, Cronobacter sakazakii, Cronobacter muytjensii, Arcobacter butzleri, Arcobacter cryaerophilus, Campylobacter jejuni, and Campylobacter coli. To detect biofilm formation the microtiter plate assay, as described by Christensen and culture on stainless steel coupons were used. Results: The biofilm forming capacity was confirmed in all microorganisms tested, both on the microtiter plates and stainless steel coupons. Biofilm formation was influenced by the culture medium, material used, and culture duration as well as by the test microorganism. It was found that different species and strains of the same genus differ in biofilm formation. Differences were also found between the collection strains and isolates from the environment. Some bacteria tended to form biofilm more readily on the surface of the polyethylene microtiter plates and less readily on stainless steel coupons while others appeared to have an opposite tendency. Some pathogens were able to increase the planktonic cell density in the initial suspension even by three orders of magnitude within 72 hours while producing plenty of biofilm. Conclusions: The study of biofilm formation by high risk pathogens is of utmost importance, not only to the food industry. From the obtained results, it is evident that bacterial biofilms form rapidly (within 24 hours in the present study). Due to their architecture, these biofilms are difficult to eradi-cate, and therefore, it is crucial to prevent biofilm formation. Keywords: stainless steel – biofilm – pathogens – planktonic cells – microtiter plate

• Keywords
• planktonní buňky, mikrotitrační destička,
• MeSH
• Arcobacter physiology   growth & development   MeSH
• Bacterial Adhesion MeSH
• Biofilms * growth & development   MeSH
• Campylobacter coli physiology   growth & development   MeSH
• Campylobacter jejuni physiology   growth & development   MeSH
• Time Factors MeSH
• Cronobacter sakazakii physiology   growth & development   MeSH
• Cronobacter physiology   growth & development   MeSH
• Culture Techniques methods   statistics & numerical data   MeSH
• Listeria physiology   growth & development   MeSH
• Stainless Steel * MeSH
• Food Microbiology * MeSH
• Food Industry MeSH
• Surface Properties MeSH
• Staphylococcus aureus physiology   growth & development   MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH

This study provides information on the occurrence of Arcobacter in several types of water and food products of animal origin in the Czech Republic. We processed 190 samples using the modified method, and the occurrence of Arcobacter spp. was confirmed in 36.8 % of these. This total incidence consisted of Arcobacter butzleri (27.3 %), Arcobacter cryaerophilus (8.4 %) and Arcobacter skirrowii (1.1 %). We newly described the common presence of Arcobacter spp. in sewage water in the Czech Republic that is released into waterways after processing in water treatment plants (86.7 %). All the acquired isolates were subject to detailed confirmation with subsequent species classification using multiplex PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In this study, we used a modification of a method using passive filtration of an enriched sample, which could be suitable for the isolation of Arcobacter, especially in combination with Campylobacter selective agar chromogenic medium. Our studies have shown this agar to be quite suited to the isolation of Arcobacter and that it can be an appropriate instrument for accelerating culture diagnostics.

• MeSH
• Arcobacter genetics   growth & development   isolation & purification   metabolism   MeSH
• Culture Media metabolism   MeSH
• Sewage microbiology   MeSH
• Colony Count, Microbial instrumentation   methods   MeSH
• Food Microbiology * MeSH
• Publication type
• Journal Article MeSH
• Evaluation Study MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic MeSH

The genus Arcobacter is related to the well-known human pathogen, Campylobacter jejuni, and has been linked to human diseases. In this study, the survival of Arcobacter spp. in various concentrations of ethanol, in various samples of beers, and in a model stomach has been investigated. For most of these bacteria, a concentration of 10 % ethanol was determined to be the minimum inhibitory concentration. The fact that these organisms are able to survive under these conditions may have an impact in the food processing industry. We studied the activity of beer against arcobacters. These bacteria were killed in all samples of beer within 30 min. A model stomach, containing a food matrix and a synthetic gastric fluid, was used to deduce the effect of beer against Arcobacter spp. during food consumption. Complete inactivation of all monitored arcobacters was detected usually within 15 min. However, the presence of beer does not potentiate the effect of gastric fluid against these bacteria. This is apparently the first study focusing upon the effect of beer on Arcobacter spp.

• MeSH
• Arcobacter drug effects   physiology   MeSH
• Time Factors MeSH
• Ethanol toxicity   MeSH
• Humans MeSH
• Microbial Sensitivity Tests MeSH
• Microbial Viability drug effects   MeSH
• Beer analysis   microbiology   MeSH
• Models, Theoretical MeSH
• Stomach microbiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The inhibitory effects of 17 organic acids (C₂-C₁₆ fatty acids, sorbic, benzoic, phenylacetic, fumaric, succinic, lactic, malic and citric) on Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii were investigated by determining their IC₅₀ values, defined as the concentration of acid at which the target DNA sequence was expressed at 50% of the positive control level in cultures incubated at 30°C for 24 h. DNA was analysed by real-time PCR. The Arcobacter strains tested were inhibited by all the organic acids, with the sensitivities in the order A. skirrowii > A. cryaerophilus > A. butzleri. Eight acids with IC₅₀ values of <1 mg/mL against A. butzleri were tested for their effects on A. butzleri inoculated on chicken carcasses at a concentration of 5 log CFU/g of skin. Inoculated halved carcasses were immersed in solutions of the acids at 5 mg/mL for 1 min. Samples of skin were collected from carcass halves after storage at 4°C for 0, 1, 2 or 3 days for enumeration of arcobacters on Muller-Hinton agar. All eight tested acids suppressed bacterial proliferation. The highest inhibitory activities were observed for benzoic, citric, malic and sorbic acids. Subsequent sensory analysis revealed benzoic acid to be the most suitable organic acid for chicken skin treatment.

• MeSH
• Arcobacter drug effects   growth & development   MeSH
• Inhibitory Concentration 50 MeSH
• Chickens MeSH
• Skin microbiology   MeSH
• Acids pharmacology   MeSH
• Food Handling MeSH
• Meat microbiology   standards   MeSH
• Organic Chemicals pharmacology   MeSH
• Food Microbiology methods   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH