Smoothie Drinks: Possible Source of Resistant and Biofilm-Forming Microorganisms.

Smoothie drinks are currently very popular drinks sold especially in fast food establishments. However, smoothies are a significant source of microorganisms. The aim of this study was to evaluate the microbiological quality of smoothies purchased in Eastern Bohemia. A higher prevalence of mesophilic aerobic bacteria (5.4-7.2 log CFU/mL), yeast (4.4-5.9 log CFU/mL) and coliform bacteria (3.1-6.0 log CFU/mL) was observed in vegetable smoothies, in which even the occurrence of enterococci (1.6-3.3 log CFU/mL) was observed. However, the occurrence of S. aureus, Salmonella spp. and Listeria spp. was not observed in any samples. Nevertheless, antimicrobial resistance was observed in 71.8% of the isolated strains. The highest level of resistance was found in isolates from smoothie drinks with predominantly vegetable contents (green smoothie drinks). Considerable resistance was observed in Gram-negative rods, especially to amoxicillin (82.2%) and amoxicillin with clavulanic acid (55.6%). Among enterococci, only one vancomycin-resistant strain was detected. The vast majority of isolated strains were able to form biofilms at a significant level, which increases the clinical importance of these microorganisms. The highest biofilm production was found in Pseudomonas aeruginosa, Kocuria kristinae and Klebsiella pneumoniae. Overall, significant biofilm production was also noted among isolates of Candida spp.

In Vitro Activity of Water Extracts of Olive Oil against Planktonic Cells and Biofilm Formation of Arcobacter-like Species.

Extra-virgin olive oils contain many bioactive substances that are phenolic compounds. The survival of Arcobacter-like strains in non-buffered (WEOO) and buffered (BEOO) extracts of olive oils were studied. Time kill curves of different strains were measured in the environment of olive oil extracts of different grades. The activity of the extracts was also monitored for biofilm formation using the Christensen method. In vitro results revealed that extra-virgin olive oil extracts exhibited the strongest antimicrobial effects, especially non-buffered extracts, which exhibited strain inhibition after only 5 min of exposure. The weakest inhibitory effects were observed for olive oil extracts. A decrease in biofilm formation was observed in the environment of higher WEOO concentrations, although at lower concentrations of extracts, increased biofilm formation occurred due to stress conditions. The dialdehydic forms of oleuropein derivatives, hydroxytyrosol, and tyrosol were the main compounds detected by HPLC-CoulArray. The results indicate that not all olive oils had a similar bactericidal effect, and that bioactivity primarily depended on the content of certain phenolic compounds.

The Effect of Antibiotics on Planktonic Cells and Biofilm Formation Ability of Collected Arcobacter-like Strains and Strains Isolated within the Czech Republic.

The purpose of this study was to test the in vitro effects of ampicillin, ciprofloxacin, clindamycin, erythromycin, gentamicin, and tetracycline on planktonic cells of Arcobacter-like microorganisms and on their biofilm formation ability. The minimum inhibitory concentrations (MICs) were determined by the microdilution method. Further, biofilm formation ability in the presence of various concentrations of antibiotics was evaluated by a modified Christensen method. Most of the 60 strains exhibited high susceptibility to gentamicin (98.3%), ciprofloxacin (95.0%), and erythromycin (100.0%). High level of resistance was observed to clindamycin and tetracycline with MIC50 and MIC90 in range of 4-32 mg/L and 32-128 mg/L, respectively. Combined resistance to both clindamycin and tetracycline was found in 38.3% of tested strains. In general, higher biofilm formation was observed especially at lower concentrations of antibiotics (0.13-2 mg/L). However, a significant decrease in biofilm formation ability of Pseudarcobacter defluvii LMG 25694 was exhibited with ampicillin and clindamycin at concentrations above 32 or 8 mg/L, respectively. Biofilm formation represents a potential danger of infection and also a risk to human health, in particular due to antimicrobial-resistant strains and the ability to form a biofilm structure at a concentration that is approximately the MIC determined for planktonic cells.

Volatiles Composition and Antimicrobial Activities of Areca Nut Extracts Obtained by Simultaneous Distillation-Extraction and Headspace Solid-Phase Microextraction.

The volatile components of areca nuts were isolated by headspace solid-phase microextraction (HS-SPME, DVB/CAR/PDMS fiber extraction) and simultaneous hydrodistillation-extraction (SHDE) and analyzed by gas chromatography/mass spectrometry. Furthermore, all SHDE fractions were tested for antimicrobial activity using the disk diffusion method on nine Gram-negative and Gram-positive bacteria (Bacillus subtilis, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus canis, Streptococcus pyogenes, and Candida albicans). In total, 98 compounds (mainly alcohols, carbonyl compounds, fatty acids, esters, terpenes, terpenoids, and aliphatic hydrocarbons) were identified in SHDE fractions and by using SPME extraction Fatty acids were the main group of volatile constituents detected in all types of extracts. The microorganism most sensitive to the extract of the areca nut was Streptococcus canis. The results can provide essential information for the application of different treatments of areca nuts in the canning industry or as natural antibiotics.

Biofilm Formation Ability of Arcobacter-like and Campylobacter Strains under Different Conditions and on Food Processing Materials.

Campylobacter jejuni is the most frequent cause of bacterial gastrointestinal food-borne infection worldwide. The transmission of Campylobacter and Arcobacter-like species is often made possible by their ability to adhere to various abiotic surfaces. This study is focused on monitoring the biofilm ability of 69 strains of Campylobacter spp. and lesser described species of the Arcobacteraceae family isolated from food, water, and clinical samples within the Czech Republic. Biofilm formation was monitored and evaluated under an aerobic/microaerophilic atmosphere after cultivation for 24 or 72 h depending on the surface material. An overall higher adhesion ability was observed in arcobacters. A chi-squared test showed no association between the origin of the strains and biofilm activity (p > 0.05). Arcobacter-like species are able to form biofilms under microaerophilic and aerobic conditions; however, they prefer microaerophilic environments. Biofilm formation has already been demonstrated at refrigerator temperatures (5 °C). Arcobacters also showed higher biofilm formation ability at the temperature of 30 °C. This is in contrast to Campylobacter jejuni NP 2896, which showed higher biofilm formation ability at temperatures of 5-30 °C. Overall, the results demonstrated the biofilm formation ability of many strains, which poses a considerable risk to the food industry, medical practice, and human health.

Chemical Composition of Natural Hydrolates and Their Antimicrobial Activity on Arcobacter-Like Cells in Comparison with Other Microorganisms.

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.

Comparison of Chemical Composition and Biological Properties of Essential Oils Obtained by Hydrodistillation and Steam Distillation of Laurus nobilis L.

The purpose of this study was to compare the yield, chemical composition, antimicrobial and antioxidant properties of essential oils isolated from leaves of Laurus nobilis L. by two different distillation methods. The essential oils isolated by hydrodistillation (HD) and steam distillation (SD) were analyzed by gas chromatography coupled to mass spectrometry (GC-MS) and gas chromatography with flame ionization detector (GC-FID). Hydrodistillation produced a yield of 0.95 ± 0.06% which is slightly higher than yield obtained by steam distillation 0.79 ± 0.07%. Seventy three compounds in the bay leaves oil obtained by steam distillation were identified while in essential oil obtained by hydrodistillation were identified only 54 compounds. The antioxidant activity was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical method. Antimicrobial activity of obtained essential oils was evaluated by disc diffusion method in comparison with several chosen antimicrobials. The antimicrobial activity was tested on five microorganisms - Escherichia coli, Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Candida albicans. In general, oils produced by steam distillation had higher antimicrobial and antioxidant activities than hydrodistillation extracts. It seems that hydrodistillation is better for higher yield while steam distillation is better to use for more quality oils with stronger biological properties.

Chemical Composition and Determination of the Antibacterial Activity of Essential Oils in Liquid and Vapor Phases Extracted from Two Different Southeast Asian Herbs-Houttuynia cordata (Saururaceae) and Persicaria odorata (Polygonaceae).

Essential oils obtained via the hydrodistillation of two Asian herbs (Houttuynia cordata and Persicaria odorata) were analyzed by gas chromatography coupled to mass spectrometry (GC-MS) and gas chromatography with flame ionization detector (GC-FID). Additionally, both the liquid and vapor phase of essential oil were tested on antimicrobial activity using the broth microdilution volatilization method. Antimicrobial activity was tested on Gram-negative and Gram-positive bacteria-Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis, Streptococcus pyogenes, Klebsiella pneumoniae, Seratia marcescense and Bacillus subtilis. Hydrodistillation produced a yield of 0.34% (Houttuynia cordata) and 0.40% (Persicaria odorata). 41 compounds were identified in both essential oils. Essential oils contained monoterpenes and their oxidized forms, sesquiterpenes and their oxidized forms, oxidized diterpenes, derivates of phenylpropene and other groups, such as, for example, aldehydes, alcohols or fatty acids. Both essential oils were antimicrobial active in both vapor and liquid phases at least in case of one bacterium. They expressed various antimicrobial activity in the range of 128-1024 µg∙mL-1, 512-1024 µg∙mL-1 in broth and 1024 µg∙mL-1, 512-1024 µg∙mL-1 in agar, respectively. Research showed new interesting information about P. odorata and H. cordata essential oils and demonstrated that both essential oils could be possibly used in the field of natural medicine or natural food preservation.

Occurrence of virulence-associated genes in Arcobacter butzleri and Arcobacter cryaerophilus isolates from foodstuff, water, and clinical samples within the Czech Republic.

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.

Susceptibility to 18 drugs and multidrug resistance of Arcobacter isolates from different sources within the Czech Republic.

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 ß-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.

Modified isolation method of Arcobacter spp. from different environmental and food samples.

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.

Effect of selected types of beer on bacteria of the genus Arcobacter.

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.