In recent years, increased rates of yeast infections in humans and animals have been recognized worldwide. Since animals may represent a source of yeast infections for humans, knowing the antifungal susceptibility profile of yeast isolates from milk and evaluating their pathogenic potential would be of great medical importance. Therefore, the aim of this survey was to study yeast diversity in milk samples, analyze the hemolytic and phospholipase activities of isolates and determine minimal inhibition concentration (MIC) for fluconazole, voriconazole and flucytosine. Out of 66 yeast isolates obtained from 910 individual raw milk samples from subclinically infected cows, 26 different yeast species were determined based on sequencing of the D1/D2 and ITS regions. Among them, Pichia kudriavzevii (formerly known as Candida krusei), Kluyveromyces marxianus (formerly known as Candida kefyr) and Debaryomyces hansenii (formerly known as Candida famata) were the most commonly identified. Hemolysin and/or phospholipase activity was observed in 66.7% of isolates. The elevated MIC for fluconazole was determined in 16 isolates from 11 species. The findings of this study demonstrate that yeast isolates from raw milk have the potential to express virulence attributes such as hemolysin and phospholipase, and additionally, some of these strains showed elevated MIC to fluconazole or to flucytosine.LAY SUMMARY: We identified 66 yeast isolates, including 26 different yeast species from 910 individual milk samples. Our results indicate that individual milk samples may serve as a source of yeasts with the potential to trigger infection and may have reduced sensitivity to tested antifungal agents.
- MeSH
- Antifungal Agents * pharmacology MeSH
- Virulence Factors * genetics MeSH
- Fluconazole pharmacology MeSH
- Microbial Sensitivity Tests veterinary MeSH
- Milk MeSH
- Cattle MeSH
- Voriconazole MeSH
- Animals MeSH
- Check Tag
- Cattle MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Animal or human protothecosis belongs to rather rare, endemic, pro-inflammatory infections. It is caused by achlorophyllous algae of the genus Prototheca. Especially, P. bovis (formerly P. zopfii genotype 2) is often inflected as a non-bacterial causative agent of dairy cattle mastitis. In this study, we present a multiplex real-time PCR (qPCR) system for rapid and exact Prototheca spp. detection and quantification. Limit of detection, diagnostic sensitivity, and specificity were determined. For the first time, specific sequences of AccD (encoding acetyl CoA reductase) for P. bovis, cox1 (encoding cytochrome C oxidase subunit 1) for P. wickerhamii, cytB (encoding cytochrome B) for P. blashkeae and atp6 (encoding transporting ATPase F0 subunit 6) for P. ciferrii (formerly P. zopfii genotype 1) were used for species identification and quantification together with 28S rRNA sequence detecting genus Prototheca. The developed qPCR assay was applied to 55 individual cow milk samples from a herd suspected of protothecosis, 41 bulk milk samples from different Czech farms, 16 boxed milk samples purchased in supermarkets and 21 environmental samples originating from a farm suspected of protothecosis. Our work thus offers the possibility to diagnose protothecosis in the samples, where bacterial mastitis is the most commonly presumed and thereby assisting adequate corrective measures to be taken.
- MeSH
- DNA chemistry isolation & purification MeSH
- Farms MeSH
- Cloning, Molecular MeSH
- Real-Time Polymerase Chain Reaction methods MeSH
- Limit of Detection MeSH
- Environmental Microbiology MeSH
- Dairying MeSH
- Milk microbiology MeSH
- Multiplex Polymerase Chain Reaction methods MeSH
- Plasmids genetics MeSH
- Prototheca genetics growth & development isolation & purification MeSH
- Sensitivity and Specificity MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czech Republic MeSH
The absence of acquired resistance to antimicrobials has become an important criterion in evaluation of the biosafety of lactobacilli used as industrial starter or probiotic cultures. The aim of this study was to assess antibiotic resistance in starter and non-starter lactobacilli of food origin. Minimal inhibitory concentrations of ampicillin, chloramphenicol, clindamycin, erythromycin, gentamicin, kanamycin, streptomycin, tetracycline and vancomycin were established in 81 strains of lactobacilli (L. acidophilus, L. animalis, L. brevis, L. curvatus, L. delbrueckii, L. fermentum, L. helveticus, L. paracasei, L. plantarum, L. rhamnosus and L. sakei) by the microdilution method. The strains were classified as susceptible or resistant to antimicrobials based on the cut-off values according to the EFSA guideline. Sixty-two strains (77% food isolates, 76% starter or adjunct cultures) were resistant to at least one antimicrobial agent (the most frequently to aminoglycosides). Adjunct cultures showed a higher antibiotic resistance (80%) than starters (60%). Four multiresistant strains (3 food isolates, 1 adjunct culture) were analyzed by whole genome sequencing. One potentially transferable aadE gene (responsible for streptomycin resistance) was detected only in one multi-drug resistant strain of L. animalis originating from an adjunct culture. Thus, there is a risk of horizontal transmission of this gene. It is necessary to eliminate such strains from use in the food industry. This study provides relevant data concerning the use of lactobacilli in safe food production. To ensure food safety, detailed characterization of resistance to antimicrobials is necessary not only in starter strains but also in non-starter lactic acid bacteria isolated from food products.
- MeSH
- Drug Resistance, Microbial * genetics MeSH
- Lactobacillus * classification metabolism drug effects MeSH
- Microbial Sensitivity Tests methods MeSH
- Food Microbiology methods MeSH
- Probiotics classification MeSH
- Food Security methods MeSH
- Publication type
- Clinical Study MeSH
- Research Support, Non-U.S. Gov't MeSH
Infection with Toxoplasma gondii has usually been connected with consumption of improperly treated meat. However, contaminated water and products of plant origin have emerged as new sources of infection in the last few years. Here, 292 vegetable samples-carrot, cucumber and lettuce-obtained from nine farms in the Czech Republic were examined using triplex real time PCR targeting two specific T. gondii sequences. Irrigation water and water used for washing of vegetables were also included. Overall, a positivity rate of 9.6% was found in vegetables. The concentration varied between 1.31 × 100 and 9.00 × 102 oocysts/g of sample. A significant difference was found between the positivity of vegetables collected directly from fields and that of vegetables collected from farm storage rooms (4.4-8.6% vs 10-24.1%, respectively). All samples of irrigation water and water used to rinse vegetables were negative. Genotyping based on restriction fragment length polymorphism (RFLP) analysis using seven markers revealed the exclusive presence of genotype II.
- MeSH
- Food Safety MeSH
- Farms * MeSH
- Genotype MeSH
- Real-Time Polymerase Chain Reaction MeSH
- Food Parasitology * MeSH
- Toxoplasma * classification genetics isolation & purification MeSH
- Vegetables parasitology MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Geographicals
- Czech Republic MeSH
Field-grown strawberries, the environment of strawberry farms and fresh strawberries from marketplaces were examined for bacterial, viral, and protozoan pathogens. The presence of bacteria was determined using culture and real-time PCR (qPCR), presence of protozoa and viruses using qPCR and reverse transcription qPCR, respectively. The highest proportion of positivity was observed for Escherichia coli both in field and purchased strawberries (up to 48.6%). Finding of Cronobacter ranged from 0.6% to 9% both for field and market strawberries. The prevalence of other pathogens (Listeria monocytogenes, Giardia intestinalis, Cryptosporidium sp., and Norovirus) in strawberries was below 4.5%; HAV was not detected at all. Positivity of the environment was determined to be lower than 2.1% for all microorganisms, except for E. coli. The concentration of pathogens in most samples did not exceed 100 CFU/g using culture and 1.8 × 102 GE/g of strawberries or swabbing area 6.1 × 102 GE/mL or swabbing area of environmental samples using qPCR. All studied farms applied preventive measures such as drip irrigation, avoidance of organic fertilizers, and use of mulch foils or gloves for workers to decrease contamination of strawberries. Despite this, certain pathogens were found in fresh strawberries. Even at low concentrations, these pathogens can be a source of infection for consumers. Thus, their presence in strawberries is of particular significance as these are mostly consumed fresh and without any thermal processing. PRACTICAL APPLICATION: Nonlegislatively monitored pathogens of bacterial, viral and parasitic origin were found in strawberries. Monitoring the presence of these pathogens in ready-to-eat food is therefore meaningful and important in terms of food safety, especially in relation to pathogens with low infectious dose (for example, viruses, parasites).
- MeSH
- Food Safety MeSH
- Cryptosporidium isolation & purification MeSH
- Escherichia coli isolation & purification MeSH
- Farms MeSH
- Giardia lamblia isolation & purification MeSH
- Fragaria microbiology parasitology virology MeSH
- Food Contamination analysis MeSH
- Humans MeSH
- Listeria monocytogenes isolation & purification MeSH
- Norovirus isolation & purification MeSH
- Colony Count, Microbial MeSH
- Food Microbiology * MeSH
- Food Parasitology MeSH
- Fast Foods microbiology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Environmental matrices and food products are hypothesized to be sources of Cronobacter spp. The severity of neonatal infections, increasing number of cases in elderly and immunocompromised individuals, as well as isolation of Cronobacter spp. from clinical materials demands that more attention should be paid to Cronobacter spp. detection and occurrence of the bacteria in food products. Here, a total of 175 samples of ready-to-eat vegetables, frozen vegetables, and sprouted seeds were collected during a period of 1 year and examined for the presence of Cronobacter spp. using a cultivation method with two different sample preparations and real-time polymerase chain reaction (qPCR). In total, Cronobacter spp. were detected in 22.3% of tested samples using cultivation. In comparison, direct qPCR detected Cronobacter spp. in 37.7% of these samples (p < 0.01; Fisher's exact test) and the numbers of genome equivalents per gram reached 108 in some samples of sprouts. Cronobacter spp. were isolated from 51.4%, 37.2%, and 5.2% samples of sprouts, frozen vegetables, and cut green leaves/salads, respectively. Using qPCR, the most frequently contaminated sample types were sprouts (91.4%) and frozen vegetables (60.5%), whereas the rate of positivity for cut green leaves/salads was, in comparison, only 8.2% (p < 0.01; χ2 -test for independence). PRACTICAL APPLICATION: This study provided valuable information on the occurrence of Cronobacter spp. in ready-to-eat vegetables using cultivation and qPCR. Cronobacter spp. are emerging opportunistic pathogens that can be present in food of plant origin. Cronobacter spp. were isolated from sprouts, frozen vegetables, and cut green leaves/salads, and the numbers of genome equivalents per gram reached 108 in some samples of sprouts.
- MeSH
- Cronobacter isolation & purification MeSH
- DNA, Bacterial isolation & purification MeSH
- Food Contamination analysis MeSH
- Real-Time Polymerase Chain Reaction MeSH
- Food Microbiology MeSH
- Fast Foods microbiology MeSH
- Vegetable Products microbiology MeSH
- Vegetables microbiology MeSH
- Publication type
- Journal Article MeSH
Fresh vegetables and herbs are usually prepared and eaten raw without cooking or heating, which leads to a high risk of foodborne infection. The aim of the present study was to assess the contamination of raw vegetables, herbs, and the environment of food chains. Vegetable and herb samples originating both from the Czech Republic as well as from other countries were examined. The work was focused on the detection of commonly found, but also less frequently monitored foodborne pathogens, including viruses of the genus Norovirus (NoVs), hepatitis A virus (HAV), Listeria monocytogenes and Cronobacter spp. bacteria, and the parasites Cryptosporidium spp. and Giardia intestinalis. All samples were analyzed using individual RT-qPCR/qPCR assays; bacterial pathogens were also simultaneously detected using culture methods. The prevalence of the studied microorganisms in 623 samples ranged from 0.6% to 44.3% for individual pathogens. None of the samples were positive for the presence of HAV. Analysis of 157 environmental samples from 12 farms revealed the presence of NoVs in the environment of four farms. NoVs were detected in water samples as well as on the hands and gloves of workers. Escherichia coli was detected in all farms in the environmental samples and in eight farms in water samples. However, no sample of water exceeded the level of 100 CFU/mL for E. coli. None of the samples of water were positive for the presence of the studied parasites. Vegetables and herbs available from Czech markets and farms may pose a certain risk of foodborne disease, especially in the case of NoVs and parasites. PRACTICAL APPLICATION: This study provides valuable information on the microbiological quality of raw vegetables and herbs available from Czech markets and farms. Good hygienic practices aimed at reducing the incidence of pathogenic agents on fresh produce should not be neglected. Emphasis should be placed on the control of irrigation water, especially with respect to norovirus contamination. It is appropriate to combine culture methods and qPCR methods for the detection of bacterial agents.
- MeSH
- Cronobacter isolation & purification MeSH
- Cryptosporidium isolation & purification MeSH
- Escherichia coli isolation & purification MeSH
- Farms MeSH
- Giardia lamblia isolation & purification MeSH
- Food Contamination analysis MeSH
- Culture Media chemistry MeSH
- Food Quality MeSH
- Humans MeSH
- Listeria monocytogenes isolation & purification MeSH
- Norovirus isolation & purification MeSH
- Colony Count, Microbial MeSH
- Food Microbiology MeSH
- Food Parasitology MeSH
- Hepatitis A virus isolation & purification MeSH
- Vegetables microbiology parasitology virology MeSH
- Farmers MeSH
- Crops, Agricultural microbiology parasitology virology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Czech Republic MeSH
The consumption of fruits and vegetables is increasing worldwide because of the positive impact of these foods on human health. Ready-to-eat, raw whole, and frozen fruits and vegetables were purchased from markets and examined for the presence of nontuberculous mycobacteria (NTM) using culture, real-time PCR (qPCR), and sequencing. Using qPCR, Mycobacterium sp. at 10(0) to 10(4) ge/g (genome equivalents per gram) was found in almost all of the 178 samples; members of the M. avium complex were found only sporadically. Culture and sequencing revealed the presence of 22 viable NTM isolates in 17 samples. In addition to NTM commonly found in the environment, several rarely described isolates of viable NTM were recovered. The presence of Mycobacterium shigaense, which has been previously isolated only from human patients, was found in lettuce, the first time that this species has been found in an environmental sample. Mycobacterium parmense, Mycobacterium palustre, and Mycobacterium llatzerense, which have been previously isolated from human patients and occasionally from soil and water, were recovered from leafy green vegetables. Strawberries and cut salad mixes contained Mycobacterium algericum, Mycobacterium fallax, and Mycobacterium minnesotense. NTM are primarily nonpathogenic. However, consumption of fruits or vegetables contaminated with NTM could represent a health risk for immunocompromised people, children, and the elderly.
- MeSH
- Humans MeSH
- Nontuberculous Mycobacteria * MeSH
- Fruit MeSH
- Vegetables * MeSH
- Environment MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
