Sequence Analysis of Changes in Microbial Composition in Different Milk Products During Fermentation and Storage.

The objective of this study was to analyze the changes in the microbiota of milk products during fermentation and storage. Two kinds of Yoghurt, one Kefir, and one Acidophilus milk were observed during the fermentation process and storage using 16S rDNA amplicon sequencing. Cow's, goat's, raw and pasteurized milk were also examined. The most represented organisms in all manufactured products were shown to be those of the phylum Firmicutes. In some products, Proteobacteria, Bacteroidetes and Actinobacteria were also present in high amounts.

Resistance to the tetracyclines and macrolide-lincosamide-streptogramin group of antibiotics and its genetic linkage - a review.

An excessive use of antimicrobial agents poses a risk for the selection of resistant bacteria. Of particular interest are antibiotics that have large consumption rates in both veterinary and human medicine, such as the tetracyclines and macrolide-lincosamide-streptogramin (MLS) group of antibiotics. A high load of these agents increases the risk of transmission of resistant bacteria and/or resistance determinants to humans, leading to a subsequent therapeutic failure. An increasing incidence of bacteria resistant to both tetracyclines and MLS antibiotics has been recently observed. This review summarizes the current knowledge on different tetracycline and MLS resistance genes that can be linked together on transposable elements.

Nontuberculous Mycobacteria on Ready-to-Eat, Raw and Frozen Fruits and Vegetables.

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.

Seasonal changes in microbial community composition in river water studied using 454-pyrosequencing.

The aims of this study were to determine the microbial community in five rivers in the proximity of a city in the Czech Republic using 454-pyrosequencing, as well as to assess seasonal variability over the course of 1 year and to identify the factors influencing the structure of bacterial communities. Samples from five rivers around the city of Brno were obtained during four seasons and analysed using 454 pyrosequencing of the 16S rRNA gene. The core composition of bacterial communities consisted of Actinobacteria, Bacteroidetes, Proteobacteria, Firmicutes, Fusobacteria, TM7 and others. Our approach enabled us to more closely study the correlation between the abundance of different families and environmental factors. Overall, Actinobacteria negatively correlated with phosphorus, sulphate, dissolved particle and chloride levels. In contrast, Proteobacteria positively correlated with sulphate, dissolved particle, chloride, dissolved oxygen and nitrite levels. Future work should focus on the dynamics of bacterial communities present in river water and their relation to the overall stability of the water ecosystem.

Faecal bacterial composition in dairy cows shedding Mycobacterium avium subsp. paratuberculosis in faeces in comparison with nonshedding cows.

The aim of this study was to determine possible differences in the faecal microbiota of dairy cows infected with Mycobacterium avium subsp. paratuberculosis (Johne's disease) in comparison with noninfected cows from the same herds. Faecal samples from cows in 4 herds were tested for M. avium subsp. paratuberculosis by real-time PCR, and faecal bacterial populations were analysed by 454 pyrosequencing of the 16S rRNA gene. The most notable differences between shedding and nonshedding cows were an increase in the genus Psychrobacter and a decrease in the genera Oscillospira, Ruminococcus, and Bifidobacterium in cows infected with M. avium subsp. paratuberculosis. The present study is the first to report the faecal microbial composition in dairy cows infected with M. avium subsp. paratuberculosis.

Changes in Microbial Composition of Wastewater During Treatment in a Full-Scale Plant.

The monitoring of wastewater treatment plants is important for their proper functioning as well as for re-use of water and also to avoid possible circulation of human or animal pathogens in our environment. The samples in this study originated from a full-scale wastewater treatment plant where the structure of the bacterial community was monitored using 454-pyrosequencing. The composition differed in different parts of the plant. In the effluent, bacteria belonging to phyla Proteobacteria, Actinobacteria, TM7 and Bacteroidetes were most frequently detected. The presence of Mycobacterium sp., Mycobacterium avium, Norovirus, Hepatitis A and E viruses was examined using quantitative real-time PCR. Mycobacterium sp. was detected in the effluent in quantities of up to 10(4) cells/ml. Mycobacterium avium subsp. paratuberculosis and subsp. hominissuis were detected in amounts of up to 10(3) cells/ml, and Norovirus group 1 and 2 were also detected. Our findings show the importance of monitoring and controlling the occurrence of specific pathogens in effluent, mainly because of the negative impact on human health when the water is reused.

Comparison of filtering methods, filter processing and DNA extraction kits for detection of mycobacteria in water.

INTRODUCTION AND OBJECTIVE: Mycobacteria have been isolated from almost all types of natural waters, as well as from man-made water distribution systems. Detection of mycobacteria using PCR has been described in different types of water; however, currently, there is no standardised protocol for the processing of large volumes of water. MATERIAL AND METHODS: In the present study, different filtering methods are tested and optimised for tap or river water filtration up to 10 L, as well as filter processing and DNA isolation using four commercially available kits. RESULTS: The PowerWater DNA isolation kit (MoBio, USA), together with a kit used for soil and other environmental samples (PowerSoil DNA isolation kit, MoBio), had the highest efficiency. Filtration of 10 L of water and elution of the filter in PBS with the addition of 0.05% of Tween 80 is suggested. CONCLUSIONS: The described protocol for filter elution is recommended, and the use of the PowerWater DNA isolation kit for the highest mycobacterial DNA yield from water samples. The described protocol is suitable for parallel detection of mycobacteria using cultivation.

Spread of Mycobacterium avium subsp. paratuberculosis through soil and grass on a mouflon (Ovis aries) pasture.

The aims of this study were to describe spatial contamination of the environment on a mouflon pasture, as well as to assess the contamination of grass and roots after surface contamination and in depth contamination with feces and buried tissues from animals infected with Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis). Samples of soil, roots, and aerial parts of plants were collected from different locations inside the mouflon pasture, and one control sample site was chosen outside the area where the animals are living. M. a. paratuberculosis DNA was present in all the examined sites and was more often detected in roots than in soil. DNA was detected at up to 80 cm of depth and was spatially more widespread than the initial hypothesis of M. a. paratuberculosis leaching vertically into deeper layers of soil. This study broadens our knowledge of the spread and persistence of M. a. paratuberculosis in an environment with highly infected animals.

Mycobacterium avium subsp. avium in lymph nodes and diaphragms of pigs from one infected herd in the Czech Republic.

This study was performed on 40 finished pigs from one herd naturally infected with Mycobacterium avium subsp. avium. The aim was to investigate the presence and amount of M. a. avium in samples of lymph nodes and diaphragm tissues collected during routine postmortem inspection using the triplex quantitative real time PCR (qPCR) method. We collected, in total, 107 samples: various lymph nodes affected by gross tuberculosis (TB)-like lesions from 17 pig carcasses, as well as samples of head and mesenteric lymph nodes from 23 carcasses without TB-like lesions. Samples of diaphragm tissues were collected from all carcasses. M. a. avium was detected in one or more tissue samples collected from half of the slaughtered pigs tested. Samples of diaphragm tissues of three pigs with detected TB-like lesions contained M. a. avium (10(2) to 10(3) cells per g of sample); the organism was not detected in diaphragm tissues from pigs without TB-like lesions. The qPCR method may be useful for quantification of M. a. avium in pigs for the purposes of foodborne risk assessment.

Mycobacterium avium subsp. avium found in raptors exposed to infected domestic fowl.

We report a case of a falcon breeding facility, where raptors (both diurnal and nocturnal) were raised in contact with domestic fowl (Gallus gallus f. domesticus) infected by Mycobacterium avium subsp. avium. Fecal and environmental samples from 20 raptors and four common ravens (Corvus corax) were collected. Mycobacterium a. avium DNA was detected in feces of four raptors (bald eagle [Haliaeetus leucocephalus], eagle owl [Bubo bubo], barn owl [Tyto alba], and little owl [Athene noctua]) using triplex quantitative real-time PCR. As both the flock of domestic fowl and one of the infected raptors had the same origin (zoological collection), they might have had a common source of colonization/infection. However, the detection of M. a. avium in feces of three other raptors may point at transmission of the agent between the birds in the facility. Contact of raptors with domestic fowl infected by M. a. avium may pose a risk for transmission of the infection for them; however, raptors from the falcon breeding facility seemed to be relatively resistant to the infection.

Usability of a gamma interferon release assay in the diagnosis of naturally infected pigs with Mycobacterium avium subspecies hominissuis.

In the current study, the results of an intradermal tuberculin test and a gamma interferon (IFN-γ) release assay were compared. IFN-γ release assay is based on the detection of IFN-γ production after in vitro stimulation with Mycobacterium avium subsp. avium-specific antigen for the discrimination of pigs naturally infected with M. avium subsp. hominissuis. Fifty-five clinically healthy pigs were used in the study. Three of these were proven by culture and real-time quantitative polymerase chain reaction methods to be infected with M. avium subsp. hominissuis (2 animals) and Mycobacterium xenopi (1 animal). No animals were positive by the tuberculin test. Both M. avium subsp. hominissuis-positive pigs were evaluated as positive by the IFN-γ release assay. Bacteriologically negative and M. xenopi-positive pigs were unresponsive in the IFN-γ release assay, indicating the specificity of the method. The results suggest that the IFN-γ release assay has a higher sensitivity than the tuberculin test and that the assay can be used for diagnosis of M. avium infections in live, naturally infected pigs.

Cell-mediated immune response in swine infected with Mycobacterium avium subsp. avium.

The zoonotic characteristic of Mycobacterium avium subsp. avium (MAA) represents a veterinary and economic problem in infected pigs. In this study, we analysed cell-mediated immunity six months after experimental infection by measuring interferon-γ (IFN-γ) production and by performing lymphocyte transformation tests after in vitro re-stimulation with the MAA-derived antigen. At the same time, IFN-γ-producing cells were characterised by flow cytometry. In MAA-infected animals, the production of IFN-γ increased in response to the MAA antigen in the blood, spleen and mesenteric lymph nodes. Similarly, a positive antigen-driven response was detected by the proliferation assay. In contrast, IFN-γ production and proliferation was undetectable after stimulation with the MAA antigen in uninfected control animals. These results indicate that both methods can be used for the identification of individual MAA-infected pigs. Using flow cytometry, we found that double-positive CD4(+)CD8(+) lymphocytes were the major T lymphocyte subset producing IFN-γ after in vitro re-stimulation.

Soil and plant contamination with Mycobacterium avium subsp. paratuberculosis after exposure to naturally contaminated mouflon feces.

The aim of this study was to demonstrate the persistence of Mycobacterium avium subsp. paratuberculosis (MAP) in soil and colonization of different plant parts after deliberate exposure to mouflon feces naturally contaminated with different amounts of MAP. Samples of aerial parts of plants, their roots, and the soil below the roots were collected after 15 weeks and examined using IS900 real-time quantitative PCR (qPCR) and cultivation. Although the presence of viable MAP cells was not demonstrated, almost all samples were found to be positive using qPCR. MAP IS900 was not only found in the upper green parts, but also in the roots and soil samples (from 1.00 × 10(0) to 6.43 × 10(3)). The level of soil and plant contamination was influenced mainly by moisture, clay content, and the depth from which the samples were collected, rather than by the initial concentration of MAP in the feces at the beginning of the experiment.

"Mycobacterium avium subsp. hominissuis" in neck lymph nodes of children and their environment examined by culture and triplex quantitative real-time PCR.

"Mycobacterium avium subsp. hominissuis" often causes cervical lymphadenitis in children; its prompt and accurate identification enables adequate therapy, tracing, and prevention. The aims of this study were to determine the causative agent of lymphadenitis using culture, PCR, and triplex quantitative real-time PCR (qPCR) methods with DNA directly isolated from tissue, as well as to identify possible sources of infection from the environment. We confirmed the diagnoses by detecting M. avium subsp. hominissuis using qPCR with DNA directly isolated from lymph node biopsy specimens of two patients. In order to trace the source of infection from the environment, a method of DNA isolation from soil and other environmental samples, such as dust, cobwebs, and compost, was developed. The triplex qPCR examination revealed the presence of M. avium subsp. hominissuis in a high proportion of the environmental samples (42.8% in the first patient's house and 47.6% in the second patient's house). Both patients were also exposed to M. avium subsp. avium, which was present due to the breeding of infected domestic hens. The high infectious dose of M. avium subsp. hominissuis or the increased susceptibility of humans to M. avium subsp. hominissuis compared to M. avium subsp. avium could be the reason why the children were infected with M. avium subsp. hominissuis.