Comparative Genome Analysis and Characterization of the Probiotic Properties of Lactic Acid Bacteria Isolated from the Gastrointestinal Tract of Wild Boars in the Czech Republic.

Probiotics are crucial components for maintaining a healthy gut microbiota in pigs, especially during the weaning period. Lactic acid bacteria (LAB) derived from the gastrointestinal tract of wild boars can serve as an abundant source of beneficial probiotic strains with suitable properties for use in pig husbandry. In this study, we analyzed and characterized 15 strains of Limosilactobacillus mucosae obtained from the gut contents of wild boars to assess their safety and suitability as probiotic candidates. The strains were compared using pan-genomic analysis with 49 L. mucosae strains obtained from the NCBI database. All isolated strains demonstrated their safety by showing an absence of transferrable antimicrobial resistance genes and hemolysin activity. Based on the presence of beneficial genes, five candidates with probiotic properties were selected and subjected to phenotypic profiling. These five selected isolates exhibited the ability to survive conditions mimicking passage through the host's digestive tract, such as low pH and the presence of bile salts. Furthermore, five selected strains demonstrated the presence of corresponding carbohydrate-active enzymes and the ability to utilize various carbohydrate substrates. These strains can enhance the digestibility of oligosaccharide or polysaccharide substrates found in food or feed, specifically resistant starch, α-galactosides, cellobiose, gentiobiose, and arabinoxylans. Based on the results obtained, the L. mucosae isolates tested in this study appear to be promising candidates for use as probiotics in pigs.

First Evidence of the Presence of the Causative Agent of Caseous Lymphadenitis-Corynebacterium pseudotuberculosis in Dairy Products Produced from the Milk of Small Ruminants.

This study focused on the detection and quantification of selected bacteria and on the presence of enterotoxin genes in milk and dairy products from sheep and goat farms in the Czech Republic using quantitative real-time PCR (qPCR) and multiplex PCR (PCR). The presence of Corynebacterium pseudotuberculosis (CP), Mycobacterium avium subsp. paratuberculosis (MAP), Listeria monocytogenes, Staphylococcus aureus, S. aureus enterotoxin genes and methicillin-resistant Staphylococcus aureus (MRSA) was determined in 18 milk samples, 28 fresh cheeses, 20 ripened cheeses and 14 yoghurts. The serological status of the herds in relation to CP and MAP was taken into account. The most frequently detected bacterium was S. aureus (48.8%), and subsequent PCR revealed 11 MRSA positive samples. The S. aureus enterotoxin genes seg, sei and sec were detected in two goat cheeses. Cheese samples showed a statistically higher risk of SA and MRSA occurrence. CP (8.8%) and MAP (13.8%) were detected by qPCR on two different seropositive farms. Cultivation of qPCR positive CP samples on agar plates supplemented with potassium tellurite showed the presence of viable bacterium. The results obtained confirmed the necessity of monitoring the infectious status of dairy animals and rapid diagnosis of bacterial pathogens in milk and dairy products.

Prototheca Infections and Ecology from a One Health Perspective.

Prototheca microalgae were only recognized as pathogens of both humans and animals in the 1960s; however, since then, these microbes have been drawing increasing interest in both human and veterinary medicine. The first human outbreak of protothecosis in a tertiary care chemotherapy ward in 2018 further highlighted the need to understand in more depth and detail their ecology, etiology, pathogenesis and routes of transmission between different hosts, environments and habitats from a One Health perspective. Protothecal infections have been reported in a growing number of cattle herds around the world in recent decades, and Prototheca has become an important bovine mastitis pathogen in certain countries and regions. The survival of Prototheca in the environment and its ability to spread in the herd pose a serious challenge to the management of infected dairy farms. Prevention of the disease is particularly important, as there is no effective and reliable treatment for it and the chances of self-healing are minimal. Therefore, the development of more effective drugs is needed for the treatment of human and animal protothecosis. The prudent use of antibiotics and their replacement by alternative or preventive measures, when possible, may further contribute to the control of protothecal infections.

Antibiotic Susceptibility, Resistance Gene Determinants and Corresponding Genomic Regions in Lactobacillus amylovorus Isolates Derived from Wild Boars and Domestic Pigs.

Restrictions on the use of antibiotics in pigs lead to the continuous search for new probiotics serving as an alternative to antibiotics. One of the key parameters for probiotic bacteria selection is the absence of horizontally transmissible resistance genes. The aim of our study was to determine antibiotic susceptibility profiles in 28 Lactobacillus amylovorus isolates derived from the digestive tract of wild boars and farm pigs by means of the broth microdilution method and whole genome sequencing (WGS). We revealed genetic resistance determinants and examined sequences flanking resistance genes in these strains. Our findings indicate that L. amylovorus strains from domestic pigs are predominantly resistant to tetracycline, erythromycin and ampicillin. WGS analysis of horizontally transmissible genes revealed only three genetic determinants (tetW, ermB and aadE) of which all tetW and ermB genes were present only in strains derived from domestic pigs. Sequence analysis of coding sequences (CDS) in the neighborhood of the tetW gene revealed the presence of site-specific recombinase (xerC/D), site-specific DNA recombinase (spoIVCA) or DNA-binding transcriptional regulator (xre), usually directly downstream of the tetW gene. In the case of ermB, CDS for omega transcriptional repressor or mobilization protein were detected upstream of the ermB gene.

Raw bovine milk as a reservoir of yeast with virulence factors and decreased susceptibility to antifungal agents.

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.

A novel TaqMan qPCR assay for rapid detection and quantification of pro-inflammatory microalgae Prototheca spp. in milk samples.

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.

Toxoplasma gondii in vegetables from fields and farm storage facilities in the Czech Republic.

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.

Tracing of Selected Viral, Bacterial, and Parasitic Agents on Vegetables and Herbs Originating from Farms and Markets.

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.

Occurrence of Cronobacter Spp. in Ready-to-Eat Vegetable Products, Frozen Vegetables, and Sprouts Examined Using Cultivation and Real-Time PCR Methods.

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.

Foodborne Bacterial, Viral, and Protozoan Pathogens in Field and Market Strawberries and Environment of Strawberry Farms.

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).

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.

Escherichia coli strains of phylogenetic group B2 and D and bacteriocin production are associated with advanced colorectal neoplasia.

BACKGROUND: Colorectal cancer (CRC) is the 3rd most common cancer worldwide and the Czech Republic has the 6th highest incidence of CRC worldwide. Large intestinal microbiota play in its etiopathogenesis important role. Bacteriocins are proteins, produced by bacteria from the Enterobacteriaceae family. The aim of our prospective study was to assess the colonization of large intestinal mucosa by Escherichia coli strains and to investigate their bacteriocin production. METHODS: A total of 30 consecutive patients with colorectal adenoma, CRA (17 men, 13 women, aged 39-79, mean age 63 ± 9), 30 patients with CRC (23 men, 7 women, aged 38-86, mean age 67 ± 11) and 20 healthy controls (9 men, 11 women, age 23-84, mean age 55 ± 15) were enrolled into prospective study. Mucosal biopsies were taken in the caecum, transverse colon and rectum during pancolonoscopy. Microbiological culture, isolation and identification of bacteria followed. Bacteriocin production was assessed by growth inhibition of indicator strains E. coli K12-Row, E. coli C6 (phi), and Shigella sonnei 17. Identification of bacteriocin-encoding determinants and E. coli phylogroups was performed using PCR methods. RESULTS: A total of 622 strains were isolated and further investigated. A significantly higher frequency of simultaneous production of colicins and microcins was revealed in the group of patients with CRC, when compared to patients with CRA, p = 0.031. A significantly higher frequency of E. coli phylogroup D was found in patients with CRC, when compared to controls, p = 0.044. A significantly higher prevalence of bacteriocinogeny was confirmed in patients with advanced adenoma when compared to patients with non-advanced adenoma, p = 0.010. Increasing bacteriocinogeny was associated with an increasing stage of CRC (assessed according to TNM classification). Either E. coli phylogroup B2 or E. coli phylogroup D were isolated in biopsies of patients with right-sided CRC. A statistically higher incidence of E. coli phylogroup B2 was found in patients with right-sided CRC when compared to patients with left-sided CRC, p = 0.028. CONCLUSIONS: Large intestinal mucosa of patients with more advanced colorectal neoplasia is colonized with more virulent strains of E. coli and higher production of bacteriocins is observed in these patients when compared to those with less advanced colorectal neoplasia.

High incidence of Mycobacterium avium subspecies hominissuis infection in a zoo population of bongo antelopes (Tragelaphus eurycerus).

Mycobacterium avium subsp. hominissuis (Mah) infection was diagnosed in 5 captive bongo antelopes (Tragelaphus eurycerus) originating from a collection in a zoological garden. The animals suffered from emaciation. Postmortem examination revealed nodular lesions in the lungs of all 5 examined animals. Acid-fast bacilli were observed in the lungs of 4 animals. Culture and polymerase chain reaction identification based on IS901 negativity and IS1245 positivity confirmed Mah infection in the lungs of all 5 antelopes. In 3 animals, Mah was also isolated from other organs (liver, spleen, and kidney). Molecular analysis of these isolates using IS1245 restriction fragment length polymorphism and/or mycobacterial interspersed repetitive units-variable number tandem repeat revealed that the studied antelopes were infected by 1 identical genotype. Furthermore, in 2 antelopes, other genotypes were also detected. This shows the possibility of either genetic modifications occurring during infection or polyclonal infection. Culture examination of environmental samples from the enclosures holding the bongos revealed Mah in mulch bark, peat, and soil. Genotyping of these environmental isolates determined several genotypes with 1 dominant genotype that was identical to the dominant genotype detected in antelopes.

Genetic IS901 RFLP diversity among Mycobacterium avium subsp. avium isolates from four pheasant flocks.

IS901 RFLP analysis of 36 Mycobacterium avium subsp. avium (MAA) isolates from 15 pheasants (Phasianus colchicus) and two goshawks (Accipiter gentilis) from four pheasant farms was performed. Using this method, six different IS901 RFLP types (E, F, G, M, Q, and V) were identified. The distribution of IS901 RFLP profiles was tightly linked to individual flocks. Matching IS901 RFLP profiles observed in the present study indicate MAA transmission between pheasants and goshawks in the same locality. In two flocks, different pheasants within a flock as well as in various organs of five individual pheasants were found to have two distinct IS901 RFLP profiles.

Mycobacterial screening of Czech red deer (Cervus elaphus) populations in overwintering sites, 2004-2006.

One isolate of Mycobacterium avium subsp. paratuberculosis was detected in 2,212 fecal samples of wild deer assembled in overwintering sites (OwS). Neither M. bovis nor M. a. subsp. avium was found. Therefore, congregating deer in OwSs does not automatically lead to the amplification of these pathogens among animals in OWSs.

Detection of Mycobacterium avium subsp. paratuberculosis in two brown bears in the central European Carpathians.

The incidence of mycobacterial infections was monitored in brown bears (Ursus arctos) in the National Park Low Tatras in the central European Carpathians in Slovakia. Tissue samples of 20 brown bears were examined microscopically and by culture for the presence of mycobacteria. Acid-fast rods were detected by Ziehl-Neelsen staining in a smear from the kidney of one brown bear, although the culture was negative for mycobacteria. Mycobacterium avium subsp. paratuberculosis, the causative agent of paratuberculosis in ruminants, was isolated from the intestinal mucosa of another two brown bears. The isolates were identified by polymerase chain reaction for the specific insertion sequence IS900. Using standardized IS900 restriction fragment length polymorphism (RFLP) analysis, the M. a. paratuberculosis isolates were classified as RFLP type B-C1, which also were detected in the infected cattle in surrounding area. This study describes the first isolation of M. a. paratuberculosis from a brown bear. Our results confirm that animal species other than ruminants can become infected with M. a. paratuberculosis and can act as potential vectors and/or reservoirs of the infection.

Identification of members of Mycobacterium avium species by Accu-Probes, serotyping, and single IS900, IS901, IS1245 and IS901-flanking region PCR with internal standards.

From Mycobacterium avium species Mycobacterium avium subsp. paratuberculosis (n=961), Mycobacterium a. avium (n=677), Mycobacterium a. silvaticum (n=5), and Mycobacterium a. hominissuis (n=1566) were examined, and from Mycobacterium tuberculosis complex M. tuberculosis (n=2), Mycobacterium bovis (n=13), M. bovis BCG (n=4), and Mycobacterium caprae (n=10) were examined. From other mycobacterial species Mycobacterium intracellulare (n=60) and atypical mycobacteria (n=256) including Mycobacterium fortuitum, Mycobacterium chelonae, Mycobacterium scrofulaceum, Mycobacterium gastri and other species of conditionally pathogenic mycobacteria were analysed. The internal standard molecules corresponding to insertion sequences IS900, IS901, IS1245, and flanking region (FR300) of IS901 were produced by PCR of alfalfa genome segment and inserted into plasmid vector. The resulting recombinant plasmid molecules were used as internal standards in coamplification with a total of 4729 mycobacterial collection strains and field isolates between 1996 and 2003. The size differences between amplicons obtained from IS900 (258 bp), IS901 (1108 bp), IS1245 (427 bp), and FR300 (300 bp) and from corresponding internal standard molecules ISIS900 (591 bp), ISIS901 (1 336 bp), ISIS1245 (583 bp), and IS901 flanking region of 300 bp ISFR300 (488 bp), respectively, allowed easy discrimination. The internal amplicons were visible by naked aye on agarose gel when 10(1), 10(3), 10(2), and 10(2) molecules for ISIS900, ISIS901, ISIS1245, and ISFR300 were used in the PCR, respectively, when no bacterial DNA was added to the reaction. The system was tested to define the amount of internal standards that could be used in the PCR without affecting the amplification of the specific segment. Non-specific amplifications were observed in M. fortuitum with IS1245 PCR and mixed infections with M. a. avium and M. a. hominissuis from pigs and cattle were found. PCR results of typing were compared with serotyping and Accu-Probes analyses in selected field isolates.