Antimicrobial Peptides (AMP) in the Cell-Free Culture Media of Xenorhabdus budapestensis and X. szentirmaii Exert Anti-Protist Activity against Eukaryotic Vertebrate Pathogens including Histomonas meleagridis and Leishmania donovani Species.

Anti-microbial peptides provide a powerful toolkit for combating multidrug resistance. Combating eukaryotic pathogens is complicated because the intracellular drug targets in the eukaryotic pathogen are frequently homologs of cellular structures of vital importance in the host organism. The entomopathogenic bacteria (EPB), symbionts of entomopathogenic-nematode species, release a series of non-ribosomal templated anti-microbial peptides. Some may be potential drug candidates. The ability of an entomopathogenic-nematode/entomopathogenic bacterium symbiotic complex to survive in a given polyxenic milieu is a coevolutionary product. This explains that those gene complexes that are responsible for the biosynthesis of different non-ribosomal templated anti-microbial protective peptides (including those that are potently capable of inactivating the protist mammalian pathogen Leishmania donovanii and the gallinaceous bird pathogen Histomonas meleagridis) are co-regulated. Our approach is based on comparative anti-microbial bioassays of the culture media of the wild-type and regulatory mutant strains. We concluded that Xenorhabdus budapestensis and X. szentirmaii are excellent sources of non-ribosomal templated anti-microbial peptides that are efficient antagonists of the mentioned pathogens. Data on selective cytotoxicity of different cell-free culture media encourage us to forecast that the recently discovered "easy-PACId" research strategy is suitable for constructing entomopathogenic-bacterium (EPB) strains producing and releasing single, harmless, non-ribosomal templated anti-microbial peptides with considerable drug, (probiotic)-candidate potential.

XENOFOOD-An Autoclaved Feed Supplement Containing Autoclavable Antimicrobial Peptides-Exerts Anticoccidial GI Activity, and Causes Bursa Enlargement, but Has No Detectable Harmful Effects in Broiler Cockerels despite In Vitro Detectable Cytotoxicity on LHM Cells.

Entomopathogenic bacteria are obligate symbionts of entomopathogenic nematode (EPN) species. These bacteria biosynthesize and release non-ribosomal-templated hybrid peptides (NR-AMPs), with strong, and large-spectral antimicrobial potential, capable of inactivating pathogens belonging to different prokaryote, and eukaryote taxa. The cell-free conditioned culture media (CFCM) of Xenorhabdus budapestensis and X. szentirmaii efficiently inactivate poultry pathogens like Clostridium, Histomonas, and Eimeria. To learn whether a bio-preparation containing antimicrobial peptides of Xenorhabdus origin with accompanying (in vitro detectable) cytotoxic effects could be considered a safely applicable preventive feed supplement, we conducted a 42-day feeding experiment on freshly hatched broiler cockerels. XENOFOOD (containing autoclaved X. budapestensis, and X. szentirmaii cultures developed on chicken food) were consumed by the birds. The XENOFOOD exerted detectable gastrointestinal (GI) activity (reducing the numbers of the colony-forming Clostridium perfringens units in the lower jejunum. No animal was lost in the experiment. Neither the body weight, growth rate, feed-conversion ratio, nor organ-weight data differed between the control (C) and treated (T) groups, indicating that the XENOFOOD diet did not result in any detectable adverse effects. We suppose that the parameters indicating a moderate enlargement of bursas of Fabricius (average weight, size, and individual bursa/spleen weight-ratios) in the XENOFOOD-fed group must be an indirect indication that the bursa-controlled humoral immune system neutralized the cytotoxic ingredients of the XENOFOOD in the blood, not allowing to reach their critical cytotoxic concentration in the sensitive tissues.

Genetic diversity and pathogenic potential of Shiga toxin-producing Escherichia coli (STEC) derived from German flour.

Shiga toxin-producing Escherichia coli (STEC) can cause severe human illness, which are frequently linked to the consumption of contaminated beef or dairy products. However, recent outbreaks associated with contaminated flour and undercooked dough in the United States and Canada, highlight the potential of plant based food as transmission routes for STEC. In Germany STEC has been isolated from flour, but no cases of illness have been linked to flour. In this study, we characterized 123 STEC strains isolated from flour and flour products collected between 2015 and 2019 across Germany. In addition to determination of serotype and Shiga toxin subtype, whole genome sequencing (WGS) was used for isolates collected in 2018 to determine phylogenetic relationships, sequence type (ST), and virulence-associated genes (VAGs). We found a high diversity of serotypes including those frequently associated with human illness and outbreaks, such as O157:H7 (stx2c/d, eae), O145:H28 (stx2a, eae), O146:H28 (stx2b), and O103:H2 (stx1a, eae). Serotypes O187:H28 (ST200, stx2g) and O154:H31 (ST1892, stx1d) were most prevalent, but are rarely linked to human cases. However, WGS analysis revealed that these strains, as well as, O156:H25 (ST300, stx1a) harbour high numbers of VAGs, including eae, nleB and est1a/sta1. Although STEC-contaminated flour products have yet not been epidemiologically linked to human clinical cases in Germany, this study revealed that flour can serve as a vector for STEC strains with a high pathogenic potential. Further investigation is needed to determine the sources of STEC contamination in flour and flour products particularly in regards to these rare serotypes.

Environment-Specific Probiotic Supernatants Modify the Metabolic Activity and Survival of Streptococcus mutans in vitro.

A range of studies showed probiotics like Streptococcus oligofermentans and Limosilactobacillus reuteri to inhibit the cariogenic activity and survival of Streptococcus mutans, possibly via the production of substances like H2O2, reuterin, ammonia and organic acids. We aimed to assess the environment-specific mechanisms underlying this inhibition. We cultured L. reuteri and S. oligofermentans in various environments; minimal medium (MM), MM containing glucose (MM+Glu), glycerol (MM+Gly), lactic acid (MM+Lac), arginine (MM+Arg) and all four substances (MM+all) in vitro. Culture supernatants were obtained and metabolite concentrations (reuterin, ammonia, H2O2, lactate) measured. S. mutans was similarly cultivated in the above six different MM variation media, with glucose being additionally added to the MM+Gly, MM+Lac, and MM+Arg group, with (test groups) and without (control groups) the addition of the supernatants of the described probiotic cultures. Lactate production by S. mutans was measured and its survival (as colony-forming-units/mL) assessed. L. reuteri environment-specifically produced reuterin, H2O2, ammonia and lactate, as did S. oligofermentans. When cultured in S. oligofermentans supernatants, lactate production by S. mutans was significantly reduced (p < 0.01), especially in MM+Lac+Glu and MM+all, with no detectable lactate production at all (controls means ± SD: 4.46 ± 0.41 mM and 6.00 ± 0.29 mM, respectively, p < 0.001). A similar reduction in lactate production was found when S. mutans was cultured in L. reuteri supernatants (p < 0.05) for all groups except MM+Lac+Glu. Survival of S. mutans cultured in S. oligofermentans supernatants in MM+Lac+Glu and MM+all was significantly reduced by 0.6-log10 and 0.5-log10, respectively. Treatment with the supernatant of L. reuteri resulted in a reduction in the viability of S. mutans in MM+Gly+Glu and MM+all by 6.1-log10 and 7.1-log10, respectively. Probiotic effects on the metabolic activity and survival of S. mutans were environment-specific through different pathways.

Membrane associated proteins of two Trichomonas gallinae clones vary with the virulence.

Oropharyngeal avian trichomonosis is mainly caused by Trichomonas gallinae, a protozoan parasite that affects the upper digestive tract of birds. Lesions of the disease are characterized by severe inflammation which may result in fatality by starvation. Two genotypes of T. gallinae were found to be widely distributed in different bird species all over the world. Differences in the host distribution and association with lesions of both genotypes have been reported. However, so far no distinct virulence factors of this parasite have been described and studies might suffer from possible co-infections of different genotypes. Therefore, in this paper, we analyzed the virulence capacity of seven clones of the parasite, established by micromanipulation, representing the two most frequent genotypes. Clones of both genotypes caused the maximum score of virulence at day 3 post-inoculation in LMH cells, although significant higher cytopathogenic score was found in ITS-OBT-Tg-1 genotype clones at days 1 and 2, as compared to clones with ITS-OBT-Tg-2. By using one representative clone of each genotype, a comparative proteomic analysis of the membrane proteins enriched fraction has been carried out by a label free approach (Data available via ProteomeXchange: PXD013115). The analysis resulted in 302 proteins of varying abundance. In the clone with the highest initial virulence, proteins related to cell adhesion, such as an immuno-dominant variable surface antigen, a GP63-like protein, an armadillo/beta-catenin-like repeat protein were found more abundant. Additionally, Ras superfamily proteins and calmodulins were more abundant, which might be related to an increased activity in the cytoskeleton re-organization. On the contrary, in the clone with the lowest initial virulence, larger numbers of the identified proteins were related to the carbohydrate metabolism. The results of the present work deliver substantial differences between both clones that could be related to feeding processes and morphological changes, similarly to the closely related pathogen Trichomonas vaginalis.

Effect of reduced nutritional supply on the metabolic activity and survival of cariogenic bacteria in vitro.

Sealed cariogenic bacteria are deprived from dietary carbohydrate, but could be provided with nutrients by pulpal fluids, with adaptive strain-specific activities being possible. We investigated survival and metabolic activity of the cariogenic bacteria Streptococcus sobrinus, Actinomyces naeslundii and Lactobacillus rhamnosus in different carbohydrate-limited media without carbon source (CLM), or containing glucose (CLM-G), albumin (CLM-A), or α1-acid glycoprotein (CLM-AGP) in vitro. Bacterial metabolite concentrations (lactate, pyruvate, oxaloacetate, citrate, acetate, formate, ethanol, acetoin) after 20 and 4 hours incubation, and bacterial numbers (CFU) after 24 hours incubation were analyzed using multivariate-analysis-of-variance (MANOVA). The medium (p = 0.02/MANOVA), strain and incubation-time (both p < 0.001) had significant impact on metabolite concentrations. Bacteria secreted mainly lactate (80.3 µg/106 bacteria S. sobrinus) and acetate (54.5 µg/106 bacteria A. naeslundii). Nearly all metabolites were produced in higher concentrations in S. sobrinus than in A. naeslundii or L. rhamnosus (p < 0.05/HSD). Metabolite concentration was significantly higher in CLM-G than in other media for most metabolites (p < 0.05). L. rhamnosus showed significantly lower survival than S. sobrinus and A. naeslundii (p < 0.05/HSD) regardless of the media, while S. sobrinus and A. naeslundii showed medium-specific survival. Survival of carbon starvation was strain- and medium-specific. Sustained organic acid production was found for all strains and media.

Bacterial reduction in sealed caries lesions is strain- and material-specific.

Sealing can arrest caries lesions. We aimed to evaluate if sealing effects and kinetics are bacterial-strain and sealing-material specific. Human dentin discs were mounted in a dual-chamber device. Caries lesions were induced chemically and contaminated with either Lactobacillus rhamnosus (LR) or Streptococcus sobrinus (SS). For (1) kinetics assessment, the initial bacterial load and the sealing period were varied, and lesions sealed using a self-etch adhesive and composite. For (2) comparing materials, six sealing protocols (#1-#6) were evaluated: 1# Self-etch adhesive plus composite placed without a liner, or #2 calcium hydroxide, or #3 mineral trioxide aggregate, or #4 Biodentine liners; #5 antibacterial adhesive plus composite; #6 glass ionomer cement. Pulpal fluid flow was simulated during sealing. The outcome was the number of surviving bacteria (CFU) per g dentin. For LR, bacterial survival increased significantly with increasing initial bacterial load and decreased with longer sealing periods. The relative reduction followed a first-order kinetics. More LR survived under calcium hydroxide or MTA than other materials (p < 0.001). For SS, nearly no bacteria survived sealing regardless of sealing period, initial bacterial load or sealing material. In conclusion, sealing effects and kinetics were strain- and material-specific.

Long-term in vitro cultivation of Histomonas meleagridis coincides with the dominance of a very distinct phenotype of the parasite exhibiting increased tenacity and improved cell yields.

The majority of research on Histomonas meleagridis was performed in the first half of the last century, especially those on morphological aspects. In the present study identical monoxenic settings for cultures of the same H. meleagridis clonal strain in its virulent low passage and attenuated high passage form enabled a comparative analysis of parasite characteristics. For the first time, it could be shown that long-term in vitro cultivation led to a severe shift in cell morphology, with the occurrence of a very distinct phenotype expressing a flagellated and highly amoebic cell morphology. Furthermore, the attenuated parasites showed better growth rates and a higher tenacity when confronted with adverse conditions. During these experiments up to 100% of the parasites, both virulent and attenuated, assumed a completely rounded morphology elucidated by electron microscopy. The findings indicate that such previously reported cyst-like stages are a defence strategy of H. meleagridis, independent of the passage level in vitro and pathogenicity in vivo. In conclusion, long-term in vitro passaging of H. meleagridis led not only to an attenuation of the parasite, as previously demonstrated, but also to a shift in the parasite's phenotype regarding morphology, growth behaviour and a higher level of tenacity.

Histomonas meleagridis--new insights into an old pathogen.

The protozoan flagellate Histomonas meleagridis is the etiological agent of histomonosis, first described in 1893. It is a fastidious disease in turkeys, with pathological lesions in the caeca and liver, sometimes with high mortality. In chickens the disease is less fatal and lesions are often confined to the caeca. The disease was well controlled by applying nitroimidazoles and nitrofurans for therapy or prophylaxis. Since their introduction into the market in the middle of the previous century, research nearly ceased as outbreaks of histomonosis occurred only very rarely. With the ban of these drugs in the last two decades in North America, the European Union and elsewhere, in combination with the changes in animal husbandry, the disease re-emerged. Consequently, research programs were set up in various places focusing on different features of the parasite and the disease. For the first time studies were performed to elucidate the molecular repertoire of the parasite. In addition, research has been started to investigate the parasite's interaction with its host. New diagnostic methods and tools were developed and tested with samples obtained from field outbreaks or experimental infections. Some of these studies aimed to clarify the introduction of the protozoan parasite into a flock and the transmission between birds. Finally, a strong focus was placed on research concentrated on the development of new treatment and prophylactic strategies, urgently needed to combat the disease. This review aims to summarize recent research activities and place them into context with older literature.

Multi-locus sequence typing confirms the clonality of Trichomonas gallinae isolates circulating in European finches.

In recent years, Trichomonas gallinae emerged as the causative agent of an infectious disease of passerine birds in Europe leading to epidemic mortality of especially greenfinches Chloris chloris and chaffinches Fringilla coelebs. After the appearance of finch trichomonosis in the UK and Fennoscandia, the disease spread to Central Europe. Finch trichomonosis first reached Austria and Slovenia in 2012. In the present study the genetic heterogeneity of T. gallinae isolates from incidents in Austria and Slovenia were investigated and compared with British isolates. For this purpose comparative sequence analyses of the four genomic loci ITS1-5.8S-ITS2, 18S rRNA, rpb1 and Fe-hydrogenase were performed. The results corroborate that one clonal T. gallinae strain caused the emerging infectious disease within passerine birds and that the disease is continuing to spread in Europe. The same clonal strain was also found in a columbid bird from Austria. Additionally, the present study demonstrates clearly the importance of multi-locus sequence typing for discrimination of circulating T. gallinae strains.

Escherichia coli strongly supports the growth of Histomonas meleagridis, in a monoxenic culture, without influence on its pathogenicity.

Based on clonal cultures of Histomonas meleagridis, monoxenic cultures have, to our knowledge for the first time, been established in a liquid medium. The faecal flora was exchanged for defined bacterial strains by selective destruction of the initial bacteria with a variety of antibiotics, keeping the flagellate alive. The growth of the protozoan parasite was found to depend on the bacteria, especially on their energy metabolism. Escherichia coli was found to strongly support the growth of the parasite, whereas Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa were less efficient. Confocal laser microscopy showed that H. meleagridis could take up green fluorescent protein-tagged E. coli DH5α, suggesting that bacteria serve as a food supply for the protozoa. By exchanging the bacterial flora for E. coli strain DH5α in H. meleagridis cultures that underwent continuous in vitro passages, it was possible to show that the in vivo attenuation process was independent of the bacteria. Furthermore, the gut flora in infected turkeys had no negative effect on the protozoan's virulence. Consequently, attenuation depends not on the bacteria in the culture but on the in vitro passages. Finally, the experiments provided evidence that the infection of turkeys with H. meleagridis enabled infection of the liver with E. coli.

Homologous gene clusters of nicotine catabolism, including a new ω-amidase for α-ketoglutaramate, in species of three genera of Gram-positive bacteria.

Gram-positive soil bacteria Arthrobacter nicotinovorans, Nocardioides sp. JS614 and Rhodococcus opacus were shown to contain similarly organized clusters of homologous genes for nicotine catabolism. An uncharacterized gene of a predicted nitrilase within these gene clusters was cloned from A. nicotinovorans and biochemical data unexpectedly showed that the protein exhibited ω-amidase activity toward α-ketoglutaramate. Structural modelling of the protein suggested the presence of the catalytic triad Cys-Glu-Lys, characteristic of this class of enzymes, and supported α-ketoglutaramate as substrate. A-ketoglutaramate could be generated by hydrolytic cleavage of the C-N bond of the trihydroxypyridine ring produced by nicotine catabolism in these bacteria. This ω-amidase, together with glutamate dehydrogenase, may form a physiologically relevant enzyme couple, leading to transformation of metabolically inert α-ketoglutaramate derived from trihydroxypyridine into glutamate, a central compound of nitrogen metabolism.

Uptake of L-nicotine and of 6-hydroxy-L-nicotine by Arthrobacter nicotinovorans and by Escherichia coli is mediated by facilitated diffusion and not by passive diffusion or active transport.

The mechanism by which l-nicotine is taken up by bacteria that are able to grow on it is unknown. Nicotine degradation by Arthrobacter nicotinovorans, a Gram-positive soil bacterium, is linked to the presence of the catabolic megaplasmid pAO1. l-[(14)C]Nicotine uptake assays with A. nicotinovorans showed transport of nicotine across the cell membrane to be energy-independent and saturable with a K(m) of 6.2+/-0.1 microM and a V(max) of 0.70+/-0.08 micromol min(-1) (mg protein)(-1). This is in accord with a mechanism of facilitated diffusion, driven by the nicotine concentration gradient. Nicotine uptake was coupled to its intracellular degradation, and an A. nicotinovorans strain unable to degrade nicotine (pAO1(-)) showed no nicotine import. However, when the nicotine dehydrogenase genes were expressed in this strain, import of l-[(14)C]nicotine took place. A. nicotinovorans pAO1(-) and Escherichia coli were also unable to import 6-hydroxy-l-nicotine, but expression of the 6-hydroxy-l-nicotine oxidase gene allowed both bacteria to take up this compound. l-Nicotine uptake was inhibited by d-nicotine, 6-hydroxy-l-nicotine and 2-amino-l-nicotine, which may indicate transport of these nicotine derivatives by a common permease. Attempts to correlate nicotine uptake with pAO1 genes possessing similarity to amino acid transporters failed. In contrast to the situation at the blood-brain barrier, nicotine transport across the cell membrane by these bacteria was not by passive diffusion or active transport but by facilitated diffusion.

Two closely related pathways of nicotine catabolism in Arthrobacter nicotinovorans and Nocardioides sp. strain JS614.

A virtually identical nicotine catabolic pathway including the heterotrimeric molybdenum enzyme nicotine and 6-hydroxy-pseudo-oxynicotine dehydrogenase, 6-hydroxy-L: -nicotine oxidase, 2,6-dihydroxy-pseudo-oxynicotine hydrolase, and 2,6-dihydroxypyridine hydroxylase have been identified in A. nicotinovorans and Nocardioides sp. JS614. Enzymes catalyzing the same reactions and similar protein antigens were detected in the extracts of the two microorganisms. Nicotine blue and methylamine, two end products of nicotine catabolism were detected in the growth medium of both bacterial species. Nicotine catabolic genes are clustered on pAO1 in A. nicotinovorans, but located chromosomally in Nocardioides sp. JS614.

A two-component small multidrug resistance pump functions as a metabolic valve during nicotine catabolism by Arthrobacter nicotinovorans.

The genes nepAB of a small multidrug resistance (SMR) pump were identified as part of the pAO1-encoded nicotine regulon responsible for nicotine catabolism in Arthrobacter nicotinovorans. When [(14)C]nicotine was added to the growth medium the bacteria exported the (14)C-labelled end product of nicotine catabolism, methylamine. In the presence of the proton-motive force inhibitors 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenylhydrazone (CCCP) or the proton ionophore nigericin, export of methylamine was inhibited and radioactivity accumulated inside the bacteria. Efflux of [(14)C]nicotine-derived radioactivity from bacteria was also inhibited in a pmfR : cmx strain with downregulated nepAB expression. Because of low amine oxidase levels in the pmfR : cmx strain, gamma-N-methylaminobutyrate, the methylamine precursor, accumulated. Complementation of this strain with the nepAB genes, carried on a plasmid, restored the efflux of nicotine breakdown products. Both NepA and NepB were required for full export activity, indicating that they form a two-component efflux pump. NepAB may function as a metabolic valve by exporting methylamine, the end product of nicotine catabolism, and, in conditions under which it accumulates, the intermediate gamma-N-methylaminobutyrate.

Final steps in the catabolism of nicotine.

New enzymes of nicotine catabolism instrumental in the detoxification of the tobacco alkaloid by Arthrobacter nicotinovorans pAO1 have been identified and characterized. Nicotine breakdown leads to the formation of nicotine blue from the hydroxylated pyridine ring and of gamma-N-methylaminobutyrate (CH(3)-4-aminobutyrate) from the pyrrolidine ring of the molecule. Surprisingly, two alternative pathways for the final steps in the catabolism of CH(3)-4-aminobutyrate could be identified. CH(3)-4-aminobutyrate may be demethylated to gamma-N-aminobutyrate by the recently identified gamma-N-methylaminobutyrate oxidase. In an alternative pathway, an amine oxidase with noncovalently bound FAD and of novel substrate specificity removed methylamine from CH(3)-4-aminobutyrate with the formation of succinic semialdehyde. Succinic semialdehyde was converted to succinate by a NADP(+)-dependent succinic semialdehyde dehydrogenase. Succinate may enter the citric acid cycle completing the catabolism of the pyrrolidine moiety of nicotine. Expression of the genes of these enzymes was dependent on the presence of nicotine in the growth medium. Thus, two enzymes of the nicotine regulon, gamma-N-methylaminobutyrate oxidase and amine oxidase share the same substrate. The K(m) of 2.5 mM and k(cat) of 1230 s(-1) for amine oxidase vs. K(m) of 140 microM and k(cat) of 800 s(-1) for gamma-N-methylaminobutyrate oxidase, determined in vitro with the purified recombinant enzymes, may suggest that demethylation predominates over deamination of CH(3)-4-aminobutyrate. However, bacteria grown on [(14)C]nicotine secreted [(14)C]methylamine into the medium, indicating that the pathway to succinate is active in vivo.