Co-occurrence of mycotoxins and other fungal metabolites in total mixed rations of cows from dairy farms in Punjab, Pakistan.

After India and the USA, Pakistan is the third country leading in global dairy production, a sector of very high socioeconomic relevance in Asia. Mycotoxins can affect animal health, reproduction and productivity. This study analysed a broad range of co-occurring mycotoxins and fungal secondary metabolites derived from Alternaria, Aspergillus, Fusarium, Penicillium and other fungal species. To complete this, a validated multi-metabolite liquid chromatography/electrospray ionization-tandem mass spectrometric (LC/ESI-MS/MS) method was employed, detecting 96 of > 500 tested secondary fungal metabolites. This first preliminary study demonstrated that total mixed rations (TMRs) (n = 30) from big commercial dairy cattle farms (> 200 lactating cows) in Punjab, Pakistan, presented ubiquitous contamination with mixtures of mycotoxins. The mean of mycotoxins per sample was 14, ranging from 11 to 20 mycotoxins among all TMR samples. Metabolites derived from other fungi and Fusarium spp. showed the highest levels, frequency and diversity among the detected fungal compounds. Among the most prevalent mycotoxins were Fusarium toxins like fumonisins B1 (FB1) (93%), B2 (FB2) (100%) and B3 (FB3) (77%) and others. Aflatoxin B1 (AFB1) was evidenced in 40% of the samples, and 7% exceeded the EU maximum limit for feeding dairy cattle (5 µg/kg at 88% dry matter). No other mycotoxin exceeds the EU guidance values (GVs). Additionally, we found that dietary ingredients like corn grain, soybean meal and canola meal were related to increased contamination of some mycotoxins (like FB1, FB2 and FB3) in TMR from the province of Punjab, Pakistan. Among typical forage sources, the content of maize silage was ubiquitous. Individually, the detected mycotoxins represented relatively low levels. However, under a realistic scenario, long-term exposure to multiple mycotoxins and other fungal secondary metabolites can exert unpredictable effects on animal health, reproduction and productivity. Except for ergot alkaloids (73%), all the groups of metabolites (i.e. derived from Alternaria spp., Aspergillus spp., Fusarium spp., Penicillium spp. and other fungi) occurred in 100% of the TMR samples. At individual levels, no other mycotoxins than AFB1 represented a considerable risk; however, the high levels of co-occurrence with several mycotoxins/metabolites suggest that long-term exposure should be considered because of their potential toxicological interactions (additive or synergistic effects).

Isoflavones in Animals: Metabolism and Effects in Livestock and Occurrence in Feed.

Soybeans are a common ingredient of animal feed. They contain isoflavones, which are known to act as phytoestrogens in animals. Isoflavones were described to have beneficial effects on farm animals. However, there are also reports of negative outcomes after the consumption of isoflavones. This review summarizes the current knowledge of metabolization of isoflavones (including the influence of the microbiome, phase I and phase II metabolism), as well as the distribution of isoflavones and their metabolites in tissues. Furthermore, published studies on effects of isoflavones in livestock species (pigs, poultry, ruminants, fish) are reviewed. Moreover, published studies on occurrence of isoflavones in feed materials and co-occurrence with zearalenone are presented and are supplemented with our own survey data.

A clinical Acanthamoeba isolate harboring two distinct bacterial endosymbionts.

Acanthamoebae feed on bacteria but are also frequent hosts of bacterial symbionts. Here, we describe the stable co-occurrence of two symbionts, one affiliated to the genus Parachlamydia and the other to the candidate genus Paracaedibacter (Alphaproteobacteria), within a clinical isolate of Acanthamoeba hatchetti genotype T4. We performed fluorescence in situ hybridization (FISH) and transmission electron microscopy (TEM) to describe this symbiosis. Our study adds to other reports of simultaneous co-occurrence of two symbionts within one Acanthamoeba cell.

Genomes of sequence type 121 Listeria monocytogenes strains harbor highly conserved plasmids and prophages.

The food-borne pathogen Listeria (L.) monocytogenes is often found in food production environments. Thus, controlling the occurrence of L. monocytogenes in food production is a great challenge for food safety. Among a great diversity of L. monocytogenes strains from food production, particularly strains belonging to sequence type (ST)121 are prevalent. The molecular reasons for the abundance of ST121 strains are however currently unknown. We therefore determined the genome sequences of three L. monocytogenes ST121 strains: 6179 and 4423, which persisted for up to 8 years in food production plants in Ireland and Austria, and of the strain 3253 and compared them with available L. monocytogenes ST121 genomes. Our results show that the ST121 genomes are highly similar to each other and show a tremendously high degree of conservation among some of their prophages and particularly among their plasmids. This remarkably high level of conservation among prophages and plasmids suggests that strong selective pressure is acting on them. We thus hypothesize that plasmids and prophages are providing important adaptations for survival in food production environments. In addition, the ST121 genomes share common adaptations which might be related to their persistence in food production environments such as the presence of Tn6188, a transposon responsible for increased tolerance against quaternary ammonium compounds, a yet undescribed insertion harboring recombination hotspot (RHS) repeat proteins, which are most likely involved in competition against other bacteria, and presence of homologs of the L. innocua genes lin0464 and lin0465.

The Listeria monocytogenes transposon Tn6188 provides increased tolerance to various quaternary ammonium compounds and ethidium bromide.

Tolerance of the foodborne pathogen Listeria monocytogenes to sublethal concentrations of disinfectants has been frequently reported. Particularly, quaternary ammonium compounds (QACs) such as benzalkonium chloride (BC) are often used in disinfectants and also as antiseptics in food industry and hospitals. Recently, we described Tn6188, a novel transposon in L. monocytogenes harbouring the transporter QacH, a molecular mechanism leading to increased tolerance to BC. In this study, we investigated the presence of Tn6188 within the genus Listeria spp. Our screening indicates that the distribution of Tn6188 may be limited to L. monocytogenes. We confirm that QacH is responsible for the observed increase in tolerance by complementation of a qacH deletion mutant and introducing qacH in a Tn6188 negative strain. We investigated the transporter's substrate spectrum by determining minimal inhibitory concentrations (MICs) and showed that QacH also confers higher tolerance towards other QACs and ethidium bromide (EtBr). This result was supported by increased expression of qacH in the presence of the various substrates as determined by quantitative reverse transcriptase PCR (qRT-PCR). In addition, we detected expression of a Tn6188 transposase gene and circular forms of Tn6188, suggesting activity and possible transfer of this transposon.

Genome sequencing of Listeria monocytogenes "Quargel" listeriosis outbreak strains reveals two different strains with distinct in vitro virulence potential.

A large listeriosis outbreak occurred in Austria, Germany and the Czech Republic in 2009 and 2010. The outbreak was traced back to a traditional Austrian curd cheese called "Quargel" which was contaminated with two distinct serovar 1/2a Listeria monocytogenes strains (QOC1 and QOC2). In this study we sequenced and analysed the genomes of both outbreak strains in order to investigate the extent of genetic diversity between the two strains belonging to MLST sequence types 398 (QOC2) and 403 (QOC1). Both genomes are highly similar, but also display distinct properties: The QOC1 genome is approximately 74 kbp larger than the QOC2 genome. In addition, the strains harbour 93 (QOC1) and 45 (QOC2) genes encoding strain-specific proteins. A 21 kbp region showing highest similarity to plasmid pLMIV encoding three putative internalins is integrated in the QOC1 genome. In contrast to QOC1, strain QOC2 harbours a vip homologue, which encodes a LPXTG surface protein involved in cell invasion. In accordance, in vitro virulence assays revealed distinct differences in invasion efficiency and intracellular proliferation within different cell types. The higher virulence potential of QOC1 in non-phagocytic cells may be explained by the presence of additional internalins in the pLMIV-like region, whereas the higher invasion capability of QOC2 into phagocytic cells may be due to the presence of a vip homologue. In addition, both strains show differences in stress-related gene content. Strain QOC1 encodes a so-called stress survival islet 1, whereas strain QOC2 harbours a homologue of the uncharacterized LMOf2365_0481 gene. Consistently, QOC1 shows higher resistance to acidic, alkaline and gastric stress. In conclusion, our results show that strain QOC1 and QOC2 are distinct and did not recently evolve from a common ancestor.

Tn6188 - a novel transposon in Listeria monocytogenes responsible for tolerance to benzalkonium chloride.

Controlling the food-borne pathogen Listeria (L.) monocytogenes is of great importance from a food safety perspective, and thus for human health. The consequences of failures in this regard have been exemplified by recent large listeriosis outbreaks in the USA and Europe. It is thus particularly notable that tolerance to quaternary ammonium compounds such as benzalkonium chloride (BC) has been observed in many L. monocytogenes strains. However, the molecular determinants and mechanisms of BC tolerance of L. monocytogenes are still largely unknown. Here we describe Tn6188, a novel transposon in L. monocytogenes conferring tolerance to BC. Tn6188 is related to Tn554 from Staphylococcus (S.) aureus and other Tn554-like transposons such as Tn558, Tn559 and Tn5406 found in various Firmicutes. Tn6188 comprises 5117 bp, is integrated chromosomally within the radC gene and consists of three transposase genes (tnpABC) as well as genes encoding a putative transcriptional regulator and QacH, a small multidrug resistance protein family (SMR) transporter putatively associated with export of BC that shows high amino acid identity to Smr/QacC from S. aureus and to EmrE from Escherichia coli. We screened 91 L. monocytogenes strains for the presence of Tn6188 by PCR and found Tn6188 in 10 of the analyzed strains. These isolates were from food and food processing environments and predominantly from serovar 1/2a. L. monocytogenes strains harboring Tn6188 had significantly higher BC minimum inhibitory concentrations (MICs) (28.5 ± 4.7 mg/l) than strains without Tn6188 (14 ± 3.2 mg/l). Using quantitative reverse transcriptase PCR we could show a significant increase in qacH expression in the presence of BC. QacH deletion mutants were generated in two L. monocytogenes strains and growth analysis revealed that ΔqacH strains had lower BC MICs than wildtype strains. In conclusion, our results provide evidence that Tn6188 is responsible for BC tolerance in various L. monocytogenes strains.

Comparative genomics suggests an independent origin of cytoplasmic incompatibility in Cardinium hertigii.

Terrestrial arthropods are commonly infected with maternally inherited bacterial symbionts that cause cytoplasmic incompatibility (CI). In CI, the outcome of crosses between symbiont-infected males and uninfected females is reproductive failure, increasing the relative fitness of infected females and leading to spread of the symbiont in the host population. CI symbionts have profound impacts on host genetic structure and ecology and may lead to speciation and the rapid evolution of sex determination systems. Cardinium hertigii, a member of the Bacteroidetes and symbiont of the parasitic wasp Encarsia pergandiella, is the only known bacterium other than the Alphaproteobacteria Wolbachia to cause CI. Here we report the genome sequence of Cardinium hertigii cEper1. Comparison with the genomes of CI-inducing Wolbachia pipientis strains wMel, wRi, and wPip provides a unique opportunity to pinpoint shared proteins mediating host cell interaction, including some candidate proteins for CI that have not previously been investigated. The genome of Cardinium lacks all major biosynthetic pathways but harbors a complete biotin biosynthesis pathway, suggesting a potential role for Cardinium in host nutrition. Cardinium lacks known protein secretion systems but encodes a putative phage-derived secretion system distantly related to the antifeeding prophage of the entomopathogen Serratia entomophila. Lastly, while Cardinium and Wolbachia genomes show only a functional overlap of proteins, they show no evidence of laterally transferred elements that would suggest common ancestry of CI in both lineages. Instead, comparative genomics suggests an independent evolution of CI in Cardinium and Wolbachia and provides a novel context for understanding the mechanistic basis of CI.

Thaumarchaeotes abundant in refinery nitrifying sludges express amoA but are not obligate autotrophic ammonia oxidizers.

Nitrification is a core process in the global nitrogen cycle that is essential for the functioning of many ecosystems. The discovery of autotrophic ammonia-oxidizing archaea (AOA) within the phylum Thaumarchaeota has changed our perception of the microbiology of nitrification, in particular since their numerical dominance over ammonia-oxidizing bacteria (AOB) in many environments has been revealed. These and other data have led to a widely held assumption that all amoA-encoding members of the Thaumarchaeota (AEA) are autotrophic nitrifiers. In this study, 52 municipal and industrial wastewater treatment plants were screened for the presence of AEA and AOB. Thaumarchaeota carrying amoA were detected in high abundance only in four industrial plants. In one plant, thaumarchaeotes closely related to soil group I.1b outnumbered AOB up to 10,000-fold, and their numbers, which can only be explained by active growth in this continuous culture system, were two to three orders of magnitude higher than could be sustained by autotrophic ammonia oxidation. Consistently, (14)CO(2) fixation could only be detected in AOB but not in AEA in actively nitrifying sludge from this plant via FISH combined with microautoradiography. Furthermore, in situ transcription of archaeal amoA, and very weak in situ labeling of crenarchaeol after addition of (13)CO(2), was independent of the addition of ammonium. These data demonstrate that some amoA-carrying group I.1b Thaumarchaeota are not obligate chemolithoautotrophs.