Novel clinically relevant antibiotic resistance genes associated with sewage sludge and industrial waste streams revealed by functional metagenomic screening.

A growing body of evidence indicates that anthropogenic activities can result in increased prevalence of antimicrobial resistance genes (ARGs) in bacteria in natural environments. Many environmental studies have used next-generation sequencing methods to sequence the metagenome. However, this approach is limited as it does not identify divergent uncharacterized genes or demonstrate activity. Characterization of ARGs in environmental metagenomes is important for understanding the evolution and dissemination of resistance, as there are several examples of clinically important resistance genes originating in environmental species. The current study employed a functional metagenomic approach to detect genes encoding resistance to extended spectrum ß-lactams (ESBLs) and carbapenems in sewage sludge, sludge amended soil, quaternary ammonium compound (QAC) impacted reed bed sediment and less impacted long term curated grassland soil. ESBL and carbapenemase genes were detected in sewage sludge, sludge amended soils and QAC impacted soil with varying degrees of homology to clinically important ß-lactamase genes. The flanking regions were sequenced to identify potential host background and genetic context. Novel ß-lactamase genes were found in Gram negative bacteria, with one gene adjacent to an insertion sequence ISPme1, suggesting a recent mobilization event and/ the potential for future transfer. Sewage sludge and quaternary ammonium compound (QAC) rich industrial effluent appear to disseminate and/or select for ESBL genes which were not detected in long term curated grassland soils. This work confirms the natural environment as a reservoir of novel and mobilizable resistance genes, which may pose a threat to human and animal health.

Dog skin parasite load, TLR-2, IL-10 and TNF-α expression and infectiousness.

Visceral leishmaniosis is a zoonotic disease that is transmitted by Lutzomyia longipalpis sandflies. Dogs are the main peri-urban reservoir of the disease, and progression of canine leishmaniosis is dependent on the type of immune response elaborated against the parasite. Type 1 immunity is characterized by effective cellular response, with production of pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF-α). In contrast, Type 2 immunity is predominantly humoral, associated with progression of the disease and mediated by anti-inflammatory cytokines such as interleukin 10 (IL-10). Although seemly important in the dynamics of leishmaniosis, other gene products such as toll-like receptor 2 (TRL-2) and inducible nitric oxide synthase (iNOS) exert unclear roles in the determination of the type of immune response. Given that the dog skin serves as a micro-environment for the multiplication of Leishmania spp., we investigated the parasite load and the expression of TLR-2, iNOS, IL-10 and TNF-α in the skin of 29 infected and 8 control dogs. We found that increased parasite load leads to upregulation of TLR-2, IL-10 and TNF-α, indicating that abundance of these transcripts is associated with infection. We also performed a xenodiagnosis to demonstrate that increased parasitism is a risk factor for infectiousness to sandflies.

Correlations between peripheral parasite load and common clinical and laboratory alterations in dogs with visceral leishmaniasis.

Intensity of peripheral parasite infection has an important role in the transmission of Leishmania spp. from one host to another. As parasite load quantification is still an expensive procedure to be used routinely in epidemiological surveillance, the use of surrogate predictors may be an important asset in the identification of dogs with high transmitting ability. The present study examined whether common clinical and laboratory alterations can serve as predictors of peripheral parasitism in dogs naturally infected with Leishmania spp. Thirty-seven dogs were examined in order to establish correlations between parasite load (PL) in multiple peripheral tissues and common clinical and laboratory findings in canine visceral leishmaniasis (CVL). Quantitative polymerase chain reaction was employed to determine PL in conjunctival swabs, ear skin, peripheral blood and buffy coat. Additionally, a series of hematological, biochemical and oxidative stress markers were quantified. Correlations between net peripheral infection and severity of clinical alterations and variation in laboratory parameters were assessed through a new analytical approach, namely Compressed Parasite Load Data (CPLD), which uses dimension reduction techniques from multivariate statistics to summarize PL across tissues into a single variable. The analysis revealed that elevation in PL is positively correlated with severity of clinical sings commonly observed in CVL, such as skin lesions, ophthalmic alterations, onycogriphosis, popliteal lymphadenomegaly and low body mass. Furthermore, increase in PL was found to be followed by intensification of non-regenerative anemia, neutrophilia, eosinopenia, hepatic injury and oxidative imbalance. These results suggest that routinely used clinical and laboratory exams can be predictive of intensity of peripheral parasite infection, which has an important implication in the identification of dogs with high transmitting ability.

Factors associated with changes of state of foot conformation and lameness in a flock of sheep.

The aim of this research was to investigate transitions between foot conformation, lameness and footrot in sheep. Data came from one lowland flock of approximately 700 ewes studied for 18 months. Multilevel multistate analyses of transitions between good and poor foot conformation states in ewes, and lame and non-lame states in ewes and lambs were conducted. Key results were that the longer sheep had feet in good conformation, the more likely they were to stay in this state; similarly, the longer a ewe was not lame the more likely she was not to become lame. Ewes with poor foot conformation were more likely to become lame (OR: 1.83 (1.24-2.67)) and to be >4 years (OR: 1.50 (1.09-2.05)). Ewes with footrot were less likely to move to good foot conformation (OR: 0.48 (0.31-0.75)) and were more likely to become lame (OR: 3.81 (2.60-5.59)). Ewes lame for >4 days and not treated with parenteral antibacterials had a higher risk of developing (OR: 2.00 (1-3.61)), or remaining in (OR: 0.49 (0.29-0.95)), poor foot conformation compared with ewes never lame. Treatment of ewes lame with footrot with parenteral antibacterials increased the probability of transition from a lame to a non-lame state (OR: 1.46 (1.05-2.02)) and these ewes, even if lame for >4 days, were not more likely to develop poor foot conformation. The risk of a ewe becoming lame increased when at least one of her offspring was lame (OR: 2.03 (1.42-2.92)) and when the prevalence of lameness in the group was ≥5% (OR: 1.42 (1.06-1.92)). Lambs were at increased risk of becoming lame when they were male (OR: 1.42 (1.01-2.01)), single (OR: 1.86 (1.34-2.59)) or had a lame dam or sibling (OR: 3.10 (1.81-5.32)). There were no explanatory variables associated with lambs recovering from lameness. We conclude that poor foot conformation in ewes increases the susceptibility of ewes to become lame and that this can arise from untreated footrot. Treatment of ewes lame with footrot with parenteral antibacterials leads to recovery from lameness and prevents or resolves poor foot conformation which then reduces the susceptibility to further lameness with footrot.

Microbial community responses associated with the development of oomycete plant pathogens on tomato roots in soilless growing systems.

AIMS: To determine the spread of different oomycete pathogens in hydroponic, soilless tomato growing systems and their impact on established microbial communities, as baseline studies prior to future introduction of microbial inoculants for disease suppression. METHODS AND RESULTS: The oomycete pathogens, Pythium group F, Pythium aphanidermatum and Phytophthora cryptogea, were introduced into small-scale recirculating tomato growing systems containing rockwool 6 weeks after set-up when roots were well-established. Two weeks later, half of the systems were switched over to run-to-waste. Pythium aphanidermatum spreads the fastest, Pythium group F the slowest and Ph. cryptogea was intermediate in its spread. The switch to run-to-waste had no effect on pathogen recovery. Microbial communities, monitored by dilution plating, were well-established at the first sampling, 6 weeks after set-up and although differences in community levels were found between experiments, changes during any one experiment were small, generally less than 1 log10 CFU g(-1) for bacteria. Pathogen introduction increased microbial community levels in roots but the switch to run-to-waste had no effect. Analysis of bacterial communities through amplification of a fragment of the 16S rRNA gene and DGGE profiling showed that different communities were established within each pathogen experiment and that different communities were established on roots, rockwool and in nutrient solutions. However, no significant changes in microbial profiles were found over time in any experiment. CONCLUSIONS: In these systems, the microbial communities were well-established 6 weeks after set-up and were resistant to biological and physical perturbation. SIGNIFICANCE AND IMPACT OF THE STUDY: The implication for microbial inoculation of such systems for disease suppression is that the micro-organisms would either have to be introduced very early during the set-up of the system or be able to replace an established but variable community.

Sexuality and genetic identity in the Agaricus section Arvenses.

Twelve wild collections and one commercial strain were used to characterize breeding systems and to develop molecular identities in the Arvenses section of the genus Agaricus, which includes the "horse mushroom" A. arvensis. Two morphotypes were identified based on macro- and micromorphological features. However, not all collections could be delimited by conventional taxonomic characters. Sequencing of the small subunit intergenic spacer (ITS) region (368 to 370 bp) of the rRNA genes clearly resolved the 13 collections into two clusters consistent with the identified morphotypes. Single-spore progenies and mating type testers were established and used to test intra- and interstock compatibility. The two compatibility groups identified were consistent with ITS clusters. Compatibility group I stocks readily interbred within the constraints of a unifactorial heterothallic system with a multiallelic mating type factor. Compatibility group II had a more restricted breeding pattern, and interactions were difficult to predict on the basis of mating type. Morphological data, ITS sequences, and the ability to interbreed suggest that these collections are part of a complex of interrelated species. Single-spore, homokaryotic isolates from both compatibility groups were able to fruit in compost culture, and two of the collections may represent natural homokaryotic fruiting. We conclude that species from the section Arvenses have versatile unifactorial heterothallic life cycles that permit both interbreeding and homokaryotic fruiting.

Modified release matrix prepared by compaction of spheres containing waxy material.

In this study, chlorpheniramine maleate spheres were prepared by the extruder/marumerizer. A new waxy material, Gelucire 50/02 at three levels (10%, 30% and 50%) was added and Avicel PH-101 was used as spheronizing material. The drug was incorporated into the waxy material by two methods. The first was the direct method, in which the drug (10%), wax and Avicel PH-101 were mixed together. The second was the fusion method, in which the drug was dispersed in the melted wax and the solidified mass was milled and mixed with Avicel PH-101. The data obtained indicated that simple addition of waxy material into chlorpheniramine maleate-Avicel PH-101 spheres interrupted matrix formation and increased drug release. Also in this study, a multiparticulate delivery system was prepared successfully by compaction of spheres into tablets. Tablets compacted from spheres prepared by fusion method gave less drug release than those compacted from spheres of the same composition but prepared with direct method. As the level of wax was increased in tablet formulation, drug release was decreased.