A local-scale One Health genomic surveillance of Clostridioides difficile demonstrates highly related strains from humans, canines, and the environment.

Although infections caused by Clostridioides difficile have historically been attributed to hospital acquisition, growing evidence supports the role of community acquisition in C. difficile infection (CDI). Symptoms of CDI can range from mild, self-resolving diarrhoea to toxic megacolon, pseudomembranous colitis, and death. In this study, we sampled C. difficile from clinical, environmental, and canine reservoirs in Flagstaff, Arizona, USA, to understand the distribution and transmission of the pathogen in a One Health framework; Flagstaff is a medium-sized, geographically isolated city with a single hospital system, making it an ideal site to characterize genomic overlap between sequenced C. difficile isolates across reservoirs. An analysis of 562 genomes from Flagstaff isolates identified 65 sequence types (STs), with eight STs being found across all three reservoirs and another nine found across two reservoirs. A screen of toxin genes in the pathogenicity locus identified nine STs where all isolates lost the toxin genes needed for CDI manifestation (tcdB, tcdA), demonstrating the widespread distribution of non-toxigenic C. difficile (NTCD) isolates in all three reservoirs; 15 NTCD genomes were sequenced from symptomatic, clinical samples, including two from mixed infections that contained both tcdB+ and tcdB- isolates. A comparative single nucleotide polymorphism (SNP) analysis of clinically derived isolates identified 78 genomes falling within clusters separated by ≤2 SNPs, indicating that ~19 % of clinical isolates are associated with potential healthcare-associated transmission clusters; only symptomatic cases were sampled in this study, and we did not sample asymptomatic transmission. Using this same SNP threshold, we identified genomic overlap between canine and soil isolates, as well as putative transmission between environmental and human reservoirs. The core genome of isolates sequenced in this study plus a representative set of public C. difficile genomes (n=136), was 2690 coding region sequences, which constitutes ~70 % of an individual C. difficile genome; this number is significantly higher than has been published in some other studies, suggesting that genome data quality is important in understanding the minimal number of genes needed by C. difficile. This study demonstrates the close genomic overlap among isolates sampled across reservoirs, which was facilitated by maximizing the genomic search space used for comprehensive identification of potential transmission events. Understanding the distribution of toxigenic and non-toxigenic C. difficile across reservoirs has implications for surveillance sampling strategies, characterizing routes of infections, and implementing mitigation measures to limit human infection.

Identification of novel, cryptic Clostridioides species isolates from environmental samples collected from diverse geographical locations.

Clostridioides difficile is a pathogen often associated with hospital-acquired infection or antimicrobial-induced disease; however, increasing evidence indicates infections can result from community or environmental sources. Most genomic sequencing of C. difficile has focused on clinical strains, although evidence is growing that C. difficile spores are widespread in soil and water in the environment. In this study, we sequenced 38 genomes collected from soil and water isolates in Flagstaff (AZ, USA) and Slovenia in an effort targeted towards environmental surveillance of C. difficile. At the average nucleotide identity (ANI) level, the genomes were divergent to C. difficile at a threshold consistent with different species. A phylogenetic analysis of these divergent genomes together with Clostridioides genomes available in public repositories confirmed the presence of three previously described, cryptic Clostridioides species and added two additional clades. One of the cryptic species (C-III) was almost entirely composed of Arizona and Slovenia genomes, and contained distinct sub-groups from each region (evidenced by SNP and gene-content differences). A comparative genomics analysis identified multiple unique coding sequences per clade, which can serve as markers for subsequent environmental surveys of these cryptic species. Homologues to the C. difficile toxin genes, tcdA and tcdB, were found in cryptic species genomes, although they were not part of the typical pathogenicity locus observed in C. difficile, and in silico PCR suggested that some would not amplify with widely used PCR diagnostic tests. We also identified gene homologues in the binary toxin cluster, including some present on phage and, for what is believed to be the first time, on a plasmid. All isolates were obtained from environmental samples, so the function and disease potential of these toxin homologues is currently unknown. Enzymatic profiles of a subset of cryptic isolates (n=5) demonstrated differences, suggesting that these isolates contain substantial metabolic diversity. Antimicrobial resistance (AMR) was observed across a subset of isolates (n=4), suggesting that AMR mechanisms are intrinsic to the genus, perhaps originating from a shared environmental origin. This study greatly expands our understanding of the genomic diversity of Clostridioides. These results have implications for C. difficile One Health research, for more sensitive C. difficile diagnostics, as well as for understanding the evolutionary history of C. difficile and the development of pathogenesis.

Tall Pinus luzmariae trees with genes from P. herrerae.

CONTEXT: Pinus herrerae and P. luzmariae are endemic to western Mexico, where they cover an area of more than 1 million hectares. Pinus herrerae is also cultivated in field trials in South Africa and South America, because of its considerable economic importance as a source of timber and resin. Seed quality, afforestation success and desirable traits may all be influenced by the presence of hybrid trees in seed stands. AIMS: We aimed to determine the degree of hybridization between P. herrerae and P. luzmariae in seed stands of each species located in the Sierra Madre Occidental, Durango, Mexico. METHODS: AFLP molecular markers from samples of 171 trees across five populations were analyzed with STRUCTURE and NewHybrids software to determine the degree of introgressive hybridization. The accuracy of STRUCTURE and NewHybrids in detecting hybrids was quantified using the software Hybridlab 1.0. Morphological analysis of 131 samples from two populations of P. herrerae and two populations of P. luzmariae was also conducted by Random Forest classification. The data were compared by Principal Coordinate Analysis (PCoA) in GenAlex 6.501. RESULTS: Hybridization between Pinus herrerae and P. luzmariae was observed in all seed stands under study and resulted in enhancement of desirable silvicultural traits in the latter species. In P. luzmariae, only about 16% molecularly detected hybrids correspond to those identified on a morphological basis. However, the morphology of P. herrerae is not consistent with the molecularly identified hybrids from one population and is only consistent with 3.3 of those from the other population. CONCLUSIONS: This is the first report of hybrid vigour (heterosis) in Mexican pines. Information about hybridization and introgression is essential for developing effective future breeding programs, successful establishment of plantations and management of natural forest stands. Understanding how natural hybridization may influence the evolution and adaptation of pines to climate change is a cornerstone to sustainable forest management including adaptive silviculture.

Discrimination of Picea chihuahuana Martinez populations on the basis of climatic, edaphic, dendrometric, genetic and population traits.

BACKGROUND: Picea chihuahuana, which is endemic to Mexico, is currently listed as "Endangered" on the Red List. Chihuahua spruce is only found in the Sierra Madre Occidental (SMO), Mexico. About 42,600 individuals are distributed in forty populations. These populations are fragmented and can be classified into three geographically distinct clusters in the SMO. The total area covered by P. chihuahuana populations is less than 300 ha. A recent study suggested assisted migration as an alternative to the ex situ conservation of P. chihuahuana, taking into consideration the genetic structure and diversity of the populations and the predictions regarding the future climate of the habitat. However, detailed background information is required to enable development of plans for protecting and conserving species and for successful assisted migration. Thus, it is important to identify differences between populations in relation to environmental conditions. The genetic diversity of populations, which affect vigor, evolution and adaptability of the species, must also be considered. In this study, we examined 14 populations of P. chihuahuana, with the overall aim of discriminating the populations and form clusters of this species. METHODS: Each population was represented by one 50 × 50 m plot established in the center of its respective location. Climate, soil, dasometric, density variables and genetic and species diversities were assessed in these plots for further analyses. The putatively neutral and adaptive AFLP markers were used to calculate genetic diversity. Affinity Propagation (AP) clustering technique and k-means clustering algorithm were used to classify the populations in the optimal number of clusters. Later stepwise binomial logistic regression was applied to test for significant differences in variables of the southern and northern P. chihuahuana populations. Spearman's correlation test was used to analyze the relationships among all variables studied. RESULTS: The binomial logistic regression analysis revealed that seven climate variables, the geographical longitude and sand proportion in the soil separated the southern from northern populations. The northern populations grow in more arid and continental conditions and on soils with lower sand proportion. The mean genetic diversity using all AFLP studied of P. chihuahuana was significantly correlated with the mean temperature in the warmest month, where warmer temperatures are associated to larger genetic diversity. Genetic diversity of P. chihuahuana calculated with putatively adaptive AFLP was not statistically significantly correlated with any environmental factor. DISCUSSION: Future reforestation programs should take into account that at least two different groups (the northern and southern cluster) of P. chihuahuana exist, as local adaptation takes place because of different environmental conditions.

Evaluación de fenoles y limonoides en hojas de Cedrela odorata (Meliaceae) de una plantación experimental establecida en Tezonapa Veracruz, México / Evaluation of phenols and limonoids in leaves of Cedrela odorata (Meliaceae) from an experimental plantation established in Tezonapa Veracruz, Mexico

Cedrela odorata (Meliaceae) is a native timber tree to Tropical America, known for its high-quality wood, unfortunately, plantations of this species are severely attacked by Hypsipyla grandella. The attraction or repellency of this pest is related to secondary metabolites such as phenols and limonoids (triterpenes); therefore, it is important to study these compounds to understand the phytochemical phenomena behind this problem. With this aim, the concentration of total phenols and limonoides was evaluated in C. odorata leaves from a plantation established in Tezonapa Veracruz, Mexico. For this, a total of 66 tree leaves samples, from seven sites, were analyzed. Phenols and limonoids concentration showed significant differences not only among different provenances, but also among individual trees of the same site (Tukey, p≤0.05). Phenols concentration was variable and in the range from 49 to 223mg EAG/g e for total phenols, from 7 to 158mg EC/g e for flavonoids and from 4 to 104mg EC/g e for proanthocyanidins. Limonoids concentration was also variable, ranging between 227 and 748mg EL/g e. A major compound was found by High-Performance Liquid Chromatography with Ultraviolet Diode Array Detection (HPLC-UV-DAD), which corresponded to a flavonol kaempferol glycoside derivative; additionally, a flavanol catechin was also detected at low concentrations. GC-MS allowed the identification of the sesquiterpenoids β-elemene, E-caryophyllene, aromadendrene, α-humulene, γ-cadinene, D-germacrene, bicyclogermacrene, and the poly terpenoids D-α-tocopherol and β-sitosterol. Our results suggest that the evaluation of phenols may play an important role as a selection parameter for improvement and conservation programs, if they are complemented with conventional breeding practices.

Cedrela odorata (Meliaceae) es una especie forestal maderable nativa de América Tropical, conocida por la alta calidad de su madera. Plantaciones de esta especie son atacadas severamente por Hypsipyla grandella; la atracción o repelencia de la plaga está relacionada con metabolitos secundarios tipo fenoles y limonoides (triterpenos), por lo que el estudio de estos compuestos es importante para comprender algunos fenómenos fitoquímicos. Se evaluó la concentración de fenoles totales y limonoides en hojas de C. odorata (Meliaceae) de una plantación establecida en Tezonapa Veracruz México, se analizaron 66 individuos de siete procedencias. La concentración de fenoles y limonoides mostró diferencias significativas, no solo entre las procedencias sino también entre los árboles de una determinada procedencia (Tukey, p≤0.05). La concentración de fenoles totales varió de 49 a 223mg EAG/g e, los flavonoides de 7 a 158mg EC/g e y las proantocianidinas de 4 a 104mg EC/g e, mientras que en limonoides se obtuvieron valores de 227 a 748mg EL/g e. Mediante Cromatografía Líquida de Alta Resolución con detector UV-Arreglo de Diodos (HPLC-UV-DAD) se encontró un compuesto mayoritario que corresponde a un flavonol de tipo glicósido de Kaempferol y se identificó el flavanol catequina a bajas concentraciones. Por medio de Cromatografía de Gases-Espectrometría de Masas (CG-MS) se identificaron los sesquiterpenos β-elemeno, E-cariofileno, aromadendreno, humuleno, gama-cadineno, D-germacreno, biciclogermacreno y los poli terpenos Di-α-Tocoferol y β-sitosterol. Nuestros resultados sugieren que la evaluación de los fenoles puede desempeñar un papel importante como parámetro de selección en programas de mejora y conservación, si se complementan con las prácticas convencionales de mejoramiento genético.