The use of flow cytometry to enumerate microorganisms is gaining traction over the traditional plate count technique on the basis of superior accuracy, precision and time-to-result. Here, we assessed the suitability of live/dead flow cytometry for the enumeration of mixed populations of probiotic bacteria (L. acidophilus, L. paracasei, L. plantarum, L. salivarius, B. lactis and B. bifidum) whilst comparing outcomes with plate counting. Using a novel gating strategy designed specifically for the enumeration of mixed populations, the application of flow cytometry resulted in the detection of higher numbers of viable bacteria with a greater level of repeatability than plate counting (RSD of 6.82 and 13.14% respectively). Across all multi-species blends tested, viable cell input was more accurately recovered by flow cytometry (101.8 ± 6.95%) than plate counts (81.37 ± 16.03%). However, when certain probiotic mixtures contained preparations with high numbers of non-viable cells in their total population, flow cytometry had the potential for overestimation of the viable population. Nevertheless, the comparative plate counts of these mixtures were low and variable, thus supporting the use of flow cytometry for the enumeration of viable bacteria in mixed populations.

BACKGROUND: The emergence of carbapenem-resistant Enterobacterales (CRE) continues to threaten public health due to limited therapeutic options. In the current study the incidence of carbapenem resistance among the 104 clinical isolates of Escherichia coli and the genomic features of carbapenem resistant isolates were investigated. METHODS: The susceptibility to imipenem, tigecycline and colistin was tested by broth dilution method. Susceptibility to other classes of antimicrobials was examined by disk diffusion test. The presence of blaOXA-48, blaKPC, blaNDM, and blaVIM carbapenemase genes was examined by PCR. Molecular characteristics of carbapenem resistant isolates were further investigated by whole-genome sequencing (WGS) using Illumina and Nanopore platforms. RESULTS: Four isolates (3.8%) revealed imipenem MIC of ≥32 mg/L and positive results for modified carbapenem inactivation method and categorized as carbapenem resistant E. coli (CREC). Colistin, nitrofurantoin, fosfomycin, and tigecycline were the most active agents against all isolates (total susceptibility rate of 99, 99, 96 and 95.2% respectively) with the last three compounds being found as the most active antimicrobials for carbapenem resistant isolates (susceptibility rate of 100%). According to Multilocus Sequence Type (MLST) analysis the 4 CREC isolates belonged to ST167 (n = 2), ST361 (n = 1) and ST648 (n = 1). NDM was detected in all CREC isolates (NDM-1 (n = 1) and NMD-5 (n = 3)) among which one isolate co-harbored NDM-5 and OXA-181 carbapenemases. WGS further detected blaCTX-M-15, blaCMY-145, blaCMY-42 and blaTEM-1 (with different frequencies) among CREC isolates. Co-occurrence of NDM-type carbapenemase and 16S rRNA methyltransferase RmtB and RmtC was found in two isolates belonging to ST167 and ST648. A colistin-carbapenem resistant isolate which was mcr-negative, revealed various amino acid substitutions in PmrB, PmrD and PhoPQ proteins. CONCLUSION: About 1.9% of E. coli isolates studied here were resistant to imipenem, colistin and/or amikacin which raises the concern about the outbreaks of difficult-to-treat infection by these emerging superbugs in the future.

Sediment bacterial communities play a critical role in biogeochemical cycling in alpine lake ecosystems. However, little is known about the sediment microbial communities in these lakes. In this study, the bacterial community composition (BCC) and their relationships with environmental factors of the sediment in Sayram Lake, the largest alpine and cold-water inland lake, China was analyzed using Illumina MiSeq sequencing. In total, we obtained 618,271 high quality sequences. The results showed that the bacterial communities with 30 phyla and 546 genera, were spread out among the 5 furface sediment samples, respectively. The communities were dominated by Proteobacteria, Acidobacteria, Planctomycetes, Gemmatimonadetes, Chloroflexi, Actinobacteria, Verrucomicrobia and Bacteroidetes, accounting for 48.15 ± 8.10%, 11.23 ± 3.10%, 8.42 ± 2.15%, 8.37 ± 2.26%, 7.40 ± 3.05%, 5.62 ± 1.25%, 4.18 ± 2.12% and 2.24 ± 1.10% of the total reads, respectively. At the genus level, the communities were dominated by Aquabacterium, Pseudomonas, Woeseia, MND1, Ignavibacterium and Truepera, accounting for 7.89% ± 8.24%, 2.32% ± 1.05%, 2.14% ± 0.94%, 2% ± 1.22%, 0.94% ± 0.14% and 0.80% ± 0.14% of the total reads, respectively. Statistical analyses showed the similarity of the sediment bacterial communities at our field sites was considerably low, far below 35%, and total organic carbon (TOC) was the dominant environmental factor affecting the spatial changes of BCC in the sediment. Thus, this study greatly improving our understanding of the microbial ecology of alpine lake in the arid and semi-arid ecosystems today so seriously threatened.

BACKGROUND: Impaired respiratory and intestinal microbiome composition is linked to cystic fibrosis lung disease severity. In people with cystic fibrosis (pwCF), regular exercise is recommended to delay disease progression and preserve a stable lung function. An optimal nutritional status is vital for best clinical outcomes. Our study investigated whether regular and monitored exercise and nutritional support promotes CF microbiome health. METHODS: A personalized nutrition and exercise program promoted nutritional intake and physical fitness in 18 pwCF for 12 months. Throughout the study, patients performed strength and endurance training monitored by a sports scientist via an internet platform. After three months, food supplementation with Lactobacillus rhamnosus LGG was introduced. Nutritional status and physical fitness were assessed before the study started, after three and nine months. Sputum and stool were collected, and microbial composition was analyzed by 16S rRNA gene sequencing. RESULTS: Sputum and stool microbiome composition remained stable and highly specific to each patient during the study period. Disease-associated pathogens dominated sputum composition. Lung disease severity and recent antibiotic treatment had the highest impact on taxonomic composition in stool and sputum microbiome. Strikingly, the long-term antibiotic treatment burden had only a minor influence. CONCLUSION: Despite the exercise and nutritional intervention, respiratory and intestinal microbiomes proved to be resilient. Dominant pathogens drove the composition and functionality of the microbiome. Further studies are required to understand which therapy could destabilize the dominant disease-associated microbial composition of pwCF.

BACKGROUND: The phylum Chloroflexi is highly abundant in a wide variety of wastewater treatment bioreactors. It has been suggested that they play relevant roles in these ecosystems, particularly in degrading carbon compounds and on structuring flocs or granules. Nevertheless, their function is not yet well understood as most species have not been isolated in axenic cultures. Here we used a metagenomic approach to investigate Chloroflexi diversity and their metabolic potential in three environmentally different bioreactors: a methanogenic full-scale reactor, a full-scale activated sludge reactor and a lab scale anammox reactor. RESULTS: Differential coverage binning approach was used to assemble the genomes of 17 new Chloroflexi species, two of which are proposed as new Candidatus genus. In addition, we recovered the first representative genome belonging to the genus 'Ca. Villigracilis'. Even though samples analyzed were collected from bioreactors operating under different environmental conditions, the assembled genomes share several metabolic features: anaerobic metabolism, fermentative pathways and several genes coding for hydrolytic enzymes. Interestingly, genome analysis from the anammox reactor indicated a putative role of Chloroflexi in nitrogen conversion. Genes related to adhesiveness and exopolysaccharides production were also detected. Complementing sequencing analysis, filamentous morphology was detected by Fluorescent in situ hybridization. CONCLUSION: Our results suggest that Chloroflexi participate in organic matter degradation, nitrogen removal and biofilm aggregation, playing different roles according to the environmental conditions.

BACKGROUND: Elevated carbon dioxide concentrations (eCO2), one of the main causes of climate change, have several consequences for both vine and cover crops in vineyards and potentially also for the soil microbiome. Hence soil samples were taken from a vineyard free-air CO2 enrichment (VineyardFACE) study in Geisenheim and examined for possible changes in the soil active bacterial composition (cDNA of 16S rRNA) using a metabarcoding approach. Soil samples were taken from the areas between the rows of vines with and without cover cropping from plots exposed to either eCO2 or ambient CO2 (aCO2). RESULTS: Diversity indices and redundancy analysis (RDA) demonstrated that eCO2 changed the active soil bacterial diversity in grapevine soil with cover crops (p-value 0.007). In contrast, the bacterial composition in bare soil was unaffected. In addition, the microbial soil respiration (p-values 0.04-0.003) and the ammonium concentration (p-value 0.003) were significantly different in the samples where cover crops were present and exposed to eCO2. Moreover, under eCO2 conditions, qPCR results showed a significant decrease in 16S rRNA copy numbers and transcripts for enzymes involved in N2 fixation and NO2- reduction were observed using qPCR. Co-occurrence analysis revealed a shift in the number, strength, and patterns of microbial interactions under eCO2 conditions, mainly represented by a reduction in the number of interacting ASVs and the number of interactions. CONCLUSIONS: The results of this study demonstrate that eCO2 concentrations changed the active soil bacterial composition, which could have future influence on both soil properties and wine quality.

BACKGROUND: Staphylococcus aureus is one of the prevalent etiological agents of contagious bovine mastitis, causing a significant economic burden on the global dairy industry. Given the emergence of antibiotic resistance (ABR) and possible zoonotic spillovers, S aureus from mastitic cattle pose threat to both veterinary and public health. Therefore, assessment of their ABR status and pathogenic translation in human infection models is crucial. RESULTS: In this study, 43 S. aureus isolates associated with bovine mastitis obtained from four different Canadian provinces (Alberta, Ontario, Quebec, and Atlantic provinces) were tested for ABR and virulence through phenotypic and genotypic profiling. All 43 isolates exhibited crucial virulence characteristics such as hemolysis, and biofilm formation, and six isolates from ST151, ST352, and ST8 categories showed ABR. Genes associated with ABR (tetK, tetM, aac6', norA, norB, lmrS, blaR, blaZ, etc.), toxin production (hla, hlab, lukD, etc.), adherence (fmbA, fnbB, clfA, clfB, icaABCD, etc.), and host immune invasion (spa, sbi, cap, adsA, etc.) were identified by analyzing whole-genome sequences. Although none of the isolates possessed human adaptation genes, both groups of ABR and antibiotic-susceptible isolates demonstrated intracellular invasion, colonization, infection, and death of human intestinal epithelial cells (Caco-2), and Caenorhabditis elegans. Notably, the susceptibilities of S. aureus towards antibiotics such as streptomycin, kanamycin, and ampicillin were altered when the bacteria were internalized in Caco-2 cells and C. elegans. Meanwhile, tetracycline, chloramphenicol, and ceftiofur were comparatively more effective with ≤ 2.5 log10 reductions of intracellular S. aureus. CONCLUSIONS: This study demonstrated the potential of S. aureus isolated from mastitis cows to possess virulence characteristics enabling invasion of intestinal cells thus calling for developing therapeutics capable of targeting drug-resistant intracellular pathogens for effective disease management.

BACKGROUND: Artisanal and small-scale gold mining activities are producing contamination with heavy metals and metalloids (HMM) into soils and water worldwide. The HMM are considered as one of the major abiotic stresses due to their long-term persistence in soil. In this context, arbuscular mycorrhizal fungi (AMF) confer resistance to a variety of abiotic plant stressors including HMM. However, little is known regarding the diversity and composition of AMF communities in heavy metal polluted sites in Ecuador. METHODS: In order to investigate the AMF diversity, root samples and associated soil of six plant species were collected from two sites polluted by heavy metals, located in Zamora-Chinchipe province, Ecuador. The AMF 18S nrDNA genetic region was analyzed and sequenced, and fungal OTUs were defined based on 99% sequence similarity. Results were contrasted with AMF communities from a natural forest and from reforestation sites located in the same province and with available sequences in GenBank. RESULTS: The main pollutants in soils were Pb, Zn, Hg, Cd and Cu with concentrations exceeding the soil reference value for agricultural use. Molecular phylogeny and OTU delimitation showed 19 OTUs, the family Glomeraceae was the most OTU-rich followed by Archaeosporaceae, Acaulosporaceae, Ambisporaceae and Paraglomeraceae. Most of the OTUs (11 of 19) have been found at other locations worldwide, 14 OTUs were proven from nearby non-contaminated sites in Zamora-Chinchipe. CONCLUSION: Our study showed that there are no specialized OTUs at the studied HMM polluted sites, but rather generalists adapted to a wide variety of habitats. Their potential role in phytoremediation approaches remains to be investigated.

BACKGROUND: Cotton Verticillium wilt, causing by Verticillium dahliae, has seriously affected the yield and quality of cotton. The incidence of Verticillium wilt in cotton fields has been on the rise for many years, especially after straw has been returned to the fields. Intercropping can reduce the incidence of soil borne diseases and is often used to control crop diseases, but the relationship between the effects of intercropping on microbial communities and the occurrence of plant diseases is unclear. This research explored the relationship between soil microbial community structure and Cotton Verticillium wilt in interplanting of cotton-onion, cotton-garlic, cotton-wheat and cotton monocultures. Amplicon sequencing applied to the profile of bacterial and fungal communities. RESULTS: The results showed that the disease index of Cotton Verticillium wilt was significantly reduced after intercropping with cotton-garlic and cotton-onion. Chao1 and Sobs indices were not significantly different in the rhizosphere soil and pre-plant soils of the four planting patterns, but the pre-plant fungal shannon index was significantly lower in the cotton-onion intercropping plot than in the other three plots. PCoA analysis showed that the soil microbial communities changed to a certain extent after intercropping, with large differences in the microbial communities under different cropping patterns. The abundance of Chaetomium was highest in the cotton-garlic intercropping before planting; the abundance of Penicillium was significantly higher in the cotton-wheat intercropping than in the other three systems. CONCLUSION: Cotton-garlic and cotton-onion interplanting can control Cotton Verticillium wilt by affecting the soil microbial community. Fungi of the genera Chaetomium and Penicillium may be associated with plant disease resistance.