Microbial Ecology

Taurine Is a Major Carbon and Energy Source for Marine Prokaryotes in the North Atlantic Ocean off the Iberian Peninsula Abstract Taurine, an amino acid-like compound, acts as an osmostress protectant in many marine metazoans and algae and is released via various processes into the oceanic dissolved organic matter pool. Taurine transporters are widespread among members of the marine prokaryotic community, tentatively indicating that taurine might be an important substrate for prokaryotes in the ocean. In this study, we determined prokaryotic taurine assimilation and respiration throughout the water column along two transects in the North Atlantic off the Iberian Peninsula. Taurine assimilation efficiency decreased from the epipelagic waters from 55 ± 14% to 27 ± 20% in the bathypelagic layers (means of both transects). Members of the ubiquitous alphaproteobacterial SAR11 clade accounted for a large fraction of cells taking up taurine, especially in surface waters. Archaea (Thaumarchaeota + Euryarchaeota) were also able to take up taurine in the upper water column, but to a lower extent than Bacteria. The contribution of taurine assimilation to the heterotrophic prokaryotic carbon biomass production ranged from 21% in the epipelagic layer to 16% in the bathypelagic layer. Hence, we conclude that dissolved free taurine is a significant carbon and energy source for prokaryotes throughout the oceanic water column being utilized with similar efficiencies as dissolved free amino acids.

The Oral Bacterium Fusobacterium nucleatum Binds Staphylococcus aureus and Alters Expression of the Staphylococcal Accessory Regulator sarA Abstract Staphylococcus aureus, an opportunistic pathogen member of the nasal and skin microbiota, can also be found in human oral samples and has been linked to infectious diseases of the oral cavity. As the nasal and oral cavities are anatomically connected, it is currently unclear whether S. aureus can colonize the oral cavity and become part of the oral microbiota, or if its presence in the oral cavity is simply transient. To start addressing this question, we assessed S. aureus ability to directly bind selected members of the oral microbiota as well as its ability to integrate into a human-derived complex oral microbial community in vitro. Our data show that S. aureus forms aggregates with Fusobacterium nucleatum and Porphyromonas gingivalis and that it can incorporate into the human-derived in vitro oral community. Further analysis of the F. nucleatum-S. aureus interaction revealed that the outer-membrane adhesin RadD is partially involved in aggregate formation and that the RadD-mediated interaction leads to an increase in expression of the staphylococcal global regulator gene sarA. Our findings lend support to the notion that S. aureus can become part of the complex microbiota of the human mouth, which could serve as a reservoir for S. aureus. Furthermore, direct interaction with key members of the oral microbiota could affect S. aureus pathogenicity contributing to the development of several S. aureus associated oral infections.

Detection of MRSA of Lineages CC130- mecC and CC398- mecA and Staphylococcus delphini - lnu (A) in Magpies and Cinereous Vultures in Spain Abstract The aim of this study was to determine the carriage rate of coagulase-positive staphylococci (CoPS) in wild birds and to characterize recovered isolates. Tracheal samples from 324 wild birds, obtained in different Spanish regions during 2015–2016, were screened for CoPS carriage. The antimicrobial resistance profile and the virulence gene content were investigated. Molecular typing was performed by spa, agr, MLST, SCCmec, and S. delphini group classification. CoPS were recovered from 26 samples of wild birds (8.3%), and 27 isolates were further characterized. Two CoPS species were detected: S. aureus (n = 15; eight cinereous vultures and seven magpies) and S. delphini(n = 12; 11 cinereous vultures and one red kite). Thirteen S. aureus were methicillin-resistant (MRSA) and the remaining two strains were methicillin-susceptible (MSSA). Twelve MRSA were mecC-positive, typed as t843-ST1583/ST1945/ST1581/ST1571 (n = 11) and t1535-ST1945 (n = 1) (all of clonal-complex CC130); they were susceptible to the non-β-lactams tested. The remaining MRSA strain carried the mecA gene, was typed as t011-ST398-CC398-agrI-SCCmec-V, and showed a multiresistance phenotype. MSSA isolates were ascribed to lineages ST97-CC97 and ST425-CC425. All S. aureus lacked the studied virulence genes (lukS/F-PV, tst, eta, etb, and etd), and the IEC type E (with scn and sak genes) was detected in four mecC-positive and one MSSA isolates. S. delphini strains were methicillin-susceptible but showed resistance to at least one of the antimicrobials tested, with high penicillin (75%, with blaZ gene) and tetracycline [58%, with tet(K)± tet(L)] resistance rates. All S. delphini isolates presented the virulence genes lukS-I, siet, and se-int, and four carried the clindamycin-resistance lnu(A) gene.

Dynamics of Biofilm Formation by Salmonella Typhimurium and Beef Processing Plant Bacteria in Mono- and Dual-Species Cultures Abstract This study aimed to determine the impact of bacteria from a beef plant conveyor belt on the biofilm formation of Salmonella in dual-species cultures. Beef plant isolates (50) including 18 Gram-negative aerobes (GNA), 8 Gram-positive aerobes (GPA), 5 lactic acid bacteria (LAB), 9 Enterobacteriaceae (EB), and 10 generic Escherichia coli (GEC) were included for developing biofilms in mono- and co-culture with S. Typhimurium at 15 °C for 6 days. Five selected cultures in planktonic form and in biofilms were tested for susceptibility to two commonly used sanitizers (i.e. E-San and Perox-E Plus). In mono-cultures, ≥ 80, 67, 61, 20, and 13% of GEC, EB, GNA, LAB, and GPA, respectively, developed measurable biofilms after 2 days, while all co-culture pairings with S. Typhimurium achieved some level of biofilm production. The predominant effect of EB and only effect of GEC strains on the biofilm formation of S.Typhimurium was antagonistic, while that of Gram-positive bacteria was synergistic, with the effect being more prominent on day 6. The effect was highly variable for the GNA isolates. Six aerobic isolates that formed moderate/strong biofilms by day 2 greatly boosted the co-culture biofilm formation. Seven Gram-negative bacteria were antagonistic against the biofilm formation of the co-cultures. Both sanitizers completely inactivated the selected planktonic cultures, but were largely ineffective against biofilms. In conclusion, all beef plant isolates assessed formed biofilms when paired with S. Typhimurium. Aerobic biofilm formers may create a more favorable condition for Salmonella biofilm formation, while some beef plant isolates have potential as a biocontrol strategy for Salmonella biofilms.

Penicillin Trunk Injection Affects Bacterial Community Structure in Citrus Trees Abstract Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), an uncultured α-proteobacterium, is the most destructive disease of citrus trees worldwide. In previous studies, trunk injections of penicillin reduced CLas titers and HLB symptoms in citrus. However, antibiotic effects on the whole plant microbial community, which include effects on taxa that interact with CLas, have not yet been addressed. In this study, we investigated the effects of penicillin injection (0, 1000, and 6000 mg L−1) on rhizospheric and endophytic bacterial communities of grapefruit trees in field and greenhouse experiments through culture-independent high-throughput sequencing. DNA extractions from petioles and roots were subjected to 16S rRNA high-throughput sequencing, and reads were clustered by sequence similarity into operational taxonomic units (OTUs). Principal coordinates analysis based on weighted-UniFrac distances did not reveal differences in bacterial communities among treatments in any of the sample sources. However, pairwise linear discriminant analysis indicated significant differences in relative abundance of some taxa (including CLas) among treatments. Network analysis showed that penicillin produced major changes in root bacterial community structure by affecting interspecific microbial associations. This study provides new knowledge of the effect of antimicrobial treatments on interspecific relationships in citrus microbial communities.

Biogeography and Diversity of Freshwater Bacteria on a River Catchment Scale Abstract To illustrate how freshwater bacterial community changes with geographic gradient, we investigated the spatial changes of bacterial abundance and community structures from over 200 samples on a catchment scale in the Songhua River using heterotrophic plate counts, flow cytometry, denaturing gradient gel electrophoresis, and pyrosequencing analysis. The results showed that the mainstream had higher cultivable bacteria and total bacterial concentration than tributaries in the Songhua River catchment. Response model analysis demonstrated that the bacterial community exhibits a biogeographical signature even in an interconnected river network system, and the total bacterial concentration and biodiversity were significantly correlated to latitude (p < 0.001) and longitude (p < 0.001). Multivariate redundancy analysis indicated that temperature was the most important factor driving bacterial community structure in the Songhua River, which accounts for 35.30% variance of communities, then dissolved oxygen (17.60%), latitude (17.60%), longitude (11.80%), and pH (5.88%). High-throughput pyrosequencing revealed that at the phylum level, Proteobacteria was numerically dominant (89.6%) in river catchment, followed by Bacteroidetes (8.1%) and Cyanobacteria (1.2%). The overall results revealed that the bacterial community was driven by geographical distance regardless of the continuum of the river on a catchment scale.

Lower Respiratory Tract Microbiome and Resistome of Bovine Respiratory Disease Mortalities Abstract Bovine respiratory disease (BRD) continues to be a serious health problem in beef cattle production. A multifactorial condition, BRD encompasses several types of pneumonia that are associated with multiple viral and bacterial agents. Comprehensive identification of microbes associated with BRD fatalities could enhance our understanding of the range of pathogens that contribute to the disease and identify new therapeutic targets. This study used metagenomic analysis to describe the lower respiratory tract microbiome and resistome of 15 feedlot cattle BRD and 3 non-BRD mortalities along with any affiliated integrative and conjugative elements (ICEs). Known bacterial pathogens associated with BRD, including Histophilus somni, Mannheimia haemolytica, and Mycoplasma bovis, were relatively abundant (> 5%) in most, but not all samples. Other relatively abundant genera (> 1%) included Acinetobacter, Bacillus, Bacteroides, Clostridium, Enterococcus, and Pseudomonas. Antimicrobial resistance genes (ARGs) comprised up to 0.5% of sequences and many of these genes were associated with ICEs previously described within the Pasteurellaceae family. A total of 20 putative ICEs were detected among 16 samples. These results document the wide diversity of microorganisms in the lower respiratory tract of cattle that have succumbed to BRD. The data also strongly suggest that antimicrobial-resistant Pasteurellaceae strains are prevalent in BRD cases in Alberta and that the resistance observed is associated with ICEs. The presence of ICEs harboring a wide array of ARGs holds significant consequence for the effectiveness of drug therapies for the control of BRD in beef cattle.

Comparative Analysis of Anuran Amphibian Skin Microbiomes Across Inland and Coastal Wetlands Abstract Amphibians host a community of microbes on their skin that helps resist infectious disease via the dual influence of anti-pathogenic microbial species and emergent community dynamics. Many frogs rely on freshwater habitats, but salinization is rapidly increasing saltwater concentrations in wetlands around the globe, increasing the likelihood that frogs will come into contact with salt-contaminated habitats. Currently, we know little about how increased salt exposure will affect the symbiotic relationship between the skin microbes and frog hosts. To better understand how salt exposure in a natural context affects the frog skin microbiome, we use Hyla cinerea, a North American treefrog species that can inhabit brackish wetlands, to explore three questions. First, we determine the extent that microbial communities in the environment and on frog skin are similar across populations. Second, we assess the microbial species richness and relative abundance on frogs from habitats with different salinity levels to determine how salinity affects the microbiome. Third, we test whether the relative abundances of putatively pathogen-resistant bacterial species differ between frogs from inland and coastal environments. We found that the frog microbiome is more similar among frogs than to the microbial communities found in surface water and soil, but there is overlap between frog skin and the environmental samples. Skin microbial community richness did not differ among populations, but the relative abundances of microbes were different across populations and salinities. We found no differences in the relative abundances of the anti-fungal bacteria Janthinobacterium lividum, the genus Pseudomonas, and Serratia marcescens, suggesting that environmental exposure to saltwater has a limited influence on these putatively beneficial bacterial taxa.

Rhizosphere Microbiomes from Root Knot Nematode Non-infested Plants Suppress Nematode Infection Abstract Root knot nematodes (RKN, Meloidogyne spp.) are serious pathogens of numerous crops worldwide. Understanding the roles plant rhizosphere soil microbiome play during RKN infection is very important. The current study aims at investigating the impacts of soil microbiome on the activity of RKN. In this study, the 16S rRNA genes of the bacterial communities from nematode-infested and non-infested rhizosphere soils from four different plants were sequenced on the Illumina Hi-Seq platform. The soil microbiome effects on RKN infection were tested in a greenhouse assay. The non-infested soils had more microbial diversity than the infested soils from all plant rhizospheres, and both soil types had exclusive microbial communities. The inoculation of the microbiomes from eggplant and cucumber non-infested soils to tomato plants significantly alleviated the RKN infection, while the microbiome from infested soil showed increased the RKN infection. Furthermore, bacteria Pseudomonas sp. and Bacillus sp. were screened out from non-infested eggplant soil and exhibited biocontrol activity to RKN on tomato. Our findings suggest that microbes may regulate RKN infection in plants and are involved in biocontrol of RKN.

High-Throughput Sequencing of the 16S rRNA Gene as a Survey to Analyze the Microbiomes of Free-Living Ciliates Paramecium Abstract Ciliates are the largest group of ubiquitous aquatic bacterivorous protists, and many species are easily cultivated. However, only few studies reported prokaryotic communities naturally associated with ciliate cells. Herein, we analyzed the microbiome composition of several strains of Paramecium (Ciliophora) originating from different locations and belonging to two morpho-species by high-throughput sequencing (HTS) of the 16S rRNA gene. Possible reasons of HTS results bias were addressed comparing DNA libraries obtained using different primers and different number of ciliate cells. Microbiomes associated with ciliates and their environments were always significantly different by prokaryotic taxonomic composition and bacterial richness. There were also pronounced differences between Paramecium strains. Interestingly, potentially pathogenic bacteria were revealed in Paramecium microbiomes.