High-Throughput Screen for Inhibitors of the Type IV Pilus Assembly ATPase PilB.

The bacterial type IV pilus (T4P) is a prominent virulence factor in many significant human pathogens, some of which have become increasingly antibiotic resistant. Antivirulence chemotherapeutics are considered a promising alternative to antibiotics because they target the disease process instead of bacterial viability. However, a roadblock to the discovery of anti-T4P compounds is the lack of a high-throughput screen (HTS) that can be implemented relatively easily and economically. Here, we describe the first HTS for the identification of inhibitors specifically against the T4P assembly ATPase PilB in vitroChloracidobacterium thermophilum PilB (CtPilB) had been demonstrated to have robust ATPase activity and the ability to bind its expected ligands in vitro. We utilized CtPilB and MANT-ATP, a fluorescent ATP analog, to develop a binding assay and adapted it for an HTS. As a proof of principle, we performed a pilot screen with a small compound library of kinase inhibitors and identified quercetin as a PilB inhibitor in vitro Using Myxococcus xanthus as a model bacterium, we found quercetin to reduce its T4P-dependent motility and T4P assembly in vivo. These results validated our HTS as effective in identifying PilB inhibitors. This assay may prove valuable in seeking leads for the development of antivirulence chemotherapeutics against PilB, an essential and universal component of all bacterial T4P systems.IMPORTANCE Many bacterial pathogens use their type IV pili (T4P) to facilitate and maintain infection of a human host. Small chemical compounds that inhibit the production or assembly of T4P hold promise in the treatment and prevention of infections, especially in the era of increasing threats from antibiotic-resistant bacteria. However, few chemicals are known to have inhibitory or anti-T4P activity. Their identification has not been easy due to the lack of a method for the screening of compound collections or libraries on a large scale. Here, we report the development of an assay that can be scaled up to screen compound libraries for inhibitors of a critical T4P assembly protein. We further demonstrate that it is feasible to use whole cells to examine potential inhibitors for their activity against T4P assembly in a bacterium.

Soil Acidobacteria Strain AB23 Resistance to Oxidative Stress Through Production of Carotenoids.

Metagenomic studies revealed the prevalence of Acidobacteria in soils, but the physiological and ecological reasons for their success are not well understood. Many Acidobacteria exhibit carotenoid-related pigments, which may be involved in their tolerance of environmental stress. The aim of this work was to investigate the role of the orange pigments produced by Acidobacteria strain AB23 isolated from a savannah-like soil and to identify putative carotenoid genes in Acidobacteria genomes. Phylogenetic analysis revealed that strain AB23 belongs to the Occallatibacter genus from the class Acidobacteriia (subdivision 1). Strain AB23 produced carotenoids in the presence of light and vitamins; however, the growth rate and biomass decreased when cells were exposed to light. The presence of carotenoids resulted in tolerance to hydrogen peroxide. Comparative genomics revealed that all members of Acidobacteriia with available genomes possess the complete gene cluster for phytoene production. Some Acidobacteriia members have an additional gene cluster that may be involved in the production of colored carotenoids. Both colored and colorless carotenoids are involved in tolerance to oxidative stress. These results show that the presence of carotenoid genes is widespread among Acidobacteriia. Light and atmospheric oxygen stimulate carotenoid synthesis, but there are other natural sources of oxidative stress in soils. Tolerance to environmental oxidative stress provided by carotenoids may offer a competitive advantage for Acidobacteria in soils.

Antibiotic resistance and microbiota in the gut of Chinese four major freshwater carp from retail markets.

Fish-associated antibiotic resistance genes (ARGs) have attracted increasing attention due to their potential risks to human beings via the food chain. However, data are scarce regarding the antibiotic resistance in fish themselves. Herein, the antibiotic resistance genes (ARGs) were assessed in the gut of four major Chinese freshwater carp (i.e., silver carp, grass carp, bighead carp, and crucian carp) from food retail markets. Results show that the abundances of target ARGs (e.g., tetA, tetO, tetQ, tetW, sulI, sulII, and blaTEM-1) and class 1 integrase (intI1) were in the range 9.4 × 10-6 - 1.6 × 10-1 and 6.7 × 10-5 - 5.2 × 10-2 gene copies per 16S rRNA gene, respectively. The sulI, sulII, and tetQ strongly correlated with silver and mercury resistance genes (e.g., silE and merR). The microbial taxa of fish gut could be partly separated among retail markets based on the PCA analysis. About 15.0% of the OTUs in fish gut were shared and 74.5% of the shared OTUs were identified as Acidobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Firmicutes, and Proteobacteria. These phyla may constitute the core microbiota in the guts of the four Chinese freshwater carp. The possible ARG hosts were revealed based on the network analysis, and the presence of pathogen-associated resistant genera in fish gut highlights the need to fully understand their potential human health risks.

Bacterial community variations in paddy soils induced by application of veterinary antibiotics in plant-soil systems.

Soil bacterial communities have complex regulatory networks, which are mainly associated with soil fertility and ecological functions, and are likely to be disturbed due to antibiotics applications. The impact of antibiotics, particularly in mixtures form, on bacterial communities in different paddy soils is poorly understood. Using pyrosequencing techniques of 16 S rRNA genes, this study investigated the synergistic effects of veterinary antibiotics (sulfadiazine, sulfamethoxazole, trimethoprim, florfenicol, and clarithromycin) on bacterial communities in a soil-bacteria-plant system. Rice was grown under controlled greenhouse conditions where unplanted and planted treatments were doped with 200 µg kg-1 of combined antibiotics over a period of 3 months. Bacterial richness remained unaltered, while a significant decline was observed in bacterial diversity due to antibiotics in the four paddy soils. Bacteroidetes and Acidobacteria were increased, while Actinobacteria and Firmicutes decreased under antibiotics exposure. Despite antibiotics perturbation, compositional variations were mainly attributed to the different paddy soils which harbor distinct bacterial communities. Haliangium and Gaiella were among the sensitive genera that were negatively correlated to antibiotics perturbation. Additionally, electrical conductivity, total organic carbon, and total nitrogen of soil solution were the key physiochemical indices which significantly influenced the structure of bacterial communities in the paddy soils. These findings expanded our knowledge of effects from synergistic antibiotics application and variations in bacterial communities among different paddy soils.

Toxicity responses of bacterial community as a biological indicator after repeated exposure to lead (Pb) in the presence of decabromodiphenyl ether (BDE209).

Continuous exposure of chemicals could cause various environmental impacts. Decabromodiphenyl ether (BDE209) and lead (Pb) can co-exist and are discharged simultaneously at e-waste recycling sites (EWRSs). Extensive concerns have been attracted by their toxic effects on soil microorganisms. Thus, by using high-throughput sequencing, this study explored bacterial community responses in a soil system after repeated Pb exposure in the presence of BDE209 in the laboratory during 90-day indoor incubation period. Gene sequencing of 16S rDNA performed on an Illumina MiSeq platform proved that one-off Pb exposure caused higher microbial abundance and community diversity. Additionally, both repetitive Pb treatment and exogenous BDE209 input could change bacterial community composition. Twenty-three different bacterial phyla were detected in the soil samples, while more than 90% of the sequences in each treatment belonged to a narrow variety. The sequence analyses elucidated that Proteobacteria, Acidobacteria, and Bacteroidetes were the top three dominant phyla. Our observations could provide a few insights into the ecological risks of Pb and BDE209 co-existed contamination in soils at EWRSs.

Bacterial community changes in response to oil contamination and perennial crop cultivation.

We investigated bacterial community dynamics in response to used motor oil contamination and perennial crop cultivation by 16S rRNA gene amplicon sequencing in a 4-year field study. Actinobacteria, Proteobacteria, Chloroflexi, Acidobacteria, and Gemmatimonadetes were the major bacterial phyla, and Rhodococcus was the most abundant genus. Initially, oil contamination decreased the overall bacterial diversity. Actinobacteria, Betaproteobacteria, and Gammaproteobacteria were sensitive to oil contamination, exhibiting clear succession with time. However, bacterial communities changed over time, regardless of oil contamination and crop cultivation. The abundance difference of most OTUs between oil-contaminated and non-contaminated plots remained the same in later sampling years after the initial abundance difference induced by oil spike. The abundances of three oil-favored actinobacteria (Lysinimonas, Microbacteriaceae, and Marmoricola) and one betaproteobacterium (Aquabacterium) changed in different manner over time in oil-contaminated and non-contaminated soil. We propose that these taxa are potential bio-indicators for monitoring recovery from motor oil contamination in boreal soil. The effect of crop cultivation on bacterial communities became significant only after the crops achieved stable growth, likely associated with plant material decomposition by Bacteroidetes, Armatimonadetes and Fibrobacteres.

Mercury alters the bacterial community structure and diversity in soil even at concentrations lower than the guideline values.

This study evaluated the effect of inorganic mercury (Hg) on bacterial community and diversity in different soils. Three soils-neutral, alkaline and acidic-were spiked with six different concentrations of Hg ranging from 0 to 200 mg kg-1 and aged for 90 days. At the end of the ageing period, 18 samples from three different soils were investigated for bacterial community structure and soil physicochemical properties. Illumina MiSeq-based 16s ribosomal RNA (rRNA) amplicon sequencing revealed the alteration in the bacterial community between un-spiked control soils and Hg-spiked soils. Among the bacterial groups, Actinobacteria (22.65%) were the most abundant phyla in all samples followed by Proteobacteria (21.95%), Bacteroidetes (4.15%), Firmicutes (2.9%) and Acidobacteria (2.04%). However, the largest group showing increased abundance with higher Hg doses was the unclassified group (45.86%), followed by Proteobacteria. Mercury had a considerable negative impact on key soil functional bacteria such as ammonium oxidizers and nitrifiers. Canonical correspondence analysis (CCA) indicated that among the measured soil properties, Hg had a major influence on bacterial community structure. Furthermore, nonlinear regression analysis confirmed that Hg significantly decreased soil bacterial alpha diversity in lower organic carbon containing neutral and alkaline soils, whereas in acidic soil with higher organic carbon there was no significant correlation. EC20 values obtained by a nonlinear regression analysis indicated that Hg significantly decreased soil bacterial diversity in concentrations lower than several guideline values.

Changes in the Bacterial Community Structure of Remediated Anthracene-Contaminated Soils.

Mixing soil or adding earthworms (Eisenia fetida (Savigny, 1826)) accelerated the removal of anthracene, a polycyclic aromatic hydrocarbon, from a pasture and an arable soil, while a non-ionic surfactant (Surfynol® 485) inhibited the removal of the contaminant compared to the untreated soil. It was unclear if the treatments affected the soil bacterial community and consequently the removal of anthracene. Therefore, the bacterial community structure was monitored by means of 454 pyrosequencing of the 16S rRNA gene in the pasture and arable soil mixed weekly, amended with Surfynol® 485, E. fetida or organic material that served as food for the earthworms for 56 days. In both soils, the removal of anthracene was in the order: mixing soil weekly (100%) > earthworms applied (92%) > organic material applied (77%) > untreated soil (57%) > surfactant applied (34%) after 56 days. There was no clear link between removal of anthracene from soil and changes in the bacterial community structure. On the one hand, application of earthworms removed most of the contaminant from the arable soil and had a strong effect on the bacterial community structure, i.e. a decrease in the relative abundance of the Acidobacteria, Chloroflexi and Gemmatimonadetes, and an increase in that of the Proteobacteria compared to the unamended soil. Mixing the soil weekly removed all anthracene from the arable soil, but had little or no effect on the bacterial community structure. On the other hand, application of the surfactant inhibited the removal of anthracene from the arable soil compared to the untreated soil, but had a strong effect on the bacterial community structure, i.e. a decrease in the relative abundance of Cytophagia (Bacteroidetes), Chloroflexi, Gemmatimonadetes and Planctomycetes and an increase in that of the Flavobacteria (Bacteroidetes) and Proteobacteria. Additionally, the removal of anthracene was similar in the different treatments of both the arable and pasture soil, but the effect of application of carrot residue, earthworms or the surfactant on the bacterial community structure was more accentuated in the arable soil than in the pasture soil. It was found that removal of anthracene was not linked to changes in the bacterial community structure.

Bromochloromethane, a Methane Analogue, Affects the Microbiota and Metabolic Profiles of the Rat Gastrointestinal Tract.

Bromochloromethane (BCM), an inhibitor of methanogenesis, has been used in animal production. However, little is known about its impact on the intestinal microbiota and metabolic patterns. The present study aimed to investigate the effect of BCM on the colonic bacterial community and metabolism by establishing a Wistar rat model. Twenty male Wistar rats were randomly divided into two groups (control and treated with BCM) and raised for 6 weeks. Bacterial fermentation products in the cecum were determined, and colonic methanogens and sulfate-reducing bacteria (SRB) were quantified. The colonic microbiota was analyzed by pyrosequencing of the 16S rRNA genes, and metabolites were profiled by gas chromatography and mass spectrometry. The results showed that BCM did not affect body weight and feed intake, but it did significantly change the intestinal metabolic profiles. Cecal protein fermentation was enhanced by BCM, as methylamine, putrescine, phenylethylamine, tyramine, and skatole were significantly increased. Colonic fatty acid and carbohydrate concentrations were significantly decreased, indicating the perturbation of lipid and carbohydrate metabolism by BCM. BCM treatment decreased the abundance of methanogen populations, while SRB were increased in the colon. BCM did not affect the total colonic bacterial counts but significantly altered the bacterial community composition by decreasing the abundance of actinobacteria, acidobacteria, and proteobacteria. The results demonstrated that BCM treatment significantly altered the microbiotic and metabolite profiles in the intestines, which may provide further information on the use of BCM in animal production.

Impact of the novel neonicotinoid insecticide Paichongding on bacterial communities in yellow loam and Huangshi soils.

Insecticides are widely sprayed in modern agriculture for ensuring the crop yield, which could also lead to contamination and insecticide residue in soils. Paichongding (IPP) is a novel neonicotinoid insecticide and was developed recently in China. Soil bacterial community, diversity, and community composition vary widely depending on environmental factors. As for now, little is known about bacterial species thriving, bacterial community diversity, and structure in IPP-spraying soils. In present study, IPP degradation in yellow loam and Huangshi soils was investigated, and bacterial communities and diversity were examined in soil without IPP spray and with IPP spray through pyrosequencing of 16S ribosomal RNA (rRNA) gene amplicons. The degradation ratio of IPP at 60 days after treatment (DAT) reached 51.22 and 34.01 % in yellow loam and Huangshi soil, respectively. A higher richness of operational taxonomic units (OTUs) was found in yellow loam soil (867 OTUs) and Huangshi soil (762 OTUs) without IPP spray while OUTs were relatively low in IPP-spraying soils. The community composition also differed both in phyla and genus level between these two environmental conditions. Proteobacteria, Firmicutes, Planctomycetes, Chloroflexi, Armatimonadetes, and Chlorobi were stimulated to increase after IPP application, while IPP inhibited the phyla of Bacteroidetes, Actinobacteria, and Acidobacteria.

Effect of fumigation with 1,3-dichloropropene on soil bacterial communities.

1,3-Dichloropropene (1,3-D) is a potential candidate as a soil fumigant because of the restriction of methyl bromide (MB) in soil fumigation. So far, little is known about the bacteria diversity in 1,3-D fumigated soil. Therefore, the impact of 1,3-D on soil bacterial community was determined by the 16S rRNA gene amplicon 454 sequencing. A total of 230,617 valid reads and 19,366 OTUs were obtained from the thirteen samples. 454 sequencing results revealed that Proteobacteria, Bacteroidetes, Actinobacteria, Acidobacteria and Firmicutes were predominant phylum in soils. Bacterial diversity was affected initially, while recovered in the later treatments and soils from 1,3-D treatment plots had a higher bacterial diversity. The results of this study demonstrated that 1,3-D had only a short-term and transitory impact on the indigenous soil microbial community. Our study would provide useful information for evaluating ecological safety of 1,3-D in China.

Diversity of bacterial communities along a petroleum contamination gradient in desert soils.

Microbial communities in oil-polluted desert soils have been rarely studied compared to their counterparts from freshwater and marine environments. We investigated bacterial diversity and changes therein in five desert soils exposed to different levels of oil pollution. Automated rRNA intergenic spacer (ARISA) analysis profiles showed that the bacterial communities of the five soils were profoundly different (analysis of similarities (ANOSIM), R = 0.45, P < 0.0001) and shared less than 20 % of their operational taxonomic units (OTUs). OTU richness was relatively higher in the soils with the higher oil pollution levels. Multivariate analyses of ARISA profiles revealed that the microbial communities in the S soil, which contains the highest level of contamination, were different from the other soils and formed a completely separate cluster. A total of 16,657 ribosomal sequences were obtained, with 42-89 % of these sequences belonging to the phylum Proteobacteria. While sequences belonging to Betaproteobacteria, Gammaproteobacteria, Bacilli, and Actinobacteria were encountered in all soils, sequences belonging to anaerobic bacteria from the classes Deltaproteobacteria, Clostridia, and Anaerolineae were only detected in the S soil. Sequences belonging to the genus Terriglobus of the class Acidobacteria were only detected in the B3 soil with the lowest level of contamination. Redundancy analysis (RDA) showed that oil contamination level was the most determinant factor that explained variations in the microbial communities. We conclude that the exposure to different levels of oil contamination exerts a strong selective pressure on bacterial communities and that desert soils are rich in aerobic and anaerobic bacteria that could potentially contribute to the degradation of hydrocarbons.

Effect of copper exposure on bacterial community structure and function in the sediments of Jiaozhou Bay, China.

Microcosms were setup to investigate the possible impact of copper exposure on bacterial community structure and function in sediments of Jiaozhou Bay, China, by culture-independent microbial ecological techniques and community-level physiological profiling. Bacterial 16S rDNA libraries indicated that proportion of the bacteria in phyla Chloroflexi and Acidobacteria decreased, but that of Gammaproteobacteria and Planctomycetes slightly increased in copper-treated sediment. Denaturing gradient gel profiles showed that bacterial communities in control and copper exposed sediments developed into different directions, while the copper exposure did not change the pattern of ammonia oxidizing bacterial community. Microbial community-level physiological profiling revealed an obvious response to copper dosage. The copper pollution caused an acute decrease of carbon utilizing ability as well as bacterial functional diversity; the number of culturable heterotrophic bacteria was reduced by 90%. This study demonstrated that high copper input would obviously reduce culturable bacterial counts and seriously impact bacterial community function in marine sediments.