rRNA Gene Profiling Research Articles

Incidence and predictive biomarkers of Clostridioides difficile infection in hospitalized patients receiving broad-spectrum antibiotics

Trial enrichment using gut microbiota derived biomarkers by high-risk individuals can improve the feasibility of randomized controlled trials for prevention of Clostridioides difficile infection (CDI). Here, we report in a prospective observational cohort study the incidence of CDI and assess potential clinical characteristics and biomarkers to predict CDI in 1,007 patients ≥ 50 years receiving newly initiated antibiotic treatment with penicillins plus a beta-lactamase inhibitor, 3rd/4th generation cephalosporins, carbapenems, fluoroquinolones or clindamycin from 34 European hospitals. The estimated 90-day cumulative incidences of a first CDI episode is 1.9% (95% CI 1.1-3.0). Carbapenem treatment (Hazard Ratio (95% CI): 5.3 (1.7-16.6)), toxigenic C. difficile rectal carriage (10.3 (3.2-33.1)), high intestinal abundance of Enterococcus spp. relative to Ruminococcus spp. (5.4 (2.1-18.7)), and low Shannon alpha diversity index as determined by 16 S rRNA gene profiling (9.7 (3.2-29.7)), but not normalized urinary 3-indoxyl sulfate levels, predicts an increased CDI risk.

16S rRNA Gene and Transcript Profiling: an Application on Full-scale Anaerobic Reactors of Wastewater Sludges

Sludge anaerobic digestion is a wastewater treatment plant crucial point for the impact mitigation on environment. Such process reduces the disposable sludge volume and pollution potential, generating biogas. Despite its crucial role, sludge digestion microbiology is not clearly understood. In this work, the characterization of the microbiota into digesters fed by primary and secondary sludges was performed. Two sampling sessions were conducted and more than 120 samples were extracted and analyzed by PCR-DGGE. Cluster analysis on DGGE profiles suggested the presence of diverse communities associated with different sludge characteristics (ANOVA, p < 0.01) and Shannon’s diversity index showed higher evenness into the secondary one. Moreover, the comparison of community structure based on DNA and bacterial rRNA genes highlighted the presence of unexpressed bacterial genome, leading to a potential functionality overestimation using a DNA-based approach (Shannon index mean − 16%, t test, p < 0.05). This approach has proven to be suitable for a first useful assessment of microbial community dynamics during the anaerobic digestion process.

Microbial rRNA gene expression and co-occurrence profiles associate with biokinetics and elemental composition in full-scale anaerobic digesters.

SummaryThis study examined whether the abundance and expression of microbial 16S rRNA genes were associated with elemental concentrations and substrate conversion biokinetics in 20 full‐scale anaerobic digesters, including seven municipal sewage sludge (SS) digesters and 13 industrial codigesters. SS digester contents had higher methane production rates from acetate, propionate and phenyl acetate compared to industrial codigesters. SS digesters and industrial codigesters were distinctly clustered based on their elemental concentrations, with higher concentrations of NH 3‐N, Cl, K and Na observed in codigesters. Amplicon sequencing of 16S rRNA genes and reverse‐transcribed 16S rRNA revealed divergent grouping of microbial communities between mesophilic SS digesters, mesophilic codigesters and thermophilic digesters. Higher intradigester distances between Archaea 16S rRNA and rRNA gene profiles were observed in mesophilic codigesters, which also had the lowest acetate utilization biokinetics. Constrained ordination showed that microbial rRNA and rRNA gene profiles were significantly associated with maximum methane production rates from acetate, propionate, oleate and phenyl acetate, as well as concentrations of NH 3‐N, Fe, S, Mo and Ni. A co‐occurrence network of rRNA gene expression confirmed the three main clusters of anaerobic digester communities based on active populations. Syntrophic and methanogenic taxa were highly represented within the subnetworks, indicating that obligate energy‐sharing partnerships play critical roles in stabilizing the digester microbiome. Overall, these results provide new evidence showing that different feed substrates associate with different micronutrient compositions in anaerobic digesters, which in turn may influence microbial abundance, activity and function.

Impacts of varying durations of passive oxygen exposure on freshwater denitrifier community structure and function

Fertilizer use has dramatically increased the availability of nitrate (NO3 −) in aquatic systems. Microbe-mediated denitrification is one of the predominant means of NO3 − removal from freshwaters, yet oxygenation (O2)-induced disruptions—e.g., extreme precipitation events—can occur, resulting in a disproportional increase in nitrous oxide (N2O) production and efflux as facultative anaerobic bacterial populations use of O2 as a terminal electron acceptor increases. We examined the effects of 12- and 24-h passive O2 exposure on previously anaerobic bacterial communities focusing on denitrification enzyme activity (DEA), N2O production, and bacterial community 16S rRNA and nitrous oxide reductase gene (nosZ) profiles after 12, 24, and 48 h of anaerobic recovery. Treatments experiencing 24-h O2 exposure had significantly higher DEA 12 h into anaerobic recovery than treatments undergoing 12-h O2 exposure. Initial N2O emissions were significantly lower in the 24-h O2 exposure treatments although by 24 h a dramatic spike (tenfold relative to the 12-h O2 exposure treatments) in N2O concentrations was observed. However, within 6 h (30-h anaerobic recovery) these differences were gone. Community nosZ profiles experiencing 24-h O2 exposure exhibited reduced diversity after 24-h recovery, which corresponded with an increase in N2O emissions. However, after 48 h of anaerobic recovery, nosZ diversity had recovered. These observations highlight the effects of short-term aerobic disruption on denitrification, as well as the effects on the denitrifier community profile. Together, these data suggest that recovery to ambient N cycling is exacerbated by disturbance length due to increased lag time and subsequent loss of denitrifier community diversity.

Water management impacts rice methylmercury and the soil microbiome

Rice farmers are pressured to grow rice using less water. The impacts of water-saving rice cultivation methods on rice methylmercury concentrations are uncertain. Rice (Oryza sativa L. cv. Nipponbare) was cultivated in fields using four water management treatments, including flooded (no dry-downs), alternating wetting and drying (AWD) (with one or three dry-downs), and furrow-irrigated fields (nine dry-downs) (n=16 fields). Anoxic bulk soil was collected from rice roots during the rice maturation phase, and rice grain was harvested after fields were dried. Total mercury and methylmercury concentrations were determined in soil and polished rice samples, and the soil microbiome was analyzed using 16S (v4) rRNA gene profiling. Soil total mercury did not differ between fields. However, compared to continuously flooded fields, soil and rice methylmercury concentrations averaged 51% and 38% lower in the AWD fields, respectively, and 95% and 96% lower in the furrow-irrigated fields, respectively. Compared to flooded fields, grain yield was reduced on average by <1% in the AWD fields and 34% in the furrow-irrigated fields. Additionally, using 16S (v4) rRNA gene profiling, the relative abundance of genera (i.e., highest resolution via this method) known to contain mercury methylators averaged 2.9-fold higher in flooded and AWD fields compared to furrow-irrigated fields. These results reinforce the benefits of AWD in reducing rice methylmercury concentrations with minimal changes in rice production yields. In the furrow-irrigated fields, a lower relative abundance of genera known to contain mercury methylators suggests an association between lower concentrations of soil and rice methylmercury and specific soil microbiomes.

Ability of a haloalkaliphilic bacterium isolated from Soap Lake, Washington to generate electricity at pH 11.0 and 7% salinity

A variety of anaerobic bacteria have been shown to transfer electrons obtained from organic compound oxidation to the surface of electrodes in microbial fuel cells (MFCs) to produce current. Initial enrichments for iron (III) reducing bacteria were set up with sediments from the haloalkaline environment of Soap Lake, Washington, in batch cultures and subsequent transfers resulted in a culture that grew optimally at 7.0% salinity and pH 11.0. The culture was used to inoculate the anode chamber of a MFC with formate as the electron source. Current densities up to 12.5 mA/m2 were achieved by this bacterium. Cyclic voltammetry experiments demonstrated that an electron mediator, methylene blue, was required to transfer electrons to the anode. Scanning electron microscopic imaging of the electrode surface did not reveal heavy colonization of bacteria, providing evidence that the bacterium may be using an indirect mode of electron transfer to generate current. Molecular characterization of the 16S rRNA gene and restriction fragment length profiles (RFLP) analysis showed that the MFC enriched for a single bacterial species with a 99% similarity to the 16S rRNA gene of Halanaerobium hydrogeniformans. Though modest, electricity production was achieved by a haloalkaliphilic bacterium at pH 11.0 and 7.0% salinity.

Microbial studies of a selenium-contaminated mine site and potential for on-site remediation

Surface water Selenium (Se) concentrations are above regulatory standards at several active and inactive phosphate mine sites in the US Western Phosphate Resource Area. The focus of the present study was to examine the impacts of the microbial communities on the oxidation state of Se in overburden waste from the Smoky Canyon phosphate mine in Idaho, USA. Microbial populations were found that reduce soluble selenate (SeO (4) (2-) ) to insoluble elemental Se. Microcosm experiments were conducted for molecular genetic analysis of this microbial community by rRNA gene profiling. An acetone pretreatment step was developed to remove interfering pre-petroleum hydrocarbons from the samples prior to extraction. PCR was used to amplify 16S and 18S rRNA genes present in the microbial community DNA. The amplified products were subjected to denaturing gradient gel electrophoresis (DGGE). Isolates and excised DGGE bands were amplified and sequenced for identification to determine the relative importance of culturable isolates to the total microbial population. Analysis of samples from different sites at the mine showed how Se contamination and previous remediation treatments changed the microbial populations across the site. Members of the family Enterobacteriaceae were dominant among the selenate reducing isolates from the site containing high Se levels. In particular, Serratia fonticola was isolated repeatedly from contaminated Smoky Canyon Mine site samples. Packed column studies were performed with seleniferous waste rock fractions from Smoky Canyon Mine. Column amendments consisted of combinations of iron, compost, and whey. Eh, pH, and extractable Se measurements were taken. Tests with infiltrated water showed columns containing an organic amendment combined with iron metal were the most resistant to Se leaching. Iron-based compounds from the corroding metal are thought to strongly bind the Se reduced by microbial activity, thereby stabilizing the Se in an insoluble form. We conclude that long-term stabilization of selenium at contaminated mine sites may require reductive microbial processes combined with abiotic immobilization by iron, either natural or engineered, to stabilize the Se and retard re-oxidation and release. Iron-selenide or iron-selenite compounds are more stable and resistant to leaching, especially when removed from active weathering.

Recent emergence of new variants of Yersinia pestis in Madagascar.

Yersinia pestis, the causative agent of plague, has been responsible for at least three pandemics. During the last pandemic, which started in Hong Kong in 1894, the microorganism colonized new, previously unscathed geographical areas where it has become well established. The aim of this longitudinal study was to investigate the genetic stability of Y. pestis strains introduced into a new environment just under a century ago and to follow the epidemiology of any new genetic variant detected. In the present study, 187 strains of Y. pestis isolated between 1939 and 1996 from different regions of Madagascar and responsible mainly for human cases of bubonic and pneumonic plague were studied. Our principal genotyping method was rRNA gene profiling (ribotyping), which has previously been shown to be an effective scheme for typing Y. pestis strains of different geographical origins. We report that all studied Y. pestis strains isolated in Madagascar before 1982 were of classical ribotype B, the ribotype attributed to the Y. pestis clone that spread around the world during the third pandemic. In 1982, 1983, and 1994, strains with new ribotypes, designated R, Q, and T, respectively, were isolated on the high-plateau region of the island. Analysis of other genotypic traits such as the NotI genomic restriction profiles and the EcoRV plasmid restriction profiles revealed that the new variants could also be distinguished by specific genomic and/or plasmid profiles. A follow-up of these new variants indicated that strains of ribotypes Q and R have become well established in their ecosystem and have a tendency to spread to new geographical areas and supplant the original classical strain.