Metatranscriptomic Investigation of Adaptation in NO and N2O Production From a Lab-Scale Nitrification Process Upon Repeated Exposure to Anoxic–Aerobic Cycling

The molecular mechanisms of microbial adaptation to repeated anoxic–aerobic cycling were investigated by integrating whole community gene expression (metatranscriptomics) and physiological responses, including the production of nitric (NO) and nitrous (N2O) oxides. Anoxic–aerobic cycling was imposed for 17 days in a lab-scale full-nitrification mixed culture system. Prior to cycling, NO and N2O levels were sustained at 0.097 ± 0.006 and 0.054 ± 0.019 ppmv, respectively. Once the anoxic–aerobic cycling was initiated, peak emissions were highest on the first day (9.8 and 1.3 ppmv, respectively). By the end of day 17, NO production returned to pre-cycling levels (a peak of 0.12 ± 0.007 ppmv), while N2O production reached a new baseline (a peak of 0.32 ± 0.05 ppmv), one order of magnitude higher than steady-state conditions. Concurrently, post-cycling transcription of norBQ and nosZ returned to pre-cycling levels after an initial 5.7- and 9.5-fold increase, while nirK remained significantly expressed (1.6-fold) for the duration of and after cycling conditions. The imbalance in nirK and nosZ mRNA abundance coupled with continuous conversion of NO to N2O might explain the elevated post-cycling baseline for N2O. Metatranscriptomic investigation notably indicated possible NO production by NOB under anoxic–aerobic cycling through a significant increase in nirK expression. Opposing effects on AOB (down-regulation) and NOB (up-regulation) CO2 fixation were observed, suggesting that nitrifying bacteria are differently impacted by anoxic–aerobic cycling. Genes encoding the terminal oxidase of the electron transport chain (ccoNP, coxBC) were the most significantly transcribed, highlighting a hitherto unexplored pathway to manage high electron fluxes resulting from increased ammonia oxidation rates, and leading to overall, increased NO and N2O production. In sum, this study identified underlying metabolic processes and mechanisms contributing to NO and N2O production through a systems-level interrogation, which revealed the differential ability of specific microbial groups to adapt to sustained operational conditions in engineered biological nitrogen removal processes

Multidrug-Resistant Enterobacter cloacae Complex Emerging as a Global, Diversifying Threat

The Enterobacter cloacae complex (ECC) includes common nosocomial pathogens capable of producing a wide variety of infections. Broad-spectrum antibiotic resistance, including the recent emergence of resistance to last-resort carbapenems, has led to increased interest in this group of organisms and carbapenem-resistant E. cloacae complex (CREC) in particular. Molecular typing methods based on heat-shock protein sequence, pulsed-field gel electrophoresis, comparative genomic hybridization, and, most recently, multilocus sequence typing have led to the identification of over 1069 ECC sequence types in 18 phylogenetic clusters across the globe. Whole-genome sequencing and comparative genomics, moreover, have facilitated global analyses of clonal composition of ECC and specifically of CREC. Epidemiological and genomic studies have revealed diverse multidrug-resistant ECC clones including several potential epidemic lineages. Together with intrinsic β-lactam resistance, members of the ECC exhibit a unique ability to acquire genes encoding resistance to multiple classes of antibiotics, including a variety of carbapenemase genes. In this review, we address recent advances in the molecular epidemiology of multidrug-resistant E. cloacae complex, focusing on the global expansion of CREC

Structural and Functional Interrogation of Selected Biological Nitrogen Removal Systems in the United States, Denmark, and Singapore Using Shotgun Metagenomics

Conventional biological nitrogen removal (BNR), comprised of nitrification and denitrification, is traditionally employed in wastewater treatment plants (WWTPs) to prevent eutrophication in receiving water bodies. More recently, the combination of selective ammonia to nitrite oxidation (nitritation) and autotrophic anaerobic ammonia oxidation (anammox), collectively termed deammonification, has also emerged as a possible energy- and cost-effective BNR alternative. Herein, we analyzed microbial diversity and functional potential within 13 BNR processes in the United States, Denmark, and Singapore operated with varying reactor configuration, design, and operational parameters. Using next-generation sequencing and metagenomics, gene-coding regions were aligned against a custom protein database expanded to include all published aerobic ammonia oxidizing bacteria (AOB), nitrite oxidizing bacteria (NOB), anaerobic ammonia oxidizing bacteria (AMX), and complete ammonia oxidizing bacteria (CMX). Overall contributions of these N-cycle bacteria to the total functional potential of each reactor was determined, as well as that of several organisms associated with denitrification and/or structural integrity of microbial aggregates (biofilm or granules). The potential for these engineered processes to foster a broad spectrum of microbial catabolic, anabolic, and carbon assimilation transformations was elucidated. Seeded sidestream DEMON® deammonification systems and single-stage nitritation-anammox moving bed biofilm reactors (MBBRs) and a mainstream Cleargreen reactor designed to enrich in AOB and AMX showed lower enrichment in AMX functionality than an enriched two-stage nitritation-anammox MBBR system treating mainstream wastewater. Conventional BNR systems in Singapore and the United States had distinct metagenomes, especially relating to AOB. A hydrocyclone process designed to recycle biomass granules for mainstream BNR contained almost identical structural and functional characteristics in the overflow, underflow, and inflow of mixed liquor (ALT) rather than the expected selective enrichment of specific nitrifying or AMX organisms. Inoculum used to seed a sidestream deammonification process unexpectedly contained <10% of total coding regions assigned to AMX. These results suggest the operating conditions of engineered bioprocesses shape the resident microbial structure and function far more than the bioprocess configuration itself. We also highlight the advantage of a systems- and metagenomics-based interrogation of both the microbial structure and potential function therein over targeting of individual populations or specific genes

Klebsiella pneumoniae induces host metabolic stress that promotes tolerance to pulmonary infection

K. pneumoniae sequence type 258 (Kp ST258) is a major cause of healthcare-associated pneumonia. However, it remains unclear how it causes protracted courses of infection in spite of its expression of immunostimulatory lipopolysaccharide, which should activate a brisk inflammatory response and bacterial clearance. We predicted that the metabolic stress induced by the bacteria in the host cells shapes an immune response that tolerates infection. We combined in situ metabolic imaging and transcriptional analyses to demonstrate that Kp ST258 activates host glutaminolysis and fatty acid oxidation. This response creates an oxidant-rich microenvironment conducive to the accumulation of anti-inflammatory myeloid cells. In this setting, metabolically active Kp ST258 elicits a disease-tolerant immune response. The bacteria, in turn, adapt to airway oxidants by upregulating the type VI secretion system, which is highly conserved across ST258 strains worldwide. Thus, much of the global success of Kp ST258 in hospital settings can be explained by the metabolic activity provoked in the host that promotes disease tolerance. Keywords: immunometabolism, Klebsiella pneumoniae, immunosuppressive, anti-inflammatory, itaconate, Type Six Secretion Syste

Comammox Functionality Identified in Diverse Engineered Biological Wastewater Treatment Systems

Complete ammonia oxidation (comammox) to nitrate by certain <i>Nitrospira</i>-lineage bacteria (CMX) could contribute to overall nitrogen cycling in engineered biological nitrogen removal (BNR) processes in addition to the more well-documented nitrogen transformations by ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), and anaerobic ammonia-oxidizing (anammox) bacteria (AMX). A metagenomic survey was conducted to quantify the presence and elucidate the potential functionality of CMX in 16 full-scale BNR configurations treating mainstream or sidestream wastewater. CMX proposed to date were combined with previously published AOB, NOB, and AMX genomes to create an expanded database for alignment of metagenomic reads. CMX-assigned metagenomic reads accounted for between 0.28 and 0.64% of total coding DNA sequences in all BNR configurations. Phylogenetic analysis of key nitrification functional genes <i>amoA</i>, encoding the α-subunit of ammonia monooxygenase, <i>haoB</i>, encoding the β-subunit of hydroxylamine oxidoreductase, and <i>nxrB</i>, encoding the β-subunit of nitrite oxidoreductase, confirmed that each BNR system contained coding regions for production of these enzymes by CMX specifically. Ultimately, the ubiquitous presence of CMX bacteria and metabolic functionality in such diverse system configurations emphasizes the need to translate novel bacterial transformations to engineered biological process interrogation, operation, and design

Associations between urinary 3-indoxyl sulfate, a gut microbiome-derived biomarker, and patient outcomes after intensive care unit admission

Purpose 3-indoxyl sulfate (3-IS) is an indole metabolism byproduct produced by commensal gut bacteria and excreted in the urine; low urinary 3-IS has been associated with increased mortality in bone marrow transplant recipients. This study investigated urinary 3-IS and patient outcomes in the ICU. Materials and methods Prospective study that collected urine samples, rectal swabs, and clinical data on 78 adult ICU patients at admission and again 72 h later. Urine was analyzed for 3-IS by mass spectrometry. Results Median urinary 3-IS levels were 17.1 μmol/mmol creatinine (IQR 9.5 to 26.2) at admission and 15.6 (IQR 4.2 to 30.7) 72 h later. 22% of patients had low 3-IS (≤6.9 μmol/mmol) on ICU admission and 28% after 72 h. Low 3-IS at 72 h was associated with fewer ICU-free days (22.5 low versus 26 high, p = 0.03) and with death during one year of follow-up (36% low versus 9% high 3-IS, p < 0.01); there was no detectable difference in 30-day mortality (18% low versus 5% high, p = 0.07). Conclusions Low urinary 3-IS level 72 h after ICU admission was associated with fewer ICU-free days and with increased one-year but not 30-day mortality. Further studies should investigate urinary 3-IS as an ICU biomarker