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bioRxiv2020

Cloning and functional expression of a food-grade circular bacteriocin, plantacyclin B21AG, in probiotic Lactobacillus plantarum WCFS1

There is an increasing consumer demand for minimally processed, preservative free and microbiologically safe food. These factors, combined with risks of antibiotic resistance, have led to interest in bacteriocins produced by lactic acid bacteria (LAB) as natural food preservatives and as protein therapeutics. We previously reported the discovery of plantacyclin B21AG, a novel circular bacteriocin produced by Lactobacillus plantarum B21. Here, we describe the cloning and functional expression of the bacteriocin gene cluster in the probiotic Lactobacillus plantarum WCFS1. Genome sequencing demonstrated that the bacteriocin is encoded on a 20 kb native plasmid, designated as pB21AG01. Seven open reading frames (ORFs) putatively involved in bacteriocin production, secretion and immunity were cloned into an E. coli/Lactobacillus shuttle vector, pTRKH2. The resulting plasmid, pCycB21, was transformed into L. plantarum WCFS1. The cell free supernatants (CFS) of both B21 and WCFS1 (pCycB21) showed an antimicrobial activity of 800 AU/mL when tested against the WCFS1 (pTRKH2) indicator strain, indicating functional expression of plantacyclin B21AG. Real-time PCR analysis revealed that the relative copy number of pB21AG01 was 7.60 {+/-} 0.79 in L. plantarum B21 whilst pCycB21 and pTRKH2 was 0.51 {+/-} 0.05 and 25.19 {+/-} 2.68 copies, respectively in WCFS1. This indicates that the bacteriocin gene cluster is located on a highly stable, low copy number plasmid pB21AG01 in L. plantarum B21. Inclusion of the native promoter for the bacteriocin operon from pB21AG01 may result in similar inhibitory zones observed in both wild type and recombinant hosts despite the low copy number of pCycB21.

microbiology↗

bioRxiv2020

Investigation of a night soil compost psychrotrophic bacterium Glutamicibacter arilaitensis LJH19 for its safety, efficient hydrolytic and plant growth-promoting potential

Night soil compost (NSC) has traditionally been a source of organic manure in north-western Himalaya. Lately, this traditional method is declining due to modernization, its unhygienic conditions and social apprehensions. Reduction in the age-old traditional practice has led to excessive usage of chemical fertilizers and shortage of water in the eco-sensitive region. Microbiological intervention was attempted to obtain bacterial consortia for accelerated degradation of human faeces in cold climate to improvise this traditional knowledge. Glutamicibacter arilaitensis LJH19, a psychrotrophic bacteria was identified as one such potential candidate for the proposed consortia. The bacterium was isolated from NSC of Lahaul valley and exhibited potential hydrolytic activities, the specific activities of amylase, cellulase and xylanase was observed as 186.76 U/mg, 21.85 U/mg and 11.31 U/mg respectively. Additionally, the strain possessed multiple plant growth-promoting (PGP) traits. The bacterium produced 166.11 {micro}g/ml indole acetic acid and 85.72 % siderophore units, and solubilized 44.76 {micro}g/ml phosphate. Whole genome sequence (3,602,821 bps) endorsed the cold adaptation, polysaccharide metabolism, PGP potential of the bacterium. Genome mining revealed biosynthetic gene clusters for type III polyketide synthase (PKS), terpene, and siderophore in agreement with its potential PGP traits. Comparative genomics within the genus revealed 217 unique genes specific to hydrolytic and PGP activity. Negative haemolysis and biofilm production and susceptibility towards all 12 tested antibiotics indicated the bacterium to be a safe bioinoculant. Genomic investigation supported the bacterium safety with absence of any virulence and antibiotic resistance genes. We propose the strain LJH19 to be a potentially safe bioinoculant candidate for efficient degradation of night soil owing to its survivability in cold and its efficient hydrolytic and PGP potential.

microbiology↗

bioRxiv2020

Functional and genetic markers of niche partitioning among enigmatic members of the human oral microbiome

Microbial residents of the human oral cavity have long been a major focus of microbiology due to their influence on host health and their intriguing patterns of site specificity amidst the lack of dispersal limitation. Yet, the determinants of niche partitioning in this habitat are yet to be fully understood, especially among the taxa that belong to recently discovered branches of microbial life. Here we assembled metagenomes from daily tongue and dental plaque samples from multiple individuals and reconstructed 790 non-redundant genomes, 43 of which resolved to TM7 that formed six monophyletic clades distinctly associated either with plaque or with tongue. Both pangenomic and phylogenomic analyses grouped tongue-specific TM7 clades with other host-associated TM7 genomes. In contrast, plaque-specific TM7 grouped together with environmental TM7 genomes. Besides offering deeper insights into the ecology, evolution, and the mobilome of the cryptic members of the oral microbiome, our study reveals an intriguing resemblance between dental plaque and non-host environments indicated by the TM7 evolution, suggesting that plaque may have served as a stepping stone for environmental microbes to adapt to host environments for some clades of human associated microbes. Additionally, we report that prophages are widespread amongst oral-associated TM7, while absent from environmental TM7, suggesting that prophages may have played a role in adaptation of TM7 to the host environment.

microbiology↗

bioRxiv2020

Evaluation of the EUROIMMUN Anti-SARS-CoV-2 ELISA Assay for detection of IgA and IgG antibodies

As the Coronavirus 2019 (COVID-19) pandemic evolves, the development of immunoassays to help determine exposure and potentially predict immunity has become a pressing priority. In this report we present the performance of the EUROIMMUN enzyme-linked immunosorbent assay (ELISA) for semi-quantitative detection of IgA and IgG antibodies in serum and plasma samples using recombinant S1 domain of the SARS-CoV-2 spike protein as antigen. Specimens from patients, with and without COVID-19 infection, were tested at the University of Chicago Clinical Microbiology and Immunology Laboratory. Of 57 samples from COVID-19 PCR-negative patients, including 28 samples positive for common human coronavirus strains, 53 tested negative and 4 tested positive for IgA (93.0% agreement) while 56 tested negative and 1 tested positive for IgG (98.2% agreement). For COVID-19 PCR-positive patients, 29 of 30 (96.7%) samples collected [≥]3 days after positive PCR were positive for IgA, and 28 of 28 samples collected [≥]4 days after positive PCR were positive for IgG. The EUROIMMUN Anti-SARS-CoV-2 ELISA Assay demonstrates excellent sensitivity for detection of IgA and IgG antibodies from samples collected [≥]3 days and [≥]4 days, respectively, after COVID-19 diagnosis by PCR. This assay did not demonstrate cross reaction in any of the 28 samples from patients with common human coronaviruses, including types HKU1, NL63, CV229E, and OC43.

microbiology↗

bioRxiv2020

Differential effects of antibiotic treatment with piperacillin/tazobactam or ceftriaxone on the murine gut microbiota

Effective antimicrobial stewardship requires a better understanding of the impact of different antibiotics on the gut microflora. Studies in humans are confounded by large inter-individual variability and difficulty in identifying control cohorts. However, controlled murine models can provide valuable information. We examined the impact of a penicillin-like antibiotic (piperacillin/tazobactam, TZP) or a third-generation cephalosporin (ceftriaxone, CRO) on the murine gut microbiota. We analyzed gut microbiome composition by 16S-rRNA amplicon sequencing and effects on the Enterobacteriaceae by qPCR and standard microbiology. Colonization resistance to multidrug resistant Escherichia coli ST131 and Klebsiella pneumoniae ST258 was also tested. Changes in microbiome composition and a significant (p<0.001) decrease in diversity occurred in all treated mice, but were more marked and longer lasting after CRO exposure with a persistent rise in Proteobacteria levels. Increases in the Enterobacteriaceae occurred in all antibiotic treated mice, but were transient and associated with direct antibiotic pressure. Co-habitation of treated and untreated mice attenuated the detrimental effect of antibiotics on treated animals, but also caused disturbance in untreated co-habitants. At the height of dysbiosis after antibiotic termination, the murine gut was highly susceptible to colonization with both multidrug resistant pathogens. The administration of a third-generation cephalosporin caused a significantly prolonged dysbiosis in the murine gut microflora, when compared to a penicillin/{beta}-lactam inhibitor combination with comparable activity against medically important virulent bacteria. At the height of dysbiosis, both antibiotic treatments equally led to microbial imbalance associated with loss of resistance to gut colonization by antibiotic-resistant pathogens.

microbiology↗

bioRxiv2020

Deep sequencing reveals Campylobacter in commercial meat chickens less than 8 days old.

Campylobacter from contaminated poultry meat is a major source of human gastroenteritis worldwide. To date, attempts to control this zoonotic infection with on-farm biosecurity measures have been inconsistent. A cornerstone of these efforts has been the detection of chicken infection with microbiological culture, which typically does not occur until birds are at least 21 days old. Using molecular methods for detecting Campylobacter presence, 16S microbiome analysis and deep sequencing of the Campylobacter porA gene, Campylobacter can be identified at very low levels in most or all flocks fewer than 8 days old. These young chicks exhibit a much greater diversity of porA types than older birds testing positive for Campylobacter by culture or qPCR. This suggests that, as the bacteria multiply sufficiently to be detected by culture methods, one or two strains, as indicated by porA type, dominate the infection. The findings that (i) most young chicks carry some Campylobacter and (ii) not all flocks become Campylobacter positive by culture, suggests that efforts to control infection should concentrate on how to maintain Campylobacter at low levels by the prevention of the overgrowth of single strains, which ultimately leads to the contamination of food.

microbiology↗

bioRxiv2020

Gnotobiotic rainbow trout (Oncorhynchus mykiss) model reveals endogenous bacteria that protect against Flavobacterium columnare infection

The health and environmental risks associated with antibiotic use in aquaculture have promoted bacterial probiotics as an alternative approach to control fish infections in vulnerable larval and juvenile stages. However, evidence-based identification of probiotics is often hindered by the complexity of bacteria-host interactions and host variability in microbiologically uncontrolled conditions. While these difficulties can be partially resolved using gnotobiotic models harboring no or reduced microbiota, most host-microbe interaction studies are carried out in animal models with little relevance for fish farming. Here we studied host-microbiota-pathogen interactions in a germ-free and gnotobiotic model of rainbow trout (Oncorhynchus mykiss), one of the most widely cultured salmonids. We demonstrated that germ-free larvae raised in sterile conditions displayed no significant difference in growth after 35 days compared to conventionally-raised larvae, but were extremely sensitive to infection by Flavobacterium columnare, a common freshwater fish pathogen causing major economic losses worldwide. Furthermore, re-conventionalization with 11 culturable species from the conventional trout microbiota conferred resistance to F. columnare infection. Using mono-re-conventionalized germ-free trout, we identified that this protection is determined by a commensal Flavobacterium strain displaying antibacterial activity against F. columnare. Finally, we demonstrated that use of gnotobiotic trout is a suitable approach for the systematic identification of both endogenous and exogenous probiotic bacterial strains that may protect teleostean hosts against F. columnare and other pathogens. This study establishes a novel and ecologically-relevant gnotobiotic model that will improve the sustainability and health of aquaculture.

microbiology↗

bioRxiv2020

ANALYSIS OF MICROBIAL DIVERSITY IN RAW FISH CEVICHE

Ceviche is a traditional dish made from raw fish meat marinated in lime juice without any heat cooking step throughout its preparation process. Although the use of organic acids as antibacterial agents is well known; recent research indicates that lime juice actually can reduce the risk of V. parahemolyticus infections but it is ineffective against other potential pathogens. Despite the fact that fresh fish meat is safe; exposed organs including skin, gills and guts represent a potential source of bacterial contamination. In Mexico, diarrheal diseases are caused mainly by contaminated food; it is estimated that almost 67% of infections are due the presence of bacterial agents mainly in frozen and fresh fish. The main objective of this study was to estimate the taxonomic diversity of microbial species present in ready-to-eat ceviche using a metagenomic approach. Six samples of commercially available ceviche were subjected to DNA high throughput sequencing and bioinformatics analyses, we identified between 65,000 and 131,000 reads per sample. The predominant phyla identified through the samples were Proteobacteria, Bacteroidetes and Firmicutes. We discuss the factors involved in the microbiological quality of this kind of raw foods and how they influence the bacterial diversity within the analyzed samples.

microbiology↗

bioRxiv2020

Grad-seq shines light on unrecognized RNA and protein complexes in the model bacterium Escherichia coli

Stable protein complexes, including those formed with RNA, are major building blocks of every living cell. Escherichia coli has been the leading bacterial organism with respect to global protein-protein networks. Yet, there has been no global census of RNA/protein complexes in this model species of microbiology. Here, we performed Grad-seq to establish an RNA/protein complexome, reconstructing sedimentation profiles in a glycerol gradient for ~85% of all E. coli transcripts and ~49% of the proteins. These include the majority of small noncoding RNAs (sRNAs) detectable in this bacterium as well as the general sRNA-binding proteins, CsrA, Hfq and ProQ. In presenting use cases for utilization of these RNA and protein maps, we show that a stable association of RyeG with 30S ribosomes gives this seemingly noncoding RNA of prophage origin away as an mRNA of a toxic small protein. Similarly, we show that the broadly conserved uncharacterized protein YggL is a 50S subunit factor in assembled 70S ribosomes. Overall, this study crucially extends our knowledge about the cellular interactome of the primary model bacterium E. coli through providing global RNA/protein complexome information and should facilitate functional discovery in this and related species.

microbiology↗

bioRxiv2020

Overexpression of blaOXA-58 Gene Driven by ISAba3 is Associated with Imipenem Resistance in a Clinical Acinetobacter baumannii Isolate from Vietnam

The aim of this study was to investigate genetic structures and expression of blaOXA-58 gene in five Acinetobacter baumannii clinical isolates recovered from two hospitals in southern Vietnam during 2012-2014. A. baumannii isolates were identified by automated microbiology systems and confirmed by PCR. All isolates were characterized as multidrug resistant by antimicrobials testing using the disk diffusion method. Four imipenem susceptible and one non-susceptible isolates (MIC > 32 g.ml-1) were identified by E-test. PCR amplification of blaOXA-58 gene upstream and downstream sequences revealed the presence of ISAba3 at both locations in one multidrug resistant isolate. Semi quantitation of blaOXA-51 and blaOXA-58 gene expression was performed by the 2-{Delta}{Delta}Ct method. The blaOXA-51 gene expression of five isolates showed little difference but the isolate bearing ISAba3-blaOXA-58-ISAba3 exhibited significant higher blaOXA-58 mRNA level. Higher {beta}-lactamases activity in periplasmic than cytoplasmic fraction was found in most isolates. The isolate overexpressing blaOXA-58 gene possessed very high periplasmic enzyme activity. In conclusion, the A. baumannii isolate bearing ISAba3-blaOXA-58 gene exhibited high resistance to imipenem, corresponding to an overexpression of blaOXA-58 gene and very high periplasmic {beta}-lactamases activity.

microbiology↗

bioRxiv2020

An inducible AraC that responds to blue light instead of arabinose

In Escherichia coli, the operon responsible for the catabolism of L-arabinose is regulated by the dimeric DNA-binding protein AraC. In the absence of L-arabinose, AraC binds to the distal I1 and O2 half-sites, leading to repression of the downstream PBAD promoter. In the presence of the sugar, the dimer changes conformation and binds to the adjacent I1 and I2 half-sites, resulting in the activation of PBAD. Here we engineer blue light-inducible AraC dimers in Escherichia coli (BLADE) by swapping the dimerization domain of AraC with blue light-inducible dimerization domains. Using BLADE to overexpress proteins important for cell shape and division site selection, we reversibly control cell morphology with light. We demonstrate the exquisite light responsiveness of BLADE by employing it to create bacteriographs with an unprecedented quality. We then employ it to perform a medium-throughput characterization of 39 E. coli genes with poorly defined or completely unknown function. Finally, we expand the initial library and create a whole family of BLADE transcription factors (TFs), which we characterize using a novel 96-well light induction setup. Since the PBAD promoter is commonly used by microbiologists, we envisage that the BLADE TFs will bring the many advantages of optogenetic gene expression to the field of microbiology.

microbiology↗

bioRxiv2020

Hydrogen peroxide production by Streptococcus pneumoniae results in alpha-hemolysis by oxidation of oxy-hemoglobin to met-hemoglobin

Streptococcus pneumoniae (Spn) and other streptococci produce a greenish halo on blood agar plates referred to as -hemolysis. This phenotype is utilized by clinical microbiology laboratories to report culture findings of -hemolytic streptococci, including Spn, and other bacteria. The -hemolysis halo on blood agar plates has been related to the hemolytic activity of pneumococcal pneumolysin (Ply), or to a lesser extent, to lysis of erythrocytes by Spn-produced hydrogen peroxide. We investigated the molecular basis of the -hemolysis halo produced by Spn. Wild-type strains TIGR4, D39, R6, and EF3030, and isogenic derivative {Delta}ply mutants, produced a similar -hemolytic halo on blood agar plates while cultures of hydrogen peroxide knockout {Delta}spxB/{Delta}lctO mutants lacked this characteristic halo. Spectroscopic studies demonstrated that culture supernatants of TIGR4 released hemoglobin-bound heme (heme-hemoglobin) from erythrocytes and oxidized oxy-hemoglobin to met-hemoglobin within 30 min of incubation. As expected, given Ply hemolytic activity, and that hydrogen peroxide contributes to the release of Ply, TIGR4 isogenic mutants {Delta}ply and {Delta}spxB/{Delta}lctO had a significantly decreased release of heme-hemoglobin from erythrocytes. However, TIGR4{Delta}ply that produces hydrogen peroxide oxidized oxy-hemoglobin to met-hemoglobin, whereas TIGR4{Delta}spxB/{Delta}lctO failed to produce oxidation of oxy-hemoglobin. We demonstrated that the so-called -hemolysis halo is caused by the oxidation oxy-hemoglobin (Fe+2) to a non-oxygen binding met-hemoglobin (Fe+3) by Spn-produced hydrogen peroxide. Since Spn colonizes the human lung, oxidation of oxy-hemoglobin might have important implications for pathogenesis. ImportanceThere is a misconception that -hemolysis observed on blood agar plates cultures of Streptococcus pneumoniae (Spn), and other -hemolytic streptococci is produced by a hemolysin, or alternatively, by lysis of erythrocytes caused by hydrogen peroxide. We noticed in the course of our investigations that wild-type Spn strains and hemolysin (e.g., pneumolysin) knockout mutants, produced the -hemolytic halo on blood agar plates. In contrast, hydrogen peroxide defective mutants prepared in four different strains lacked the characteristic -hemolysis halo. We also demonstrated that wild-type strains and pneumolysin mutants oxidized oxy-hemoglobin to met-hemoglobin. Hydrogen peroxide knockout mutants, however, failed to oxidize oxy-hemoglobin. Therefore, the greenish halo formed on cultures of Spn and other so-called -hemolytic streptococci is caused by the oxidation of oxy-hemoglobin produced by hydrogen peroxide. Oxidation of oxy-hemoglobin to the non-binding oxygen form, met-hemoglobin, might occur in the lungs during pneumococcal pneumonia.

microbiology↗

bioRxiv2020

POPULATION GENOMICS REVEALS MOLECULAR DETERMINANTS OF SPECIALIZATION TO TOMATO IN THE POLYPHAGOUS FUNGAL PATHOGEN BOTRYTIS CINEREA

Many fungal plant pathogens encompass multiple populations specialized on different plant species. Understanding the factors underlying pathogen adaptation to their hosts is a major challenge of evolutionary microbiology, and it should help preventing the emergence of new specialized pathogens on novel hosts. Previous studies have shown that French populations of the grey mould pathogen Botrytis cinerea parasitizing tomato and grapevine are differentiated from each other, and have higher aggressiveness on their host-of-origin than on other hosts, indicating some degree of host specialization in this polyphagous pathogen. Here, we aimed at identifying the genomic features underlying the specialization of B. cinerea populations to tomato and grapevine. Based on whole genome sequences of 32 isolates, we confirmed the subdivision of B. cinerea pathogens into two genetic clusters on grapevine and another, single cluster on tomato. Levels of genetic variation in the different clusters were similar, suggesting that the tomato-specific cluster has not recently emerged following a bottleneck. Using genome scans for selective sweeps and divergent selection, tests of positive selection based on polymorphism and divergence at synonymous and non-synonymous sites and analyses of presence/absence variation, we identified several candidate genes that represent possible determinants of host specialization in the tomato-associated population. This work deepens our understanding of the genomic changes underlying the specialization of fungal pathogen populations.

microbiology↗

bioRxiv2020

Loss of RpoS results in attenuated Escherichia coli colonization of human intestinal organoids and a competitive disadvantage within the germ-free mouse intestine

Pluripotent stem-cell-derived human intestinal organoids (HIOs) are three-dimensional, multicellular structures that model a previously uncolonized, naive intestinal epithelium in an in vitro system. We recently demonstrated that microinjection of the non-pathogenic Escherichia coli strain, ECOR2, into HIOs induced morphological and functional maturation of the HIO epithelium, including increased secretion of mucins and cationic antimicrobial peptides. In the current work, we use ECOR2 as a biological probe to investigate the bacterial response to colonization of the HIO lumen. In E. coli and other Gram-negative bacteria, adaptation to environmental stress is regulated by the general stress response sigma factor, RpoS. We generated an isogenic {triangleup}rpoS ECOR2 mutant to compare challenges faced by a bacterium during colonization of the HIO lumen relative to the germ-free mouse intestine, which is currently the best available system for studying the initial establishment of bacterial populations within the gut. We demonstrate that loss of RpoS significantly decreases the ability of ECOR2 to colonize HIOs, though it does not prevent colonization of germ-free mice. Rather, the {triangleup}rpoS ECOR2 exhibits a fitness defect in the germ-free mouse intestine only in the context of microbial competition. These results indicate that HIOs pose a differentially restrictive luminal environment to E. coli during colonization, thus increasing our understanding of the HIO model system as it pertains to studying the establishment of intestinal host-microbe symbioses. ImportanceTechnological advancements have and will continue to drive the adoption of organoid-based systems for investigating host-microbe interactions within the human intestinal ecosystem. Using E. coli deficient in the RpoS-mediated general stress response, we demonstrate that the type or severity of microbial stressors within the HIO lumen differ from those of the in vivo environment of the germ-free mouse gut. This study provides important insight into the nature of the HIO microenvironment from a microbiological standpoint.

microbiology↗

bioRxiv2020

Genomic and chemical diversity of Bacillus subtilis secondary metabolites against plant pathogenic fungi

Bacillus subtilis produces a wide range of secondary metabolites providing diverse plant-growth-promoting and biocontrol abilities. These secondary metabolites include non-ribosomal peptides (NRPs) with strong antimicrobial properties, causing either cell lysis, pore formation in fungal membranes, inhibition of certain enzymes, or bacterial protein synthesis. However, the natural products of B. subtilis are mostly studied either in laboratory strains or in individual isolates and therefore, a comparative overview of B. subtilis secondary metabolites is missing. In this study, we have isolated 23 B. subtilis strains from eleven sampling sites, compared the fungal inhibition profiles of wild types and their NRPs mutants, followed the production of targeted lipopeptides, and determined the complete genomes of 13 soil isolates. We discovered that non-ribosomal peptide production varied among B. subtilis strains co-isolated from the same soil samples. In vitro antagonism assays revealed that biocontrol properties depend on the targeted plant pathogenic fungus and the tested B. subtilis isolate. While plipastatin alone is sufficient to inhibit Fusarium sp., a combination of plipastatin and surfactin is required to hinder the growth of Botrytis cinerea. Detailed genomic analysis revealed that altered NRP production profiles in certain isolates is due to missing core genes, nonsense mutation, or potentially altered gene regulation. Our study combines microbiological antagonism assays with chemical NRPs detection and biosynthetic gene cluster predictions in diverse B. subtilis soil isolates to provide a broader overview of the secondary metabolite chemodiversity of B. subtilis. IMPORTANCESecondary or specialized metabolites with antimicrobial activities define the biocontrol properties of microorganisms. Members of the Bacillus genus produce a plethora of secondary metabolites, of which non-ribosomally produced lipopeptides in particular display strong antifungal activity. To facilitate prediction of the biocontrol potential of new Bacillus subtilis isolates, we have explored the in vitro antifungal inhibitory profiles of recent B. subtilis isolates, combined with analytical natural product chemistry, mutational analysis, and detailed genome analysis of biosynthetic gene clusters. Such a comparative analysis helped to explain why selected B. subtilis isolates lack production of certain secondary metabolites.

microbiology↗

bioRxiv2020

Longitudinal metatranscriptomic analysis of a meat spoilage microbiome detects abundant continued fermentation and environmental stress responses during shelf life and beyond.

Microbial food spoilage is a complex phenomenon associated with the succession of the specific spoilage organisms (SSO) over the course of time. We performed a longitudinal metatranscriptomic study on a modified atmosphere packaged (MAP) beef product to increase understanding of the longitudinal behavior of a spoilage microbiome during shelf life and onward. Based on the annotation of the mRNA reads, we recognized three stages related to the active microbiome that were descriptive for the sensory quality of the beef: acceptable product (AP), early spoilage (ES) and late spoilage (LS). Both the 16S RNA taxonomic assignments from the total RNA and functional annotations of the active genes showed that these stages were significantly different from each other. However, the functional gene annotations showed more pronounced difference than the taxonomy assignments. Psychrotrophic lactic acid bacteria (LAB) formed the core of the SSO according to the transcribed reads. Leuconostoc species were the most abundant active LAB throughout the study period, whereas the activity of Streptococcaceae (mainly Lactococcus) increased after the product was spoiled. In the beginning of the experiment, the community managed environmental stress by cold-shock responses which were followed by the expression of the genes involved in managing oxidative stress. Glycolysis, pentose phosphate pathway and pyruvate metabolism were active throughout the study at a relatively stable level. However, the proportional activity of the enzymes in these pathways changed over time. For example, acetate kinase activity was characteristic for the AP stage whereas formate C-acetyltransferase transcription was associated with spoilage. ImportanceIt is generally known which organisms are the typical SSO in foods, whereas the actively transcribed genes and pathways during microbial succession are poorly understood. This knowledge is important since better approaches to food quality evaluation and shelf life determination are needed. Thus, we conducted this study to find longitudinal markers that are connected to quality deterioration. These kind of RNA markers could be used to develop novel type of rapid quality analysis tools in the future. New tools are needed since even though SSO can be detected and their concentrations determined using the current microbiological methods, results from these analyses cannot predict how close timewise a spoilage community is from production of clear sensory defects. Main reason for this is that the species composition of a spoilage community does not change dramatically during late shelf life, whereas the ongoing metabolic activities lead to the development of notable sensory deterioration.

microbiology↗

bioRxiv2020

From the Andes to the desert: First characterization of bacterial communities in the Rimac river, the main source of water for Lima, Peru

BackgroundThe Rimac river is the main source of water for Lima, Perus capital megacity. The river is constantly affected by different types of contamination including mine tailings in the Andes and urban sewage in the metropolitan area. We aim to produce the first characterization of bacterial communities in the Rimac river using a 16S rRNA amplicon sequencing approach which would be useful to identify bacterial diversity and potential understudied pathogens. ResultsWe report a higher diversity in bacterial communities from the Upper and, especially, Middle Rimac compared to the Lower Rimac (Metropolitan zone). Samples were generally grouped according to their geographical location. Bacterial classes Alphaproteobacteria, Bacteroidia, Campylobacteria, Fusobacteriia, and Gammaproteobacteria were the most frequent along the river. Arcobacter cryaerophilus (Campylobacteria) was the most frequent species in the Lower Rimac while Flavobacterium succinicans (Bacteroidia) and Hypnocyclicus (Fusobacteriia) were the most predominant in the Upper Rimac. Predicted metabolic functions in the microbiota include bacterial motility, quorum sensing and xenobiotics metabolism. Additional metabolomic analyses showed the presence natural flavonoids and antibiotics in the Upper Rimac, and herbicides in the Lower Rimac. ConclusionsThe dominance in the Metropolitan area of Arcobacter cryaerophilus, an emergent pathogen associated with fecal contamination and antibiotic multiresistance, but that is not usually reported in traditional microbiological quality assessments, highlights the necessity to apply next-generation sequencing tools to improve pathogen surveillance. We believe that our study will encourage the integration of omics sciences in Peru and its application on current environmental and public health issues.

microbiology↗

bioRxiv2020

Microbially-mediated pretreatment of aerated oil and gas produced water by addition of phosphorous or activated sludge

Produced Water (PW) from oil and gas (O&G) producing wells is a unique source of water in water-stressed regions. Microbiota within O&G formations have been well-studied on site/in-situ, but few applied works have considered their role in the treatment of PW in engineered water treatment systems. Herein, we operated a simple aeration/mixing bench-scale bioreactor fed with produced water under three conditions: 1) PW alone (control; hereafter referred to as baseline), 2) phosphorous dosed daily as KH2PO4, and 3) activated sludge (AS) dosed daily from a sequence batch membrane bioreactor (SB-MBR). Aerated and mixed PW alone (baseline) was able to attenuate PW chemistry with removal of soluble chemical oxygen demand (sCOD) and ammonia by 27.6% and 17.8%, respectively. Further KH2PO4 and AS additions improved water treatment efficiency markedly; in the KH2PO4 addition reactor, sCOD and ammonia were reduced by 50.0% and 61.5%, respectively, and in the AS addition reactor by 52.5% and 59.2%, respectively. Microbial consortia determined via 16S rRNA gene amplicons differ in composition between raw PW and all reactors; order Kiloniellales was most common in raw PW while orders Rhodobacterales, Pseudomonadales, and Caulobacterales were most abundant amongst AS, KH2PO4, and baseline conditions, respectively. Several different microbial consortia are capable of treating raw PW which suggests that functional redundancy amongst microbiota in engineered treatment systems may be underappreciated. With simple addition of phosphorous and/or activated sludge to PW as part of a treatment strategy, a higher quality water can then be subjected to conventional treatment and/or local reuse. ImportanceMultiple microbiological communities are capable of treating O&G PW in a simple, applied, engineered setting. The broad possibility of PW treatment by multiple different microbial consortia elucidates the potential for easy, effective, water reuse processes in the hydraulically-stressed arid west as well as any region generating PW from O&G operations.

microbiology↗