Environmental sources along natural cave ripening shape the microbiome and metabolome of artisanal blue-veined cheeses

Microorganisms colonising processing environments can significantly impact food quality and safety. Here we describe a detailed longitudinal study assessing the impact of cave ripening on the microbial succession and quality markers across different producers of blue-veined cheese. Both the producer and cave in which cheeses were ripened significantly influenced the cheese microbiome and metabolome. The cheese microbiome was significantly determined by the microbiome of caves, which were a source of Brevibacterium, Corynebacterium, Staphylococcus, Tetragenococcus and Yaniella, among others, as demonstrated through source tracking and the characterization of 613 metagenome assembled genomes. Tetragenococcus koreensis and T. halophilus were detected at high abundance in cheese for the first time, associated with the occurrence of various metabolites, and showed high levels of horizontal gene transfer with other members of the cheese microbiome. Overall, we demonstrated that processing environments can be a source of non-starter microorganisms of relevance to ripening of artisanal fermented foods.

Aqueous system-level processes and prokaryote assemblages in the ferruginous and sulfate-rich bottom waters of a post-mining lake

In the aqueous oligotrophic ecosystem of a post-mining lake (Lake Medard, Czechia), reductive Fe(II) dissolution outpaces sulfide generation from microbial sulfate reduction (MSR), and ferruginous conditions occur without quantitative sulfate depletion. An isotopically constrained estimate of the rates of sulfate reduction (SRR) suggests that despite a high genetic potential, this respiration pathway is limited by the rather low amounts of metabolizable organic carbon. This points to substrate competition exerted by iron and nitrogen respiring prokaryotes. Yet, the microbial succession across the nitrogenous and ferruginous zones of the bottom water column also indicates sustained genetic potential for chemolithotrophic sulfur oxidation. Therefore, our isotopic SRR estimates could be rather portraying high rates of anoxic sulfide oxidation to sulfate, probably accompanied by microbially induced disproportionation of S intermediates. Near and at the anoxic sediment-water interface, vigorous sulfur cycling can be fuelled by ferric and manganic particulate matter and redeposited siderite stocks. Sulfur oxidation and disproportionation then appear to prevent substantial stabilization of iron monosulfides as pyrite but can enable the interstitial precipitation of small proportions of equant microcrystalline gypsum. This latter mineral isotopically fingerprints sulfur oxidation proceeding at near equilibrium with the ambient anoxic waters, whilst authigenic pyrite-sulfur displays a 38 to 27 ‰ isotopic offset from ambient sulfate, suggestive of incomplete MSR and likely reflective also of an open sulfur cycling system. Pyrite-sulfur fractionation decreases with increased reducible reactive iron in the sediment. In the absence of ferruginous coastal zones today, the current water column redox stratification in the post-mining Lake Medard has scientific value for (i) testing emerging hypotheses on how a few interlinked biogeochemical cycles operated in nearshore paleoenvironments during redox transitional states; and (ii) to acquire insight on how similar early diagenetic redox proxy signals developed in sediments affected by analogue transitional states in ancient water columns.

Characterising the benthic Phormidium autumnale-dominated biofilm community and anatoxin production throughout biofilm succession

<p>There has been an increase in the prevalence and intensity of Phormidium autumnale-dominated benthic blooms in New Zealand over the last decade. This species produces the potent neurotoxins Anatoxin-a, Homoanatoxin-a and their derivatives, and consumption of P. autumnale biofilms has led to over 70 dog deaths since 2005. The mechanisms regulating the dominance and toxicity of P. autumnale are still unclear, as these blooms can reach high biomass in low nutrient conditions. Benthic biofilms are composed of multiple taxa and usually harbor a complex community of bacteria and other microbes, which can change over time and interact to facilitate biofilm development and metabolic processing. Prior to this thesis, the microbial composition of P. autumnale-dominated biofilms was unknown. This study provides insights into the relationships of this neurotoxic cyanobacterium with microbial components of the biofilm community. Benthic biofilms were sampled every two to four days for 32 days from three sites in the Hutt River (Wellington) following a high flow event. A combination of microscopy and molecular techniques (bacterial ARISA and Illumina™ sequencing) were used to identify the micro-algal and bacterial components of the biofilm throughout its development. Variation in total anatoxin production was measured using LC-MS and changes in toxic P. autumnale cell numbers were quantified using QPCR. A suite of environmental variables (point velocity, depth, flow, conductivity, temperature and nutrients) were also monitored throughout the study period. Three distinct phases of microbial succession were identified (early, mid and late) using non-metric multidimensional cluster analyses. The micro-algal community composition (including P. autumnale) shifted from early to mid-phase ca. 16 days after the flushing flow and from mid to late phase at ca. day 30. The ARISA and Illumina™ sequencing showed the bacterial community shifts occurred ca. 4 and 9 days before the respective micro-algal community shifts. These analyses indicate a close coupling of the micro-algal and bacterial communities and may suggest bacterial driven succession. However, bacteria are likely to depend on micro-algal by-products for nutrition from the mid-phases onward and assessment of the metabolic processes occurring within the biofilms is needed to clarify this. Phormidium autumnale was dominant in the biofilm from an early stage in development and grew exponentially despite an influx of diatoms at day 20. None of the environmental parameters measured could explain the temporal variation in micro-algal and bacterial communities, which suggested that intrinsic rather than extrinsic factors were more important in regulating succession. This further supports the hypothesis that biofilm microbes may facilitate P. autumnale dominance. There was a significant variation in anatoxins per cell over time (p = 0.034). Production of anatoxins was greatest in the mid-phase of succession (208 fg cell⁻¹), coinciding with an increase in diatom biomass, which could implicate anatoxins as allelopathic chemicals that alleviate the effects of competition on P. autumnale. Changes in proportions of the different anatoxin variants produced over time also aligned with the three successional phases in both the micro-algal and bacterial communities, providing further evidence of a relationship between anatoxin production and microbial biofilm components. Bacterial taxa of the Alphaproteobacteria were dominant within the early bacterial community, but were surpassed by the Betaproteobacteria and Flavobacteria in mid and late phases. Bacterial genera involved in exopolysaccharide production, alkaline phosphatase activity and biopolymer degradation were identified. These attributes are important in the formation, maintenance and break-down of biofilms and therefore strengthen the likelihood of linkages between the micro-algal and bacterial community. Further investigations into functional roles of the biofilm components are needed to infer relationships between P. autumnale and the bacterial community. A clear pattern of microbial succession is described here and linkages between the micro-algal and bacterial communities are evident. Future work should focus on the functional attributes of microbes occurring at different stages of succession to further understand how P. autumnale dominates these benthic communities.</p>

Characterising the benthic Phormidium autumnale-dominated biofilm community and anatoxin production throughout biofilm succession

<p>There has been an increase in the prevalence and intensity of Phormidium autumnale-dominated benthic blooms in New Zealand over the last decade. This species produces the potent neurotoxins Anatoxin-a, Homoanatoxin-a and their derivatives, and consumption of P. autumnale biofilms has led to over 70 dog deaths since 2005. The mechanisms regulating the dominance and toxicity of P. autumnale are still unclear, as these blooms can reach high biomass in low nutrient conditions. Benthic biofilms are composed of multiple taxa and usually harbor a complex community of bacteria and other microbes, which can change over time and interact to facilitate biofilm development and metabolic processing. Prior to this thesis, the microbial composition of P. autumnale-dominated biofilms was unknown. This study provides insights into the relationships of this neurotoxic cyanobacterium with microbial components of the biofilm community. Benthic biofilms were sampled every two to four days for 32 days from three sites in the Hutt River (Wellington) following a high flow event. A combination of microscopy and molecular techniques (bacterial ARISA and Illumina™ sequencing) were used to identify the micro-algal and bacterial components of the biofilm throughout its development. Variation in total anatoxin production was measured using LC-MS and changes in toxic P. autumnale cell numbers were quantified using QPCR. A suite of environmental variables (point velocity, depth, flow, conductivity, temperature and nutrients) were also monitored throughout the study period. Three distinct phases of microbial succession were identified (early, mid and late) using non-metric multidimensional cluster analyses. The micro-algal community composition (including P. autumnale) shifted from early to mid-phase ca. 16 days after the flushing flow and from mid to late phase at ca. day 30. The ARISA and Illumina™ sequencing showed the bacterial community shifts occurred ca. 4 and 9 days before the respective micro-algal community shifts. These analyses indicate a close coupling of the micro-algal and bacterial communities and may suggest bacterial driven succession. However, bacteria are likely to depend on micro-algal by-products for nutrition from the mid-phases onward and assessment of the metabolic processes occurring within the biofilms is needed to clarify this. Phormidium autumnale was dominant in the biofilm from an early stage in development and grew exponentially despite an influx of diatoms at day 20. None of the environmental parameters measured could explain the temporal variation in micro-algal and bacterial communities, which suggested that intrinsic rather than extrinsic factors were more important in regulating succession. This further supports the hypothesis that biofilm microbes may facilitate P. autumnale dominance. There was a significant variation in anatoxins per cell over time (p = 0.034). Production of anatoxins was greatest in the mid-phase of succession (208 fg cell⁻¹), coinciding with an increase in diatom biomass, which could implicate anatoxins as allelopathic chemicals that alleviate the effects of competition on P. autumnale. Changes in proportions of the different anatoxin variants produced over time also aligned with the three successional phases in both the micro-algal and bacterial communities, providing further evidence of a relationship between anatoxin production and microbial biofilm components. Bacterial taxa of the Alphaproteobacteria were dominant within the early bacterial community, but were surpassed by the Betaproteobacteria and Flavobacteria in mid and late phases. Bacterial genera involved in exopolysaccharide production, alkaline phosphatase activity and biopolymer degradation were identified. These attributes are important in the formation, maintenance and break-down of biofilms and therefore strengthen the likelihood of linkages between the micro-algal and bacterial community. Further investigations into functional roles of the biofilm components are needed to infer relationships between P. autumnale and the bacterial community. A clear pattern of microbial succession is described here and linkages between the micro-algal and bacterial communities are evident. Future work should focus on the functional attributes of microbes occurring at different stages of succession to further understand how P. autumnale dominates these benthic communities.</p>

Turnover in life strategies recapitulates microbial succession on synthetic marine particles

Particulate organic matter (POM) in the ocean sustains diverse communities of bacteria that mediate the remineralization of organic complex matter. However, the variability of these particles and of the environmental conditions surrounding them present a challenge to the study of the ecological processes shaping particle-associated communities and their function. In this work, we utilise data from experiments in which coastal water communities were grown on synthetic particles to ask which are the most important ecological drivers of their assembly and associated traits. Combining 16S rRNA amplicon sequencing with shotgun metagenomics, together with an analysis of the full genomes of a subset of isolated strains, we were able to identify two-to-three distinct community classes, corresponding to early vs. late colonizers. We show that these classes are shaped by environmental selection (early colonizers) and facilitation (late colonizers), and find distinctive traits associated with each class. While early colonizers have a larger proportion of genes related to uptake of nutrients, motility and environmental sensing with few pathways enriched for metabolism, late colonizers devote a higher proportion of genes for metabolism, comprising a wide array of different pathways including metabolism of carbohydrates, amino acids and xenobiotics We find evidence in selected metabolic pathways for the existence of a trophic-chain topology connecting both classes. The interpretation of these traits suggests a distinction between early and late colonizers analogous to other classifications found in the literature, and we discuss connections with the classical distinction between r- and K-strategists.

Terrestrial connectivity, upstream aquatic history and seasonality shape bacterial community assembly within a large boreal aquatic network

During transit from soils to the ocean, microbial communities are modified and re-assembled, generating complex patterns of ecological succession. The potential effect of upstream assembly on downstream microbial community composition is seldom considered within aquatic networks. Here, we reconstructed the microbial succession along a land-freshwater-estuary continuum within La Romaine river watershed in Northeastern Canada. We captured hydrological seasonality and differentiated the total and reactive community by sequencing both 16 S rRNA genes and transcripts. By examining how DNA- and RNA-based assemblages diverge and converge along the continuum, we inferred temporal shifts in the relative importance of assembly processes, with mass effects dominant in spring, and species selection becoming stronger in summer. The location of strongest selection within the network differed between seasons, suggesting that selection hotspots shift depending on hydrological conditions. The unreactive fraction (no/minor RNA contribution) was composed of taxa with diverse potential origins along the whole aquatic network, while the majority of the reactive pool (major RNA contribution) could be traced to soil/soilwater-derived taxa, which were distributed along the entire rank-abundance curve. Overall, our findings highlight the importance of considering upstream history, hydrological seasonality and the reactive microbial fraction to fully understand microbial community assembly on a network scale.

Microbial colonization and resistome dynamics in food processing environments of a newly opened pork cutting industry during 1.5 years of activity

Background The microorganisms that inhabit food processing environments (FPE) can strongly influence the associated food quality and safety. In particular, the possibility that FPE may act as a reservoir of antibiotic-resistant microorganisms, and a hotspot for the transmission of antibiotic resistance genes (ARGs) is a concern in meat processing plants. Here, we monitor microbial succession and resistome dynamics relating to FPE through a detailed analysis of a newly opened pork cutting plant over 1.5 years of activity. Results We identified a relatively restricted principal microbiota dominated by Pseudomonas during the first 2 months, while a higher taxonomic diversity, an increased representation of other taxa (e.g., Acinetobacter, Psychrobacter), and a certain degree of microbiome specialization on different surfaces was recorded later on. An increase in total abundance, alpha diversity, and β-dispersion of ARGs, which were predominantly assigned to Acinetobacter and associated with resistance to certain antimicrobials frequently used on pig farms of the region, was detected over time. Moreover, a sharp increase in the occurrence of extended-spectrum β-lactamase-producing Enterobacteriaceae and vancomycin-resistant Enterococcaceae was observed when cutting activities started. ARGs associated with resistance to β-lactams, tetracyclines, aminoglycosides, and sulphonamides frequently co-occurred, and mobile genetic elements (i.e., plasmids, integrons) and lateral gene transfer events were mainly detected at the later sampling times in drains. Conclusions The observations made suggest that pig carcasses were a source of resistant bacteria that then colonized FPE and that drains, together with some food-contact surfaces, such as equipment and table surfaces, represented a reservoir for the spread of ARGs in the meat processing facility.

Phage Therapy Related Microbial Succession Associated with Successful Clinical Outcome for a Recurrent Urinary Tract Infection

We rationally designed a bacteriophage cocktail to treat a 56-year-old male liver transplant patient with complex, recurrent prostate and urinary tract infections caused by an extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli) (UCS1). We screened our library for phages that killed UCS1, with four promising candidates chosen for their virulence, mucolytic properties, and ability to reduce bacterial resistance. The patient received 2 weeks of intravenous phage cocktail with concomitant ertapenem for 6 weeks. Weekly serum and urine samples were collected to track the patient’s response. The patient tolerated the phage therapy without any adverse events with symptom resolution. The neutralization of the phage activity occurred with sera collected 1 to 4 weeks after the first phage treatment. This was consistent with immunoassays that detected the upregulation of immune stimulatory analytes. The patient developed asymptomatic recurrent bacteriuria 6 and 11 weeks following the end of phage therapy—a condition that did not require antibiotic treatment. The bacteriuria was caused by a sister strain of E. coli (UCS1.1) that remained susceptible to the original phage cocktail and possessed putative mutations in the proteins involved in adhesion and invasion compared to UCS1. This study highlights the utility of rationally designed phage cocktails with antibiotics at controlling E. coli infection and suggests that microbial succession, without complete eradication, may produce desirable clinical outcomes.