Environmentally Relevant Antibiotic Concentrations Exert Stronger Selection Pressure on River Biofilm Resistomes than AMR-Reservoir Effluents.

Freshwater environments are primary receiving systems of wastewater and effluents, which carry low concentrations of antibiotics and antimicrobial-resistant (AMR) bacteria and genes. Aquatic microbial communities are thus exposed to environmentally relevant concentrations of antibiotics (ERCA) that presumably influence the acquisition and spread of environmental AMR. Here, we analyzed ERCA exposure with and without the additional presence of municipal wastewater treatment plant effluent (W) and swine manure run-off (M) on aquatic biofilm resistomes. Microscopic analyses revealed decreased taxonomic diversity and biofilm structural integrity, while metagenomic analysis revealed an increased abundance of resistance, virulence, and mobile element-related genes at the highest ERCA exposure levels, with less notable impacts observed when solely exposed to W or M effluents. Microbial function predictions indicated increased gene abundance associated with energy and cell membrane metabolism and heavy metal resistance under ERCA conditions. In silico predictions of increased resistance mechanisms did not correlate with observed phenotypic resistance patterns when whole communities were exposed to antimicrobial susceptibility testing. This reveals important insight into the complexity of whole-community coordination of physical and genetic responses to selective pressures. Lastly, the environmental AMR risk assessment of metagenomic data revealed a higher risk score for biofilms grown at sub-MIC antibiotic conditions.

Impact of wastewater treatment upgrade and nitrogen removal on bacterial communities and their interactions in eutrophic prairie streams.

Eutrophication can impact bacteria by altering fluxes and processing of nutrients and organic matter. However, relatively little is known of how bacterial communities, diversity, and interactions with phytoplankton might respond to nutrient management. We used 16S rRNA amplicon sequencing to compare bacterial assemblages in the water column upstream (control) and downstream (impact) of a wastewater treatment plant (WWTP) located on a eutrophic prairie stream. Sampling occurred before (2012) and after (2018) the 2016 biological nutrient removal (BNR) upgrade that removed >90% of nitrogen (N, mainly NH4+). Multivariate ordination suggested that effluent-impacted bacterial communities were associated mainly with elevated NH4+ concentrations before the upgrade, whereas those after BNR were characteristic of reference systems (low NO3-, diverse regulation). Genera such as Betaproteobacteria and Rhodocyclacea were abundant at impacted sites in 2012, whereas Flavobacterium and a potential pathogen (Legionella) were common at impacted sites in 2018. Nitrifier bacteria (Nitrospira and Nitrosomonas) were present but rare at all sites in 2012, but recorded only downstream of the WWTP in 2018. Generalized additive models showed that BNR reduced bacterial diversity, with ∼70% of the deviance in diversity explained by hydrology, pH, nutrients, and phytoplankton abundance. Overall, NH4+ removal reduced symptoms of cultural eutrophication in microbe assemblages.

Sub-MIC antibiotics influence the microbiome, resistome and structure of riverine biofilm communities.

The effects of sub-minimum inhibitory concentrations (sub-MICs) of antibiotics on aquatic environments is not yet fully understood. Here, we explore these effects by employing a replicated microcosm system fed with river water where biofilm communities were continuously exposed over an eight-week period to sub-MIC exposure (1/10, 1/50, and 1/100 MIC) to a mix of common antibiotics (ciprofloxacin, streptomycin, and oxytetracycline). Biofilms were examined using a structure-function approach entailing microscopy and metagenomic techniques, revealing details on the microbiome, resistome, virulome, and functional prediction. A comparison of three commonly used microbiome and resistome databases was also performed. Differences in biofilm architecture were observed between sub-MIC antibiotic treatments, with an overall reduction of extracellular polymeric substances and autotroph (algal and cyanobacteria) and protozoan biomass, particularly at the 1/10 sub-MIC condition. While metagenomic analyses demonstrated that microbial diversity was lowest at the sub-MIC 1/10 antibiotic treatment, resistome diversity was highest at sub-MIC 1/50. This study also notes the importance of benchmarking analysis tools and careful selection of reference databases, given the disparity in detected antimicrobial resistance genes (ARGs) identity and abundance across methods. Ultimately, the most detected ARGs in sub-MICs exposed biofilms were those that conferred resistance to aminoglycosides, tetracyclines, ß-lactams, sulfonamides, and trimethoprim. Co-occurrence of microbiome and resistome features consistently showed a relationship between Proteobacteria genera and aminoglycoside ARGs. Our results support the hypothesis that constant exposure to sub-MICs antibiotics facilitate the transmission and promote prevalence of antibiotic resistance in riverine biofilms communities, and additionally shift overall microbial community metabolic function.

Environmental Biofilms as Reservoirs for Antimicrobial Resistance.

Characterizing the response of microbial communities to a range of antibiotic concentrations is one of the strategies used to understand the impact of antibiotic resistance. Many studies have described the occurrence and prevalence of antibiotic resistance in microbial communities from reservoirs such as hospitals, sewage, and farm feedlots, where bacteria are often exposed to high and/or constant concentrations of antibiotics. Outside of these sources, antibiotics generally occur at lower, sub-minimum inhibitory concentrations (sub-MICs). The constant exposure to low concentrations of antibiotics may serve as a chemical "cue" that drives development of antibiotic resistance. Low concentrations of antibiotics have not yet been broadly described in reservoirs outside of the aforementioned environments, nor is the transfer and dissemination of antibiotic resistant bacteria and genes within natural microbial communities fully understood. This review will thus focus on low antibiotic-concentration environmental reservoirs and mechanisms that are important in the dissemination of antibiotic resistance to help identify key knowledge gaps concerning the environmental resistome.

Metagenomic and metatranscriptomic analysis reveals enrichment for xenobiotic-degrading bacterial specialists and xenobiotic-degrading genes in a Canadian Prairie two-cell biobed system.

Biobeds are agriculture-based bioremediation tools used to safely contain and microbially degrade on-farm pesticide waste and rinsate, thereby reducing the negative environmental impacts associated with pesticide use. While these engineered ecosystems demonstrate efficient pesticide removal, the microbiomes in these environments remain largely understudied both taxonomically and functionally. This study used metagenomic and metatranscriptomic techniques to characterize the microbial community in a two-cell Canadian biobed system before and after a field season of pesticide application. These culture-independent approaches identified an enrichment of xenobiotic-degrading bacteria, such as Afipia, Sphingopyxis and Pseudomonas, and enrichment and transcription of xenobiotic-degrading genes, such as peroxidases, oxygenases, and hydroxylases, among others; we were able to directly link the transcription of these genes to Pseudomonas, Oligotropha, Mesorhizobium, Rhodopseudomonas, and Stenotrophomonas taxa.

Impact of sample collection on prokaryotic and eukaryotic diversity of niche environments of the oil-sand mining impacted Athabasca River.

Microbial communities are an important aspect of overall riverine ecology; however, appreciation of the effects of anthropogenic activities on unique riverine microbial niches, and how the collection of these samples affects the observed diversity and community profile is lacking. We analyzed prokaryotic and eukaryotic communities from surface water, biofilms, and suspended load niches along a gradient of oil sands-related contamination in the Athabasca River (Alberta, Canada), with suspended load or particle-associated communities collected either via Kenney Sampler or centrifugation manifold. At the phylum level, different niche communities were highly similar to each other and across locations. However, there were significant differences in the abundance of specific genera among the different niches and across sampling locations. A generalized linear model revealed that use of the Kenney Sampler resulted in more diverse bacterial and eukaryotic suspended load community than centrifugal collection, though suspended load communities collected by any means remained stably diverse across locations. Although there was an influence of water quality parameters on community composition, all sampled sites support diverse bacterial and eukaryotic communities regardless of the degree of contamination, highlighting the need to look beyond ecological diversity as a means of assessing ecological perturbations, and consider collecting samples from multiple niche environments.

How Do the Players Play? A Post-Genomic Analysis Paradigm to Understand Aquatic Ecosystem Processes.

Culture-independent and meta-omics sequencing methods have shed considerable light on the so-called "microbial dark matter" of Earth's environmental microbiome, improving our understanding of phylogeny, the tree of life, and the vast functional diversity of microorganisms. This influx of sequence data has led to refined and reimagined hypotheses about the role and importance of microbial biomass, that paradoxically, sequencing approaches alone are unable to effectively test. Post-genomic approaches such as metabolomics are providing more sensitive and insightful data to unravel the fundamental operations and intricacies of microbial communities within aquatic systems. We assert that the implementation of integrated post-genomic approaches, specifically metabolomics and metatranscriptomics, is the new frontier of environmental microbiology and ecology, expanding conventional assessments toward a holistic systems biology understanding. Progressing beyond siloed phylogenetic assessments and cataloging of metabolites, toward integrated analysis of expression (metatranscriptomics) and activity (metabolomics) is the most effective approach to provide true insight into microbial contributions toward local and global ecosystem functions. This data in turn creates opportunity for improved regulatory guidelines, biomarker discovery and better integration of modeling frameworks. To that end, critical aquatic environmental issues related to climate change, such as ocean warming and acidification, contamination mitigation, and macro-organism health have reasonable opportunity of being addressed through such an integrative approach. Lastly, we argue that the "post-genomics" paradigm is well served to proactively address the systemic technical issues experienced throughout the genomics revolution and focus on collaborative assessment of field-wide experimental standards of sampling, bioinformatics and statistical treatments.

Transcriptomics reveal core activities of the plant growth-promoting bacterium Delftia acidovorans RAY209 during interaction with canola and soybean roots.

The plant growth-promoting rhizobacterium Delftia acidovorans RAY209 is capable of establishing strong root attachment during early plant development at 7 days post-inoculation. The transcriptional response of RAY209 was measured using RNA-seq during early (day 2) and sustained (day 7) root colonization of canola plants, capturing RAY209 differentiation from a medium-suspended cell state to a strongly root-attached cell state. Transcriptomic data was collected in an identical manner during RAY209 interaction with soybean roots to explore the putative root colonization response to this globally relevant crop. Analysis indicated there is an increased number of significantly differentially expressed genes between medium-suspended and root-attached cells during early soybean root colonization relative to sustained colonization, while the opposite temporal pattern was observed for canola root colonization. Regardless of the plant host, root-attached RAY209 cells exhibited the least amount of differential gene expression between early and sustained root colonization. Root-attached cells of either canola or soybean roots expressed high levels of a fasciclin gene homolog encoding an adhesion protein, as well as genes encoding hydrolases, multiple biosynthetic processes, and membrane transport. Notably, while RAY209 ABC transporter genes of similar function were transcribed during attachment to either canola or soybean roots, several transporter genes were uniquely differentially expressed during colonization of the respective plant hosts. In turn, both canola and soybean plants expressed genes encoding pectin lyase and hydrolases - enzymes with purported function in remodelling extracellular matrices in response to RAY209 colonization. RAY209 exhibited both a core regulatory response and a planthost-specific regulatory response to root colonization, indicating that RAY209 specifically adjusts its cellular activities to adapt to the canola and soybean root environments. This transcriptomic data defines the basic RAY209 response as both a canola and soybean commercial crop and seed inoculant.

Metatranscriptomic Insights Into the Response of River Biofilm Communities to Ionic and Nano-Zinc Oxide Exposures.

Manufactured Zn oxide nanoparticle (ZnO-NP) are extensively used world-wide in personal care and industrial products and are important contaminants of aquatic environments. To understand the overall impact of ZnO-NP contamination on aquatic ecosystems, investigation of their toxicity on aquatic biofilms is of particular consequence, given biofilms are known sinks for NP contaminants. In order to assess alterations in the functional activity of river microbial biofilm communities as a result of environmentally-relevant ZnO-NP exposure, biofilms were exposed to ionic zinc salt or ZnOPs that were uncoated (hydrophilic), coated with silane (hydrophobic) or stearic acid (lipophilic), at a total concentration of 188 µg l-1 Zn. ICP-MS analyses of biofilms indicated ZnO-NP concentrated in the biofilms, with hydrophilic, hydrophobic, and lipophilic treatments reaching 0.310, 0.250, and 0.220 µg Zn cm-2 of biofilm, respectively, while scanning transmission X-ray microspectroscopy (STXM) analyses of biofilms confirmed that Zn was extensively- and differentially-sorbed to biofilm material. Microbial community composition, based on taxonomic affiliation of mRNA sequences and enumeration of protozoa and micrometazoa, was not affected by these treatments, and the total transcriptional response of biofilms to all experimental exposures was not indicative of a global toxic-response, as cellular processes involved in general cell maintenance and housekeeping were abundantly transcribed. Transcripts related to major biological processes, including photosynthesis, energy metabolism, nitrogen metabolism, lipid metabolism, membrane transport, antibiotic resistance and xenobiotic degradation, were differentially expressed in Zn-exposures relative to controls. Notably, transcripts involved in nitrogen fixation and photosynthesis were decreased in abundance in response to Zn-exposure, while transcripts related to lipid degradation and motility-chemotaxis were increased, suggesting a potential role of Zn in biofilm dissolution. ZnO-NP and ionic Zn exposures elicited generally overlapping transcriptional responses, however hydrophilic and hydrophobic ZnO-NPs induced a more distinct effect than that of lipophilic ZnO-NPs, which had an effect similar to that of low ionic Zn exposure. While the physical coating of ZnO-NP may not induce specific toxicity observable at a community level, alteration of ecologically important processes of photosynthesis and nitrogen cycling are an important potential consequence of exposure to ionic Zn and Zn oxides.

Spatial analysis of a hydrocarbon waste-remediating landfarm demonstrates influence of management practices on bacterial and fungal community structure.

Cultivation of dedicated soil plots called 'landfarms' is an effective technology for bioremediation of hydrocarbon waste generated by various industrial practices. To understand the influence of soil conditions on landfarm microbial communities, analysis of bacterial and fungal community structure using next-generation sequencing at different sections and depths was performed across a hydrocarbon-waste landfarm in Regina, Saskatchewan, Canada. While a core set of hydrocarbon-associated bacterial and fungal taxa are present throughout the landfarm, unique bacterial and fungal operational taxonomic units are differentially abundant at sections within the landfarm, which correlate with differences in soil physiochemical properties and management practices. Increased frequency of waste application resulted in strong positive correlations between bacterial community assemblages and elevated amounts of oil, grease and F3 - F4 hydrocarbon fractions. In areas of standing water and lower application of hydrocarbon, microbial community structure correlated with soil pH, trace nutrients and metals. Overall, diversity and structure of bacterial communities remain relatively stable across the landfarm, while in contrast, fungal community structure appears more responsive to soil oxygen conditions. Results are consistent with the hypothesis that years of bioremediation activity have shaped microbial communities; however, several management practices can be undertaken to increase efficiency of remediation, including the removal of standing water and soil tilling across the landfarm.

Lactobacillus backii and Pediococcus damnosus isolated from 170-year-old beer recovered from a shipwreck lack the metabolic activities required to grow in modern lager beer.

In 2010, bottles of beer containing viable bacteria of the common beer-spoilage species Lactobacillus backii and Pediococcus damnosus were recovered from a shipwreck near the Åland Islands, Finland. The 170-year quiescent state maintained by the shipwreck bacteria presented a unique opportunity to study lactic acid bacteria (LAB) evolution vis-a-vis growth and survival in the beer environment. Three shipwreck bacteria (one L. backii strain and two P. damnosus strains) and modern-day beer-spoilage isolates of the same two species were genome sequenced, characterized for hop iso-α-acid tolerance, and growth in degassed lager and wheat beer. In addition, plasmid variants of the modern-day P. damnosus strain were analyzed for the effect of plasmid-encoded genes on growth in lager beer. Coding content on two plasmids was identified as essential for LAB growth in modern lager beer. Three chromosomal regions containing genes related to sugar transport and cell wall polysaccharides were shared by pediococci able to grow in beer. Our results show that the three shipwreck bacteria lack the necessary plasmid-located genetic content to grow in modern lager beer, but carry additional genes related to acid tolerance and biofilm formation compared to their modern counterparts.

Complete Genome Sequence of Delftia acidovorans RAY209, a Plant Growth-Promoting Rhizobacterium for Canola and Soybean.

Herein, we report the genome sequence of Delftia acidovorans strain RAY209, a plant growth-promoting rhizobacterium that is used in commercial inoculants for canola and soybean. The genome of RAY209 has a consensus of 6,528,879 bp and an estimated 5,721 coding sequences.

Comparative genomic and plasmid analysis of beer-spoiling and non-beer-spoiling Lactobacillus brevis isolates.

Beer-spoilage-related lactic acid bacteria (BSR LAB) belong to multiple genera and species; however, beer-spoilage capacity is isolate-specific and partially acquired via horizontal gene transfer within the brewing environment. Thus, the extent to which genus-, species-, or environment- (i.e., brewery-) level genetic variability influences beer-spoilage phenotype is unknown. Publicly available Lactobacillus brevis genomes were analyzed via BlAst Diagnostic Gene findEr (BADGE) for BSR genes and assessed for pangenomic relationships. Also analyzed were functional coding capacities of plasmids of LAB inhabiting extreme niche environments. Considerable genetic variation was observed in L. brevis isolated from clinical samples, whereas 16 candidate genes distinguish BSR and non-BSR L. brevis genomes. These genes are related to nutrient scavenging of gluconate or pentoses, mannose, and metabolism of pectin. BSR L. brevis isolates also have higher average nucleotide identity and stronger pangenome association with one another, though isolation source (i.e., specific brewery) also appears to influence the plasmid coding capacity of BSR LAB. Finally, it is shown that niche-specific adaptation and phenotype are plasmid-encoded for both BSR and non-BSR LAB. The ultimate combination of plasmid-encoded genes dictates the ability of L. brevis to survive in the most extreme beer environment, namely, gassed (i.e., pressurized) beer.

Genome Sequence of Megasphaera cerevisiae NSB1, a Bacterium Isolated from a Canning Line and Able To Grow in Beer with High Alcohol Content.

The genome sequence of the brewery isolate Megasphaera cerevisiae NSB1 was determined. Strain NSB1 tolerates 5% (vol/vol) alcohol, which is higher than previously reported for M. cerevisiae The NSB1 genome will help elucidate genetics required for alcohol tolerance and niche adaptation of this Gram-negative beer-spoilage bacterium.

Next-generation sequencing approaches for improvement of lactic acid bacteria-fermented plant-based beverages.

Plant-based beverages and milk alternatives produced from cereals and legumes have grown in popularity in recent years due to a range of consumer concerns over dairy products. These plant-based products can often have undesirable physiochemical properties related to flavour, texture, and nutrient availability and/or deficiencies. Lactic acid bacteria (LAB) fermentation offers potential remediation for many of these issues, and allows consumers to retain their perception of the resultant products as natural and additive-free. Using next-generation sequencing (NGS) or omics approaches to characterize LAB isolates to find those that will improve properties of plant-based beverages is the most direct way to product improvement. Although NGS/omics approaches have been extensively used for selection of LAB for use in the dairy industry, a comparable effort has not occurred for selecting LAB for fermenting plant raw substrates, save those used in producing wine and certain types of beer. Here we review the few and recent applications of NGS/omics to profile and improve LAB fermentation of various plant-based substrates for beverage production. We also identify specific issues in the production of various LAB fermented plant-based beverages that such NGS/omics applications have the power to resolve.

Influence of commercial (Fluka) naphthenic acids on acid volatile sulfide (AVS) production and divalent metal precipitation.

Energy-derived waters containing naphthenic acids (NAs) are complex mixtures often comprising a suite of potentially problematic constituents (e.g. organics, metals, and metalloids) that need treatment prior to beneficial use, including release to receiving aquatic systems. It has previously been suggested that NAs can have biostatic or biocidal properties that could inhibit microbially driven processes (e.g. dissimilatory sulfate reduction) used to transfer or transform metals in passive treatment systems (i.e. constructed wetlands). The overall objective of this study was to measure the effects of a commercially available (Fluka) NA on sulfate-reducing bacteria (SRB), production of sulfides (as acid-volatile sulfides [AVS]), and precipitation of divalent metals (i.e. Cu, Ni, Zn). These endpoints were assessed following 21-d aqueous exposures of NAs using bench-scale reactors. After 21-days, AVS molar concentrations were not statistically different (p<0.0001; α=0.05) among NA treatments (10, 20, 40, 60, and 80mg NA/L) and an untreated control (no NAs). Extent of AVS production was sufficient in all NA treatments to achieve ∑SEM:AVS <1, indicating that conditions were conducive for treatment of metals, with sulfide ligands in excess of SEM (Cu, Ni, and Zn). In addition, no adverse effects to SRB (in terms of density, relative abundance, and diversity) were measured following exposures of a commercial NA. In this bench-scale study, dissimilatory sulfate reduction and subsequent metal precipitation were not vulnerable to NAs, indicating passive treatment systems utilizing sulfide production (AVS) could be used to treat metals occurring in NAs affected waters.

Transcriptome analysis of beer-spoiling Lactobacillus brevis BSO 464 during growth in degassed and gassed beer.

Lactobacillus brevis BSO 464 (Lb464) is a beer-spoilage-related (BSR) isolate of interest given its unique physiological attributes; specifically, it is highly hop-tolerant and exhibits very rapid growth in pressurized/gassed beer. RNA sequencing was performed on Lb464 grown in pressurized and non-pressurized beer to determine important genetic mechanisms for growth in these environments. The data generated were compared against data in a previous transcriptional study of another lactic acid bacterium (LAB) during growth in beer, namely, Pediococcus claussenii ATCC BAA-344(T) (Pc344). Results revealed that the most important genetic elements for Lb464 growth in beer are related to biogenic amine metabolism, membrane transport and fortification, nutrient scavenging, and efficient transcriptional regulation. Comparison with the previous transcriptional study of Pc344 indicated that the total coding capacity (plasmid profile and genome size) of a LAB isolate allows for beer-spoilage virulence and adaptation to different beer environments, i.e., the ability to grow in degassed beer (during production) or gassed beer (packaged product). Further, differences in gene expression of Lb464 and Pc344 during mid-exponential growth in beer may dictate how rapidly each isolate exhausts particular carbon sources during. The presence of headspace pressure/dissolved CO2 was found to drive Lb464 transcription during mid-exponential growth in beer towards increasing cell wall and membrane modification, transport, osmoregulation, and DNA metabolism and transposition events. This transcriptional activity resembles transcriptional patterns or signatures observed in a viable, but non-culturable state established by non-related organisms, suggesting that Lb464 overall uses complex cellular regulation to maintain cell division and growth in the stressful beer environment. Additionally, increased expression of several hypothetical proteins, the hop-tolerance gene horC, and DNA repair and recombination genes from plasmids pLb464-2, -4, and -8 were observed in the gassed beer environment. Thus, plasmids can harbor genes with specific (gassed) beer growth advantages, and confirm that plasmid transfer and acquisition as important activities for adaptation to the beer environment.

Genome Sequence of Rapid Beer-Spoiling Isolate Lactobacillus brevis BSO 464.

The genome of brewery-isolate Lactobacillus brevis BSO 464 was sequenced and assembly produced a chromosome and eight plasmids. This bacterium tolerates dissolved CO2/pressure and can rapidly spoil packaged beer. This genome is useful for analyzing the genetics associated with beer spoilage by lactic acid bacteria.

Mapping microbial ecosystems and spoilage-gene flow in breweries highlights patterns of contamination and resistance.

Distinct microbial ecosystems have evolved to meet the challenges of indoor environments, shaping the microbial communities that interact most with modern human activities. Microbial transmission in food-processing facilities has an enormous impact on the qualities and healthfulness of foods, beneficially or detrimentally interacting with food products. To explore modes of microbial transmission and spoilage-gene frequency in a commercial food-production scenario, we profiled hop-resistance gene frequencies and bacterial and fungal communities in a brewery. We employed a Bayesian approach for predicting routes of contamination, revealing critical control points for microbial management. Physically mapping microbial populations over time illustrates patterns of dispersal and identifies potential contaminant reservoirs within this environment. Habitual exposure to beer is associated with increased abundance of spoilage genes, predicting greater contamination risk. Elucidating the genetic landscapes of indoor environments poses important practical implications for food-production systems and these concepts are translatable to other built environments.

Role of plasmids in Lactobacillus brevis BSO 464 hop tolerance and beer spoilage.

Specific isolates of lactic acid bacteria (LAB) can grow in the harsh beer environment, thus posing a threat to brew quality and the economic success of breweries worldwide. Plasmid-localized genes, such as horA, horC, and hitA, have been suggested to confer hop tolerance, a trait required for LAB survival in beer. The presence and expression of these genes among LAB, however, do not universally correlate with the ability to grow in beer. Genome sequencing of the virulent beer spoilage organism Lactobacillus brevis BSO 464 revealed the presence of eight plasmids, with plasmids 1, 2, and 3 containing horA, horC, and hitA, respectively. To investigate the roles that these and the other five plasmids play in L. brevis BSO 464 growth in beer, plasmid curing with novobiocin was used to derive 10 plasmid variants. Multiplex PCRs were utilized to determine the presence or absence of each plasmid, and how plasmid loss affected hop tolerance and growth in degassed (noncarbonated) beer was assessed. Loss of three of the eight plasmids was found to affect hop tolerance and growth in beer. Loss of plasmid 2 (horC and 28 other genes) had the most dramatic effect, with loss of plasmid 4 (120 genes) and plasmid 8 (47 genes) having significant, but smaller, impacts. These results support the contention that genes on mobile genetic elements are essential for bacterial growth in beer and that beer spoilage ability is not dependent solely on the three previously described hop tolerance genes or on the chromosome of a beer spoilage LAB isolate.