Global epistasis and the emergence of function in microbial consortia.

The many functions of microbial communities emerge from a complex web of interactions between organisms and their environment. This poses a significant obstacle to engineering microbial consortia, hindering our ability to harness the potential of microorganisms for biotechnological applications. In this study, we demonstrate that the collective effect of ecological interactions between microbes in a community can be captured by simple statistical models that predict how adding a new species to a community will affect its function. These predictive models mirror the patterns of global epistasis reported in genetics, and they can be quantitatively interpreted in terms of pairwise interactions between community members. Our results illuminate an unexplored path to quantitatively predicting the function of microbial consortia from their composition, paving the way to optimizing desirable community properties and bringing the tasks of predicting biological function at the genetic, organismal, and ecological scales under the same quantitative formalism.

Anaerobic Degradation of Alkanes by Marine Archaea.

Alkanes are saturated apolar hydrocarbons that range from their simplest form, methane, to high-molecular-weight compounds. Although alkanes were once considered biologically recalcitrant under anaerobic conditions, microbiological investigations have now identified several microbial taxa that can anaerobically degrade alkanes. Here we review recent discoveries in the anaerobic oxidation of alkanes with a specific focus on archaea that use specific methyl coenzyme M reductases to activate their substrates. Our understanding of the diversity of uncultured alkane-oxidizing archaea has expanded through the use of environmental metagenomics and enrichment cultures of syntrophic methane-, ethane-, propane-, and butane-oxidizing marine archaea with sulfate-reducing bacteria. A recently cultured group of archaea directly couples long-chain alkane degradation with methane formation, expanding the range of substrates used for methanogenesis. This article summarizes the rapidly growing knowledge of the diversity, physiology, and habitat distribution of alkane-degrading archaea.

The optimization of microbial functions through rational environmental manipulations.

Microorganisms play a central role in biotechnology and it is key that we develop strategies to engineer and optimize their functionality. To this end, most efforts have focused on introducing genetic manipulations in microorganisms which are then grown either in monoculture or in mixed-species consortia. An alternative strategy to optimize microbial processes is to rationally engineer the environment in which microbes grow. The microbial environment is multidimensional, including factors such as temperature, pH, salinity, nutrient composition, etc. These environmental factors all influence the growth and phenotypes of microorganisms and they generally "interact" with one another, combining their effects in complex, non-additive ways. In this piece, we overview the origins and consequences of these "interactions" between environmental factors and discuss how they have been built into statistical, bottom-up predictive models of microbial function to identify optimal environmental conditions for monocultures and microbial consortia. We also overview alternative "top-down" approaches, such as genetic algorithms, to finding optimal combinations of environmental factors. By providing a brief summary of the state of this field, we hope to stimulate further work on the rational manipulation and optimization of the microbial environment.

Transcriptomic responses of Mediterranean sponges upon encounter with symbiont microbial consortia.

BACKGROUND: Sponges (phylum Porifera) constantly interact with microbes. They graze on microbes from the water column by filter-feeding and they harbor symbiotic partners within their bodies. In experimental setups, sponges take up symbionts at lower rates compared with seawater microbes. This suggests that sponges have the capacity to differentiate between microbes and preferentially graze in non-symbiotic microbes, although the underlying mechanisms of discrimination are still poorly understood. Genomic studies showed that, compared to other animal groups, sponges present an extended repertoire of immune receptors, in particular NLRs, SRCRs, and GPCRs, and a handful of experiments showed that sponges regulate the expression of these receptors upon encounter with microbial elicitors. We hypothesize that sponges may rely on differential expression of their diverse repertoire of poriferan immune receptors to sense different microbial consortia while filter-feeding. To test this, we characterized the transcriptomic response of two sponge species, Aplysina aerophoba and Dysidea avara, upon incubation with microbial consortia extracted from A. aerophoba in comparison with incubation with seawater microbes. The sponges were sampled after 1 h, 3 h, and 5 h for RNA-Seq differential gene expression analysis. RESULTS: D. avara incubated with A. aerophoba-symbionts regulated the expression of genes related to immunity, ubiquitination, and signaling. Within the set of differentially-expressed immune genes we identified different families of Nucleotide Oligomerization Domain (NOD)-Like Receptors (NLRs). These results represent the first experimental evidence that different types of NLRs are involved in microbial discrimination in a sponge. In contrast, the transcriptomic response of A. aerophoba to its own symbionts involved comparatively fewer genes and lacked genes encoding for immune receptors. CONCLUSION: Our work suggests that: (i) the transcriptomic response of sponges upon microbial exposure may imply "fine-tuning" of baseline gene expression as a result of their interaction with microbes, (ii) the differential response of sponges to microbial encounters varied between the species, probably due to species-specific characteristics or related to host's traits, and (iii) immune receptors belonging to different families of NLR-like genes played a role in the differential response to microbes, whether symbionts or food bacteria. The regulation of these receptors in sponges provides further evidence of the potential role of NLRs in invertebrate host-microbe interactions. The study of sponge responses to microbes exemplifies how investigating different animal groups broadens our knowledge of the evolution of immune specificity and symbiosis.

Excessive Ozonation Stress Triggers Severe Membrane Biofilm Accumulation and Fouling.

The established benefits of ozone on microbial pathogen inactivation, natural organic matter degradation, and inorganic/organic contaminant oxidation have favored its application in drinking water treatment. However, viable bacteria are still present after the ozonation of raw water, bringing a potential risk to membrane filtration systems in terms of biofilm accumulation and fouling. In this study, we shed light on the role of the specific ozone dose (0.5 mg-O3/mg-C) in biofilm accumulation during long-term membrane ultrafiltration. Results demonstrated that ozonation transformed the molecular structure of influent dissolved organic matter (DOM), producing fractions that were highly bioavailable at a specific ozone dose of 0.5, which was inferred to be a turning point. With the increase of the specific ozone dose, the biofilm microbial consortium was substantially shifted, demonstrating a decrease in richness and diversity. Unexpectedly, the opportunistic pathogen Legionella was stimulated and occurred in approximately 40% relative abundance at the higher specific ozone dose of 1. Accordingly, the membrane filtration system with a specific ozone dose of 0.5 presented a lower biofilm thickness, a weaker fluorescence intensity, smaller concentrations of polysaccharides and proteins, and a lower Raman activity, leading to a lower hydraulic resistance, compared to that with a specific ozone dose of 1. Our findings highlight the interaction mechanism between molecular-level DOM composition, biofilm microbial consortium, and membrane filtration performance, which provides an in-depth understanding of the impact of ozonation on biofilm accumulation.

Responses of composition and metabolism of microbial communities during the remediation of black and odorous water using bioaugmentation and aeration.

This study elucidated the effect patterns of aeration and bioaugmentation on indigenous microbial communities, metabolites, and metabolic pathways in the remediation of black and odorous water. This is crucial for the precise formulation and targeted development of effective microbial consortia, as well as for tracking and forecasting the bioremediation of black and odorous water. The results confirmed that combining bioaugmentation with aeration markedly enhanced the degradation of COD, NH4+-N, and TN and the conversion of Fe and Mn. Aeration significantly increased the relative abundance of Flavobacterium and Diaphorobacter, and the positive interbacterial interaction in the effective microbial consortia EM31 gave the constituent strain Klebsiella and Bacillus a dominant niche in the bioaugmentation. Furthermore, bioaugmentation improved the capacity of the indigenous microbial consortia to utilize basic carbon source, particularly the utilization of L-glycerol, I-erythritol, glucose-1-phosphate, and the catabolism of cysteine and methionine. Moreover, during the remediation of black and odorous water by aeration and bioaugmentation, Glucosinolate biosynthesis (map00966), Steroid hormone biosynthesis (map00140), Folate biosynthesis (map00790), One carbon pool by folate (map00670), and Tyrosine metabolism (map00350) were identified as key functional metabolic pathways in microbial communities.

Top-down and bottom-up microbiome engineering approaches to enable biomanufacturing from waste biomass.

Growing environmental concerns and the need to adopt a circular economy have highlighted the importance of waste valorization for resource recovery. Microbial consortia-enabled biotechnologies have made significant developments in the biomanufacturing of valuable resources from waste biomass that serve as suitable alternatives to petrochemical-derived products. These microbial consortia-based processes are designed following a top-down or bottom-up engineering approach. The top-down approach is a classical method that uses environmental variables to selectively steer an existing microbial consortium to achieve a target function. While high-throughput sequencing has enabled microbial community characterization, the major challenge is to disentangle complex microbial interactions and manipulate the structure and function accordingly. The bottom-up approach uses prior knowledge of the metabolic pathway and possible interactions among consortium partners to design and engineer synthetic microbial consortia. This strategy offers some control over the composition and function of the consortium for targeted bioprocesses, but challenges remain in optimal assembly methods and long-term stability. In this review, we present the recent advancements, challenges, and opportunities for further improvement using top-down and bottom-up approaches for microbiome engineering. As the bottom-up approach is relatively a new concept for waste valorization, this review explores the assembly and design of synthetic microbial consortia, ecological engineering principles to optimize microbial consortia, and metabolic engineering approaches for efficient conversion. Integration of top-down and bottom-up approaches along with developments in metabolic modeling to predict and optimize consortia function are also highlighted. ONE-SENTENCE SUMMARY: This review highlights the microbial consortia-driven waste valorization for biomanufacturing through top-down and bottom-up design approaches and describes strategies, tools, and unexplored opportunities to optimize the design and stability of such consortia.

Degradation of multiple PAHs and co-contaminants by microbial consortia and their toxicity assessment.

The anthropogenic activities toward meeting the energy requirements have resulted in an alarming rise in environmental pollution levels. Among pollutants, polycyclic aromatic hydrocarbons (PAHs) are the most predominant due to their persistent and toxic nature. Amidst the several pollutants depuration methods, bioremediation utilizing biodegradation is the most viable alternative. This study investigated the biodegradation efficacy using developed microbial consortium PBR-21 for 2-4 ringed PAHs named naphthalene (NAP), anthracene (ANT), fluorene (FLU), and pyrene (PYR). The removal efficiency was observed up to 100 ± 0.0%, 70.26 ± 4.2%, 64.23 ± 2.3%, and 61.50 ± 2.6%, respectively, for initial concentrations of 400 mg L-1 for NAP, ANT, FLU, and PYR respectively. Degradation followed first-order kinetics with rate constants of 0.39 d-1, 0.10 d-1, 0.08 d-1, and 0.07 d-1 and half-life t 1 / 2  of 1.8 h, 7.2 h, 8.5 h, and 10 h, respectively. The microbial consortia were found to be efficient towards the co-contaminants with 1 mM concentration. Toxicity examination indicated that microbial-treated PAHs resulted in lesser toxicity in aquatic crustaceans (Artemia salina) than untreated PAHs. Also, the study suggests that indigenous microbial consortia PBR-21 has the potential to be used in the bioremediation of PAH-contaminated environment.

Synergic association of the consortium Arthrospira maxima with the microalga growth-promoting bacterium Azospirillum cultured under the stressful biogas composition.

The present study evaluates the association of the blue-green microalga Arthrospira maxima (Spirulina), which is known for its CO2 fixation, biomass, and high-value metabolite production, with the microalga growth-promoting bacterium Azospirillum brasilense under the stressful composition of biogas. The results demonstrated that A. maxima co-cultured with A. brasilense under the high CO2 (25%) and methane (CH4; 75%) concentrations of biogas recorded a CO2 fixation rate of 0.24 ± 0.03 g L-1 days-1, thereby attaining a biomass production of 1.8 ± 0.03 g L-1. Similarly, the biochemical composition quality of this microalga enhanced the attainment of higher contents of carbohydrates, proteins, and phycocyanin than cultured alone. However, metabolites other than tryptophan (Trp) and indole-3-acetic acid could have supported this beneficial interaction. Overall, the results demonstrate that this prokaryotic consortium of A. maxima-A. brasilense established a synergic association under biogas, which represents a sustainable strategy to improve the bio-refinery capacity of this microalga and increase the usefulness of A. brasilense in multiple economic sectors.

Effect of loading rate and pH on glycerol fermentation and microbial population in an upflow anaerobic filter reactor.

A reactor with silicone tubes as support medium was used for glycerol fermentation. The experimental set-up consisted of three phases. In P1, the applied glycerol loading rate (gly-LR) was in the range of 6-10 g.L-1.d-1 at an influent pH of 7.9 ± 0.4. In P2, gly-LR was kept constant (18.0 ± 1.8 g.L-1.d-1) with different doses of NaHCO3. Finally in P3, two different gly-LR (9 and 18 g.L-1.d-1) were evaluated, dosing 1 g-NaHCO3 per g-COD of glycerol. Glycerol consumption was close 90%. The main end-product was 1,3-propanediol (1,3-PDO) (0.40 mol.mol-gly-1), but ethanol was also generated, particularly at pH above 8 and low gly-LR (0.20 mol.mol-gly-1). After 1-year operation with glycerol as the only carbon source, a drastic shift in the bacterial community was observed. The 1,3-PDO producers Lacrimispora and Clostridium became dominant, although non-glycerol-degrading fermentative genera, e.g., Actinomyces and Eubacterium, thrived at the expense of cellular breakdown products.

Metagenomic investigations into the microbial consortia, degradation pathways, and enzyme systems involved in the biodegradation of plastics in a tropical lentic pond sediment.

The exploitation of exciting features of plastics for diverse applications has resulted in significant plastic waste generation, which negatively impacts environmental compartments, metabolic processes, and the well-being of aquatic ecosystems biota. A shotgun metagenomic approach was deployed to investigate the microbial consortia, degradation pathways, and enzyme systems involved in the degradation of plastics in a tropical lentic pond sediment (APS). Functional annotation of the APS proteome (ORFs) using the PlasticDB database revealed annotation of 1015 proteins of enzymes such as depolymerase, esterase, lipase, hydrolase, nitrobenzylesterase, chitinase, carboxylesterase, polyesterase, oxidoreductase, polyamidase, PETase, MHETase, laccase, alkane monooxygenase, among others involved in the depolymerization of the plastic polymers. It also revealed that polyethylene glycol (PEG), polyhydroxyalkanoates (PHA), polyhydroxybutyrate (PHB), polylactic acid (PLA), polybutylene adipate terephthalate (PBAT), polyethylene terephthalate (PET), and nylon have the highest number of annotated enzymes. Further annotation using the KEGG GhostKOALA revealed that except for terephthalate, all the other degradation products of the plastic polymers depolymerization such as glyoxylate, adipate, succinate, 1,4-butanediol, ethylene glycol, lactate, and acetaldehyde were further metabolized to intermediates of the tricarboxylic acid cycle. Taxonomic characterization of the annotated proteins using the AAI Profiler and BLASTP revealed that Pseudomonadota members dominate most plastic types, followed by Actinomycetota and Acidobacteriota. The study reveals novel plastic degraders from diverse phyla hitherto not reported to be involved in plastic degradation. This suggests that plastic pollution in aquatic environments is prevalent with well-adapted degrading communities and could be the silver lining in mitigating the impacts of plastic pollution in aquatic environments.

Inter-species interaction of bradyrhizobia affects their colonization and plant growth promotion in Arachis hypogaea.

Bradyrhizobia are the principal symbiotic partner of the leguminous plant and take active part in biological nitrogen-fixation. The present investigation explores the underlying competition among different strains during colonization in host roots. Six distinct GFP and RFP-tagged Bradyrhizobium strains were engineered to track them inside the peanut roots either independently or in combination. The Bradyrhizobium strains require different time-spans ranging from 4 to 21 days post-infection (dpi) for successful colonization which further varies in presence of another strain. While most of the individual strains enhanced the shoot and root dry weight, number of nodules, and nitrogen fixation capabilities of the host plants, no significant enhancement of plant growth and nodulation efficiency was observed when they were allowed to colonize in combinations. However, if among the combinations one strains is SEMIA 6144, the co-infection results in higher growth and nodulation efficiency of the hosts. From the competition experiments it has been found that Bradyrhizobium japonicum SEMIA 6144 was found to be the most dominant strain for effective nodulation in peanut. The extent of biofilm and exopolysaccharide (EPS) production by these isolates, individually or in combinations, were envisaged to correlate whether these parameters have any impact on the symbiotic association. But the extent of colonization, growth-promotion and nitrogen-fixation ability drastically lowered when a strain present together with other Bradyrhizobium strain. Therefore, it is imperative to understand the interaction between two co-inoculating Bradyrhizobium species for nodulation followed by plant growth promotion to develop suitable consortia for enhancing BNF in peanut and possibly for other legumes.

Polycyclic aromatic hydrocarbon: underpinning the contribution of specialist microbial species to contaminant mitigation in the soil.

The persistence of PAHs poses a significant challenge for conventional remediation approaches, necessitating the exploration of alternative, sustainable strategies for their mitigation. This review underscores the vital role of specialized microbial species (nitrogen-fixing, phosphate-solubilizing, and biosurfactant-producing bacteria) in tackling the environmental impact of polycyclic aromatic hydrocarbons (PAHs). These resistant compounds demand innovative remediation strategies. The study explores microbial metabolic capabilities for converting complex PAHs into less harmful byproducts, ensuring sustainable mitigation. Synthesizing literature from 2016 to 2023, it covers PAH characteristics, sources, and associated risks. Degradation mechanisms by bacteria and fungi, key species, and enzymatic processes are examined. Nitrogen-fixing and phosphate-solubilizing bacteria contributions in symbiotic relationships with plants are highlighted. Biosurfactant-producing bacteria enhance PAH solubility, expanding microbial accessibility for degradation. Cutting-edge trends in omics technologies, synthetic biology, genetic engineering, and nano-remediation offer promising avenues. Recommendations emphasize genetic regulation, field-scale studies, sustainability assessments, interdisciplinary collaboration, and knowledge dissemination. These insights pave the way for innovative, sustainable PAH-contaminated environment restoration.

Metabolic engineering of human gut microbiome: Recent developments and future perspectives.

Many studies have demonstrated that the gut microbiota is associated with human health and disease. Manipulation of the gut microbiota, e.g. supplementation of probiotics, has been suggested to be feasible, but subject to limited therapeutic efficacy. To develop efficient microbiota-targeted diagnostic and therapeutic strategies, metabolic engineering has been applied to construct genetically modified probiotics and synthetic microbial consortia. This review mainly discusses commonly adopted strategies for metabolic engineering in the human gut microbiome, including the use of in silico, in vitro, or in vivo approaches for iterative design and construction of engineered probiotics or microbial consortia. Especially, we highlight how genome-scale metabolic models can be applied to advance our understanding of the gut microbiota. Also, we review the recent applications of metabolic engineering in gut microbiome studies as well as discuss important challenges and opportunities.

Co-cultivation of Saccharomyces cerevisiae strains combines advantages of different metabolic engineering strategies for improved ethanol yield.

Glycerol is the major organic byproduct of industrial ethanol production with the yeast Saccharomyces cerevisiae. Improved ethanol yields have been achieved with engineered S. cerevisiae strains in which heterologous pathways replace glycerol formation as the predominant mechanism for anaerobic re-oxidation of surplus NADH generated in biosynthetic reactions. Functional expression of heterologous phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) genes enables yeast cells to couple a net oxidation of NADH to the conversion of glucose to ethanol. In another strategy, NADH-dependent reduction of exogenous acetate to ethanol is enabled by introduction of a heterologous acetylating acetaldehyde dehydrogenase (A-ALD). This study explores potential advantages of co-cultivating engineered PRK-RuBisCO-based and A-ALD-based strains in anaerobic bioreactor batch cultures. Co-cultivation of these strains, which in monocultures showed reduced glycerol yields and improved ethanol yields, strongly reduced the formation of acetaldehyde and acetate, two byproducts that were formed in anaerobic monocultures of a PRK-RuBisCO-based strain. In addition, co-cultures on medium with low acetate-to-glucose ratios that mimicked those in industrial feedstocks completely removed acetate from the medium. Kinetics of co-cultivation processes and glycerol production could be optimized by tuning the relative inoculum sizes of the two strains. Co-cultivation of a PRK-RuBisCO strain with a Δgpd1 Δgpd2 A-ALD strain, which was unable to grow in the absence of acetate and evolved for faster anaerobic growth in acetate-supplemented batch cultures, further reduced glycerol formation but led to extended fermentation times. These results demonstrate the potential of using defined consortia of engineered S. cerevisiae strains for high-yield, minimal-waste ethanol production.

Biocontrol of plant pathogens in omics era-with special focus on endophytic bacilli.

Nearly all plants and their organs are inhabited by endophytic microbes which play a crucial role in plant fitness and stress resilience. Harnessing endophytic services can provide effective solutions for a sustainable increase in agriculture productivity and can be used as a complement or alternative to agrochemicals. Shifting agriculture practices toward the use of nature-based solutions can contribute directly to the global challenges of food security and environmental sustainability. However, microbial inoculants have been used in agriculture for several decades with inconsistent efficacy. Key reasons of this inconsistent efficacy are linked to competition with indigenous soil microflora and inability to colonize plants. Endophytic microbes provide solutions to both of these issues which potentially make them better candidates for microbial inoculants. This article outlines the current advancements in endophytic research with special focus on endophytic bacilli. A better understanding of diverse mechanisms of disease control by bacilli is essential to achieve maximum biocontrol efficacy against multiple phytopathogens. Furthermore, we argue that integration of emerging technologies with strong theoretical frameworks have the potential to revolutionize biocontrol approaches based on endophytic microbes.

Electrogenetic signaling and information propagation for controlling microbial consortia via programmed lysis.

To probe signal propagation and genetic actuation in microbial consortia, we have coopted the components of both redox and quorum sensing (QS) signaling into a communication network for guiding composition by "programming" cell lysis. Here, we use an electrode to generate hydrogen peroxide as a redox cue that determines consortia composition. The oxidative stress regulon of Escherichia coli, OxyR, is employed to receive and transform this signal into a QS signal that coordinates the lysis of a subpopulation of cells. We examine a suite of information transfer modalities including "monoculture" and "transmitter-receiver" models, as well as a series of genetic circuits that introduce time-delays for altering information relay, thereby expanding design space. A simple mathematical model aids in developing communication schemes that accommodate the transient nature of redox signals and the "collective" attributes of QS signals. We suggest this platform methodology will be useful in understanding and controlling synthetic microbial consortia for a variety of applications, including biomanufacturing and biocontainment.

Recent advances in research on biocontrol of postharvest fungal decay in apples.

Apple is the largest fruit crop produced in temperate regions and is a popular fruit worldwide. It is, however, susceptible to a variety of postharvest fungal pathogens, including Penicillium expansum, Botrytis cinerea, Botryosphaeria dothidea, Monilia spp., and Alternaria spp. Decays resulting from fungal infections severely reduce apple quality and marketable yield. Biological control utilizing bacterial and fungal antagonists is an eco-friendly and effective method of managing postharvest decay in horticultural crops. In the current review, research on the pathogenesis of major decay fungi and isolation of antagonists used to manage postharvest decay in apple is presented. The mode of action of postharvest biocontrol agents (BCAs), including recent molecular and genomic studies, is also discussed. Recent research on the apple microbiome and its relationship to disease management is highlighted, and the use of additives and physical treatments to enhance biocontrol efficacy of BCAs is reviewed. Biological control is a critical component of an integrated management system for the sustainable approaches to apple production. Additional research will be required to explore the feasibility of developing beneficial microbial consortia and novel antimicrobial compounds derived from BCAs for postharvest disease management, as well as genetic approaches, such as the use of CRISPR/Cas9 technology.

Enrichment of microbial consortia for MEOR in crude oil phase of reservoir-produced liquid and their response to environmental disturbance.

Developing microbial consortiums is necessary for microbial enhanced oil recovery (MEOR) in heavy crude oil production. The aqueous phase of produced fluid has long been considered an ideal source of microorganisms for MEOR. However, it is recently found that rich microorganisms (including hydrocarbon-degrading bacteria) are present in the crude oil phase, which is completely different from the aqueous phase of produced fluid. So, in this study, the microbial consortia from the crude oil phase of produced fluids derived from four wells were enriched, respectively. The microbial community structure during passage was dynamically tracked, and the response of enriched consortia to successive disturbance of environmental factors was investigated. The results showed the crude oil phase had high microbial diversity, and the original microbial community structure from four wells was significantly different. After ten generations of consecutive enrichment, different genera were observed in the four enriched microbial consortia, namely, Geobacillus, Bacillus, Brevibacillus, Chelativorans, Ureibacillus, and Ornithinicoccus. In addition, two enriched consortia (eG1614 and eP30) exhibited robustness to temperature and oxygen perturbations. These results further suggested that the crude oil phase of produced fluids can serve as a potential microbial source for MEOR.

Correlation between Bacterial Cell Density and Abundance of Antibiotic Resistance on Milking Machine Surfaces Assessed by Cultivation and Direct qPCR Methods.

The relative abundance of antibiotic-resistant bacteria and antibiotic-resistance genes was surveyed for different parts of a milking machine. A cultivation approach based on swab samples showed a highly diverse microbiota, harboring resistances against cloxacillin, ampicillin, penicillin, and tetracycline. This approach demonstrated a substantial cloxacillin resistance of numerous taxa within milking machine microbiota coming along with regular use of cloxacillin for dry-off therapy of dairy cows. For the less abundant tetracycline-resistant bacteria we found a positive correlation between microbial cell density and relative abundance of tetracycline-resistant microorganisms (R2 = 0.73). This indicated an accelerated dispersion of resistant cells for sampling locations with high cell density. However, the direct quantification of the tetM gene from the swap samples by qPCR showed the reverse relation to bacterial density if normalized against the abundance of 16S rRNA genes (R2 = 0.88). The abundance of 16S rRNA genes was analyzed by qPCR combined with a propidium monoazide treatment, which eliminates 16S rRNA gene signals in negative controls.