Natural Microbial Populations Research Articles

Ecological drivers of CRISPR immune systems.

Microbes must constantly defend themselves against viral pathogens, and a large proportion of prokaryotes do so using the highly effective CRISPR-Cas adaptive immune system. However, many prokaryotes do not. We investigated the ecological factors behind this uneven distribution of CRISPR-Cas immune systems in natural microbial populations. We found strong patterns linking CRISPR-Cas systems to prokaryotic density within ocean environments and to prokaryotic diversity within human oral environments. Our study validates previous within-lab experimental results that suggested these factors might be important and confirms that local environment and ecological context interact to select for CRISPR immunity.

Extremely halophilic brine community manipulation shows higher robustness of microbiomes inhabiting human-driven solar saltern than naturally driven lake.

Viruses greatly influence succession and diversification of their hosts, yet the effects of viral infection on the ecological dynamics of natural microbial populations remain poorly understood, especially at finer scales of diversity. By manipulating the viral predation pressure by autochthonous and allochthonous viruses, we uncovered potential phage-host interaction, and their important role in structuring the prokaryote community at an ecotype level.

Engineering plasmid copy number heterogeneity for dynamic microbial adaptation.

Natural microbial populations exploit phenotypic heterogeneity for survival and adaptation. However, in engineering biology, limiting the sources of variability is a major focus. Here we show that intentionally coupling distinct plasmids via shared replication mechanisms enables bacterial populations to adapt to their environment. We demonstrate that plasmid coupling of carbon-metabolizing operons facilitates copy number tuning of an essential but burdensome construct through the action of a stably maintained, non-essential plasmid. For specific cost-benefit situations, incompatible two-plasmid systems can stably persist longer than compatible ones. We also show using microfluidics that plasmid coupling of synthetic constructs generates population-state memory of previous environmental adaptation without additional regulatory control. This work should help to improve the design of synthetic populations by enabling adaptive engineered strains to function under changing growth conditions without strict fine-tuning of the genetic circuitry.

Ecological drivers of CRISPR immune systems.

2Microbes must constantly defend themselves against viral pathogens, and a large proportion of prokaryotes do so using the highly effective CRISPR-Cas adaptive immune system. However, many prokaryotes do not. We investigated the ecological factors behind this uneven distribution of CRISPR-Cas immune systems in natural microbial populations. We found strong patterns linking CRISPR-Cas systems to prokaryotic density within ocean environments and to prokaryotic diversity within human oral environments. Our study validates previous within-lab experimental results that suggested these factors might be important and confirms that local environment and ecological context interact to select for CRISPR immunity.

Antimicrobial screening of pecan shell extract and efficacy of pecan shell extract-pullulan coating against Listeria monocytogenes, Salmonella enterica, and Staphylococcus aureus on blueberries

Pecan shell is considered an agricultural waste; however, it contains various bioactive compounds with potential inhibitory effect against microorganisms. This study evaluated the antimicrobial efficacy of pecan shell extract (PSE) in vitro using disc-diffusion method and in vivo on blueberries as an antimicrobial coating using pullulan. For in vitro study, 5 and 10 % of aqueous (A-PSE) or ethanol pecan shell extract (E-PSE) incorporated into pullulan film were tested against different bacterial and fungal strains. Pullulan film disc was used as control. The diameter of growth inhibition (mm) around discs was measured. For in vivo study, PSE-P (5 % w/v aqueous pecan shell extract+5 % w/v pullulan), P (5 % w/v pullulan) or control (water) were spray coated on blueberries inoculated (∼5 log CFU/g) with Listeria monocytogenes, Salmonella enterica or Staphylococcus aureus; and stored at 4 °C, 50 ± 10 % RH for 15 days. The effect of antimicrobial coating against pathogens and its impact on quality during storage were determined. A-PSE and E-PSE films were more effective against Gram-positive bacteria and showed no antifungal effect at tested concentrations when evaluated in vitro. Immediately after coating on blueberries, PSE-P significantly reduced Listeria monocytogenes by 2 log CFU/g and lowered survival until day 5 than control or P. Native aerobic microbial population was reduced (P < 0.05) by 0.7 log CFU/g immediately after coating. PSE-P coating or storage time had no significant effect (P > 0.05) on the survival of S. enterica, S. aureus and native fungal population. PSE-P maintained the firmness of blueberry with no significant effect on its color, TSS, and pH during 15 days storage. PSE-P significantly reduced (P < 0.05) the spoilage rate by 21 % than control (28.5 %) and P (37 %); and minimized (P < 0.05) weight loss during storage. Pecan shell extracts show promise as a potential antimicrobial compound whose application on various food products or packaging material could be further explored.

From Microbes to Immunity: A Comprehensive Review of Microbiome Modulation

The host environment and the native microbial population, or microbiota, make up the microbiome, which is changing how medical professionals see pathogens in connection to human disease and health. The discovery that the majority of bacteria in human bodies perform ecosystem-critical tasks that benefit the entire microbial host system is perhaps the most fundamental development. The microbiome is the broad term for the diverse and abundant population of bacteria found in the gastrointestinal system. This ecosystem contains billions of microbial cells, the majority of which are essential to the preservation of human health. Nutrient consumption, immunology, digestion, and metabolism have all been related to the microbiome. Scientific research has recently established a correlation between alterations in the microbiome and the development of cancer, obesity, inflammatory pulmonary disease, and cardiovascular complications. Epithelial-intestinal microbiome modifications have a substantial impact on the development of diseases and human health. Numerous factors contribute to these changes, such as underlying medical issues and lifestyle decisions. Depending on where in the body it occurs, dysbiosis increases an organism's susceptibility to various threats. Due to the inherent diversity of the human microbiota, these bacteria carry out specific metabolic tasks and play unique roles in each anatomical location. It follows that knowledge of the microbial makeup and activities of the human microbiome in connection to health and disease is essential.

Strategies to extend the shelf life of sheep and goat cheese whey under refrigeration: Nisin, bioprotective culture, and acidification

The valorization of sheep and goat cheese whey are important from economic, social and environmental perspectives. However, their low microbiological and physical stabilities are major challenges, especially for artisanal productions. In this study, the effectiveness of nisin addition, bioprotective culture (Lacticaseibacillus casei) inoculation and direct acidification (pH 2.5–4.5) was evaluated to improve the stability of pasteurized (75 °C/5 min) sheep and goat cheese whey. All strategies were effective in controlling the native microbial population, with minimal or no change in the pH or acidity during 28 days/7 °C, whereas samples only pasteurized reached counts of up to 8 log CFU/mL. Phase separation occurred in all samples, but at different rates and intensities. Acidification at pH 3.5 and 4.5 resulted in higher creaming rates (up to 30%), even without change in fat globule size, and sedimentation (up to 6%), due to protein particle agglomeration - that increased D[3,2] values by up to 27% and reduced the specific surface area by around 20%. Conversely, acidification at pH 2.5 or the addition of nisin or L. casei resulted in samples with smaller particle size and better stability to creaming and sedimentation. The results indicated that nisin addition or L. casei inoculation are possible strategies to improve the shelf life of sheep and goat pasteurized whey, which can be used even by artisanal producers, and thus contributing for their valorization.

Frequent asymmetric migrations suppress natural selection in spatially structured populations.

Natural microbial populations often have complex spatial structures. This can impact their evolution, in particular the ability of mutants to take over. While mutant fixation probabilities are known to be unaffected by sufficiently symmetric structures, evolutionary graph theory has shown that some graphs can amplify or suppress natural selection, in a way that depends on microscopic update rules. We propose a model of spatially structured populations on graphs directly inspired by batch culture experiments, alternating within-deme growth on nodes and migration-dilution steps, and yielding successive bottlenecks. This setting bridges models from evolutionary graph theory with Wright-Fisher models. Using a branching process approach, we show that spatial structure with frequent migrations can only yield suppression of natural selection. More precisely, in this regime, circulation graphs, where the total incoming migration flow equals the total outgoing one in each deme, do not impact fixation probability, while all other graphs strictly suppress selection. Suppression becomes stronger as the asymmetry between incoming and outgoing migrations grows. Amplification of natural selection can nevertheless exist in a restricted regime of rare migrations and very small fitness advantages, where we recover the predictions of evolutionary graph theory for the star graph.

Distribution and quantification of antibiotic resistance genes in a large subalpine lake (Lugano Lake) and tributary rivers

Antibiotic resistance genes (ARGs) are causing increasing problems, especially in clinical settings. Nowadays, they are considered important environmental contaminants, but little is known about their fate in the environment or how they affect natural microbial populations. In the environment, especially in water affected by anthropic activities such as discharge of hospital, urban, and industrial wastewater treatment plant (WWTP) and agricultural runoff, antibiotic determinants may become part of the environmental gene pool, spread horizontally, and be ingested by humans and animals via contaminated food and drinking water. The aim of this work was to monitor long-term the presence of antibiotic resistance determinants in water samples collected from a subalpine lake and some tributary rivers located in the southern part of Switzerland, and to assess if anthropic activities could influence the distribution of antibiotic resistance genes present in water environments. We analysed water samples by qPCR to quantify five antibiotic resistance genes that confer resistance to the major classes of antibiotics used in clinical and veterinary settings (β-lactams, macrolides, tetracycline, quinolones, and sulphonamides). Water samples were collected from January 2016 to December 2021, from three rivers located in south Switzerland and from five different sites of Lugano Lake. The most abundant genes were sulII, followed by ermB, qnrS, and tetA; they were found especially in the river influenced by wastewater treatment plants and in the lake near the potable water uptake plant. We observed an overall decrease in the number of resistance genes during the three years. Our results indicate that the aquatic ecosystems monitored in this study are a reservoir of ARGs and could potentially be a setting for the transmission of resistance from the environment to humans.

Effect of soil solarization on tomato (Solanum lycopersicum L.) growth and impact on native microbial diversity of farm soil in Nigeria

BackgroundTomato plant leaves can be wilted by the presence of various species of soil-residing bacteria, especially Ralstonia solanacearum. Soil solarization has proven to be an environment-friendly method for disease management in various crops. Therefore, this study aimed to evaluate solarization as an effective and non-chemical way to control R. solanacearum population in farm soil cultivated with tomato plants. The tomato variety UC 82 was raised on a nursery bedding for 3 weeks, after which four solarization-based treatments were applied to the field plots where tomato plants were cultivated subsequently. Agronomic, pathological, and soil temperature data were recorded from the soil samples, while isolation, Gram staining, morphological, biochemical, and physicochemical analyses were carried out on the same soil samples.ResultsThe bacterial species identified from the pre-experiment soil included Enterobacter cloacae, Serratia marcescens, and Proteus mirabilis, while for the post-experiment were Citrobacter freundii, Klebsiella pneumoniae, P. mirabilis, Salmonella sp., and Citrobacter diversus. Occurrences of bacteria and fungi populations in solarized soils were R. solanacearum, Aspergillus niger, Aspergillus flavus, Penicillium, Rhizopus spp., Actinomycetes, and yeast.ConclusionsThe results obtained showed that solarization reduced the native soil microbial populations since the solarized soils had a lower occurrence of bacteria and fungi than the non-solarized soils. Thus, the present study suggests that solarization is effective in reducing the pathogenic bacteria population on farm soils.

Growth Substrate and Prophage Induction Collectively Influence Metabolite and Lipid Profiles in a Marine Bacterium.

ABSTRACTBacterial growth substrates influence a variety of biological functions, including the biosynthesis and regulation of lipid intermediates. The extent of this rewiring is not well understood nor has it been considered in the context of virally infected cells. Here, we used a one-host-two-temperate phage model system to probe the combined influence of growth substrate and phage infection on host carbon and lipid metabolism. Using untargeted metabolomics and lipidomics, we reported the detection of a suite of metabolites and lipid classes for two Sulfitobacter lysogens provided with three growth substrates of differing complexity and nutrient composition (yeast extract/tryptone [complex], glutamate and acetate). The growth medium led to dramatic differences in the detectable intracellular metabolites, with only 15% of 175 measured metabolites showing overlap across the three growth substrates. Between-strain differences were most evident in the cultures grown on acetate, followed by glutamate then complex medium. Lipid distribution profiles were also distinct between cultures grown on different substrates as well as between the two lysogens grown in the same medium. Five phospholipids, three aminolipid, and one class of unknown lipid-like features were identified. Most (≥94%) of these 75 lipids were quantifiable in all samples. Metabolite and lipid profiles were strongly determined by growth medium composition and modestly by strain type. Because fluctuations in availability and form of carbon substrates and nutrients, as well as virus pressure, are common features of natural systems, the influence of these intersecting factors will undoubtedly be imprinted in the metabolome and lipidome of resident bacteria.IMPORTANCE Community-level metabolomics approaches are increasingly used to characterize natural microbial populations. These approaches typically depend upon temporal snapshots from which the status and function of communities are often inferred. Such inferences are typically drawn from lab-based studies of select model organisms raised under limited growth conditions. To better interpret community-level data, the extent to which ecologically relevant bacteria demonstrate metabolic flexibility requires elucidation. Herein, we used an environmentally relevant model heterotrophic marine bacterium to assess the relationship between growth determinants and metabolome. We also aimed to assess the contribution of phage activity to the host metabolome. Striking differences in primary metabolite and lipid profiles appeared to be driven primarily by growth regime and, secondarily, by phage type. These findings demonstrated the malleable nature of metabolomes and lipidomes and lay the foundation for future studies that relate cellular composition with function in complex environmental microbial communities.

Effect of jambu mawar [Syzygium jambos (L.) Alston] leaves extract on natural microbial populations in food

Raw food materials can be contaminated with foodborne pathogens and foodborne spoilage. Washing raw food material with water can reduce microbial loading in raw food material prior to the process, but not eliminate it. Due to food safety issues, synthetic food sanitisers are currently being avoided. Natural food sanitisers such as plant extracts are now widely researched and may be used. Jambu mawar [Syzygium jambos (L.) Alston] leaves extract has been reported for its medicinal uses and treatment of a variety of illnesses. The current study aimed to evaluate the effectiveness of jambu mawar leaves extract in order to determine its suitability as a natural food sanitiser. There were three different concentrations (0.05%, 0.50% and 5.00%) of jambu mawar leaves extract solutions used to treat shrimp and cherry tomatoes with immersion method at different exposure times (5 mins and 15 mins). Moreover, the treated samples were stored at different temperatures (25±2oC, 4±2oC, and -18±2oC) for 23 days. The results showed that washing with tap water for 5 mins and 15 mins, was not able to reduce the population of total plate count (TPC), Vibrio parahaemolyticus, Staphylococcus aureus and Klebsiella pneumoniae in food samples. In contrast, washing with 0.05% for 5 mins can significantly reduce the microorganism population in food samples. If the concentration is increased to 5.00% the microbial population in the food samples can be removed completely. Correspondingly, the extract can also inhibit or kill the microbial population in raw food materials at different temperatures. It can be concluded that S. jambos (L.) Alston leaves extract can reduce or eliminate the microbial population in raw food materials. These results suggested that this extract might be developed as a natural food sanitiser.

Recombination resolves the cost of horizontal gene transfer in experimental populations of Helicobacter pylori

Horizontal gene transfer (HGT) is important for microbial evolution, yet we know little about the fitness effects and dynamics of horizontally transferred genetic variants. In this study, we evolve laboratory populations of Helicobacter pylori, which take up DNA from their environment by natural transformation, and measure the fitness effects of thousands of transferred genetic variants. We find that natural transformation increases the rate of adaptation but comes at the cost of significant genetic load. We show that this cost is circumvented by recombination, which increases the efficiency of selection by decoupling deleterious and beneficial genetic variants. Our results show that adaptation with HGT, pervasive in natural microbial populations, is shaped by a combination of selection, recombination, and genetic drift not accounted for in existing models of evolution.

Development of a bacterial consortium from Variovorax paradoxus and Pseudomonas veronii isolates applicable in the removal of BTEX

In this study, we report on the development of a novel bacterial consortium, consisting of Variovorax paradoxus and Pseudomonas veronii isolates, applicable in the biodegradation of all six BTEX compounds (benzene, toluene, ethylbenzene, o-, m- and p-xylene) and the bioremediation of contaminated sites. The co-cultivability of the selected bacterial isolates was determined in nutrient-rich medium, as well as in BTEX amended mineral salts solution using Terminal Restriction Fragment Length Polymorphism (T-RFLP) and CFU determinations. BTEX biodegradation capacity of the two-strain consortium was assessed in mineral salts solution, where a series of BTEX depletions and supplementations occurred, as well as in a real, BTEX polluted environmental sample (contaminated groundwater) in the presence of the autochthonous bacterial community. The obtained results indicated that the developed bacterial consortium is very efficient in BTEX biodegradation. Under laboratory conditions, the acclimatized bacterial consortium completely degraded the BTEX mixture with a concentration as high as 20 mg l−1 in a mineral salt medium within a short span of 6 h. Close to in situ groundwater conditions (incubated at 15 °C under static conditions in the absence of light), groundwater microcosms containing the autochthonous bacterial community inoculated with the developed bacterial consortium showed more efficient toluene, o-, m-and p-xylene biodegradation capacity than microcosms containing solely the native microbial population originally found in the groundwater. In the inoculated microcosms, after 115 h of incubation the concentration (~ 1.7 mg l−1 each) of o-, m- and p-xylene decreased to zero, whereas in the non-inoculated microcosms the concentration of xylene isomers was still 0.2, 0.3 and 0.3 mg l−1, respectively. The allochthonous bioaugmentation of the contaminated groundwater with the obtained inoculant was successful and manifested in a better BTEX degradation rate. Our results suggest that the obtained bacterial consortium can be a new, stable and efficient bioremediation agent applicable in the synergistic elimination of BTEX compounds from contaminated sites.

A three-step approach to assess efficacy of alternative chemical treatments to preserve fresh fruit juices: Application to pineapple (Ananas comosus ‘Queen Victoria’)

Fresh pineapple juice exhibits a shelflife of a few days limited by the development of yeasts and molds. An approach was implemented to assess the efficacy of treatments of juice, based on three successive steps. The first step was to validate the use of a microbial cocktail which leads to juice spoilage. In a second step, the efficacy of treatments was assessed on the growth of fungi in two conditions. The treatment chosen was the addition of Thymus leptobotrys (Tl) and Thymus maroccanus (Tm) essential oils (EO). These extracts were mainly composed of the monoterpene carvacrol, and inhibited fungal growth in culture medium. The addition of EO to pasteurized juice inoculated with a fungal cocktail limited fungal population below 4.6 log CFU/mL during 14 days. The low pH of pineapple juice probably strengthened the effect of antifungal compounds acting on proton gradient across plasma membrane. Lastly, the efficacy of the treatments was assessed in fresh pineapple juice harboring its natural microbial population. The extracts TlEO at 0.25‰ or TmEO at 0.05‰ limited fungal growth below 5 log CFU/mL during 14 days. Thyme scent was detected after EO addition but the characteristic descriptors of pineapple and sugary were maintained.

Effects of radio frequency heating on microbial populations and physicochemical properties of buckwheat

Microbial contamination is a persistent problem for grain industry. Many studies have shown that radio frequency (RF) heating can effectively reduce pathogens populations in low moisture foods, but there is a lack on the efficacy to decontaminate natural microbiome. The main objectives of this study were to investigate the effects of different RF heating conditions on natural microbial populations and physicochemical properties of buckwheat. In this study, 30 buckwheat samples collected from 10 different Provinces in China were analyzed for their microbial loads, and the samples with the highest microbial populations were used for further study to select the suitable RF heating conditions. The results showed that microbial loads in tested buckwheat kernels were in the range of 3.4–6.2 log CFU/g. Samples from Shanxi (SX-3) had significantly higher microbial counts than other samples. The selected four temperature-time combinations: 75 °C-20 min, 80 °C-10 min, 85 °C-5 min, and 90 °C-0 min of RF heating could reduce microbial counts to <3.0 log CFU/g in buckwheat kernels at 16.5% w.b. moisture content. Furthermore, the reduction populations of the inoculated pathogens (Salmonella Typhimurium, Escherichia coli, Cronobacter sakazakii, and Bacillus cereus) reached 4.0 log CFU/g under the above conditions, and almost 5.0 log CFU/g especially at high temperature-short holding time combinations (85 °C-5 min and 90 °C-0 min). Besides, physicochemical properties evaluation also showed the insignificant color changes and nutrients loss after RF treatment at 90 °C-0 min. Therefore, the RF heating at 90 °C-0 min holds greater potential than the other lower temperature-longer holding time combinations for applications in buckwheat pasteurization.

Heterotrophic bacteria outcompete diazotrophs for orthophosphate in the Mediterranean Sea

AbstractUnicellular photoautotrophic diazotrophs such as Crocosphaera spp. are ubiquitous in many oligotrophic and N‐limited oceans, as they can reduce N2 into bioavailable ammonia. The Mediterranean Sea is potentially an ideal environment for photoautotrophic diazotrophic activity, and yet N2‐fixation rates measured in the last two decades are typically very low and no diazotrophic blooms have been recorded in its offshore waters. Previous studies suggest that diazotrophs, as well as nondiazotrophic phytoplankton and heterotrophic bacteria, may be P‐limited, hence their low biomass and activity. Here, we amended surface seawater from six stations across a nutrient gradient in the Mediterranean Sea (east to west transect) with dissolved inorganic phosphorus (DIP), and with seawater‐acclimated inocula of Crocosphaera watsonii, a unicellular cyanobacterial diazotroph, to examine if DIP can stimulate diazotrophy. Our results demonstrate that C. watsonii are poor competitors for DIP relative to native nondiazotrophic heterotrophic microbial populations, especially in the ultraoligotrophic eastern Mediterranean basin, resulting in low N2‐fixation rates. Moreover, the results indicate that when the ambient DIP concentrations are &lt; 35 nmol L−1, unicellular photoautotrophic diazotrophs such as C. watsonii will likely be outcompeted for this macronutrient in the Mediterranean Sea, whereas above 35 nmol L−1 diazotrophy can be stimulated. Our findings support the “bypass theory” stating that photoautotrophs may be outcompeted by heterotrophic bacteria for DIP in nutrient‐poor regions such as the Mediterranean Sea.

Effectiveness of Permeable Reactive Bio-Barriers for Bioremediation of an Organohalide-Polluted Aquifer by Natural-Occurring Microbial Community

In this study, a bioremediation approach was evaluated for the decontamination of an aquifer affected by the release of organohalides by an industrial landfill. After preliminary physicochemical and microbiological characterization of the landfill groundwater, the stimulation of natural organohalide respiration by the addition of a reducing substrate (i.e., molasse) was tested both at microcosm and at field scales, by the placement of an anaerobic permeable reactive bio-barrier. Illumina sequencing of cDNA 16S rRNA gene revealed that organohalide-respiring bacteria of genera Geobacter, Sulfurospirillum, Dehalococcoides, Clostridium and Shewanella were present within the aquifer microbial community, along with fermentative Firmicutes and Parvarchaeota. Microcosm experiments confirmed the presence of an active natural attenuation, which was boosted by the addition of the reducing substrate. Field tests showed that the bio-barrier decreased the concentration of chloroethenes at a rate of 23.74 kg d−1. Monitoring of organohalide respiration biomarkers by qPCR and Illumina sequencing revealed that native microbial populations were involved in the dechlorination process, although their specific role still needs to be clarified. The accumulation of lower-chloroethenes suggested the need of future improvement of the present approach by supporting bacterial vinyl-chloride oxidation, to achieve a complete degradation of chloroethenes.

Effect of the Dry-Cured Fermented Sausage "Salchichón" Processing with a Selected Lactobacillus sakei in Listeria monocytogenes and Microbial Population.

In the present work, the effect of processing of dry-cured fermented sausage “salchichón” spiked with the selected Lactobacillus sakei 205 was challenge-tested with low and high levels of L. monocytogenes. The evolution of the natural microbial population throughout the “salchichón” ripening was also evaluated. For this, a total of 150 “salchichón” were elaborated and divided into six equal cases which were inoculated with different levels of L. monocytogenes, and L. sakei 205. Afterwards, sausages were ripened for 90 days according to a typical industrial process. Moisture content (%) and water activity (aw) decreased throughout the ripening up to values around 26% and 0.78, respectively. No differences for moisture content, aw, pH, NaCl and nitrite concentration were observed between the analyzed cases. Lactic acid bacteria counts in the L. sakei 205 inoculated cases were always higher than 6 log CFU g−1 during ripening. Enterobacteriaceae counts were reduced during ripening until non-detectable levels at the end of processing. Reductions in L. monocytogenes counts ranged from 1.6 to 2.2 log CFU g−1; therefore, the processing of “salchichón” itself did not allow the growth of this pathogen. Reduction in L. monocytogenes was significantly higher in the cases inoculated with L. sakei 205.

Soil microbial community characteristics in a vineyard ten years after fumigation

AbstractCalifornia is the number one producer of grapes in the United States. Soil fumigation has been used to control soilborne pests and pathogens. However, fumigants can potentially have a negative impact on native soil microbial populations. Given the roles of microorganisms in the soil system, it is important to assess the long‐term impact of fumigation on soil microbial communities, which is currently lacking. The objective of this research was to study the long‐term impact of microbial communities in grapevine (Vitis vinifera L.) replanting situations after fumigation. Soil treatments included methyl bromide (MeBr), Telone C35, InLine, a nonfumigated control, and a mustard (Sinapis alba L.) cover crop in two side‐by‐side field experiments fumigated in 2007 and 2008. Microbial community biomass in 2017 and 2018 did not differ between fumigated and nonfumigated plots at the 0‐ to 30‐cm depth. However, plots with shank‐injected MeBr and Telone C35 applied in 2008 showed significantly reduced microbial biomass in 2018 at deeper depths than subsurface drip‐applied InLine plots. The results show the impact of fumigation on bacterial communities; however, the impact on fungal and arbuscular mycorrhizal fungi (AMF) communities was not evident after 10 years. Furthermore, the effect of depth was apparent on bacterial communities compared with fungal and AMF communities. The data suggest that deep‐rooted grapevines influenced fungal populations, which probably supplied labile C in fumigated and nonfumigated plots. Future research needs to focus collectively on the long‐term effects of fumigation on fruit quality and yield in vineyards, and on soil microbial communities.